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Destructive
characteristics of Th17 cells
A joint venture
Renoud J. Marijnissen
The research presented in this thesis was performed at the Laboratory of
Rheumatology Research & Advanced Therapeutics, Department of Rheumatology,
NCMLS, Radboud University Nijmegen Medical Centre, the Netherlands. This work was
supported by MSD (Oss, the Netherlands) and Pfizer Inc (Cambridge, MA, USA)
Financial support for publication of this thesis was kindly provided by
The Dutch Arthritis Foundation (DAF)
ISBN 978-94-6191-801-7
Design cover: Promotie In Zicht, Arnhem
Geïnspireerd op een design van Anne Besselink
Lay-out: Promotie In Zicht, Arnhem
Print: Ipskamp Drukkers, Nijmegen
Copyright © R.J. Marijnissen
All rights reserved. No parts of this publication may be reported or transmitted,
in any form or by any means, without permission of the author.
Destructive
characteristics of Th17 cells
A joint venture
Proefschrift
ter verkrijging van de graad van doctor
aan de Radboud Universiteit Nijmegen
op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann,
volgens besluit van het college van decanen
in het openbaar te verdedigen op woensdag 28 augustus 2013
om 10.30 uur precies
door
Renoud Johannes Marijnissen
geboren op 4 mei 1981
te Breda
Promotoren
Prof. dr. W.B. van den Berg
Prof. dr. A.M.H. Boots (Universitair Medisch Centrum Groningen)
Copromotoren
Dr. M.I. Koenders
Dr. L.A.B. Joosten
Dr. A.A.J. van de Loo
Manuscriptcommissie
Prof. dr. P.C.M. van de Kerkhof (voorzitter)
Prof. dr. J.W.M. van der Meer
Dr. E. Lubberts (Erasmus Medisch Centrum)
Destructive
characteristics of Th17 cells
A joint venture
Doctoral Thesis
to obtain the degree of doctor
from Radboud University Nijmegen
on the authority of the Rector Magnificus prof. dr. S.C.J.J. Kortmann,
according to the decision of the Council of Deans
to be defended in public on Wednesday, August 28, 2013
at 10.30 hours
by
Renoud Johannes Marijnissen
Born on May 4, 1981
in Breda (the Netherlands)
Supervisors
Prof. dr. W.B. van den Berg
Prof. dr. A.M.H. Boots (University Medical Center Groningen)
Co-supervisors
Dr. M.I. Koenders
Dr. L.A.B. Joosten
Dr. A.A.J. van de Loo
Doctoral Thesis Committee
Prof. dr. P.C.M. van de Kerhof (chair)
Prof. dr. J.W.M. van der Meer
Dr. E. Lubberts (Erasmus Medical Center)
Contents
Chapter 1
General introduction
Chapter 2
Exposure to Candida albicans polarizes a T-cell driven arthritis
model towards Th17 responses, resulting in a more destructive
arthritis.
PLoS ONE, 2012, 7(6): e38889
23
Chapter 3
The macrophage mannose receptor induces IL-17 in response to
Candida Albicans.
Cell Host & Microbe, 2009, 5(4): 329-340
43
Chapter 4
Tumor Necrosis Factor–Interleukin-17 interplay induces S100A8,
interleukin-1β and matrix metalloproteinases, and drives
irreversible cartilage destruction in murine arthritis.
Arthritis & Rheumatism, 2011, 63(8): 2329–2339
69
Chapter 5
Dual role of IL-21 in experimental arthritis: IL-21R-deficiency
protects against joint pathology by reducing Th1/17 cells, but
increases local expression of inflammatory mediators.
Submitted for publication
91
Chapter 6
Increased expression of interleukin-22 by synovial Th17 cells
during late stages of murine experimental arthritis is controlled by
interleukin-1 and enhances bone degradation.
Arthritis & Rheumatism, 2011, 64(10): 2939–2948
111
Chapter 7
T cell lessons from the Rheumatoid Arthritis synovium SCID mouse
model: CD3-rich synovium lacks response to CTLA-4Ig but is
successfully treated by Interleukin-17 neutralization.
Arthritis & Rheumatism, 2012, 64(6): 1762–1770
133
Chapter 8
The functional role of the Th17 cytokines in Rheumatoid Arthritis.
Manuscript in preparation
151
Chapter 9
Summary and final considerations
169
Chapter 10
Nederlandse samenvatting
181
List of abbreviations
188
Dankwoord
191
List of publications
199
Curriculum vitae
200
9
Chapter 1
General introduction
10 | Chapter 1
General introduction | 11
Introduction
Rheumatoid Arthritis (RA) is a systemic disorder, characterized by a chronic inflammatory
infiltration of the synovial membrane in multiple joints, affecting approximately 1% of the
population worldwide. Although the disease is associated with autoimmunity, the
etiology remains unknown. In many patients the arthritis is characterized by exacerbations
and remissions. A hallmark of RA is the progressive destruction of cartilage and bone due
to chronic inflammation (1). In the synovial membrane from joints of patients suffering
from RA you will typically find hyperplasia, increased vascularity and influx of inflammatory
cells, like macrophages, plasma cells and T cells (figure 1) (2). Among the T-cells present in
the inflamed synovium of RA patients, CD4+ T cells (T lymphocytes expressing CD4
glycoprotein on their cell surface) are the dominant T-cells (3).
Under physiological conditions, CD4+ T cells instruct immune cells to respond to
infections and are therefore named T helper or Th cells. T helper cells can be divided into
three effector subsets, categorized as Th1, Th2, and Th17 cells, which each in their own way
activate the immune system and protect against pathogens. In addition, T helper cells can
differentiate into regulatory subsets (Tregs) that control effector T cell responses and
Healthy joint
Arthritic joint
Immune cell
infiltration
Muscle
Hyperplasia
Fibroblast
Tendon
Macrophage
Synovium
Angiogenesis
Cartilage
Cyto/Chemokine
secretion
Osteoclast Pannus
Chondrocyte
Bone
Figure 1 | Schematic presentation of a healthy knee joint and the processes that take place during
the development of arthritis, ultimately leading to joint destruction and loss of function (2).
12 | Chapter 1
prevent autoimmunity (figure 2). After the activation of naïve T-cells by antigen presenting
cells, the cytokine micro-environment determines the differentiation of the cell, accompanied
by its own key transcription factors (4). Th1 cells are induced by Interleukin-12 (IL-12) that
consists of an IL-12p35 and an IL-12p40 subunit, and induces the lineage-defining
transcription factor T-Bet (and Eomes) (5;6). They are characterized by the production of
high levels of interferon-γ (IFN-γ) and function by enhancing the activation of macrophages
of the innate immunity, thus playing an important role in the protection against
intracellular pathogens. The second Th subset, hence Th2 cells, are induced by IL-4, which
upregulates the lineage-defining transcription factor GATA3. Th2 cells produce IL-4, IL-5,
and IL-13 and help in the activation and class switching of B cells, and are mainly involved
in the protection against parasitic worms (helminths) (7). Subsequently, the orphan
nuclear receptor RORγT specifies the Th17 cells, which are induced by TGF-β, IL-6, IL-1 and
IL-23 (8;9). Th17 cells produce IL-17A (IL-17), IL-17F, IL-21 and IL-22 and are involved in the
clearance of certain extracellular bacteria and fungi particularly in the gut (figure 2).
Effector T helper cells
Eomes
T-BET
IL-12
IL-27
IFN-γ
Protection against intracullar
pathogens (Viruses, bacteria)
Autoimmunity, delayed-type
hypersensitivity
Th1
TCR
IL-4
IL-25
CD4
TGF-β1
IL-6
Naive T helper cell
IL-1
IL-23
GATA3
Protection against extracullar
pathogens (Parasites, bacteria)
Allergy, asthma
Th2
RORγT
TGF-β1
IL-2
IL-4
IL-5
IL-17
IL-21
IL-22
Protection against extratracullar
pathogens (Fungi, bacteria)
Autoimmunity
Th17
Regulatory T helper cells
FOXP3
TGF-β1
IL-10
Immonusuppression
Treg
Figure 2 | Differentiation of T helper cell subsets. Following activation, naïve CD4 T cells differentiate
towards Th1 in the presence of IL-12, which eventually induces the Th1 lineage transcription factor
Tbet. Th2 by contrast differentiates in response to IL-4, resulting in the induction of GATA3. The
Th17 subset develops in response to IL-6 and TGF-β and is amplified and stabilized by IL-1 and IL-23.
Signaling activates the lineage determining transcription factor RORγT. Differentiation to Treg cells
results from expression of the transcription factor FOXP3.
General introduction | 13
In addition to their protective functions against invading pathogens, T helper cells
contribute to the development of numerous human disorders. If an inflammatory
response is not adequate and/or an infection is not cleared properly, persistence of T
helper cells can eventually result in chronic or even auto-immune inflammation.
Considerable evidence supports a role for T helper cells both in the initiation as well as the
perturbation of the chronic inflammation in RA (1;10-12). In addition, the importance of T
helper cells has been strengthened by data provided from experimental arthritis models,
in which disease can be transferred to a naïve host by injecting T helper cells from an
affected animal (13). Moreover, clinical data on the therapeutic effects of Abatacept, which
targets the costimulatory binding of B7/CD28 (between APC’s and T cells), supports the
role of T helper cells in the pathogenesis of RA (14). In this thesis, we focus on the relative
contribution of Th17 cells in various experimental arthritis models and the pathogenesis in RA.
Origin and regulation of Th17 cells
The discovery of Th17 cells came from research in murine models of autoimmunity. Since
anti-IL-12 antibodies, directed against the p40 subunit of IL-12, ameloriated disease activity
in several experimental autoimmune models, arthritis was previously considered as being
mainly Th1-driven (15;16). Surprisingly, induction of experimental autoimmune encephalomyelitis (EAE) in an IFN-γ -/- and IL-12p35-/- background resulted in an increased disease
severity, while IL-12p40-/- mice did not develop disease at all (17;18). Soon it was discovered
that the IL-12p40 subunit can also form a complex with the IL-23p19 subunit, forming IL-23
(19). This clarified the contradictory data and revealed that the IL-12p40 antibody studies
had blocked both the IL-12 and IL-23 signaling pathways. Gene analysis of the IL-23
deficiency finally led to the discovery of IL-17 producing T helper cells, named Th17 cells (9).
Since then the role of Th17 cells has been investigated in many inflammatory disorders (20).
Although IL-23 is required for the maintenance, survival and expansion of Th17 cells
and crucial for the Th17 induced immunopathology in vivo, it is dispensable for IL-17
induction in vitro. In mice, activation of naïve CD4+ T cells in the presence of IL-6 and TGF-β
is sufficient to induce high levels of IL-17 production (8). IL-6 upregulates IL-21 and IL-23R in
a STAT3/ RORγT dependent manner, which finally results in the induction of IL-17A (IL-17),
IL-17F, IL-21 and IL-22 (21;22) . IL-1 drives Th17 pathogenicity, which was shown in vivo and
during the early phase of Th17 differentiation in vitro (23;24). Subsequently, IL-23 stabilizes
the Th17 phenotype during the late phase of the differentiation (25).
Features of Th17 cytokines in the pathogenesis of Rheumatoid Arthritis
IL-17A (IL-17), the key cytokine of Th17 cells, is a highly pleiotrophic cytokine that affects a
variety of cell types including macrophages, synovial fibroblasts, chondrocytes and
osteoclasts (26). IL-17 activates several MAP kinase pathways resulting in transcriptional
NFκB activity, which contributes to the induction of pro-inflammatory cytokines (27).
Depending on the target cell population, IL-17 induces chemokines like IL-8 (CXCL-8),
14 | Chapter 1
G-CSF, MCP-1 (CCL-2) and MIP-1α (CCL-3) that recruit polymorphonuclear leukocytes
(PMNs) and moncocytes/macrophages to the site of inflammation (28;29). Direct and
indirect effects result in increased angiogenesis, osteoclastogenesis and eventually
breakdown of cartilage and bone (figure 3). Several studies demonstrated that elevated
levels of IL-17 in blood and synovium of RA patients correlate with joint damage (30;31).
Deletion or blocking of IL-17 during experimental arthritis results in protection against
joint inflammation and erosion (32;33). Of most interest is the synergy between IL-17 and
TNF-α on cytokine induction and tissue destruction, since anti-TNF treatment shows great
beneficial results during the treatment of patients with RA (34-36).
Besides IL-17, Th17 cells coproduce IL-17F. IL-17 and IL-17F both bind the same IL-17 receptor
(IL-17R, a complex of the IL-17RA and IL-17RC receptor subunits) and have similar function.
Although the role of IL-17 in tissue inflammation has been studied extensively, the role of
the other Th17 cytokines remains undecided.
IL-21 is a member of the IL-2 cytokine family and is produced by activated T helper cells
and NK T cells. It signals via the IL-21R complex, which consists of the distinctive IL-21Rα
subunit and the common IL-2Rγc subunit. Binding of IL-21 to its receptor leads to the
Inflammation
TNF-α, IL-1, IL-6
Macrophage
IL-12, IL-23,
IL-6, TGF-β
IL-6, IL-8, GM-CSF
IL-1, IL-6,
IL-12, IL-23
Fibroblast
MMP’s
Cartilage erosion
Dendritic cell
NO, MMP’s
Naive T-cell
Treg
IL-6,
IL-10
Chondrocyte
Th17 (or Th1)
B cell
Bone erosion
RANKL
Osteoblast
Plasma cell
Osteoclastogenesis
Osteoclast
Autoantibodies
Figure 3 | Effector T cell interactions in the synovium of Rheumatoid Arthritis. Naïve T cells are
activated by antigen presenting cells (dendritic cells, B cells, macrophages) and home to the joint
where they stimulate all sorts of cells resulting in inflammation, cartilage and bone destruction.
General introduction | 15
phosporylation of STAT1, STAT3 and STAT5 (37;38). Although IL-21 is not solely produced by
Th17 cells, it serves as an autocrine factor that amplifies the Th17 differentiation. In addition,
it can replace IL-6 during Th17 differentiation in vitro, but with less potency. Additional
studies have demonstrated that Th17 differentiation and responses are disturbed in the
absence of IL-21 or its receptor. IL-21 acts on a variety of hematopoietic immune cells,
including T, B, DC and NK cells as well as on tissue cells including endothelial cells and
fibroblasts. On B cells, IL-21 plays an important role in immunoglobulin production and
the differentiation into plasma cells. In addition, IL-21 contributes to antibody production
indirectly via its effect on T follicular helper cell generation and germinal center formation
(39;40). On T cells, it induces proliferation and the secretion of proinflammatory cytokines
like TNF-α and IFN-γ, and enhances the expression of RANKL (41;42). Synovial fibroblasts
show increased expression of various matrix metalloproteinases (MMPs) as well as RANKL
upon IL-21 stimulation (43). Furthermore, a pathogenic role for IL-21 has been shown in
animal models of RA, where blocking of IL-21 is beneficial (44). Because the IL-21R is
increased in the synovium of RA patients and blocking endogenous IL-21 activity in RA
synovium cultures decreases inflammatory cytokine production, a role for IL-21 in the
pathogenesis of RA has been suggested (41;45).
Another important Th17 cytokine is IL-22, belonging to the IL-10 superfamily, and has been
demonstrated to promote innate immunity (46). It signals via the IL-22R (a complex
consisting of IL-22receptor alpha 1 (IL-22Ra1) and IL-10R2 and induces high phosphorylation of STAT-3, but can also activate STAT-1, STAT-5, and several MAP kinases (47). IL-22 is
produced by various types of hematopoietic cells, like Th cells, γδT cells, NK and NKT cells
(48). Interestingly, the receptor has been described to be only present on resident tissue
cells and not on hematopoietic immune cells (46). Because IL-22–producing T cells are
presents in chronically inflamed tissues, it implies that the expression of IL-22R on the tissue-resident cells determines the function and response to IL-22. Until now, most studies
have focused on the effect of IL-22 on hepatocytes and keratinocytes, inducing a wide
range of antimicrobial proteins, such as beta-defensins, S100 proteins, serum amyloid A
proteins, and matrix metalloproteinases (46;49). IL-22 plays a dual role in animal
autoimmune models of psoriasis, IBD, MS (EAE) and RA, which is not yet fully understood.
While blocking of IL-22 may be beneficial in skin disease models, blocking aggravates
disease in IBD models (50-52). In addition, IL-22-/- mice are fully susceptible to EAE (53). In a
recent study, development of collagen-induced arthritis (CIA) in IL-22-/- C57BL/6 mice
reduced disease incidence and resulted in less pannus formation (54). In human RA, the
receptor is highly expressed in the inflamed synovium and increased serum levels of IL-22
correlate with increased joint destruction over time (55-57). Further work will be required
to elucidate the role of IL-22 in the pathogenesis of rheumatic diseases.
16 | Chapter 1
Relationship between Th1 and Th17 cells in immune diseases
Although there is strong evidence from animal models that IL-17 and Th17 play an
important role in experimental arthritis, it is still uncertain whether the human disease is a
real Th17 or rather a Th1 mediated disease. In RA, more often IFN-γ producing T helper cells
are found rather than IL-17 producing T helper cells (58;59). And although clinicians do
agree on the importance of IL-17 during chronic arthritic processes, the response to
anti-IL-17 therapy in clinical trials is not as impressive as compared to the blocking in
animal models. One of the explanations might be that the biology of Th17 cells differs in
important ways between mice and humans (8;60). Besides, the initiation in the murine
experimental arthritis models and therefore the role for IL-17 can be monitored narrowly
throughout the course of arthritis. For RA patients the initiation process is yet unknown
and might have already occurred during childhood, whereas the features and diagnosis of
RA occur many years later.
Recently, the plasticity and stability of T helper cell subsets in vivo are under discussion.
To support this, in 2009 in diabetes models, transfer studies demonstrated that Th17 cells
can convert into Th1 cells (61). In addition, cells recovered from inflamed joints in Juvenile
Idiopathic Arthritis (JIA) that produced IFN-γ, expressed chemokine receptors that are
intermediate in phenotype between Th1 and Th17 cells (62). More recently, Hirota et al.
have shown that during experimental autoimmune encephalomyelitis (EAE), the IFN-γ
producing T helper cells, present in the inflamed tissue, exclusively arise from IL-17
producing T-cells. These cells still possess certain Th17 specific cell markers that are not
present on ‘classical’ Th1 cells (63). Further studies are needed to unravel the ancestry and
transcriptional control of Th17 during the course of chronic inflammatory diseases, like RA.
Outline of this thesis
Despite the vast amount of knowledge about the role of IL-17 in the pathogenesis of RA at
the start of this project, it was still unclear which cells could produce the protein and how
this is regulated. The aim of this thesis was to demonstrate the presence and function of
Th17 cells in models of experimental arthritis. Furthermore, the aim was to study the
functional role of the Th17 effector cytokines in (Rheumatoid) arthritis.
The first part of this thesis (Chapter 2 and 3) focuses on the relation between innate immune
responses by Candida Albicans and the induction of Th17 cells. C. albicans is known for its
potent induction of Th17 cells. First, we tried to induce pathogenic Th17 cells in vivo and
study there effects on joint pathology (Chapter 2). We used the chronic SCW-induced arthritis
and investigated whether co-exposure to a small amount of yeast particles on top of the
repetitive local exposure to TLR2/NOD2 agonist could boost the Th17 response and
specifically could polarize the arthritis towards a more destructive phenotype.
General introduction | 17
Parallel, we addressed the question which receptors are important for the induction
of a human Th17 response after Candida exposure (Chapter 3). Freshly isolated PBMCs were
stimulated with several pathogen-associated molecular patterns (PAMPs) and whole
(heat-killed) microorganisms in the presence of human serum, without additional
costimulatory factors. We investigated the innate immune receptors and PAMPs involved
in triggering Th17 responses during pathogen-specific host defense.
The second part of this thesis (Chapter 4-9) focuses on the role of the Th17 effector
cytokines IL-17, IL-21 and IL-22 during experimental arthritis models. Cytokine networks
play a prominent role in RA, which has been confirmed by the success of many
anti-cytokine treatments in the clinical practice. Among the pro-inflammatory cytokines,
anti-TNF-α is currently the most targeted cytokine in the treatment of RA. It has been
published that TNF-α synergizes with IL-17 in vitro. Furthermore, IL-17 plays a dominant role
in enhancing inflammation and joint destruction. In Chapter 4, we examined whether
synovial IL-17 expression promotes TNF-α-induced joint pathologic processes in vivo and
analyzed the potential of neutralizing both IL-17 and TNF-α during collagen-induced
arthritis (CIA).
Apart from there IL-17 production, Th17 cells co-express other cytokines, including
IL-21 and IL-22. IL-21 is mainly known for its role in inducing and expanding Th17 cells. In
addition, IL-21 has been studied for its regulatory effects on other immune and
non-immune cells. In Chapter 5, we investigated the potential role of IL-21 in experimental
arthritis in relation to Th17 cells. We compared local versus systemic reactions during acute
and chronically induced arthritis under IL-21R deficiency.
Classically, Th17 cells produce both IL-17 and IL-22. Understanding the role of IL-22 in
Rheumatoid Arthritis is still limited. Consequently, we evaluated whether IL-22 would be a
better target apart from IL-17. In Chapter 6, we explored the regulation of IL-22 by T helper
cells in vitro and evaluated the potential for IL-22 depletion in an experimental arthritis
model using mice deficient in the IL-1 receptor antagonist (IL-1Ra-/-). Finally, in this model
we characterized the local gene expression and source of IL-22 during this spontaneous
progressive erosive arthritis model.
The introduction of biological therapies targeting specific inflammatory mediators
have revolutionized the treatment of RA. The most frequent described biological therapies
for RA, includes antibodies agains TNF-α, CD20 (B-cell specific antigen) and CD80/CD86
(targeting inhibits co-stimulation of T cells). Although our knowledge in identifying novel
therapeutic targets from preclinical experimental arthritis studies is increasing rapidly, it is
still difficult to predict the eventual therapautic responses in patients. We therefore
investigated whether a humanized model would better predict the success of new
therapeutic agents as compared to the ‘classical’ animal models. In Chapter 7, we describe
the setup and validation of a RA synovium SCID mouse model. Initially, we tested evaluated
the potency of anti-TNF and anti-IL-1 treatment in this model. In addition, the direct effect
18 | Chapter 1
of T and B cell-related therapies on the transplanted RA synovial tissue was investigated.
Finally, Chapter 8, the potential of neutralizing the Th17 cytokines in RA are discussed. In
Chapter 9, the findings described in this thesis are summarized.
General introduction | 19
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20 | Chapter 1
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Th17 cytokine interleukin-22 in collagen-induced arthritis in C57BL/6 mice. Arthritis Rheum 2009; 60(2):390-395.
(55) Ikeuchi H, Kuroiwa T, Hiramatsu N, Kaneko Y, Hiromura K, Ueki K et al. Expression of interleukin-22 in rheumatoid
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(56) Cascao R, Moura RA, Perpetuo I, Canhao H, Vieira-Sousa E, Mourao AF et al. Identification of a cytokine network
sustaining neutrophil and Th17 activation in untreated early rheumatoid arthritis. Arthritis Res Ther 2010; 12(5):R196.
(57) Leipe J, Schramm MA, Grunke M, Baeuerle M, Dechant C, Nigg AP et al. Interleukin 22 serum levels are
associated with radiographic progression in rheumatoid arthritis. Ann Rheum Dis 2011; 70(8):1453-1457.
(58) Gullick NJ, Evans HG, Church LD, Jayaraj DM, Filer A, Kirkham BW et al. Linking power Doppler ultrasound to
the presence of th17 cells in the rheumatoid arthritis joint. PLoS One 2010; 5(9).
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in the joints of patients with rheumatoid arthritis. Ann Rheum Dis 2008; 67(9):1299-1304.
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(62) Nistala K, Adams S, Cambrook H, Ursu S, Olivito B, de JW et al. Th17 plasticity in human autoimmune arthritis is
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inflammatory responses. Nat Immunol 2011; 12(3):255-263.
Chapter 2
Exposure to Candida albicans
Polarizes a T-cell driven Arthritis Model
Towards Th17 Responses, Resulting in
a More Destructive Arthritis
Renoud J. Marijnissen1, Marije I. Koenders1, Frank L. van de Veerdonk2, John Dulos3,
Mihai G. Netea2, Annemieke M.H. Boots4, Leo A.B. Joosten2, Wim B. van den Berg1.
heumatology Research & Advanced Therapeutics, Department of Rheumatology,
R
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
2
Department of Medicine and Nijmegen Institute for Infection, Inflammation & Immunity (N4i),
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
3
Department I&DL, section Pharmacology, Crucell, Leiden, The Netherlands.
4
Department of Rheumatology and Clinical Immunology, University Medical Centre Groningen,
University of Groningen, the Netherlands.
1
24 | Chapter 2
Abstract
Background
Fungal components have been shown very effective in generating Th17 responses. We
investigated whether exposure to a minute amount of C. albicans in the arthritic joint
altered the local cytokine environment, leading to enhanced Th17 expansion and resulting
in a more destructive arthritis.
Methodology
Chronic SCW arthritis was induced by repeated injection with Streptococcus pyogenes
(SCW) cell wall fragments into the knee joint of C57Bl/6 mice, alone or in combination with
the yeast of C. albicans or Zymosan A. During the chronic phase of the arthritis, the
cytokine levels, mRNA expression and histopathological analysis of the joints were
performed. To investigate the phenotype of the IL-17 producing T-cells, synovial cells were
isolated and analyzed by flowcytometry.
Principal Findings
Intra-articular injection of either Zymosan A or C. albicans on top of the SCW injection
both resulted in enhanced joint swelling and inflammation compared to the normal SCW
group. However, only the addition of C. albicans during SCW arthritis resulted in severe
chondrocyte death and enhanced destruction of cartilage and bone. Additionally,
exposure to C. albicans led to increased IL-17 in the arthritic joint, which was accompanied
by an increased synovial mRNA expression of T-bet and RORγT. Moreover, the C. albicansinjected mice had significantly more Th17 cells in the synovium, of which a large population
also produced IFN-γ.
Conclusion
This study clearly shows that minute amounts of fungal components, like C. albicans, are
very potent in interfering with the local cytokine environment in an arthritic joint, thereby
polarizing arthritis towards a more destructive phenotype.
C. albicans skews the T-cell balance in experimental arthritis | 25
Introduction
Rheumatoid arthritis (RA) is a systemic joint disease with an unknown etiology,
characterized by a chronic inflammatory infiltration of the synovial membrane, and
associated with destruction of cartilage and bone. Both genetic and environmental
factors contribute to the development of disease. Various infectious agents, such as
bacteria and viruses have long been associated with the pathogenesis of RA (1;2). The
involvement of these micro-organisms has not only been proposed in the initiation of
arthritis, but also in the progression and exacerbations of the disease.
Microorganisms are recognized by pattern recognition receptors (PRR), such as
Toll-like receptors (TLRs) and C-type lectins, which are essential components of the innate
immune system and form the bridge with adaptive immunity. If the inflammatory
response is not adequate and/or the infection is not cleared properly, persistence can
eventually result in chronic or even auto-immune inflammation (2). Particularly TLR
signaling has been shown to play an intrinsic role in the inflammatory processes of arthritis
(3-5). The concept that commensal pathogens affect the pathogenesis of RA is
strengthened by data from spontaneous murine arthritis models and the notion that
spontaneous arthritis does not develop in animals kept under germ free conditions (3;6).
This implies the importance of (memory) responses for the ‘break of tolerance’ and
induction of auto-inflammation/immunity.
Candida albicans is the most common opportunistic fungal pathogen in humans.
Infection with C. albicans induces IL-17 producing T helper (Th17) cells in vitro and in vivo in
naïve mice (7-9). Under physiological conditions, these Th17 cells produce proinflammatory cytokines like IL-17A (IL-17), IL-17F, IL-21 and IL-22, and are involved in the clearance of
several extracellular bacteria and fungi (10). In the arthritic joint, direct or indirect effects of
IL-17/Th17 result in increased inflammation, angiogenesis, and osteoclastogenesis,
resulting in enhanced breakdown of cartilage and bone (11-14). Although C. albicans or
Candida-derived β-glucan have been shown to induce and aggravate various models of
arthritis(15;16), these observations have not yet been linked to modulation of the IL-17/
Th17 pathway and increased structural damage.
The purpose of the present study was to demonstrate the ability of C. albicans to
skew the T-cell balance in the chronic murine SCW model. This model initiates as a local
TNF-dependent macrophage-driven inflammation, at which repeated antigen exposure
results in a chronic T-cell dependent arthritic process (17). A small quantity of C. albicans or
Zymosan A (< 10% of mass) was added to the cell wall fragments of Streptococcus pyogenes
(SCW) that were repeatedly injected into the knee joint. During the chronic phase of the
arthritis, the development of macroscopic joint swelling and histopathological changes in
synovium, cartilage, and bone were determined. Furthermore, the levels of antibodies,
secretion of T-cell cytokines and presence of T-cells were examined.
26 | Chapter 2
Materials and Methods
Animals. Male C57Bl/6 mice were purchased from Janvier, France. The mice were housed
in filter-top cages; water and food were provided ad libitum. The mice used were between
10 - 12 weeks of age. All animal procedures were approved by the animal ethics committee
of the Radboud University Nijmegen.
Study protocol. Steptococcus pyogenes T12 organisms were cultured and prepared as
described previously (17). For the fungal components, the blastoconidia of Candida
albicans (ATCC MYA-3573 (UC 820)) were used (8). Zymosan A (Saccharomyces Cerevisiae)
was purchased from Sigma-Aldrich and prepared as described earlier (18). Chronic
unilateral arthritis was induced by repeated intra-articular (i.a.) injection of 25 μg SCW on
days 0, 7, 14, and 21, whether or not with 1*105 particles of C. albicans (1*105≈ 1 μg) or 2 μg
Zymosan, in 7 μl phosphate buffered saline (PBS) into the right knee joint of naive mice. As
a control, additional groups were injected with the fungal particles alone. On day 22,
twenty-four hours after the last injection, a subgroup of mice was sacrificed for the
collection of synovial washouts. Accordingly, patellae with surrounding soft tissue were
isolated from inflamed knee joints and cultured 1 hour at RT in RPMI-1640 medium
containing 0.1% BSA (200 µl/patella). In addition, the draining lymph nodes (popliteal and
inguinal) were collected and cells were isolated. Then, 1*105 cells were stimulated for 72
hours with 2 µg/ml plate bound anti-CD3 (R&D systems) and 2 µg/ml plate bound
anti-CD28 (BD Biosciences). Thereafter, supernatants were collected, centrifuged and
stored for cytokine determination. On day 28, during the chronic joint inflammation, the
sera from the remaining mice were collected, the mice were sacrificed, and knee joints
were prepared for histology.
Measurement of joint swelling. Joint swelling was assessed by measuring the accumulation
of 99mTc in the inflamed joint due to increased blood flow and edema. Therefore, 0.74 MBq
of 99mTc in 200 μl of saline was injected subcutaneously. After several minutes of distribution
throughout the body, external gamma radiation in the knee joints was measured. Swelling
was expressed as the ratio of gamma counts in the right (inflamed) knee joint to gamma
counts in the left (control) knee joint. Values higher than 1.1 counts per minute were
considered to represent joint swelling.
Histopathology. For standard histological assessment, the isolated joints were fixed for 4
days in 10% formalin, decalcified in 5% formic acid, and the specimens were processed for
paraffin embedding. Tissue sections were stained with hematoxylin and eosin. The
severity of inflammation in the joints was scored on a scale of 0–3 (0 = no cells, 1 = mild
cellularity, 2 = moderate cellularity, and 3 = maximal cellularity). Bone destruction was
graded on a scale of 0–3, ranging from no damage to the complete loss of bone structure.
C. albicans skews the T-cell balance in experimental arthritis | 27
Proteoglycan (PG) depletion was determined using Safranin O staining. Loss of
proteoglycans was scored on a scale of 0–3, ranging from normal, fully stained cartilage to
destained cartilage, fully depleted of PG. The scoring of the sections was performed in a
blinded manner.
Cytokine detection. Cytokine levels were measured using Luminex multi-analyte technology
in combination with Bio-Plex cytokine kits (Bio-Rad; IL-17, IL-4, IFN-γ, IL-10) and performed
according to manufacturer’s instructions.
Quantitative PCR. RNA was isolated from synovial knee biopsies using TRI-reagent (Sigma)
and treated with DNase to remove genomic DNA. The RNA was subsequently reverse
transcribed with oligo(dT) primers in a reverse transcriptase procedure. Quantitative
real-time PCR was performed with cDNA specific primers (Biolegio) and SYBR Green PCR
Master Mix (Applied Biosystems). The ΔCt method was used to normalize transcripts to
GAPDH and to calculate relative mRNA expression (2–ΔCt), for which the expression in the
SCW injected group was arbitrarily set to 1. All primer pairs were developed using Primer
Express 2.0 (Applied Biosystems) and validated according to protocol.
Isolation and stimulation of synovial cells. After sacrificing the mice, the ankle joint
synovium was dissected for single cell isolation. In short, synovial biopsies were incubated
with enzymatic digestion buffers (Liberase Blendzyme, Roche) for 30 minutes at 37°C.
Next, a 70 μm nylon cell strainer (BD Falcon) was used to process the digested tissue. The
cell preparation was collected in RPMI with 10% FCS. To isolate the single mononuclear
cells, Lympholyte-M (Cederlane) was used according to manufacturer’s protocol. All cells
were cultured in RPMI-1640 (Gibco; Invitrogen) supplemented with 10% FCS. Subsequently,
the cells were prepared for intracellular flowcytometry.
Flow cytometry. Synovial cells were stimulated for 5 hours with PMA (50 ng/ml; Sigma)
and ionomycine (1 µg/ml; Sigma) in the presence of Golgiplug (BD Biosciences) according
to manufacturers protocol. After staining the cells extracellularly with anti-CD3 APC (BD
Biosciences), the cells were fixed and permeabilized with Cytofix/ Cytoperm solution (BD
Biosciences). Subsequently, they were intracellularly stained with anti-IFN-γ PE (BD
Biosciences) and anti-IL-17 FITC (BD Biosciences). Samples were measured on a FACS
Calibur and data were analyzed using FlowJo software.
Determination of anti-SCW antibody levels. Levels of anti-SCW antibodies in the
serum of mice with chronic SCW-induced arthritis (day 28) were analyzed according to
standard methods (17). Briefly, 10 ng of SCW fragments were coated overnight onto
96-well plates. Thereafter, plates were washed, and nonspecific binding was blocked with
1% BSA in PBS-Tween 80 (0.05%). Anti-SCW antibodies were examined in serial 2× dilutions,
28 | Chapter 2
starting with an initial dilution of 20×. After incubation for 1 hour, plates were washed and
isotype-specific goat anti-mouse Ig–HRP (1:1,000) was added for 1 hour at room temperature.
Statistical analysis. Results are expressed as the mean ± SEM. Differences between
experimental groups were tested using Mann-Whitney U-test or one-way analysis of
variance with Dunnett’s multiple comparison test, as appropriate. P-values less than 0.05
were considered significant.
Results
Exposure to fungal particles aggravates chronic SCW arthritis
Repeated intra-articular injection of SCW bacterial fragments induce arthritic flares that shift
from a predominantly macrophage-driven acute inflammation to a T-cell driven chronic
inflammation (17). To investigate the ability of C. albicans to effectively alter the local synovial
inflammatory process, heat-killed conidia of C.albicans were co-injected with 25 µg SCW
fragments in the knee joint on day 0, 7, 14 and 21. In preliminary experiments, exposure to
differential doses of C. albicans to murine peritoneal macrophages resulted in the production of
various innate cytokines like TNF-α, IL-1β and IL-6 (Supplemental figure S1). Subsequently, during
a pilot experiment, we selected a dose of 1*104 conidia of C. albicans, which we co-injected with
the SCW fragments into the knee joint. This dose did not show a significant alteration in arthritis
severity/ score (data not shown). We therefore increased the dose of C. Albicans to 1*105 on top
of the SCW dose of 25 μg. For comparison, we injected 2 µg Zymosan A, a glucan derived
from the yeast cell wall of Saccharomyces Cerevisiae, known for its adjuvant properties (19-21).
As control, mice were injected i.a. with SCW, C. albicans or Zymosan A alone.
The control mice injected with C. albicans or Zymosan A alone did not develop joint
swelling after the injections (figure 1A). Furthermore, the co-injection of C. Albicans or
Zymosan A on top of the SCW fragments did not result in an increased joint swelling
compared to the SCW injected group during the acute phase of the model. On the
contrary, one day after the final injection, on day 22, the C. Albicans co-injected group
showed a significant increase in the technetium measurements, whereas the Zymosan A
co-injected group only showed a trend for increase. Accordingly, we analyzed the joint
swelling and histology during the chronic phase of the model, on day 28. During this
phase, the joint swelling in the mice injected with SCW remained significantly increased
(figure 1B). Interestingly, exposure to minute amounts of C. albicans or Zymosan A on top
of the SCW fragments significantly aggravated the joint swelling. This increase in joint
swelling in both the C. albicans and Zymosan A injected mice was supported by a
significant increase in the influx of inflammatory cells after histopathological analysis
(figure 1 panels C and D), indicating that both fungal particles aggravated the inflammatory
process during the chronic phase of the model.
C. albicans skews the T-cell balance in experimental arthritis | 29
Increased joint destruction in SCW arthritis after co-exposure to C. albicans
To compare the ability of C. albicans and Zymosan A to affect the cartilage and bone
destruction, we performed a detailed histological analysis. Compared to the SCW-injected
mice, the increased joint inflammation at day 28 after co-injection of SCW with fungal
particles was accompanied by marked proteoglycan depletion (figure 2A). Remarkably,
although the influx of inflammatory cells was comparable after addition of either C.
albicans or Zymosan A, we observed a significantly higher increase in cartilage erosion,
chondrocyte death and bone erosion in the C. albicans co-injected group compared to
the SCW injected group (figure 2A), suggesting that Candida exposure modulates towards
a more destructive immune response.
In addition, we analyzed the mRNA expression of several inflammatory genes
involved in bone and cartilage destruction using synovial tissue collected during the
chronic phase of the arthritis. While the osteoclast marker Cathepsin K (Cat.K) was not
IL-6
9000
750
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pg/ml
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1000
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3000
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Supplemental figure S1 | Candida albicans and Zymosan A induce a different cytokine
profile in vitro. 1*105 peritoneal macrophages from naive C57Bl/6 mice were stimulated with
different concentrations Zymosan or C. Albicans for 16 hours (n=5). Levels of IL-1β, IL-6, IL-10 and
TNF-α were determined by Luminex in the supernatants. Results are mean ± SEM.
30 | Chapter 2
significantly enhanced, RANKL was upregulated in the synovium in both the C. albicans
and Zymosan A induced groups, in line with to the increase in bone erosion. Moreover,
the matrix metalloproteinase’s MMP3 and MMP13 were significantly increased in the
synovium of C. albicans inoculated mice compared to the single SCW group, corresponding
to the increased cartilage erosion (figure 2B).
A
C. Albicans
Zymosan
SCW
SCW + C. Albicans
SCW + Zymosan
ns
3.0
**
ns
ns
ns
R/L ratio
2.5
ns
ns
ns
2.0
ns
ns
ns
*
1.5
1.0
1
8
15
22
Days
Joint Swelling
B
1.5
2.5
1.4
R/L ratio
3.0
**
Histologic score (0-3)
*
Inflammation
C
1.3
1.2
1.1
**
***
2.0
1.5
1.0
0.5
1.0
D
SCW
SCW + Candida
W
SC + Ca
W nd
+ Z id
ym a
os
a
Na n
Ca ive
nd
Zy ida
mo
sa
n
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W
SC
SC
W
SC
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W nd
+ Z id
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a
Na n
Ca ive
nd
Zy ida
mo
sa
n
0.0
SCW + Zymosan
Figure 1 | Candida albicans and Zymosan A aggravate the inflammation in the chronic
SCW model. On days 0, 7, 14, and 21, streptococcal cell wall (SCW) fragments were injected intraarticularly (i.a.) into the knee joint. Joint swelling (ratio of 99mTc uptake in the treated right knee
joint to that in the untreated left knee joint) were determined 1 day after every injection (A) and
during the chronic phase of the model on day 28 (B; n=6 mice/group).. In addition, the inflammatory
cell influx (C) on histological slides was quantified on day 28. H&E stained frontal knee sections
(original magnification 100x) (D) are shown (n=8 mice/ group. Values are the mean ± SEM; ns = non
significant, * P < 0.05, ** P < 0.01, *** P < 0.001, by One-way ANOVA.
C. albicans skews the T-cell balance in experimental arthritis | 31
To support that the increase in cartilage destruction was induced by a matrix metalloproteinase–mediated process, we examined the expression of the MMP-specific
aggrecan neo-epitope VDIPEN by immunohistochemistry. Repeated SCW injections were
sufficient to induce VDIPEN expression at the cartilage sites (figure 2C). The expression of
PG depletion
*
2.5
2.0
0.25
SC
W
SC
W
+C
an
did
a
+Z
ym
os
an
SC
W
+C
an
did
a
+Z
ym
os
an
SC
W
SC
W
Chondrocyte death
*
***
1.00
Bone erosion
1.75
*
Histologic score (0-3)
Histologic score (0-3)
1.25
0.75
0.50
0.25
0.00
*
***
1.50
***
1.25
1.00
0.75
SC
SC
SC
W
W
SC
W
+C
an
did
W+
a
Zy
mo
sa
n
SC
W
+C
an
did
W+
a
Zy
mo
sa
n
0.50
40
Relative mRNA expression
***
0.50
0.00
1.5
B
ns
***
SC
W
Histologic score (0-3)
Histologic score (0-3)
**
3.0
Cartilage erosion
0.75
ns
SC
A
SCW
SCW + Candida
SCW + Zymosan
*
30
**
**
20
10
**
5
0
MMP3
MMP13
MMP14
Cat.K
RANKL
SCW
C to C. albicans
Figure 2 | Exposure
increases the 3cartilage destruction and bone erosion
ns
VDIPEN expression (overall mean)
did
a
+Z
ym
os
an
SC
W
SC
SC
W
+C
an
W
% dark area
during chronic SCW arthritis. Analysis of destructive parameters after
4 relapsing flares of arthritis.
ns
**
On day 28, knee joints (n=8/group) were harvested for histological
assessment. Knee joint sections
2
were stained using Safranin O to determine the degree of
proteoglycan (PG) depletion. H&E staining
SCW + Candida
was used to score the degree of chondrocyte death, cartilage surface erosion and bone erosion
(A). QPCR analysis of destructive related genes was performed
on synovial biopsies (n=3/group) of
1
day 22, one day after the last injection (B). Representative images of arthritic knee joints showing
immunohistochemical staining for VDIPEN after the 4 repeated injections (day 28) (C). Besides,
0
SCW + of
Zymosan
the quantitative measurement
VDIPEN expression (percentage of positively stained area) in the
cartilage of the 3 groups of mice (n=8/group) was analyzed. Values are the mean ± SEM; ns = non
significant * P < 0.05, ** P < 0.01, *** P < 0.001, by One-way ANOVA.
**
**
20
10
**
5
0
C
SCW + Zymosan
MMP3
MMP13
SCW
MMP14
Cat.K
RANKL
VDIPEN expression (overall mean)
3
ns
ns
1
+Z
ym
SC
W
+C
an
did
a
0
SC
W
SCW + Zymosan
2
SC
W
SCW + Candida
% dark area
**
os
an
Relative mRNA expression
32 | Chapter 2
30
Figure 2 | Continued.
VDIPEN after four weeks of SCW arthritis was comparable to the Zymosan A co-injected
mice. However, in line with the increase in MMP mRNA expression, the amount of VDIPEN
expression increased dramatically when C. albicans had been co-injected with SCW into
the joints. These findings show that a small amount of C. albicans is sufficient to exacerbate
the chronic murine SCW arthritis model by increasing the cartilage and bone destruction.
Exposure to C. albicans during chronic SCW arthritis skews the T cell
cytokine profile towards Th17 responses
To study modification of the adaptive immune responses, we initially studied the B-cell
compartment and determined the levels of anti-SCW antibodies of total IgG, IgG1 and
IgG3 in the sera of the mice. We previously demonstrated that in the chronic SCW model,
antigen-specific antibodies are induced after the fourth i.a. injection of SCW fragments
(22). As can be appreciated from figure 3A, we observed no differences in the serum levels
of SCW specific antibodies between the groups, indicating that the B-cell antibody
production was not altered after addition of C. albicans or Zymosan A.
Since T helper cells are crucial for the chronic phase of the SCW arthritis model, and
Candida is known for the induction of Th17 cells, we explored alterations in T-cell
responses. Pan T-cell stimulation with anti-CD3 and anti-CD28 of draining lymph node
cells on day 22 revealed no difference in the levels of IL-4 and IL-10 production. Interestingly,
a significant increase in IL-17 production and a tendency for increased IFN-γ production
were found, suggesting an enhanced helper T cell response of mainly the Th17 lineage by
the addition of C. albicans (figure 3B).
To conclude whether this peripheral increase in IL-17 and IFN-γ represented a shift in
the local T-cell balance in the arthritic joint, we measured the same T-cell derived cytokines
in the synovial washouts of the mice. IFN-γ and IL-10 levels were not altered, and IL-4 was
undetectable (figure 3C). In contrast, the IL-17 levels were significantly increased in the C.
C. albicans skews the T-cell balance in experimental arthritis | 33
albicans co-injected group, while the cytokine levels in the Zymosan A co-injected group
were not different from the SCW group. All together, these data suggest that C. albicans
induces a shift in the T cell compartment during SCW arthritis, favoring Th17.
C. albicans mainly induces IL-17-producing T cells in the joint
To establish a shift in the T-cell compartment, we subsequently determined the levels of
T-cell transcriptional lineage factors, which specify the different T helper cell lineages.
A
SCW
0.4
SCW + Candida
SCW + Zymosan
OD/50
0.3
0.2
0.1
0.0
Total IgG
B
SCW
IgG1
SCW + Candida
IgG3
SCW + Zymosan
ns
Concentration (pg/ml)
2500
*
ns
1500
500
50
25
0
IL-4
IFN γ
IL-17
IFN γ
10
0
*
100
50
SC
W
+C
an
did
W+
a
Zy
mo
sa
n
SC
W
+C
an
did
W+
a
Zy
mo
sa
n
SC
SC
W
SC
SC
W
+C
an
SC
did
W+
a
Zy
mo
sa
n
W
25
0
0
SC
50
W
20
**
75
SC
Concentration (pg/ml)
Concentration (pg/ml)
30
IL-10
ns
150
50
40
IL-10
Concentration (pg/ml)
C
IL-17
Figure 3 | C. albicans skews T-cell cytokine profile. Serum levels of anti-SCW-specific total IgG,
IgG1, and IgG3 antibodies (A). Draining lymph node cells (1*105/ well) were stimulated with antiCD3/ anti-CD28 antibodies for 72 hours (n = 6/group). Levels of IFN-γ, IL-17 and IL-10 were determined
by Luminex in the supernatants of the stimulated lymph node cells (B) and washouts of synovial
biopsies of day 22 (C). Results are the mean ± SEM; ns = non significant *= P < 0.05; **= P < 0.01, ***
= P < 0.001, by One-Way ANOVA.
34 | Chapter 2
A
ns
Relative mRNA expression
20
ns
**
SCW
SCW + Candida
SCW + Zymosan
15
ns
10
** *
5
0
T-bet
GATA-3
FOXP3
RORγt
Relative mRNA expression
B
100
ns
ns
80
60
**
ns
**
ns
ns
SCW
SCW + C. Albicans
SCW + Zymosan
* *
40
20
15
10
5
0
IFNy
C
IL-17A
IL-17F
IL-21
SCW
IL-22
IL-10
IL-17
SCW + Candida
IFNy
D
Figure 4 | Co-exposure
to C. albicans induces Th17 cells in the joint. The mRNA expression
IL-17+IFNγ-
IL-17-IFNγ+
IL-17+IFNγ+
Pecentage (%)
Pecentage (%)
Pecentage (%)
of T-bet, GATA-3, RORγT and FOXP3 in synovial biopsies of the knee joints (n=3 mice/group) were
15
10.0
2.0
measured by QPCR (A) The mRNA
* expression of IFN-γ,* IL-17A, IL-17F, IL-21, IL-22 and IL-10 are shown
(B). The knee joint synovium was dissected 1.5and prepared for enzymatic
digestion. The cells were
7.5
incubated with PMA/ 10ionomycine and Golgiplug for 5 hours before flowcytometric analysis (n= 6
1.0
mice/group) and stained for CD3, IL-17, and IFN-γ.
Representative dot5.0plots of the isolated cells gated
on CD3+ are shown (C).5 The mean percentage of isolated IFN-γ and IL-17 producing T cells (CD3+
2.5
population) are shown (D). Results are mean0.5± SEM; ns = non significant
* = P < 0.05, ** = P < 0.01,
by one-way ANOVA.
0.0
0
a
did
SC
W
+C
an
W
SC
a
did
SC
W
+C
an
W
SC
a
did
+C
an
W
SC
SC
W
0.0
C
SCW
SCW + Candida
IL-17
C. albicans skews the T-cell balance in experimental arthritis | 35
IFNy
1.0
0.5
2.5
a
did
a
did
+C
an
SC
W
a
did
+C
an
SC
W
5.0
0.0
0.0
SC
W
7.5
+C
an
0
*
1.5
SC
W
5
10.0
Pecentage (%)
Pecentage (%)
Pecentage (%)
*
10
IL-17-IFNγ+
IL-17+IFNγ+
2.0
SC
W
IL-17+IFNγ15
SC
W
D
Figure 4 | Continued.
FOXP3 (Treg) and GATA3 (Th2) mRNA expression levels were not significantly different
between the SCW-arthritis groups (figure 4A). In line with the enhanced IL-17 production
from the lymph nodes, we observed a significant increase in the mRNA expression of the
Th17 transcription factor RORγT after C. albicans injection. Remarkably, although the level
of IFN-γ was not significantly increased in the synovial washouts, we did observe an
increase in Th1 transcriptional lineage factor T-bet. Co-exposure to Zymosan A did not
influence the other transcription factors. To further confirm the Th17 profile, we analyzed
the expression of IL-17A, IL-17F, IL-21, IL-22 and IFNy (figure 4B). Here we again observed a
significant increase in the expression of IL-17A, IL-21 and IFN-γ. Although a trend for an
increase was found for the other Th17 profile cytokines, they were not significantly
increased in the C. albicans co-injected group compared to the SCW and SCW + Zymosan
A groups.
Next, we determined whether the inoculation of C. albicans in the chronic SCW
model affected the phenotype of the infiltrated T-cells. On day 22, at the peak of the
inflammation, we dissected the inflamed synovium and isolated the residing cells.
Intracellular cytokine staining revealed an increased percentage of single IL-17 producing
T-cells (figure 4B,C). Further analysis revealed that a subpopulation of these IL-17 producing
T cells, also produced IFN-γ, partly explaining the increase in the Th1 transcription factor
T-bet. This indicates that in the presence of C. albicans the T-cell balance during
SCW-arthritis is shifted towards a pronounced Th17/Th1 profile.
36 | Chapter 2
Discussion
Our main finding is that a low-grade exposure to C. albicans can skew the T cell balance in
the SCW arthritis model, by inducing Th17 cells and pushing it towards a more destructive
arthritis model. This indicates that the presence of a minute amount of C. albicans antigen,
after e.g. a mucosal infection, might be sufficient to skew an inflammatory cytokine profile.
Microbial infections have long been associated with the pathogenesis of RA.
Therefore, several fungi and fungal-derived ligands have been used to provoke or
accelerate arthritic processes. In the last decade, several PRR’s that recognize fungal PAMPs
have been identified, in particular Toll-like receptors (TLR-2 and TLR-4) and C-type lectin
family members (Dectin-1, Dectin-2 and mannose receptor) (6;15;16;18;20;23). C. albicans is
especially known for the induction of Th17 cells via Dectin-1, TLR-2, TLR4, Mannose
receptor, and complement (8;24) Earlier, it has been shown that gut colonization by C.
albicans or exposure to C. albicans derived β-glucans can initiate and aggravate collageninduced arthritis, probably via an increased and/or altered cytokine profile (16;25).
Although this shows that C. albicans can initiate and aggravate arthritis models, it was not
known whether and how Th17 responses were mechanistically involved.
To explore the potency of a minute amount of C. albicans on skewing T-cell responses
during arthritis, we investigated the chronic SCW model. In this model, chronic T cell
dependent arthritis is induced by repetitive intra-articular injection of Steptococcus
pyogenes cell wall (SCW) fragments (17). S. pyogenes is a common Gram-positive bacteria
that can induce severe rheumatic fever and/or reactive arthritis, depending on the host
response (26). Previously, we have shown that intra-articular exposure to SCW mainly
depends on macrophage activation via TLR-2/MyD88 pathway (27). Repeated exposure
results in a chronic arthritis involving SCW-specific T- and B-cell memory responses,
without the need for adjuvants like Freund’s complete adjuvant. Furthermore, although
the model involves a chronic phase that is characterized by an IL-1 and IL-17 dependent
cartilage destruction, the model is less destructive compared to collagen-induced arthritis
(CIA) and hardly involves bone erosions. We hypothesized that co-exposure to C. albicans,
would alter the macrophage-derived cytokine production during the flares, aggravating
the chronic disease.
Monocytes and macrophages stimulated with C. albicans induce pro-inflammatory
cytokine production, like TNF-α, IL-1 and IL-6 (24). To prevent an aggravation of the
SCW-induced arthritis by the addition of Candida, we choose the dose of C. albicans at
such a subtle level that it did not cause a significant increase in the joint swelling on top of
the S. pyogenes cell wall (SCW) induced local inflammation. During this acute phase of the
SCW arthritis model, joint swelling is TNF-α dependent (28). We therefore conclude that a
possible Candida-induced increase in TNF-α could not be observed by our TNF-dependent
Tc-measurement. Even three weeks of repeated co-injections with C. albicans or Zymosan
did not significantly accelerate the model, showing that the co-exposure to C. albicans did
C. albicans skews the T-cell balance in experimental arthritis | 37
not increase the innate immune response like a classical adjuvant (29), but may have
prolonged or altered the immune response.
This was further strengthened by the fact that we observed no changes in classes of
SCW-specific immunoglobulin production. These SCW specific antibodies can be detected
in sera during the late chronic stage, and are also TNF-α dependent (17). Interestingly, B-cells
were reported to be involved in the Th17 response after C. albicans exposure (30). And
although B-cells play a minor role in the repeated SCW arthritis model (17), co-exposure to C.
albicans might have altered the antigen presenting function of B-cells. The presence of
autoantibodies, like rheumatoid factor (RF) and anti-citrullinated petide antibodies (ACPA) in
the sera of patients are early predictors of disease and correlate with arthritis severity (31).
At day 22, one day after the last injection, when the model shifts from a predominately
macrophage driven model to a T cell dependent model, we observed a significantly
increased joint swelling in the Candida co-injected group, with a trend for increase in the
Zymosan co-injected mice. Furthermore, during the late chronic phase of the model (at day
28) we observed a prolonged increase in joint swelling after co-exposure to both C. albicans
or Zymosan A. Interestingly, although the increase in joint swelling and inflammatory cell
influx in the C. albicans co-injected mice was comparable to Zymosan A co-injected mice,
the chondrocyte death, bone erosion and cartilage erosion were significantly elevated by C.
albicans. Additional analysis revealed that during the chronic phase of the model, the C.
albicans exposure contributed to the increased MMP-mediated cartilage destruction and
increased bone erosion, suggesting that additional processes were involved in the C. albicans
exposed joints. This increase in joint destruction was accompanied by local synovial IL-17
expression, which is thought to/may enhance the role of TNF-α in the progression of the
destructive process (32-34). Synergy between IL-17 and TNF-α may underlie the increased
pathology in the C. albicans co-injected group. Together, we concluded that the low levels
of C. albicans and Zymosan A did not accelerate the model during the first three weeks of
repeated antigen injections, but addition of C. albicans did alter the T cell balance and
arthritis outcome at the end of the experiment.
Focusing on the T-cell cytokine production, we observed an increased Th17/Th1
cytokine profile. In line with the increase in the transcriptional factors T-bet and RORγT, this
increase was related to an increase in IL-17 single and double positive T-cells. Furthermore,
over 90% of the total IL-17 producing cells were CD3+ (data not shown). This supports that T
lymphocytes are the main source of IL-17 in this model, rather than innate immune cells
capable of producing IL-17 (35).
T helper cells are defined T lymphocytes expressing CD4 glycoprotein on their cell
surface. We chose CD3 staining over CD4 staining due to the reported sensitivity of CD4
to cleavage during enzymatic digestion of the studied tissue (36). Only recently, we have
altered the protocol of enzymatic digestion slightly, to prevent CD4 cleavage. In a yet
unpublished observation (submitted manuscript) we observed that in the SCW model
more than 80% of the IL-17+ producing cells express CD4.
38 | Chapter 2
Apparently, C. albicans does not only increase the ‘classical’ IL-17 producing T-cells, but
also promotes the transition to a Th1/Th17 mixed phenotype. The role and induction of
Th1 profile versus Th17 profiles in models have received increased attention. In human RA,
IFN-γ producing T helper cells are found at the level of the synovial tissue rather than IL-17
producing T helper cells (37;38), whereas the experimental arthritis models do illustrate an
important role for IL-17 (14). The strength of the IFN-γ response seems to determine the
dependency on IFN-γ or IL-17 (39). Remarkably, isolated IFN-γ producing T helper cells from
patients with chronic inflamed joints show more resemblance with ‘classical’ Th17 cell
surface repertoire compared to ‘classical’ Th1 cells (40;41). In addition, recent reports have
shown the plasticity of Th17 cells in vivo, that shift towards IFN-γ producing cells during
chronic autoimmune inflammation (42;43). Both IL-12 and IL-23 might be important players
for this transition (41;42;44).
On the other hand, recent studies have shown an inverse relationship between Th17
cells and regulatory T cells (45). Tregs in the joints of RA patients can inhibit the production
of IFN-γ and IL-17 (46). Although we did not observe a significant alteration in the FOXP3
levels, the increased IL-6 levels after C. albicans exposure, might contribute to the
imbalance between Th1/Th17 and Tregs cells (47). These results raise questions about the
ancestry and transcriptional control of Th17 during the course of chronic inflammatory
diseases, such as Rheumatoid Arthritis. Clarifying pathways that control the induction of
these double positive cells in vivo during arthritis will lead to new strategies for the
development of novel therapeutic interventions in the Rheumatic diseases.
In conclusion, exposure to a small amount of the fungal pathogen C. albicans is
sufficient to shift the T-cell balance of the synovial immune response towards a more
pronounced Th17/Th1 profile, thereby aggravating the model towards increased cartilage
and bone destruction. In the past others have shown that fungal particles, like C. albicans,
can accelerate or aggravate arthritic disease. This is the first report showing that this
aggravation involves the induction of Th17 cells in vivo. C. albicans is a common commensal
that can cause mucosal and systemic infections (48). Whether C. albicans can contribute to
the progression of joint destruction in RA remains to be elucidated.
Acknowledgements
We would like to thank Dr. Annemiek van Spriel for her valuable comments and suggestions.
We thank Birgitte Walgreen, Monique Helsen and Liduine van den Bersselaar for excellent
technical assistance.
C. albicans skews the T-cell balance in experimental arthritis | 39
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(37) Yamada H, Nakashima Y, Okazaki K, Mawatari T, Fukushi JI, Kaibara N et al. Th1 but not Th17 cells predominate
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C. albicans skews the T-cell balance in experimental arthritis | 41
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Chapter 3
The Macrophage Mannose
Receptor Induces IL-17 in Response
to Candida albicans
Frank L. van de Veerdonk 1,4, Renoud J. Marijnissen2, Bart Jan Kullberg1,4,
Hans J.P.M. Koenen3, Shih-Chin Cheng1,4, Irma Joosten3, Wim B. van den Berg2,
David L. Williams5, Jos W.M. van der Meer1,4, Leo A.B. Joosten1,4, Mihai G. Netea1,4
Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Department of Rheumatology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
3
Department of Blood Transfusion and Transplantation Immunology, Radboud University Nijmegen Medical
Centre, Nijmegen, The Netherlands
4
Nijmegen Institute for Infection, Inflammation and Immunity, Nijmegen, The Netherlands
5
Department of Surgery, James H. Quillen College of Medicine, East Tennessee State University,
Johnson City, TN 37614, USA
1
2
44 | Chapter 3
Abstract
The cytokine IL-17 controls neutrophil-mediated inflammatory responses. The pattern
recognition receptor(s) that induce Th17 responses during infection, in the absence of
artificial mitogenic stimulation with anti-CD3/anti-CD28 antibodies, remain obscure. We
investigated the innate immune receptors and pathogen-associated molecular patterns
involved in triggering Th17 responses during pathogen-specific host defense. The
prototypic fungal pathogen Candida albicans was found to induce IL-17 more potently
than Gram-negative bacteria. Candida mannan, but not Zymosan, β-glucans, Toll-like
receptor (TLR) agonists, or the NOD2 ligand MDP, induced IL-17 production in the absence
of anti-CD3/anti-CD28 antibodies. Candida-induced IL-17 response was dependent on
antigen-presenting cells and the macrophage mannose receptor (MR), demonstrating
that Candida mannan is not simply a mitogenic stimulus. The TLR2/dectin-1 pathway, but
not TLR4 or NOD2, amplified MR-induced IL-17 production. This study identifies the
specific pattern recognition receptors that trigger the Th17 response induced by a human
pathogen in the absence of mitogenic stimulation.
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 45
Introduction
The recently described T helper 17 (Th17) cells produce interleukin-17 (IL-17), a cytokine
that is important in the host defense against various pathogens (1), including
Gram-negative bacteria such as Kleibsiella pneumonia (2) and Citrobacter rodentium (3), the
spirochetal infection Borrelia burgdorferi (4), and the fungal pathogen C. albicans (5). Candida-­
specific Th17 responses have also been observed in peripheral blood in humans (6). IL-17
induces infiltration of polymorphonuclear leucocytes (PMNs) at the site of infection,
activation of tissue neutrophils and macrophages, and the synthesis of antimicrobial
peptides (1). These data suggest an important role for the Th17 response in the host
defense against pathogens. However, overproduction of IL-17 can also lead to autoimmune
processes (7-9), underscoring the delicate balance in regulation of the Th17 response.
Recently, much has been learned about the cytokine profiles that are needed to
differentiate naive T cells into Th17 cells and how to enable central memory T cells to secrete
IL-17. Whereas IL-1β alone is capable of inducing IL-17 secretion in human central memory
T cells (10), IL-21 and TGF-β seem to be needed for driving differentiation of naive T cells into
Th17 cells (11). More controversial are the receptor pathways that direct the immune response
toward a Th17 profile, with each of the TLRs (12), dectin-1 (13), and NOD2 (14) pathways
individually being implicated in the induction of Th17 cells by different researchers. However,
these studies have used simultaneous stimulation of these receptors with proliferation
cocktails such as anti-CD3/anti-CD28, a model with limited relevance for the in vivo situation.
Little is known about the interplay between the various receptor pathways of the innate
immune system that will eventually lead to a Th17 response during infection with human
pathogens. In the present study, we have focused on the induction of IL-17 by human
microbial pathogens and tried to clarify which innate immune receptors and pathogen-associated molecular patterns (PAMPs) are involved in triggering a Th17 response during the
pathogen-specific host defense. Surprisingly, we have identified the prototypic fungal
pathogen C. albicans as a much more potent inducer of IL-17 production compared to
Gram-negative bacteria. The most important pathway of IL-17 induction was represented by
the engagement of the macrophage mannose receptor (MR), with the TLR2/dectin-1
pathway having a secondary amplification effect on MR-induced IL-17 production.
Materials and Methods
Volunteers. Blood was collected from ten healthy, nonsmoking volunteers who were free
of infectious or inflammatory disease and from three patients who were defective in
dectin-1 expression (B. Ferwerda, G. Ferwerda, T. Plantinga, J.A. Willment, A.B. van Spriel, H.
Venselaar, A. Cambi, C. Huysamen, L. Jacobs, T. Jansen, K. Verheijen, L. Masthof, G. Vriend, D.L.W.,
L.A.B.J., J.W.M.v.d.M., G.J. Adema, B.J.K., G.D. Brown, and M.G.N., unpublished data) after
46 | Chapter 3
informed consent. This was due to a single nucleotide polymorphism at position 238,
leading to a premature stop codon and a defective transport of the truncated dectin-1
protein to the cell surface. Blood samples were collected in 10 ml ethylenediaminetetraacetic acid (EDTA) tubes (BD Vacutainer; Plymouth, UK). Serum was obtained from a
patient with XLA after informed consent.
Reagents and Ligands. Bartonella LPS was used as a potent TLR4 inhibitor (anti-TLR4)
(15). TLR2-blocking antibody (anti-TLR2) and its control IgA1 (catalog numbers maba-htlr2
and maba1-ctrl) were purchased from InvivoGen (San Diego, CA). Anti-dectin-1 antibody
GE2 (anti-dectin-1) was a kind gift of Dr. Gordon Brown (University of Capetown, South
Africa) (16). Mannose macrophage antibody (anti-MR) (catalog number 555953, clone 19.2)
was purchased from BD Biosciences (Dendermonde, Belgium). Matching mIgG1 isotype
control (anti-IgG) was purchased from R&D Systems (Minneapolis). E. coli LPS (serotype
055:B5) was purchased from Sigma (St. Louis), repurified as previously described, and used
as an ultra pure TLR4 ligand (17). TLR ligands Pam3Cys, PolyI:C, Flagellin, and ODN M362
were from InvivoGen (San Diego, CA). Synthetic MDP was purchased from Sigma (St.
Louis). Chitin was kindly provided by Professor Neil A.R. Gow (School of Medical Scienses;
Aberdeen, UK) and prepared according to previous protocols (18). Candida mannan and
particulated β-glucan were prepared as previously described (19;20). Briefly, for Candida
mannan: 100 g wet weight of yeast biomass was suspended in 400 ml 2% (wt/vol) KOH
and heated for 1 hr at 100°C. Insoluble residues were separated by centrifugation, and
mannan was precipitated from supernatant with Fehling’s reagent. The sedimented
mannan-copper complex was dissolved in a minimum volume of 3 M HCl and added
drop wise to methanol/acetic acid at a ratio of 8:1 (vol/vol). The procedure of dissolution
and precipitation was repeated twice. Finally, the sediment was separated, dissolved in
distilled water, and dialyzed for 24 hr. The purified mannan was considered to be maximally
deproteinized. Protein analysis by BCA Kit (Pierce) measured 0.77 μg/ml of protein in the
preparation of 100 μg/ml. Curdlan (Wako) is a dectin-1 ligand consisting of linear
β-1,3-glucan polymers derived from Alcaligenes faecalis (21). Zymosan and mannan (lot
16H3842 from Saccharomyces cerevisiae) were from Sigma (St. Louis). All stimulations with
fungal ligands were in a concentration of 10 μg/ml unless indicated otherwise. Beads were
coated with anti-CD3a alone or in combination with anti-CD28 and were prepared
according to the manufacturer’s instructions (Miltenyi Biotec; Utrecht, The Netherlands).
Microorganisms. C. albicans ATCC MYA-3573 (UC 820), a strain well described elsewhere
(22), was used. C. albicans was grown overnight in Sabouraud broth at 37°C; cells were
harvested by centrifugation, washed twice, and resuspended in culture medium
(RPMI-1640 Dutch modification; ICN Biomedicals; Aurora, OH) (23). To generate
pseudohyphae, C. albicans blastoconidia were grown at 37°C in culture medium adjusted
to pH 6.4 by using hydrochloric acid. Pseudohyphae were killed for 1 hr at 100°C and
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 47
resuspended in culture medium to a hyphal inoculum size that originated from 106/ml
blastoconidia (referred to as 106/ml pseudohyphae). All stimulations with C. albicans were
with a concentration of 106/ml unless indicated otherwise. Clinical isolates of S. enteritidis,
K. Pneumonia, and E. coli were heat-killed and used in a dosage of 107/ml. A heat-killed
clinical isolate of S. cerevisiae was kindly provided by Professor Neil A.R. Gow and used in a
dosage of 106/ml (School of Medical Sciences; Aberdeen, UK).
In Vitro Cytokine Production. Separation and stimulation of PBMCs was performed as
described previously (24). Briefly, the PBMC fraction was obtained by density centrifugation
of diluted blood (one part blood to one part pyrogen-free saline) over Ficoll-Paque
(Pharmacia Biotech; Uppsala). PBMCs were washed twice in saline and suspended in
culture medium supplemented with gentamicin 1%, L-glutamine 1%, and pyruvate 1%.
The cells were counted in a Bürker counting chamber, and their number was adjusted to
5 × 106 cells/ml. Five hundred thousand PBMCs in a volume of 200 μl per well were
incubated at 37°C in round-bottom 96-well plates (Greiner; Nuremberg, Germany), in the
presence of 10% human pooled serum (unless indicated otherwise), with stimuli or culture
medium alone. In blocking experiments, PBMCs were preincubated for 1 hr with various
inhibitors (anti-TLR4 100 ng/ml, anti-TLR2 10 μg/ml, anti-Dectin-1 10 μg/ml, anti-MR 5 μg/
ml, anti-IgG1 10 μg/ml) before stimulation with C. albicans. After 48 hr or 5 days of
incubation, supernatants were collected and stored at −20°C until assayed.
Cytokine Assays. Cytokine concentrations were measured by commercial ELISA kits:
IL-1β, IL-17, and TNF-α (R&D Systems); IL-6, IFN-γ, and IL-10 (Pelikine Compact, Sanquin;
Amsterdam); and IL-23 (eBioscience) according to the manufacturer’s instructions. IL-12p70
and IL-2 were measured using the Bio-Plex (Luminex) cytokine assays from Bio-Rad
(Hercules, CA), following the manufacturer’s instructions.
Intracellular Cytokine Staining. PBMC cells were stimulated for 4-6 hr with PMA (50 ng/
ml) (Sigma) and ionomycine (1 μg/ml) (Sigma) in the presence of GolgiPlug (BD Biosciences)
according to manufacturers’ protocols. Cells were first extracellularly stained using an
anti-CD4/APC antibody (BD Biosciences). Subsequently, the cells were fixed and
permeabilized with Cytofix/Cytoperm solution (BD Biosciences) and then intracellularly
stained with anti-IFN-γ/PE (eBiosciences) and anti-IL-17/FITC (eBiosciences). Samples were
measured on a FACS Calibur, and data were analyzed using the CellQuest Pro software (BD
Biosciences).
Cell Isolation, Stimulation, and Flow Cytometry. Isolation of CD4+ cells was performed
as described previously (25). Purified CD4+ T cells were labeled with CD25-PE-conjugated
mAb (MA251) (BD Biosciences) and CD45RA-ECD-conjugated mAb (2H4) (Beckman
Coulter; Mijdrecht, The Netherlands); thereafter, CD25negCD45RApos and CD25negC-
48 | Chapter 3
D45RAneg cells were isolated by high-purity flow cytometric cell sorting using an Altra
cell sorter (Beckman Coulter). A rerun was performed to analyze the cell purity of the
sorted cells; sorted cells were always more than 98% pure. Monocytes were positively
isolated from PBMCs with magnetic bead isolation using CD14 microbeads (Miltenyi-Biotec; Utrecht, The Netherlands) according to the manufacturer’s instructions. Cells were
cultured in culture medium (RPMI-1640 with glutamax supplemented with pyruvate [0.02
mM], 100 U/ml penicillin, 100 μg/ml streptomycin (all from GIBCO; Paisley, UK), and 10%
human pooled serum (HPS) at 37°C, 95% humidity, and 5% CO2 in 96-well round-bottom
plates (Greiner; Frickenhausen, Germany). Sorted naive (CD25-CD45RA+) and memory
(CD25-CD45RA-) cell (2.5 × 104) populations were cultured with or without autologous
monocytes (1.0 × 105) in 200 μl culture medium in the absence or presence of stimuli. All
stimuli were added at the start of the cultures. Cells were phenotypically analyzed by flow
cytometry. The following conjugated mAbs were used: CD3 (UCHT1) ECD-labeled
(Beckman Coulter). Intracellular analysis of cytokines was performed following stimulation
for 4 hr with PMA (12.5 ng/ml) plus ionomycin (500 ng/ml) in the presence of Brefeldin A
(5 μg/ml) (Sigma-Aldrich). Cells were fixed and permeabilized using Fix and Perm reagents
(eBioscience) and subsequently stained with anti-IL-17-Alexa Fluor 647 (64DEC17)
(eBioscience). Cell samples were measured on a FC500 flow cytometer (Beckman Coulter),
and flow cytometry data were analyzed using CXP software (Beckman Coulter).
Quantitative PCR. PBMCs were cultured for 3 days, and RNA was extracted using TRI
Reagent (Sigma). The isolated RNA was treated with DNase to remove genomic DNA and
subsequently reverse transcribed with oligo(dT) primers in a reverse transcriptase procedure
with a total volume of 20 μl. Quantitative real-time PCR was performed using the ABI/
PRISM 7000 Sequence Detection System with primer pairs and SYBR Green PCR Master
Mix (Applied Biosystems). The sequences used were as follows: IL-17A, forward 5'-TTGATTGGAAGAAACAACGATGA-3' and reverse 5'-CTCAGCAGCAGTAGCAGTGACA-3'; RORγt,
forward 5'-TGAGAAGGACAGGGAGCCAA-3' and reverse 5'-CCACAGATTTTGCAAGGGATCA-3'; MR, forward 5'-CTCAAGACAATCCACCAGTTACTGA-3' and reverse 5'-CTTCTCTTTGCTGAAATAATACTGGTAGTC-3'. Quantification of the PCR signals was performed by comparing
the cycle threshold (Ct) value of the gene of interest with the Ct values of the reference
gene GAPDH. All primers were developed using Primer Express 2.0 (Applied Biosystems)
and validated according to the protocol. Values are expressed as fold increases of mRNA
relative to that in unstimulated cells.
siRNA Transfection. PBMCs were transfected by electroporation with the Amaxa Human
Monocyte Nucleofector kit (Amaxa Inc.; Walkersville, MD) in accordance with the
manufacturer’s instructions. In brief, PBMCs (2 × 107) were harvested and resuspended in
100 μl of nucleofector solution. After addition of MR siRNA (011730) (Dharmacon, Inc.) or
control GFP siRNA (VSC-1001) (Amaxa Inc.) at final concentration of 125 nM, cells were
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 49
electroporated with Amaxa program Y-001 and recovered for 24 hr before further
stimulation.
Statistical Analysis. The differences between groups were analyzed by a two-tailed
paired t test. Differences were considered statistically significant when p ≤ 0.05, and the
actual P-value is given for each test. All experiments were performed at least twice, and
the data are presented as the cumulative result of all experiments performed, unless
otherwise indicated.
Results
Receptor- versus Proliferation-Dependent Induction of IL-17 Production
Several receptor-dependent pathways have been described to induce IL-17 production,
such as dectin-1 (6), NOD2 (14), or TLRs (12). However, all of these studies have used
combinations of receptor ligands and proliferation stimuli such as anti-CD3/anti-CD28
(aCD3/aCD28) antibodies. We wanted to differentiate the relative importance of the receptor-specific and proliferation-dependent induction of IL-17 by stimulating cells with a
known IL-17 inducer, Zymosan, in the presence or absence of aCD3/aCD28. Zymosan alone
was able to induce only very limited amounts of IL-17 in the presence of human serum,
and anti-CD3 as a costimulatory signal was needed in order to potentiate the Zymosaninduced IL-17 production (Figure 1A). The nonspecific stimuli aCD3/aCD28, in the presence
of serum, induced the highest amount of IL-17 secretion (Figure 1A). To investigate whether
TLR and NLR ligands alone were able to induce IL-17 production, PBMCs were stimulated
with a TLR2 ligand (Pam3Cys 10 μg/ml), TLR4 ligand (LPS 10 ng/ml), TLR3 ligand (PolyI:C 10
μg/ml), TLR5 ligand (flagellin 10 μg/ml), TLR9 ligand (ODN 10 μg/ml), and the NOD2 ligand
MDP (10 μg/ml) in the presence of human serum, without additional costimulatory factors.
No single ligand was able to induce IL-17 production, although all ligands were able to
induce IL-6 production (Figure 1B). Several combinations of these ligands (TLR2 with TLR4,
TLR2 with NOD2, and TLR4 with NOD2) were also not able to induce IL-17 production in
human PBMCs (data not shown). Thus, stimulation of TLRs and NOD2 pathways alone
cannot induce production of IL-17.
Pathogen-Induced Production of IL-17
IL-17 is important in the host defense against extracellular bacterial and fungal infections
(2;26). We investigated the capacity of whole microorganisms to induce IL-17 production.
Both Gram-negative and fungal pathogens induced IL-17 secretion. However, C. albicans
was by far the most potent inducer of IL-17 production, in concentrations comparable to
those induced by aCD3/aCD28 antibodies. In contrast, comparable amounts of mono-
50 | Chapter 3
A
B
Figure 1 | Receptor- versus Proliferation-Dependent Induction of IL-17 Production. (A) Human
PBMCs were stimulated for 5 days with RPMI, Zymosan 100 μg/ml, beads coated with anti-CD3, antiCD28, or various combination, in the presence or absence of human serum. Production of IL-17 in the
supernatants was measured by ELISA. (B) Human PBMCs were stimulated for 5 days in the presence
of human serum with TLR ligands (LPS 10 ng/ml, Pam3Cys 10 μg/ml, PolyI:C 10 μg/ml, Flagellin 10
μg/ml, and ODN M362 10 μg/ml), the NOD2 ligand MDP (10 μg/ml), or anti-CD3/anti-CD28 (cell:bead
ratio of 2:1). IL-6 and IL-17 were measured by ELISA. All experiments were performed at least twice
with a total of five healthy volunteers. Data are pooled and expressed as mean ± SEM.
cyte-derived IL-1β, a cytokine known to be important for the induction of IL-17, were
induced by Gram-negative bacteria and C. albicans (Figure 2A). Because bacteria and fungi
differ in size and therefore in surface area and antigen load, we compared high dosages of
Gram-negative bacteria with low dosages of fungi. Even a 1000-fold higher concentration
of Gram-negative bacteria was still less potent in the induction of IL-17 production (data
not shown). RT-PCR showed that the induced IL-17 mRNA was accompanied by upregulation
of RORγt (Figure 2B). The conidial form of C. albicans was the most potent for the induction
of IL-17 (Figure 2A), and both heat-killed and live C. albicans induced significant amounts of
IL-17 (Figure 2C). Cytokine kinetics during these stimulations showed that IL-17 increased
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 51
steadily during the 7 day period, while IL-23 production was maximal in the first 24 hr and
decreased thereafter (Figure 2D). IL-10 was present in the first 48 hr and decreased to
undetectable limits at day 3 and stayed undetectable (Figure 2D). IFN-γ was present at 48
hr and reached its maximum at 4-5 days, while IL-1β reached its maximum level at 24
hours, after which a plateau was attained (Figure 2D). At day 5 of stimulation, flow
cytometry of PBMCs stimulated with C. albicans yeast cells showed a subset of CD4+ cells
that was capable of producing IL-17 alone or in combination with IFN-γ (Figures 2E and 2F).
A
B
C
Figure 2 | Pathogen-Induced Production of IL-17. (A) Human PBMCs were stimulated for 5 days
with RPMI or several pathogens. IL-1β and IL-17 were measured by ELISA. n = 10; data are pooled and
expressed as mean ± SEM. (B) RT-PCR of the expression of RORγt and IL-17A in PBMCs stimulated
with RPMI or C. albicans for 3 days. Data are given as relative mRNA expression (2−ΔCT × 1000) and
expressed as median from two separate experiments (n = 3). (C) PBMCs stimulated for 5 days with
either live C. albicans (105/ml) or heat-killed (HK) C. albicans (105/ml). n = 10; data are pooled and
expressed as mean ± SEM. (D) Time course of IL-17 production in human PBMCs stimulated with
heat-killed C. albicans yeasts or pseudohyphae. Cytokines were measured by ELISA. Data are from
one healthy volunteer and represent the pattern observed in two separate experiments, with a total
of n = 5. (E) Intracellular cytokine staining of IL-17 and IFN-γ in human PBMCs (n = 5) stimulated for 5
days with RPMI or C. albicans and then stimulated for 4 hr with PMA and ionomycin. Cells were gated
for CD4, and data is given as percentage of total gated CD4+ cells.(F) Representative intracellular
cytokine staining of IL-17 and IFN-γ for data given in (E). Data are from one healthy volunteer and
represent the pattern observed in two separate experiments, with a total of n = 5.
52 | Chapter 3
D
E
F
Figure 2 | Continued.
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 53
The Mannose Receptor Is Crucial for the Induction of IL-17 Production by
C. albicans
The structure of the C. albicans cell wall is very different from that of bacteria, being
composed of polysaccharides such as mannans, β-glucans, and chitin (27). Single fungal
components were tested for the induction of IL-17. C. albicans mannan, but not S. cerevisiae
mannan, was able to induce IL-17 production (Figure 3A). Surprisingly, the other fungal
components, β-glucan and chitin, did not induce IL-17 production. Although zymosan
was able to induce IL-1β, it did not lead to a significant production of IL-17 (Figure 3A). C.
albicans mannan showed a dose-dependent induction of IL-17 production (Figure 3A). C.
albicans mannan did not stimulate IL-17 production in the absence of serum (data not
shown). Subsequently, we assessed the receptors leading to IL-17 production in human
PBMCs by blocking them with specific antibodies. Blocking of the MR resulted in the
strongest inhibition of IL-17 production, and quantitative RT-PCR showed that mRNA for
IL-17A and RORγt were downregulated in the presence of the MR inhibitor (Figures 3B and
3C). Blocking of the dectin-1/TLR2 pathway also led to partial inhibition of IL-17 production
(Figure 3C). Blocking of TLR4 had no effect on Candida mannan-induced IL-17 secretion
(Figure 3C). This was not due to the loss of functional blocking by the inhibitor on day 5,
since LPS-induced IL-1β production was still inhibited by TLR4 blocking after 5 days (data
not shown). Furthermore, C. albicans was able to induce pro- and anti-inflammatory
cytokines (Figure 3D). Interestingly, in the presence of the anti-MR, there was no effect on
IL-12, IL-23, IL-2, IL-1β, TNF-α, and IFN-γ production, but there was a significant reduction in
IL-10 (Figure 3D). Next, we determined if the observed lower IL-17 production was in line
with a lower induction of IL-17-producing cells. We observed that blocking the MR
inhibited the percentage of CD4+ IL-17-producing cells (Figures 3E and 3F).
Since S. cerevisiae mannan itself cannot induce IL-17 production, but is able to interact
with the MR, we preincubated PBMCs with an excess of S. cerevisiae mannan (Figure 4A).
This inhibited the IL-17 production induced by C. albicans mannan and C. albicans. To
further establish that C. albicans mannan induces a Th17 response specifically through the
MR, we investigated the effect of siRNA depletion of the MR from monocytes. Levels of MR
mRNA was strongly reduced by the siRNA (Figure 4B), and FACS analysis also demonstrated
decreased expression of MR on the cell membrane (Figure 4C). The IL-17 production
induced by C. albicans mannan was reduced by roughly 50% in PBMCs treated with MR
siRNA (Figure 4D). In contrast, immunoglobulins are not involved in the induction of IL-17
by C. albicans, as serum from both control individuals or patients with X-linked agammaglobulinemia (XLA) was equally potent to support the production of IL-17 (Figure 4E).
IL-17 Production Induced by C. albicans and Mannan Is Monocyte
Dependent
It is known that fungal mannans contain immunodominant T cell epitopes (28) and that
Candida can directly interact with lymphocytes (29). We therefore investigated whether C.
54 | Chapter 3
A
B
Figure 3 | The Mannose Receptor Is the Main Receptor Pathway for the Induction of IL-17
Production by C. albicans. (A) Human PBMCs were stimulated for 5 days with RPMI, several fungal
components (in a concentration of 10 μg/ml), or Zymosan 100 μg/ml. Mannan was given in different
concentrations (μg/ml). IL-1β and IL-17 were measured by ELISA. (B) RT-PCR of the expression of
RORγt and IL-17A in PBMCs stimulated with RPMI or C. albicans at 3 days in the presence or absence
of the blocking antibody for MR (aMR). Data are given as relative mRNA expression (2-DCT 3 1000)
and expressed as median; two separate experiments n = 3. (C) Human PBMCs were stimulated for 48
hr with RPMI as a control or heat-killed C. albicans yeast cells in the presence or absence of blocking
antibodies and isotype controls. For (A)–(C), data are pooled from at least two separate experiments
with a total of six healthy volunteers (mean ± SEM). (D) Human PBMCs were stimulated for 5 days
with heat-killed C. albicans yeast cells in the presence or absence of specific receptor inhibitors
and isotype controls. (E) Intracellular cytokine staining of IL-17 and IFN-γ in human PBMCs (n = 5)
stimulated for 5 days with C. albicans in the presence or absence of the aMR and then stimulated for
4 hr with PMA and ionomycin. Cells were gated for CD4, and data are given as percentage of total
gated CD4+ cells.(F) Representative intracellular cytokine staining of IL-17 and IFN-γ for data given in
(D). Data are from one healthy volunteer and represent the pattern observed in (E).
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 55
C
D
Figure 3 | Continued.
56 | Chapter 3
D
F
Figure 3 | Continued.
E
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 57
A
C
B
D
E
Figure 4 | Blocking the MR with S. cerevisiae Mannan and Inhibiting MR Expression Using
siRNA Transfection. (A) Human PBMCs were preincubated for 1 hr with 100 μg/ml of S. cerevisiae
mannan and stimulated for 5 days with RPMI, C. albicans mannan (10 μg/ml), or heat-killed C. albicans.
(B) RT-PCR for the expression of MR in cells transfected with control siRNA and MR siRNA. (A) and
(B) had a total of five healthy volunteers. Data are pooled and expressed as mean ± SEM. (C) Flow
cytometry: expression of the MR on the cell surface of cells stimulated for 5 days with 10 μg/ml of
C. albicans mannan that were treated with control siRNA (black line) or MR siRNA (dotted gray line).
Data are shown from one volunteer, representative for all volunteers. (D) IL-17 production in cells
transfected with control siRNA or MR siRNA and stimulated with RPMI or C. albicans mannan. (E) IL17 production induced in the presence of serum isolated from healthy volunteers or from a patient
with XLA with immunoglobulin deficiency. (D) and (E) had a total of five healthy volunteers. Data are
pooled and expressed as mean ± SEM.
albicans or purified C. albicans mannan alone was able to directly activate T cells and
would lead to the production of IL-17. Neither naive nor memory CD4+ T cells were able to
produce IL-17 when stimulated in the absence of antigen-presenting cells (APC) (Figure 5A).
We therefore explored the role of APCs for the induction of IL-17. Monocytes were isolated
and stimulated with C. albicans or C. albicans mannan in the presence or absence of naive
58 | Chapter 3
A
B
Figure 5 | IL-17 Production Induced by C. albicans and Mannan Is Monocyte Dependent. (A)
CD4+ naive (CD25-CD45RA+) and CD4+ memory (CD25-CD45RA−) T cells (2.5 × 104) were cultured
with or without autologous monocytes (1.0 × 105) and stimulated for 8 days with culture medium, C.
albicans mannan, or heat-killed C. albicans. IL-17 was measured in supernatants by ELISA. (B) Density
plots show intracellular IL-17 staining of the same experiment as shown in (A). T cells were gated
on CD3; CD14+ monocytes were CD3-negative. Data are representative for two identical, separate
experiments.
or memory T cells. We demonstrate that monocytes alone were not able to produce
IL-17, but the coculture of monocytes with memory CD4+ T cells induced a strong Th17
response (Figure 5B).
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 59
Dectin-1-Deficient PBMCs Have a Relatively Lower Th17 Response to
C. albicans Yeasts
The in vitro stimulations described above suggest that although β-glucan by itself cannot
stimulate IL-17 production, the dectin-1/TLR2 pathway can still contribute to the IL-17
induction by C. albicans, possibly by potentiating other routes of stimulation. To assess
whether this is the case in patients, we investigated IL-17 production in three patients with
a complete defect in dectin-1 expression due to an eary-stop codon mutation (Tyr238Stop)
(B. Ferwerda, G. Ferwerda, T. Plantinga, J.A. Willment, A.B. van Spriel, H. Venselaar, A. Cambi, C.
Huysamen, L. Jacobs, T. Jansen, K. Verheijen, L. Masthof, G. Vriend, D.L.W., L.A.B.J., J.W.M.v.d.M.,
G.J. Adema, B.J.K., G.D. Brown, and M.G.N., unpublished data). FACS analysis confirmed that
the cells of the three patients used in these experiments expressed no dectin-1 on their
surface (Figure 6A). Dectin-1-deficient PBMCs showed a lower IL-17 production compared
to healthy controls when stimulated with C. albicans yeasts (Figure 6B), but not in
pseudohyphae that do not express β-glucans on their surface (30). The normal pattern of
a higher IL-17 production induced by C. albicans yeast cells compared to pseudohyphae
was lost in PBMCs that were dectin-1 deficient. IL-1β and IL-6 induction by C. albicans was
also defective in these patients (data not shown). On the other hand, Candida mannan
induced significant IL-17 production in PBMC isolated from two dectin-1-deficient patients
(Figure 6C), demonstrating that the observed induction of IL-17 by Candida mannan is not
through dectin-1. Furthermore, IL-17 release induced by heat-killed C. albicans was strongly
inhibited by the blockade of MR even in the cells defective for dectin-1, demonstrating
that IL-17 production is largely dependent on the MR in the absence of dectin-1 (Figure 6D).
TLR2 Is Synergistic with the Mannose Receptor for IL-17 Production
Dectin-1 is known to associate with TLR2 and can induce synergistic effects on TLR2
responses (31;32). In order to assess whether stimulation of both receptors could induce
IL-17, we tested the combination of β-glucan with a TLR2 ligand (Pam3Cys). Although
β-glucans synergized with Pam3Cys for the production of IL-1β, IL-10, IFN-γ, and TNF-α, no
IL-17 production was induced by this combination of stimuli (Figure 7A). Preparations of
β-glucans (β-glucan or curdlan) were not able to stimulate IL-17 production either alone or
in combination with C. albicans mannan (data not shown). This is most likely due to the
requirement of spatial presentation of the β-glucans in the fungal cell wall, stimulating
dectin-1 more efficiently compared to the purified β-glucan preparations. Surprisingly,
although Pam3Cys alone was not capable of inducing IL-17, combining Pam3Cys with C.
albicans mannan resulted in strong synergism for IL-17 production (Figure 7A). RT-PCR
demonstrated that upregulation of mRNA for IL-17 was also synergistic (Figure 7B).
Interestingly, the mannan-induced IFN-γ and IL-1β was significantly inhibited by stimulating
the TLR2 pathway, while the production of IL-10 and TNF-α by Candida mannan was
unaffected by Pam3Cys (Figure 7C).
60 | Chapter 3
A
C
B
D
Figure 6 | Dectin-1-Deficient PBMCs Have a Relatively Lower Th17 Response to C. albicans
Yeasts. (A) Flow cytometry of the expression of dectin-1 on the surface of PBMCs isolated from
healthy volunteers (C1 and C2) and from three individuals homozygous for the Tyr238 stop mutation
(P1–P3). (B) PBMCs described in (A) were stimulated for 5 days with heat-killed C. albicans yeasts or
pseudohyphae. (C) Candida mannan induced significant IL-17 production in PBMCs isolated from
control volunteers (black bars) as well as from two dectin-1-deficient patients (gray bars). (D) IL-17
release induced by heat-killed C. albicans was strongly inhibited by the blockade of MR in both
control (black bars) and dectin-1-deficient (gray bars) PBMCs. For (C) and (D), data are pooled and
expressed as mean ± SEM. IL-17 was measured by ELISA.
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 61
A
C
B
Figure 7 | TLR2 Is Synergistic with the Mannose Receptor for IL-17 Production. (A) Human
PBMCs were stimulated for 5 days with RPMI, fungal components, or/and the TLR2 ligand Pam3Cys.
IL-17 was measured by ELISA. Data are pooled from at least two separate experiments with a total of
six healthy volunteers (mean ± SEM). (B) RT-PCR of the expression of IL-17A in PBMCs stimulated with
RPMI, Pam3Cys and mannan, or combinations for 3 days. Data are given as relative mRNA expression
(2−ΔCT × 1000) and are expressed as median; two separate experiments n = 3. (C) Human PBMCs
were stimulated for 48 hr with the same set of stimuli used in (B), and IL-1β, IL-10, IFN-γ, and TNF-α
were measured by ELISA. Data are pooled from at least two separate experiments with a total of six
healthy volunteers (mean ± SEM).
62 | Chapter 3
Discussion
IL-17 has emerged as one of the most important proinflammatory cytokines due to its
modulation of neutrophil-mediated responses, with important roles for both host defense
(2;5;26) and autoimmunity (7-9). While most studies to date have focused on the conditions
(cytokine profiles) necessary for generating Th17 cells in vitro (10;11) or the generation of
Th17 cells in mice (33;34), very little is known about the pathways regulating IL-17 upon the
encounter of pathogenic microorganisms by human cells. In addition, in order to stimulate
IL-17 production, these studies have used aCD3/aCD28 antibodies for inducing T cell
proliferation, artificial conditions obviously not encountered during in vivo infections or
inflammation. In the present study, we have investigated the pathways of IL-17 stimulation
by human primary cells stimulated with whole microorganisms, conditions that mimic
real-life infections. In addition, we deciphered the receptor-ligand pathways responsible
for IL-17 induction by the fungal pathogen C. albicans. We demonstrate that C. albicans is
the most potent pathogen in eliciting a pathogen-mediated Th17 response in human
PBMCs in the absence of anti-CD3 and/or anti-CD28 antibodies. The MR is the main
pathway through which C. albicans induces the Th17 response. While no single TLR or
NOD2 ligand was able to induce IL-17 in the absence of aCD3/aCD28 antibodies, mannan
from C. albicans was the only component capable of inducing IL-17 secretion. Interestingly,
the dectin-1/TLR2 pathway was able to synergize with the MR-induced IL-17 secretion,
revealing an interaction between C-type lectin receptors and TLRs. These data imply that
direct recognition through the MR is sufficient for inducing a Th17 response, and that this
ability is unique to the MR within the family of pattern recognition receptors.
Using anti-CD3 or a combination of anti-CD3 and anti-CD28 antibodies in order to
investigate the pathways leading to the generation of Th17 cells, several studies suggested
that dectin-1 (13), NOD2 (14), or TLR (12) receptors specifically induce Th17 generation. However,
these results are potentially biased by the use of anti-CD3 and anti-CD28 antibodies that can,
by themselves, induce IL-17, as clearly demonstrated by our results. In this study, we did not
use aCD3/aCD28 antibodies to investigate the IL-17 responses to specific pathogens or ligands,
but instead optimized the conditions necessary for the induction of IL-17 in human cells by
pathogens alone. Human serum alone did not induce IL-17 secretion in human PBMCs, but
was able to optimize the conditions needed for the induction of a Th17 response. Recently,
it has been speculated that serum contains small amounts of TGF-β that may be responsible for
the observed differences between IL-17 production of cells cultured in the absence or
presence of human serum (35). Furthermore, we have addressed the role of serum antibodies
in our experiments by using serum from patients with XLA. These patients cannot produce
antibodies, because they lack functional B cells. No differences were observed between pooled
serum and XLA serum devoid of antibodies, concluding that IL-17 production was not
antibody dependent. Notably, in the absence of aCD3/aCD28, we show that no single TLR or
NOD2 ligand was able to induce IL-17 production in human PBMCs.
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 63
IL-17 is important for the host defense against human pathogens (2;5). Although both
Gram-negative bacteria and the fungus S. cerevisiae were able to induce IL-17 production
in human PBMCs, C. albicans was by far the most potent inducer of IL-17 secretion. The IL-17
production corresponded with the appearance of a subset of CD4+ cells that contained
cells capable of producing IL-17 or both IFN-γ and IL-17. Since C. albicans was the most
potent inducer of IL-17 production, we wanted to further dissect the pathways responsible
for inducing the Th17 response by this fungal pathogen. When purified fungal components
were tested, we observed a strong IL-17 production in PBMCs upon stimulation with C.
albicans mannan. This is in line with the fact that live Candida was even more potent in the
induction of IL-17 production compared to heat-killed yeasts, since mainly mannoproteins
are exposed when live Candida is used as a stimulus. Interestingly, S. cerevisiae mannan
was not able to induce IL-17, and this is in line with the absent Th17 response to Zymosan,
as Zymosan is derived from S. cerevisiae cell wall. The differential recognition of mannans
from C. albicans and Saccharomyces is most likely due to the different branching of these
structures: while Candida-derived mannan has a highly branched structure, Saccharomyces
mannan mainly contains short linear chains of mannose polymers (36). These data argue
that the MR is able to coordinate the different responses to fungal pathogens, particularly
regarding the Th17 response. To investigate further the receptor pathways responsible for
IL-17 induction by Candida, the main receptors involved in fungal recognition were
blocked with specific antibodies before stimulation of IL-17 was triggered. In line with the
effects of Candida mannan, blocking of the MR resulted in the strongest inhibition of IL-17
production, confirming its important role in the Th17 response to C. albicans. Blockade of
MR did not only result in a lower amount of IL-17, but also led to a lower percentage of
CD4+ cells that were able to produce IL-17 alone or the combination of IFN-γ and IL-17.
Furthermore, blocking the MR with an excess of S. cerevisiae mannan or decreasing the MR
expression with siRNA also inhibited IL-17 production.
It has been demonstrated in the literature that mannoproteins contain immunodominant
T cell epitopes (28). Several mannoproteins, among them mannoprotein 65 kDa (MP65),
stimulate T cell proliferation through their protein parts, with the mannan component
being responsible for the cytokine induction (37). Indeed, recombinant MP65 devoid of its
mannan structure was unable to stimulate cytokine production (28). We have measured
the amount of protein in our highly purified C. albicans mannan preparation. Despite the
extensive purification procedures, we have measured a quantity of 0.77 μg/ml protein in
the C. albicans mannan preparation with a concentration of 100 μg/ml. Although this
amount is far lower than the protein content in the mannoproteins used in the literature,
which have a protein-to-polysaccharide ratio of 1.8:1 (38), we cannot exclude that these
protein components induced TCR-dependent cell proliferation. These effects, due to Candida-specific epitopes, may in fact explain why the memory T cells were by far more
effective for the release of IL-17 in comparison with the naive T cells. Therefore, we envisage
a model in which TCR-dependent effects are responsible for T cell activation/proliferation,
64 | Chapter 3
while the mannan/MR interaction induces the skewing of this population toward a Th17
phenotype. Saccharomyces mannan also contained these low amounts of protein.
However, it did not stimulate IL-17 release, most likely due to the important differences in
the branching of the mannan structures that do not allow proper stimulation of MR by
Saccharomyces mannan (36).
When cells were stimulated with a combination of TLR ligands or TLR ligands with the
NOD2 ligand MDP, no IL-17 production was observed in human PBMCs. Blockade of the
TLR2/dectin-1 pathway partially inhibited IL-17 production, while TLR4 blockade had no
effect. Stimulation of cells with both TLR2 and dectin-1 ligand, alone or in combination,
did not result in IL-17 production, although synergism for the production of other cytokines
has been previously reported (32) and confirmed in our study. Interestingly, TLR2 showed
a specific synergistic effect on the IL-17 production induced by the MR, while no such
effect was apparent on IL-1β production. The role of dectin-1 was further investigated by
stimulating PBMCs isolated from individuals homozygous for an early stop codon
mutation in the dectin-1 gene, which leads to a total loss of dectin-1 activity (data not
shown). When compared to cells isolated from healthy volunteers, the dectin-1-deficient
PBMCs released less IL-17 after stimulation with C. albicans. The role of dectin-1 for the
amplification of IL-17 production is supported by an additional line of evidence. We
observed that the Th17 response induced by C. albicans yeast cells was stronger than the
Th17 response induced by C. albicans pseudohyphae. The fact that C. albicans
pseudohyphae do not express the dectin-1 ligand β-glucan on their surface (30) and that
the observed pattern was lost in PBMCs lacking dectin-1 expression on their membrane
suggests an amplification of β-glucan/dectin-1 on IL-17 production induced by other C.
albicans components (e.g., mannan). These observations are in line with a recent study
that suggests that dectin-1 signaling through Syk-CARD9 represents an important
pathway in the Th17 response induced by C. albicans (13). However, β-glucans alone were
not able to induce IL-17, and they did not give additional IL-17 production in combination
with Candida mannan. Candida mannan induced significant IL-17 production in the dectin-1-deficient patients, demonstrating that the observed induction of IL-17 by Candida
mannan is not through dectin-1. Furthermore, IL-17 release induced by heat-killed C.
albicans was strongly inhibited by the blockade of MR, demonstrating that IL-17 production
is largely dependent on the MR in the absence of dectin-1. These data imply that the MR
is the sole receptor that can directly lead to IL-17 production in human PBMCs, and this
pathway is amplified by dectin-1/TLR2 signaling.
In the present study, we report a methodology to investigate the pathogen-specific
stimulation of Th17 cells in conditions mimicking the in vivo situation. We demonstrate the
strong capacity of C. albicans to induce IL-17 production, and we have deciphered the
receptor pathways involved. However, the data presented here also bring new questions
into light and open avenues for future research. First, one of the most interesting
observations regards the capacity of TLR2 ligation to amplify MR-induced IL-17 on the one
The macrophage mannose receptor induces IL-17 in response to Candida Albicans | 65
hand but to inhibit IFN-γ production on the other hand. We and others have previously
shown that TLR2 engagement is not able to induce Th1 cytokines (17;39;40). The data
presented here demonstrate that the TLR2 pathway shifts the immune response toward a
Th17 type, with important consequences for understanding pathophysiology of disease.
In addition, previous studies have shown the capacity of TLR2 to induce Treg proliferation
(41). It has been recently shown that FOXP3+ cells control the secondary induction of
RORγt, and Treg cells can be directed to induce differentiation of Th17 cells in the presence
of IL-6 (42) or even differentiate themselves into Th17 cells (25). Our data suggest that TLR2
may play an important role in this process, but future studies are needed to assess this
hypothesis in more detail. Second, an important aspect to be investigated regards the
pathways through which MR induces IL-17. Indeed, a very recent study has demonstrated
that MR is recruited to the phagosome and has a crucial role for cytokine induction by C.
albicans (43). However, the intracellular signaling induced by MR is yet unknown, and the
cytokine profile induced by MR that is responsible for IL-17 induction is the first aim of
future studies in our laboratory. All cytokines known to be required for the induction of
IL-17—e.g., IL-23, IL-1β, and IL-6—are stimulated by MR engagement by C. albicans or
mannan, fulfilling a first set of conditions necessary for IL-17 production. However, none of
these cytokines has been specifically induced only by or especially by MR, implying that
an additional factor important for IL-17 production is induced by MR recognition. Third,
one has to acknowledge that although C. albicans was by far the most potent inducer of
IL-17 production, the Gram-negative bacteria Escherichia coli, K. pneumonia, and Salmonella
enteritidis also stimulated small amounts of IL-17. TLR and NOD2 ligands, alone or in
combination, did not stimulate IL-17 release, and therefore the precise pathways through
which Gram-negative bacteria stimulate IL-17 production remain to be elucidated.
In conclusion, we have analyzed the innate receptors and PAMPs responsible for
inducing a Th17 response during a specific pathogen-host interaction. In anti-C. albicans
host defense, the MR takes center stage in the induction of the Th17 response, and this
pathway can be augmented by TLR2/dectin-1. These data reveal a pathway leading to the
induction of the Th17 response in human PBMCs. The essential position played by the MR
warrants further investigation for its role in the Th17 response during infection or auto­immune diseases and might lead to the design of novel immunotherapeutic strategies.
Acknowledgments
This study was supported by a Vidi Grant of the Netherlands Foundation for Scientific
Research to M.G.N. We thank Esther van Rijssen for the help with T cell isolation and flow
cytometry.
66 | Chapter 3
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(42) Xu L, Kitani A, Fuss I, Strober W. Cutting edge: regulatory T cells induce CD4+CD25-Foxp3- T cells or are
self-induced to become Th17 cells in the absence of exogenous TGF-beta. J Immunol 2007; 178(11):6725-9.
(43) Heinsbroek SE, Taylor PR, Martinez FO, Martinez-Pomares L, Brown GD, Gordon S. Stage-specific sampling
by pattern recognition receptors during Candida albicans phagocytosis. PLoS Pathog 2008; 4(11):e1000218.
Chapter 4
Tumor necrosis factor–interleukin-17
interplay induces S100A8, interleukin-1β,
and matrix metalloproteinases, and drives
irreversible cartilage destruction in
murine arthritis: Rationale for combination
treatment during arthritis
Marije I. Koenders1, Renoud J. Marijnissen1, Isabel Devesa2, Erik Lubberts3,
Leo A. B. Joosten1, Johannes Roth4, Peter L. E. M. van Lent1, Fons A. van de Loo1,
Wim B. van den Berg1
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Universidad Miguel Hernández, Alicante, Spain
3
Erasmus Medical Centre, Rotterdam, The Netherlands
4
University of Muenster, Muenster, Germany
1
2
70 | Chapter 4
Abstract
Objective
To examine whether synovial interleukin-17 (IL-17) expression promotes tumor necrosis
factor (TNF)–induced joint pathologic processes in vivo, and to analyze the surplus
ameliorative value of neutralizing IL-17 in addition to TNF during collagen-induced arthritis
(CIA).
Methods
Adenoviral vectors were used to induce overexpression of IL-17 and/or TNF in murine knee
joints. In addition, mice with CIA were treated, at different stages of arthritis, with soluble
IL-17 receptor (sIL-17R), TNF binding protein (TNFBP), or the combination.
Results
Overexpression of IL-17 and TNF resulted in joint inflammation and bone erosion in murine
knees. Interestingly, IL-17 strikingly enhanced both the joint-inflammatory and joint-destructive capacity of TNF. Further analysis revealed a strongly enhanced up-regulation of
S100A8, IL-1β, and matrix metalloproteinase (MMP) messenger RNA, only when both TNF
and IL-17 were present. Moreover, the increase in irreversible cartilage destruction was not
merely the result of enhanced inflammation, but also was associated with a direct
synergistic effect of these cytokines in the joint. S100A9 deficiency in mice protected
against IL-17/TNF–induced expression of cartilage NITEGE neoepitopes. During established
arthritis, the combination of sIL-17R and TNFBP was more effective than the anticytokine
treatments alone, and significantly inhibited further joint inflammation and cartilage
destruction.
Conclusion
Local synovial IL-17 expression enhances the role of TNF in joint destruction. Synergy
between TNF and IL-17 in vivo results in striking exaggeration of cartilage erosion, in
parallel with a synergistic up-regulation of S100A8, IL-1β, and erosive MMPs. Moreover,
neutralizing IL-17 in addition to TNF further improves protection against joint damage and
is still effective during late-stage CIA. Therefore, compared with anti-TNF alone, combination
blocking of TNF and IL-17 may have additional therapeutic value for the treatment of
destructive arthritis.
Synergistic effects of TNF-α and IL-17 on cartilage erosion in vivo | 71
Introduction
Rheumatoid arthritis (RA) is a chronic disorder with unknown etiology. It is characterized
by autoimmunity, infiltration of joint synovium by activated inflammatory cells, synovial
hyperplasia, and progressive destruction of cartilage and bone. In the last decade, tumor
necrosis factor (TNF) has been demonstrated to play a key role in the pathologic processes
of RA (1–5). In the murine model of collagen-induced arthritis (CIA), it was shown that
neutralizing TNF ameliorated CIA, especially when treatment was started before or early
after the onset of disease (6). Importantly, neutralizing TNF in RA patients considerably
improves joint inflammation and inhibits the progression of joint destruction (1–5). This
led to use of the first anti-TNF biologic agents, for the treatment of RA.
Although anti-TNF therapy leads to considerable improvement of RA, this treatment
does not stop progression of the disease in many patients. It is thus tempting to speculate
that other cytokines are critical in inducing and maintaining the chronic destructive joint
inflammation of RA, and therefore these other cytokines should be considered important
additional targets for future therapy. In addition, further improvement of the current
anti-TNF therapy to extend the beneficial effects to more RA patients is a challenge.
A recent study showed that anti-TNF treatment, despite ameliorating the arthritis,
increased the production of interleukin-17 (IL-17) during CIA (7). IL-17 is a proinflammatory
cytokine that is expressed in the synovium and synovial fluid of RA patients (8–11). Th17
cells have been identified as the main producers of this proinflammatory cytokine (12, 13),
but also γ/δ T cells, natural killer T cells, polymorphonuclear neutrophils, and mast cells
have been shown to express or release IL-17. Studies have shown that IL-17 plays a role in
many models of autoimmunity, including experimental arthritis (14, 15). For instance,
elevated levels of IL-17 were found in CIA, and neutralizing IL-17 at different stages of CIA
ameliorated the severity of the arthritis (16, 17). In addition, substantially diminished
expression of CIA was observed in IL-17–deficient mice (18).
Furthermore, spontaneous arthritis development in IL-1 receptor antagonist–deficient
mice was abolished by the inactivation of IL-17 expression, and blocking IL-17 after the
onset of arthritis could prevent further aggravation of disease (19, 20). In addition, using
IL-17 receptor (IL-17R)–deficient mice, it was shown that IL-17R signaling plays a critical role
in driving the synovial expression of proinflammatory and catabolic mediators during the
development of chronic destructive arthritis (21). Interestingly, IL-17R signaling in radiationresistant cells in the joint was required for full progression of destructive synovitis (22).
These studies indicate a significant role for IL-17 in the development and progression of
chronic destructive arthritis.
IL-17 is a potent inducer of various cytokines and chemokines (23–25). In addition, this
T cell cytokine has been shown to have additive or even synergistic effects with TNF and
IL-1 on cytokine induction and tissue destruction (26–30). Of note, an IL-1–independent
role for IL-17 in synovial inflammation and joint destruction in CIA has been reported (17),
72 | Chapter 4
whereas the bone-erosive effect of IL-17 in vivo is strongly mediated by RANKL (31). We
previously demonstrated that IL-17 acts in a manner independent of TNF under arthritic
conditions (32). In an ex vivo RA model utilizing cocultures of RA synovial tissue, a
combination of TNF blockade with blockade of IL-1 and IL-17 was more effective than
blockade of single cytokines for controlling synovial inflammation and bone resorption
(33). However, the effect of in vivo blockade of IL-17 in combination with blockade of TNF
on the pathologic processes of arthritis is still unknown.
In this study, we examined the additional pathologic contribution of synovial IL-17 in
TNF-induced joint pathologic processes in vivo. In addition, we hypothesized that blocking
IL-17 in addition to TNF should further improve the efficacy of anti-TNF therapy in CIA.
Material and Methods
Animals. Male DBA/1J mice were obtained from Janvier-Elevage. S100A9−/− mice,
backcrossed to the C57BL/6 background for 10 generations, were provided by one of the
authors (JR). Wild-type (WT) C57BL/6 mice (The Jackson Laboratory) were used as controls.
All mice were used between 10 weeks and 12 weeks of age and were housed in filter-top
cages under specific pathogen–free conditions. A standard diet and water were provided
ad libitum. All animal procedures were approved by the institutional ethics committee.
Adenoviral vectors. Adenoviral TNF (AdTNF) and AdIL-17 were kindly provided by Dr. J. K.
Kolls (Children’s Hospital of Pittsburgh, Pittsburgh, PA). Viruses were under the regulation
of a cytomegalovirus promoter and were constructed as reported previously (34). The
recombinant adenoviruses contained fewer than 1 endotoxin unit/ml, as measured by the
Limulus amebocyte lysate assay (BioWhittaker). A replication-deficient empty viral vector,
AdDel70-3, was used as the control vector throughout the study.
Study protocol. Naive mice were anesthetized with isoflurane, and a small aperture in the
skin of the knee was introduced for the intraarticular injection procedure. For local over­
expression of IL-17 and/or TNF, 1 × 107 plaque-forming units (PFU) of each adenoviral vector in
6 ml phosphate buffered saline (PBS) was injected intraarticularly into the knee joint. As a control,
the same amount of the control vector, AdDel70-3, was used. Four or 10 days thereafter,
mice were killed by cervical dislocation, and the knee joints were isolated for histology.
Histology. For standard histology, isolated joints were fixed for 4 days in 10% formalin,
decalcified in 5% formic acid, and subsequently dehydrated and embedded in paraffin.
Standard frontal sections, 7 mm in size, were mounted on SuperFrost slides (MenzelGläser). Hematoxylin and eosin staining and Safranin O staining were performed to study
the joint pathologic features. The severity of arthritis in the joints was scored on a scale of
Synergistic effects of TNF-α and IL-17 on cartilage erosion in vivo | 73
0–3 (where 0 = no pathology and 3 = maximal cellularity), as previously described (17), for
5 different parameters: joint inflammation, proteoglycan (PG) depletion from the cartilage
matrix, chondrocyte death, cartilage surface erosion, and bone erosion. Histopathologic
changes were scored on 5 semiserial sections, spaced 140 mm apart. Scoring was
performed in a blinded manner by 2 independent observers.
VDIPEN neoepitope staining. Irreversible PG damage by matrix metalloproteinase
(MMP) activity in the cartilage was assessed using immunohistochemical detection of the
VDIPEN neoepitope, as previously described (35). Briefly, joint sections were deparaffinized,
rehydrated, and digested in chondroitinase to remove chondroitin sulfate from the PGs.
Sections were treated with 1% H2O2 in methanol, followed by treatment with 0.1% Triton
X-100. After incubation with 1.5% normal goat serum, sections were incubated overnight
at 4°C with rabbit anti-VDIPEN IgG (a kind gift from Dr. John Mort, Montreal, Quebec,
Canada) or normal rabbit IgG.
RNA isolation and quantitative polymerase chain reaction (PCR) analysis. Mice
were killed by cervical dislocation, immediately followed by dissection of the patella with
adjacent synovium. RNA from the patellar cartilage and pooled synovial biopsy tissue was
isolated, as described previously (35). Real-time quantitative PCR was performed using the
ABI Prism 7000 Sequence Detection System, for quantification with SYBR Green Master
Mix and melting curve analysis. Relative quantification of the PCR signals was performed
by comparing the threshold cycle (Ct) value, in duplicate, for the gene of interest of each
sample with the Ct values for the reference gene, GAPDH. The quantitative PCR analysis for
each sample was performed in duplicate.
NF-κB luciferase bioassay. The ability of IL-17 and TNF to activate NF-κB was tested on
responsive fibroblast NF-κB reporter cells. One day prior to conducting the experiment,
NIH-3T3 reporter cells were seeded in Krystal 2000 96-well plates (Thermo Labsystems) at
a concentration of 2–4 × 104 cells/well. Cells were cultured in 5% CO2 at 37°C. Six hours
after the addition of IL-17, TNF, or the combination (both at 10 ng/ml), NF-κB activation was
measured through determination of intracellular luciferase production. Luciferase activity
was quantified using the Bright-Glo luciferase assay system (Promega), followed by
luminometric detection, according to the manufacturer’s protocol (Polarstar Galaxy; BMG).
Measurement of NITEGE neoepitopes in patellar cartilage. Patellae were derived
from S100A9−/− mice and WT mice, and the synovium was carefully removed. Patellae
were incubated for 24 hours with 100 ng/ml of IL-17, TNF, IL-17 plus TNF, or IL-1β as a positive
control (all recombinant murine cytokines from R&D Systems). Patellae were decalcified in
EDTA, and 5-μm sections were stained with anti-NITEGE antibodies, a kind gift from Dr.
John Mort, and counterstained with hematoxylin (Merck).
74 | Chapter 4
Induction of CIA. Bovine type II collagen (CII) was prepared as described previously (6). CII
was diluted in 0.05M acetic acid to a concentration of 2 mg/ml and emulsified in equal
volumes of Freund’s complete adjuvant (2 mg/ml of Mycobacterium tuberculosis strain
H37Ra; both from Difco). The DBA/1J mice were immunized intradermally at the base of
the tail with 100 μg of CII. On day 21, mice received an intraperitoneal (IP) booster injection
of 100 μg of CII dissolved in PBS, and the onset of arthritis usually occurred a few days after
the booster injection. Mice were carefully examined 3 times per week for the visual
appearance of arthritis in the peripheral joints, and scores for disease activity were given
as previously described (36). The clinical severity of arthritis (arthritis score) was graded on
a scale of 0–2 for each paw, according to changes in redness and swelling in the digits or
in other parts of the paws.
Anticytokine treatment. Recombinant murine soluble IL-17R:Fc fusion protein (sIL-17R)
(37) was kindly provided by Dr. S. D. Lyman (Immunex, Seattle, WA). To neutralize TNF, mice
were treated with dimerically linked PEGylated soluble p55 TNF receptor I (Amgen). This
so-called TNF binding protein (TNFBP) showed efficacy in murine streptococcal cell wall
(SCW)–induced arthritis (38) and CIA (39). Mice were injected IP with 3 mg/kg sIL-17R,
TNFBP, or the combination. Bovine serum albumin (BSA; Sigma-Aldrich), at a dose of 6 mg/
kg, was used as a control. A total of 4 injections were given on alternate days. Anticytokine
treatment was started before the clinical onset of disease (day 25) or during established
arthritis (macroscopic arthritis scores of 2.0–2.5, on a scale of 0–8).
Statistical analysis. Differences between experimental groups were tested using oneway analysis of variance or the Kruskal-Wallis test with Dunn’s test for multiple comparisons,
unless stated otherwise. P values less than 0.05 were considered significant. Results are
expressed as the mean ± SEM.
Results
Enhancement of the inflammatory and joint-destructive capacity
of TNF by IL-17
To investigate the individual potency of IL-17 and TNF, as well as the combination of these
2 cytokines, in the induction of joint pathologic processes, naive mice were intraarticularly injected with an adenoviral vector expressing IL-17 and/or TNF, or a control vector,
directly into the knee joint. The adenoviruses were injected at a concentration of 1 × 107
PFU in 6 μl, resulting in rapid overexpression of the cytokines in the first 24 hours (mean ±
SEM peak expression in synovial washouts reaching 544 ± 73 pg/ml for IL-17 and 589 ± 208
pg/ml for TNFα), followed by a fast decline from these levels and loss of expression within
1 week.
Synergistic effects of TNF-α and IL-17 on cartilage erosion in vivo | 75
Four and 10 days after injection, the knee joints were isolated for histology. Over­
expression of IL-17 resulted in influx of inflammatory cells into the joint, accompanied by
mild bone erosion (Figures 1 and 2A). Local TNF gene transfer resulted in slightly more
joint inflammation as well as a higher degree of cartilage PG depletion than that following
gene transfer with IL-17 (Figures 1 and 2A). Of note, although overexpression of both proinflammatory cytokines led to cartilage PG depletion, hardly any irreversible cartilage
damage (chondrocyte death and surface erosions) was found in these groups; only TNF
overexpression induced some cartilage erosion on day 10 (Figure 1).
The combination of IL-17 and TNF enhanced joint inflammation and bone erosion,
in an additive manner (Figures 1 and 2A). Intriguingly, although IL-17 itself did not induce
irreversible cartilage destruction, IL-17 in combination with TNF synergistically enhanced
chondrocyte death and cartilage surface erosions (Figures 1 and 2A). VDIPEN, a metalloproteinase-generated aggrecan neoepitope, was hardly detectable in the knee joints
of mice injected with only AdTNF or only AdIL-17. However, the amount of VDIPEN
expression increased dramatically when TNF and IL-17 were simultaneously overexpressed in the joint (Figures A and 2B). These findings show that the presence
of both TNF and IL-17 amplifies the joint-inflammatory and joint-destructive capacity
of the single cytokines.
Synergistic up-regulation of S100A8 and IL-1β by TNF and IL-17
Synovial biopsy tissue and isolated cartilage layers from the TNF/IL-17 overexpression
study were further analyzed to detect the gene regulation that accompanies aggravated
joint inflammation and, more importantly, the synergistic effects on cartilage destruction.
Quantitative PCR analysis demonstrated that, when compared to the control group, both
TNF and IL-17 induced a ±60-fold up-regulation of IL-1β messenger RNA (mRNA), and even
induced a ±350-fold increase in S100A8 mRNA, an alarmin that was recently found to
regulate severe cartilage destruction (40) (Figure 3A). Interestingly, an even stronger, more
additive up-regulation of S100A8 and IL-1β was found in the synovium of mice over-­
expressing both TNF and IL-17 (Figure 3A). In contrast, cartilage-destructive MMP-14 and
bone-protective osteoprotegerin mRNA were slightly up-regulated, to a similar extent, in
all 3 cytokine groups. The levels of mRNA for the osteoclast regulator RANKL were more
potently up-regulated by IL-17 than by TNF, while the combination of IL-17 plus TNF did
not further enhance the expression of RANKL mRNA (Figure 3A).
TNF and IL-17 synergy associated with enhanced activation of NF-κB
and up-regulation of MMPs
To study the effect of combined stimulation with TNF and IL-17 on intracellular NF-κB
signaling, we used an NF-κB reporter fibroblast that produces luciferase upon NF-κB
activation. Following stimulation of the cells with equal concentrations of TNF and IL-17,
we observed that both cytokines were able to activate NF-κB, with TNF being more
76 | Chapter 4
Figure 1 | Results of histologic analysis and VDIPEN staining to determine irreversible
cartilage destruction in murine knee joints. Histologic scores were determined for the extent
of inflammatory infiltrate, cartilage proteoglycan (PG) depletion, bone erosion, chondrocyte death,
cartilage surface erosion, and VDIPEN staining after local overexpression of interleukin-17 (IL-17)
and/or tumor necrosis factor (TNF) in the knee joints of naive mice. Adenoviruses (AdControl, AdIL17, AdTNF, or the combination of AdIL17 plus AdTNF at 1 × 107 plaque-forming units [PFU]) were
injected intraarticularly, and on days 4 and 10, the knee joints were isolated for analysis of histologic
parameters and VDIPEN expression. Results are the mean ± SEM of 4–6 mice per group. * = P < 0.05;
*** = P < 0.001 versus AdControl.
Synergistic effects of TNF-α and IL-17 on cartilage erosion in vivo | 77
A
B
Figure 2 | IL-17 enhances the joint-destructive capacity of TNF. Inflamed knee joints were
stained with hematoxylin and eosin for histologic features (A) or stained for VDIPEN expression as
a marker for matrix metalloproteinase–mediated cartilage destruction (B), at 10 days after a single
injection of an adenoviral vector expressing IL-17, TNF, the combination of IL-17 plus TNF, or a control
vector. Note the enhanced joint inflammation and joint destruction in the IL-17/TNF combination
group. Original magnification × 100. See Figure 1 for definitions.
potent than IL-17. The combined stimulation with TNF and IL-17 resulted in a significantly
enhanced NF-κB activation (Figure 3C).
The association of the TNF/IL-17 interplay with regulation of target genes was shown
not only in synovial tissue, but also in cartilage. Following overexpression of TNF or IL-17 in
cartilage chondrocytes, the mRNA expression of various MMPs was up-regulated
compared to that in the control group (Figure 3B). However, the chondrocytes showed a
much stronger up-regulation of MMP-3, MMP-13, and ADAMTS-4 when both cytokines
were present (Figure 3B). For example, the expression of MMP-13 was enhanced by
±14-fold by AdTNF or AdIL-17 compared to that in the control group, and when AdTNF
and AdIL-17 were combined, this expression showed a further increase of ±6-fold
compared to that with the single cytokines.
Direct synergistic effects of IL-17 and TNF on cartilage destruction
After injection of 1 × 107 PFU of each cytokine virus, we demonstrated that only the
combination of IL-17 and TNF induced severe cartilage destruction. In the next experiment,
we aimed to demonstrate that TNF and IL-17 act in synergy directly on cartilage destruction,
and sought to exclude the possibility that the enhancements in MMP expression, VDIPEN
staining, and cartilage destruction were caused only by increased inflammation. In this
experiment, lower doses of AdIL-17/AdTNF were used in the combination group (each 3 ×
106 PFU), which resulted in a comparable level of synovial inflammation as that with the
normal dose of each single-cytokine virus (kept at 1 × 107 PFU). As shown in Figure 4, the
combination of IL-17 and TNF at this lower dose no longer resulted in aggravated PG
78 | Chapter 4
A
B
C
Figure 3 | Effects of overexpression of TNF and/or IL-17 on gene regulation and NF-κB
activation during aggravated inflammation in murine joints. A and B, Combined overexpression
of TNF and IL-17 resulted in enhanced up-regulation of synovial S100A8 and IL-1β mRNA, with little
effect on synovial RANKL, osteoprotegerin (OPG), or matrix metalloproteinase 14 (MMP-14) mRNA
expression (A), and enhanced up-regulation of cartilage MMP-3 and MMP-13 mRNA, with little effect
on cartilage MMP-9, MMP-14, or ADAMTS-4 mRNA expression (B). Adenoviruses (AdControl, AdIL17, AdTNF, or the combination of AdIL17 plus AdTNF) were injected intraarticularly, and on day 4,
synovial biopsy tissue and patellar cartilage were isolated for quantitative polymerase chain reaction
analysis. Representative results from 1 of 2 independent in vivo experiments are shown. Values
are the mean of 2 pooled samples from 3 mice per group. C, Stimulation of a fibroblast luciferase
reporter cell line with the combination of TNF and IL-17 resulted in clear additive effects on NF-κB
activation, as compared with either cytokine alone. Bars show the mean ± SEM relative light units
(RLU) of luciferase activity. ** = P < 0.01; *** = P < 0.001, by one-way analysis of variance. See Figure 1
for other definitions.
depletion or bone erosion. Whereas the degree of inflammation appeared to be coupled
to bone erosion and showed the same relative decline after reducing the viral dose,
parameters of cartilage destruction were more strongly affected by reducing the viral
dose. Interestingly, histologic analysis of the cartilage demonstrated that, again, only the
combination of IL-17 and TNF resulted in irreversible chondrocyte death, cartilage erosion,
Synergistic effects of TNF-α and IL-17 on cartilage erosion in vivo | 79
Figure 4 | After reducing the virus dose for the combination group (IL-17 plus TNF), to reach
comparable levels of synovial inflammation among the 3 cytokine treatment groups, only
the combination of TNF and IL-17 resulted in irreversible cartilage destruction in murine
knee joints. Adenoviruses (AdControl, AdIL-17, AdTNF, or the combination of AdIL17 plus AdTNF)
were injected intraarticularly, and on day 10, the knee joints were isolated for analysis of histologic
parameters and VDIPEN expression. Results are the mean ± SEM of 6 mice per group. ** = P < 0.01;
*** = P < 0.001 versus AdControl. See Figure 1 for definitions.
80 | Chapter 4
and MMP-mediated VDIPEN expression (Figure 4), suggesting that the synergy between
TNF and IL-17 induces cartilage destruction independent of the degree of inflammation.
Protective effects of S100A9 deficiency against IL-17/TNF–induced
cartilage damage in vitro
Our overexpression studies in vivo demonstrated that IL-17 in the presence of TNF induces
irreversible cartilage destruction, associated with enhanced mRNA expression of S100A8,
MMPs, and ADAMTS. Previous studies demonstrated that S100A8 is an important inducer
and activator of MMPs and aggrecanases (40). To determine whether the up-regulation of
S100A8 by IL-17/TNF is indeed responsible for the cartilage-destructive effect, we
performed an in vitro study with cartilage of S100A9-deficient mice. Patellar cartilage was
isolated from WT and S100A9-deficient mice, the latter of which express neither S100A9
nor S100A8, probably because of the high turnover of free S100A8 in the absence of
its binding partner, S100A9. In vitro stimulation of the WT cartilage with IL-17 plus TNF
induced clear NITEGE neoepitope expression as a marker for aggrecanase-mediated
cartilage destruction (Figure 5C), to an extent comparable with that in cartilage stimulated
A
B
C
D
Figure 5 | NITEGE neoepitope expression, a measure of aggrecanase-mediated cartilage
destruction, was determined by NITEGE antibody staining of unstimulated wild-type mouse
patellae (control) (A), wild-type mouse patellae stimulated with 100 ng of IL-1β (B), or wildtype mouse patellae stimulated with the combination of IL-17 plus TNF (C) for 24 hours.
IL-17 plus TNF elevated the expression of NITEGE neoepitopes to levels comparable with those in the
positive control IL-1β–stimulated group, and this IL-17/TNF–induced NITEGE expression was almost
completely prevented in S100A9−/− mouse patellae (D). Arrows indicate clear brown staining for
NITEGE neoepitopes around chondrocytes. See Figure 1 for definitions.
Synergistic effects of TNF-α and IL-17 on cartilage erosion in vivo | 81
A
B
C
D
E
F
Figure 6 | Effects of neutralizing endogenous IL-17 and/or TNF on murine collagen-induced
arthritis (CIA). A and B, Neutralizing endogenous IL-17 and/or TNF before disease onset (A) or during
established CIA (B) reduced disease severity. DBA/1J mice were immunized with type II collagen,
and a booster injection was given on day 21. Mice were treated with intraperitoneal injections of
3 mg/kg soluble IL-17 receptor (sIL-17R), TNF binding protein (TNFBP), or the combination. Bovine
serum albumin (BSA) was used as a control. A total of 4 injections on alternate days were given. The
appearance of arthritis was assessed, and the severity of arthritis was visually scored on a scale of 0–2
for each paw. Treatment was started just before the expected onset (day 25) (A) or when an arthritis
score of 2.0–2.5 had been reached (B). C–F, The histologic parameters of influx of inflammatory
infiltrates (C), bone erosion (D), chondrocyte death (E), and cartilage erosion (F) were scored on day
8. Results in A and B are the mean ± SEM arthritis score for 10 mice per group. Results in C–F are box
plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent
the median, and the lines outside the boxes represent the 10th and 90th percentiles for 10 mice per
group. NS = not significant (see Figure 1 for other definitions).
82 | Chapter 4
with IL-1β as a positive control (Figure 5B), whereas no NITEGE neoepitope expression was
observed in unstimulated mouse patellae (Figure 5A). However, induction of NITEGE
neoepitopes by IL-17 and TNF was almost completely prevented in cartilage derived from
S100A9-deficient mice (Figure 5D), demonstrating an important role for S100A8/S100A9
proteins in this process of IL-17/TNF–mediated cartilage destruction.
Reduction in the severity of arthritis after blockade of IL-17 and TNF
during CIA
To examine the effect of individual blocking as well as combination blocking of TNF and
IL-17 during experimental arthritis, DBA/1J mice were immunized with CII in emulsified
Freund’s complete adjuvant. Before the visible onset of arthritis or during established
arthritis, mice were systemically treated on alternate days with a total of 4 injections of 3
mg/kg sIL-17R, TNFBP, the combination of sIL-17R plus TNFBP, or BSA as a control.
Neutralizing TNF before the onset of CIA significantly suppressed the severity of the
disease, as shown by a significant decrease in the macroscopic arthritis score in the TNFBP-­
treated group (Figure 6A). Nevertheless, mice that were treated with the combination of
sIL-17R and TNFBP showed even greater reductions in the severity of arthritis (Figure 6A),
suggesting that neutralization of IL-17 in addition to TNF has further therapeutic efficacy
in CIA.
In addition to cytokine blocking before arthritis onset, equal doses of sIL-17R and
TNFBP were used in the treatment of established CIA. Treatment was started in mice when
an arthritis score of 2.0–2.5 had been reached, using the same protocol of 4 IP injections
of sIL-17R, TNFBP, the combination, or BSA as a control on alternate days. In this late stage
of CIA, blocking of IL-17 or TNF alone did not significantly reduce the progression of CIA
(Figure 6B). Interestingly, however, neutralizing the combination of both IL-17 and TNF
almost completely inhibited further progression of disease (Figure 6B).
Histologic analysis revealed that there was a significant suppression of not only
inflammation but also cartilage destruction when the treatment combination of sIL-17R
and TNFBP was used (Figures 6C–F). Bone erosion in the sIL-17R/TNFBP treatment group of
arthritic mice was not significantly reduced (Figure 6D). Although sIL-17R or TNFBP alone
did not have any significant effects on the extent of inflammatory infiltrate, chondrocyte
death, and cartilage surface erosion, the combination of TNFBP plus sIL-17R was clearly
effective in preventing cartilage destruction and protecting its repair capacity (Figures 6E
and F).
Synergistic effects of TNF-α and IL-17 on cartilage erosion in vivo | 83
Discussion
In the present study, we demonstrated that the presence of IL-17 in the joint enhanced the
progression of joint destruction induced by local TNF. Moreover, neutralizing IL-17 in
addition to TNF during different stages of CIA further ameliorated the severity of arthritis
and inhibited progression of CIA. In particular, TNF and IL-17 were demonstrated to synergistically enhance cartilage destruction independent of the degree of inflammation. This
implies that combined anti–IL-17/anti-TNF therapy may have additional therapeutic value
for the treatment of destructive arthritis, compared with anti-TNF alone.
Anti-TNF therapy in RA patients leads to considerable improvement of arthritis (1–5).
However, not all patients respond to anti-TNF therapy. The cytokine cascades involved in
joint inflammation are very complicated, and many interactions may take place, leading to
a complex communication network between different cell types. Therefore, more
knowledge is needed to unravel the critical interactions between key players in the
arthritis process to further improve current therapy. In the last couple of years, ample data
have confirmed the beneficial effects of neutralizing IL-17, as shown in ex vivo studies
using human material from patients with RA as well as in experimental arthritis models. It
has also become clear that IL-17 can fine-tune the arthritis process by inducing and
working together with different cytokines and chemokines in an additive or synergistic
way (15, 41).
In this study, we combined blocking of TNF and IL-17 to enhance therapeutic efficacy.
The rationale for using this combination is as follows. TNF is produced by different cell
types, including T cells. Activated macrophages may be the predominant cell type
responsible for TNF production during arthritis. IL-17 expression is restricted and is
predominantly produced by activated Th17 cells. Since T cells play an important role in
exacerbations of (destructive) arthritis and during flares, IL-17 may be the major driving
cytokine in this process (16, 42). In addition, we previously showed that IL-17 has activities
in vivo that are either IL-1– and TNF-independent or IL-1– and TNF-dependent (16, 17, 32,
35). Therefore, blocking both TNF and IL-17 may control both the macrophage as well as T
cell activity pathways in the arthritis process.
Interestingly, a recent study demonstrated that anti-TNF treatment in CIA might cause
an expansion of IL-17–producing T cells (7), suggesting that blocking IL-17 in addition to
TNF might have enhanced therapeutic effect. Our data from the present study support
the hypothesis of effective combination therapy by showing an increased beneficial
response in CIA after neutralizing both TNF and IL-17.
Cartilage surface erosion is due to a loss of collagen structure, mediated by specific
enzymes such as MMPs. Immune complexes play an important role in this type of
destruction. Overexpression of TNF or IL-17 alone did not result in loss of cartilage
structures, and this may be due to the lack of immune complexes formed during IL-17–
and TNF-induced joint inflammation in normal mice. In contrast, overexpression of both
84 | Chapter 4
IL-17 and TNF in the knee joints of normal mice did result in cartilage surface erosion.
This implies that overexpression of IL-17 and TNF together is able to induce a process
responsible for the induction of cartilage destruction.
Quantitative PCR analysis revealed that the combination of TNF and IL-17 induced
a synergistic up-regulation of MMP-3 and MMP-13, which might well explain the MMPmediated VDIPEN expression in the knee joints, as revealed with immunohistochemistry.
In addition, S100A8 was tremendously up-regulated, especially by the combination of
these 2 inflammatory cytokines. This enhanced gene regulation was accompanied by
enhanced NF-kB activation, as demonstrated with a luciferase reporter fibroblast assay.
Other intracellular mechanisms, via the transcription factor CCAAT/enhancer binding
protein, or enhanced mRNA stability might also contribute to the TNF/IL-17 synergy, as
previously described (43–45).
Previous studies from our group have shown that S100A8 can stimulate chondrocytes
to produce MMPs, to activate latent MMPs, and to degrade the actual cartilage matrix, and
these effects were even further enhanced in the presence of IL-1 (46). These previous
findings and our current observations in S100A9−/− mice lacking IL-17/TNF–induced
NITEGE epitopes suggest that the enhanced up-regulation of S100A8 may very well
contribute to the cartilage destruction induced by TNF/IL-17 synergy.
One of the difficulties associated with anticytokine therapy in arthritis is that after
cessation of treatment, arthritis relapses. Indeed, we found that 7 days after the last sIL-17R
or TNFBP injection, treated mice with CIA no longer showed a significant difference in the
arthritis score compared with untreated mice with CIA. In contrast, neutralizing both IL-17
and TNF still resulted in significantly less disease expression 1 week after cessation of
treatment, although the combination treatment was not able to switch off CIA (results not
shown). This suggests that IL-17/TNF combination therapy is able to slow down the process
of upcoming arthritis after cessation of therapy. However, it also indicates that continuous
blocking of IL-17/TNF will be needed to keep the suppression of CIA ongoing.
TNF is hardly detectable in the later stages of CIA, and previous blocking studies with
anti–IL-1 and anti-TNF, performed in our laboratory, have shown that TNF plays an
important role in early CIA, whereas IL-1 is important in early and established CIA (6). The
results of the present study are consistent with the notion of an early beneficial effect of
anti-TNF therapy.
Of note, in the murine model of SCW-induced arthritis, it was found that TNF is a
principal cytokine contributing to the joint swelling, since TNF-deficient mice showed
hardly any increase in joint thickness (47). In these TNF-deficient mice, local injection of
SCW fragments into the knee joint resulted in local synovial expression of IL-1, indicating
that synovial IL-1 production could be induced in a TNF-independent manner (35).
Recently, we showed that IL-17 is a potent inducer of IL-1 in CIA (18), and in another study
using the murine SCW-induced arthritis, IL-17R signaling was critical for synovial IL-1
expression in the late chronic destructive phase of arthritis (21). This suggests that IL-17 is
Synergistic effects of TNF-α and IL-17 on cartilage erosion in vivo | 85
an upstream mediator of synovial IL-1. Our overexpression study showed that IL-1 was one
of the genes that were synergistically up-regulated by TNF and IL-17. Therefore, we
hypothesized that neutralizing IL-17 in addition to TNF may inhibit synovial IL-1 expression,
which is of additional benefit in the suppression of CIA. Studies are in progress to unravel
the truth of this hypothesis.
In conclusion, this study showed that neutralizing IL-17 in addition to TNF was still
effective in the late stages of CIA and was still able to suppress the progression of CIA
under conditions in which blocking of IL-17 or TNF alone had not been effective. Therefore,
it will be interesting to consider this potential new therapy for the treatment of human RA,
especially in RA patients whose condition has not responded to anti-TNF therapy alone.
Acknowledgements
We thank Birgitte Walgreen, Monique Helsen, and Liduine van den Bersselaar for providing
excellent technical assistance.
86 | Chapter 4
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Synergistic effects of TNF-α and IL-17 on cartilage erosion in vivo | 89
Chapter 5
Dual role of IL-21 in experimental arthritis:
IL-21R-deficiency protects against
joint pathology by reducing Th1/17 cells,
but increases local expression of
inflammatory mediators
Renoud J. Marijnissen, MSc1*, Marije I. Koenders, PhD1*, Deborah Young2,
Cheryl Nickerson-Nutter2, Shahla Abdollahi-Roodsaz, PhD1,
Sabrina Garcia de Aquino, MSc1,3, Fons A.J. van de Loo, PhD1, Annemieke M.H. Boots, PhD3,
Wim B. van den Berg, PhD1
adboud University Nijmegen Medical Centre, Rheumatology Research and Advanced Therapeutics,
R
Department of Rheumatology, Nijmegen, the Netherlands
2
Pfizer Inc, Cambridge, Massachusetts, USA
3
Department of Diagnosis and Oral Surgery, Periodontic Division, Araraquara Dental School,
University of São Paulo, São Paulo, Brazil
4
University Medical Center Groningen, Department of Rheumatology and Clinical Immunology,
Groningen, the Netherlands.
* Authors contributed equally to this manuscript.
1
92 | Chapter 5
Abstract
Objective
IL-21 is an immune-regulatory cytokine that can have both proinflammatory and immunosuppressive effects. The purpose of this study was to investigate the potential dual role of
IL-21 in experimental arthritis in relation to Th17 cells.
Method
In IL-21R-deficient mice and wild-type (WT) controls, antigen-induced arthritis (AIA) and
chronic Streptococcal Cell Wall (SCW) arthritis were induced. Knee joints, synovial tissue
and serum were analyzed for arthritis pathology and inflammatory markers.
Results
Our studies show that during AIA and chronic SCW-arthritis, IL-21R-deficiency protects
against severe inflammation and joint destruction. This is accompanied by suppressed
serum IgG1 levels and antigen-specific T cells responses. IL-17 was reduced during AIA,
and synovial lymphocytes isolated during SCW-arthritis for flowcytometry demonstrated
that mainly IL-17+ IFNγ+ T cells were reduced in IL-21R-deficient mice.
However, during the acute phases of SCW arthritis significantly higher joint swelling
scores were observed. Interestingly, IL-21R-/- mice were significantly less capable in
upregulating SOCS 1/3 mRNA. IL-21 stimulation also affected the TLR2/NOD2 response to
SCW fragments in vitro, indicating that impaired SOCS regulation in the absence of IL-21
signaling might contribute to the increased local activation during SCW arthritis.
Conclusion
In contrast to the proinflammatory role of IL-21 in adaptive immunity, driving IL-17/IFN
double-positive cells and joint pathology during chronic experimental arthritis, IL-21 also
has an important immunosuppressive role in innate immunity by inhibiting TLR signaling
via SOCS1/3. If this dual role of IL-21 in various immune processes is present in human
disease, it could make IL-21 a difficult therapeutic target in RA.
Dual role of IL-21 in experimental arthritis | 93
Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown etiology,
characterized by chronic joint inflammation and destruction of articular cartilage and
bone. Hyperplasia of the synovial lining and influx of proinflammatory cells leads to
thickening of the synovium, and production of soluble mediators like cytokines, enzymes
and oxygen radicals by both resident as well as the infiltrating cells contributes to the final
destruction of the joint matrix.
Tumor-necrosis factor alpha (TNFα) and interleukin-1 (IL-1) are the most extensively
studied cytokines in the arthritis process, and biological treatments neutralizing TNFα
have shown good clinical effects in many RA patients. However, for RA patients who do
not respond well to current available therapies, the search for new therapeutic targets
continues. Since non-responders to anti-TNF treatment show increased percentage of T
helper 17 (Th17) cells in circulation (1), additional new therapies targeting the Th17 pathway
might provide good candidates. Previous studies on the potential of the major Th17
cytokine IL-17 in various mouse models demonstrated arthritis suppression by anti-IL-17
treatment, especially when IL-17 blocking was combined with anti-TNF treatment in the
protection against cartilage damage (2), or when human RA synovial tissue was confirmed
to be rich in CD3+ T cells before treatment in the RA synovium SCID mouse model (3).
Another Th17-derived candidate is IL-21, a pleiotropic cytokine that binds to the IL-21
receptor (IL-21R) consisting of the common cytokine receptor gamma chain and the
specific IL-21R subunit (4). IL-21 is not solely produced by Th17, but also by follicular helper
T (Tfh) cells and NK T cells. The IL-21 receptor is expressed on a variety of hematopoietic
immune cells, including T, B, DC and NK cells as well as on tissue cells including endothelial
cells and fibroblasts. IL-21 acts on B cells and plays an important role in normal Ig
production and B cell differentiation into plasma cells (5-7). In addition, IL-21 contributes to
antibody production indirectly via its effect on T follicular helper cell generation and
germinal center formation (8). More interestingly, IL-21 also acts as an autocrine cytokine of
activated T cells, since it induces Th17 differentiation of both human and murine naïve
CD4+ T cells in the presence of TGFβ (9-11). In vivo studies also demonstrated that Th17
differentiation and responses are disturbed in the absence of IL-21 or its receptor (9-10,12).
Increased levels of IL-21 are found in serum and synovial fluid of RA patients compared to
OA patients and healthy controls (1,13), suggesting a role for IL-21 in the pathogenesis of
RA. Enhanced IL-21R expression was found in RA synovial fibroblasts and macrophages,
although regulation of IL-21R could not be induced by the most common proinflammatory cytokines IL-1 and TNF (14). IL-21 on T cells induces the secretion of proinflammatory
cytokines like TNFα and IFNγ, and enhances the expression of RANKL (13,15). Synovial
fibroblasts show increased expression of various matrix metalloproteinases (MMPs) as well
as RANKL upon IL-21 stimulation (13).
The involvement of IL-21 in autoimmune disease was previously shown in various
94 | Chapter 5
disease models. IL-21-deficiency protected against experimental autoimmune encephalomyelitis (9), and blocking IL-21 in MRL/lpr mice reduced signs of experimental systemic
lupus erythematosus (12). Neutralizing IL-21 in experimental arthritis already showed a
role for IL-21 in the rat adjuvant arthritis and murine collagen-induced arthritis, with
reduced clinical signs of disease and improved histological scores (16). However, the link
between IL-21 and Th17 cells was not made in these studies. The objective of this study
was to investigate the effect of IL-21R-deficiency on joint pathology in relation to Th17 cells
during chronic experimental arthritis.
Materials and Methods
Animals. IL-21R-deficient mice were provided by Pfizer. C57Bl/6J mice as wild-type
controls were obtained from Elevage-Janvier, Le Genest Saint Isle, France. All mice were
housed in filter-top cages under specific pathogen-free conditions and a standard diet
and water were provided ad libitum. The mice were used between 10 and 12 weeks of
age. All animal procedures were approved by the institutional ethics committee.
Materials. Methylated bovine serum albumin was purchased from Sigma-Aldrich (St. Louis,
MO). RPMI 1640 medium, TRIzol Reagent, oligo-dT primers and Moloney murine leukemia
virus reverse transcriptase (MMLV-RT) were obtained from Life Technologies (Breda, The
Netherlands). Primers were purchased from Biolegio (Malden, The Netherlands). SYBR Green
Master Mix was purchased from Applied Biosystems (ABI/PE, Foster City, CA). Cytokine kits
for the Luminex ® multi-analyte system (Milliplex) were obtained from Millipore.
Induction of Antigen-Induced Arthritis. Mice were immunized with 100 µg of mBSA
(Sigma), emulsified in 100 µl Freund’s complete adjuvant (Difco Laboratories, Detroit,
Michigan). Injections were divided over both flanks. Heat-killed Bordetella pertussis (RIVM,
Bilthoven, the Netherlands) was administered intraperitoneally as an additional adjuvant.
Two subcutaneous booster injections with in total 50 µg mBSA/FCA were given in the
neck region 1 week after the initial immunization. Three weeks after these injections,
primary AIA was induced by injecting 60 µg of mBSA in 6 µl PBS into the right knee joint,
resulting in chronic arthritis.
SCW preparation and induction of SCW arthritis. Streptococcus pyogenes T12
organisms were cultured overnight in Todd-Hewitt Broth. Cells walls were prepared as
described previously (17). The resulting 10,000 x g supernatant was used throughout the
experiments. These preparations contained 11% muramic acid. Unilateral arthritis was
induced by intra-articular injection of 25 µg of SCW (rhamnose content) in 6 µl pyrogen-free
saline into the right knee joints of naive mice.
Dual role of IL-21 in experimental arthritis | 95
Technetium uptake measurements. Joint inflammation was measured by
Technetium (99mTc) pertechnetate uptake in the knee joint. Mice were sedated with
chloralhydrate and subsequently injected intraperitoneally with 20 µCi 99mTc. Thirty
minutes thereafter, gamma radiation was assessed by use of a collimated Na-I-scintillation
crystal with the knee in a fixed position. Arthritis was scored as the ratio of the 99mTc uptake
in the arthritic right (R) and the control left (L) knee joint. R:L ratios >1.1 were taken to
indicate inflammation of the right knee joint.
99m
99m
Histology. For standard histology of the knee joints, tissue was fixed for 4 days in 10%
formalin, decalcified in 5% formic acid, and subsequently dehydrated and embedded in
paraffin. Standard frontal section of 7 µm were mounted on SuperFrost slides (MenzelGläser, Braunschweig, Germany). Hematoxylin and eosin (H&E) staining was performed to
study joint inflammation. The severity of inflammation in the knee joints was scored as the
amount of inflammatory cell influx on a scale of 0-3 (0 = no cells, 1 = mild cellularity, 2 =
moderate cellularity, and 3 = maximal cellularity). To study proteoglycan (PG) depletion
from the cartilage matrix, sections were stained with safranin O followed by counterstaining with fast green. PG depletion was determined using an arbitrary scale of 0-3, ranging
from normal, fully stained cartilage to destained cartilage, completely depleted of PGs.
The degree of chondrocyte death was scored on a scale of 0-3, ranging from no empty
lacunae to complete loss of chondrocytes in the cartilage layer. Histopathological changes
in the knee joints were scored in the patella and femur/tibia regions on five semi serial
sections of the joint spaced 70 µm apart. Scoring was performed in a blindfolded manner
by two independent observers.
Measurement of anti-mBSA and anti-SCW antibodies. Levels of anti-mBSA or
anti-SCW antibodies in sera of arthritic mice were analyzed by enzyme-linked
immunosorbent assay. Briefly, 10 ng of mBSA or SCW fragments were coated onto 96-well
plates overnight. Plates were washed, and nonspecific binding sites were blocked with 1%
BSA in PBS-Tween (0.05%). Serial 2x dilutions of sera, starting with an initial dilution of 20x,
were incubated in the plates for 1 hour. The plates were then washed, and isotype-specific horseradish peroxidase-labeled goat anti-mouse Ig (1:1,000) was added for 1 hour at
room temperature; 5-aminosalicylic acid was used as substrate. Absorbance was measured
at 450 nm.
Lymphocyte stimulation test. Spleens were isolated and disrupted, and erythrocytes
were lysed in 0.16M NH4Cl (pH 7.2). The cell suspension was washed with saline and
enriched for lymphocytes by allowing antigen-presenting cells to adhere to plastic culture
flasks for 45 minutes. Cells (2 x 105/well) were cultured for 72 hours at 37°C in an atmosphere
of 5% CO2, in RPMI 1640 (Gibco-Invitrogen, Paisley, UK) supplemented with 5% fetal calf
serum, 1 mM pyruvate, and 50 mg/liter of gentamicin in the presence of two-fold serial
96 | Chapter 5
dilutions of antigen (mBSA or SCW fragments). For the last 16 hours the cells were labeled
with 0.25 μCi 3H-thymidine. After harvesting, incorporation of 3H-thymidine was measured
and the increase in counts per minute (cpm) caused by stimulation with specific antigen
was determined.
Synovial washouts. To determine levels of several cytokines and chemokines in patellae
washouts, patellae with surrounding soft tissue consisting of the tendon and synovium
were dissected in a standardized manner. Patellae were cultured in RPMI 1640 medium
containing 0.1% BSA (200 µl/patella) for 1 hour at room temperature. Thereafter,
supernatant was harvested and centrifuged for 5 minutes at 1000 x g. Cytokine levels were
determined using the Luminex multianalyte technology.
Luminex analysis. To determine levels of the cytokines and chemokines in serum
samples and synovial washouts, Luminex multianalyte technology in combination with
multiplex cytokine kits (Milliplex, Millipore Corporation) was used. Cytokines were
measured in 25 µl of synovial washout, or in serum 1:3 diluted in assay buffer. The sensitivity
of the multiplex kit was <1 pg/ml.
Isolation of synovial cells. The protocol was kindly provided by prof. Shimon Sakaguchi
and adjusted. After sacrifice of the mice, the joint synovium was dissected. In short,
synovial biopsies were incubated with enzymetic digestion buffers (Liberase, Roche) for
30 minutes at 37°C. Next, a 70 μm nylon cell strainer (BD Falcon) was used to process the
digested tissue. The cell preparation was collected in RPMI with 10% FCS. To isolate the
single mononuclear cells, Lympholyte-M (Cederlane) was used according to the
manufacturer’s protocol. Subsequently, the isolated cells were prepared for intracellular
flowcytometry.
Intracellular flowcytometry. Cells were stimulated for 4-6 hours with PMA (50 ng/ml;
Sigma-Aldrich) and ionomycine (1 μg/ml; Sigma-Aldrich) and Golgiplug (1 μl/ml; BD
Biosciences). Next, for intracellular detection of IFNγ and IL-17, cells were washed, fixed and
permeabilized according to the manufacturer’s protocol (eBioscience) and incubated with
anti-IFNγ (BD Biosciences) and anti-IL-17 (BD Biosciences) antibodies.
RNA isolation and RT reaction. Mice were sacrificed by cervical dislocation, immediately
followed by dissection of the patella with adjacent synovium. Two biopsies with a diameter
of 3 mm were punched out using a biopsy punch (Stiefel, Wachtersbach, Germany); one
from the lateral side and one from the medial side of the synovial tissue. The synovium
samples were immediately frozen in liquid nitrogen. Total RNA was extracted in 1 ml
TRIzol reagent. Thereafter, RNA was precipitated with isopropanol, washed with 70%
ethanol and re-dissolved in water. Isolated RNA was treated with DNAse before being
Dual role of IL-21 in experimental arthritis | 97
reverse transcribed into cDNA using oligo-dT primers and Moloney murine leukemia virus
reverse transcriptase.
Quantitative PCR. Quantitative real-time PCR was performed using the ABI/PRISM 7000
Sequence Detection System for quantification with SYBR Green and melting curve analysis
(ABI/PE, Foster City, CA). PCR conditions were as follows: 2 min at 50°C and 10 min at 95°C
followed by 40 cycles of 15 s at 95°C and 1 min at 60°C, with data collection in the last 30
s. For all PCRs, SYBR Green Master Mix was used in the reaction. Primer concentrations
were 300 nmol/l. All PCRs were performed in a total volume of 25 µl. Relative quantification
of the PCR signals was performed by comparing the cycle threshold value (Ct), in duplicate,
of the gene of interest of each sample with the Ct values of the reference gene GAPDH.
Statistical analysis. Differences between experimental groups were tested using the
Mann-Whitney U test or Kruskal-Wallis test, unless stated otherwise. P-values less than 0.05 were
considered significant. Results are expressed as the mean ± SEM unless stated otherwise.
Results
IL-21R-deficiency protects against severe inflammation and bone
destruction during AIA
The role of IL-21 in experimental arthritis was first studied in the chronic antigen-induced
arthritis (AIA) model using IL-21R-deficient mice and their wild-type (WT) controls.
99m
Technetium measurements after intra-articular (i.a.) injection with the antigen mBSA
showed similar joint swelling in both groups in the first two days of arthritis. However, at
day 4 a more rapid decline was observed in IL-21R-deficient mice compared to WT controls
(Figure 1A). More important, histological analysis at day 7 of arthritis demonstrated
significantly reduced joint inflammation, cartilage destruction, and bone erosion in the
absence of the IL-21 receptor (Figure 1B and supplemental figure 1). This reduced joint
pathology in IL-21R-/- mice was accompanied by suppressed adaptive immune responses;
anti-mBSA IgG1 antibodies and antigen-specific lymphocyte proliferation were
significantly decreased by the IL-21R deficiency (Figure 1C,D), while another subclass of
antibodies, IgG2b, and total IgGs were not affected. In addition, the cytokines IL-6 and IL-17
and the chemokine CXCL-1 were significantly reduced in synovial washout of these
knockout mice at day 7 of AIA (Figure 1E). These data indicate an important proinflammatory role for IL-21 in driving adaptive immunity and chronic destructive arthritis during
murine AIA.
98 | Chapter 5
A
Joint swelling (Ratio R/L)
2.0
WT
IL-21R-/-
1.8
1.6
1.4
*
1.2
2
1
4
7
Time (Days)
2
*
*
1
PG depletion
Inflammation
0.4
WT
Chondrocyte death
D
IL-21R-/-
28000
WT
IL-21R-/-
20000
0.2
0.1
12000
4000
**
** *** *** **
**
*
3000
2000
**
1000
A
00
0.1
Co
n
00
00
0.4
0.2
00
00
1.6
0.8
50
6.2
0
.00
IgG2b
IgG1
25
IgG total
25
0
0.0
0
OD/50
Netto counts (cpm)
*
0.3
Bone erosion
.50
0
C
WT
IL-21R-/-
**
12
Histological analysis (0-3)
*
3.1
B
3
mBSA antigen concentration (µg/ml)
Synovial washouts (pg/ml)
E
2000
1500
1000
250
200
150
100
50
10
8
6
4
2
0
**
**
*
TNF-α
IL-1β
IL-6
CXCL1
IL-10
IL-17
IFNγ
Figure 1 | IL-21R deficiency results in a decrease in arthritis with less destruction paired
with reduced adaptive immune response. IL-21 receptor (IL-21R) deficiency during the induction
of a primary antigen induced arthritis (AIA) results in a more rapid decrease in joint swelling (A) and
suppressed influx of inflammatory cells, cartilage proteoglycan (PG) depletion, chondrocyte death
and bone erosion at day 28 (B). Joint Swelling was measured by 99mTc uptake at day 1,2,4 and 7
(n=6-8 mice/ group). Total knee joints were isolated for histopathological analysis at day 7 (n=8
mice/ group). Serum levels of mBSA specific IgG1 antibodies were decreased at day 7 (C). Reduced
proliferation of mBSA-specific splenic lymphocytes of WT and IL-21R deficient mice (D). Nonadherent spleen cells were stimulated with various concentrations of mBSA or 1 µg/ml Concanavalin
A (Con A) for 72 hours, followed by 18 hours incubation with 0.25 µCi 3H-thymidine (n=7 mice/
group). Results are shown as netto counts (cpm stimulation – cpm background). IL-21R deficiency
reduces the IL-6, CXCL1 (KC) and IL-17 cytokine levels in synovial washouts of arthritic mice (E). Values
are the mean ±SEM from 6 mice per group; * P < 0.05, ** P < 0.01, *** P < 0.001, by Student’s t-test.
Dual role of IL-21 in experimental arthritis | 99
Enhanced local activation in IL-21R-deficient mice during SCW-arthritis
In addition to the AIA model, the role of IL-21/IL-21R signaling was investigated in the
Streptococcal Cell wall (SCW)-induced arthritis. In this model, a single injection of SCW
fragments into the knee joint results in local acute inflammation, and subsequent repeated
A
Joint swelling (Ratio R/L)
2.5
2.0
WT
IL-21R-/-
*
***
*
***
1.5
*
1.0
7 8
1
14 15
21 22
28
Time (Days)
B
4000
WT
IL-21R-/-
3000
2000
ns
Synovial washouts (pg/ml)
1250
1000
750
500
ns
250
200
150
100
50
0
IL-1β
IL-6
CXCL1
WT
IL-21R-/-
150
100
50
0
TNF-α
IL-1β
IL-6
IL-10
Day 28
Day 22
200
CXCL-1
IL-10
Serum levels (pg/ml)
Serum levels (pg/ml)
C
TNF-α
200
P = 0.055
150
100
*
*
50
0
TNF-α
IL-1β
IL-6
CXCL-1
IL-10
Figure 2 | IL-21R deficiency in a local activated model results in enhanced local activation.
IL-21 R deficiency during chronic SCW arthritis results in an increased joint swelling (A). Joint Swelling
was measured by 99mTc uptake (n=8-9 mice/ group). IL-21R deficiency during chronic SCW arthritis
did not reduce the pro-inflammatory cytokine levels in synovial washouts of arthritic mice at day
22 (n=6-8 mice/ group) (B). On the contrary, the serum cytokine levels of IL1β, IL-6 and IL-10 were
reduced by day 28 (C). Results are mean ±SEM; ** = P < 0.01, *** = P < 0.001, ns = none significant
by Student’s t-test.
100 | Chapter 5
weekly injections turn this acute inflammation into a chronic destructive arthritis. In
contrast to our findings in the AIA model, the IL-21R-deficient mice showed enhanced
joint swelling at various time points during the SCW arthritis, most significantly as soon as
day 1 after arthritis induction (Figure 2A). This enhanced swelling was accompanied by a
trend for higher IL-6 and CXCL-1 levels in synovial washouts at day 22 (Figure 2B), indicating
an enhanced local activation in the absence of IL-21R. However, systemic cytokine levels at
this time point did not show any difference between knockouts and wild-types. More
remarkably, at time of sacrifice (day 28) the cytokine levels in IL-21R-/- mice were significantly
reduced compared to WT controls (Figure 2C), in contrast to the previous enhanced local
activation.
Reduced joint pathology and IL-17+ IFNγ+Th cells in IL-21R-/- mice
during SCW-arthritis
To study the absolute effect of IL-21R-deficiency on joint pathology during this SCW
arthritis model, knee joints were isolated at day 28 for histological analysis. As shown in
Figure 3(A,B), the arthritic joints of IL-21R-knockout mice showed a significant reduction in
joint inflammation, in contrast to the enhanced joint swelling during the course of the
arthritis. In addition, the protective effect of IL-21R-deficiency was also visible on cartilage
damage, as both proteoglycan depletion and chondrocyte death were clearly suppressed
compared to wild-type controls (Figure 3A,C). In line with the results during AIA, antigenspecific B and T cell responses were reduced in IL-21R-/- mice (Figure 4A,B).
The reduced IL-17 levels during AIA in IL-21R-/- mice suggested that T cells and in
particular Th17 cells might be affected by the absence of IL-21R signaling. Therefore, during
the chronic SCW arthritis model we dissected synovial tissue of the arthritic knee joints
and isolated the infiltrating cells for flowcytometric analysis. As demonstrated in Figure
4C, the percentage of CD4+ Th17 cells (IL-17+ IFNγ-) and Th1 cells (IFNγ+ IL-17-) were not
changed in IL-21R-knockout mice. However, the double-positive IL-17+ IFNγ+ T cells
(Th1/17) were greatly reduced by the IL-21R-deficiency (Figure 4C,D), suggesting a potential
role for IL-21 in driving these double-positive Th1/17 cells and mediating joint pathology.
IL-21 induces SOCS expression and reduces TLR/NOD response to SCW
fragments
To further investigate the enhanced local activation during SCW arthritis in the IL-21R-/- mice,
we studied the expression of TLR2 and NOD2, that are crucial for response to SCW fragments.
Basal synovial levels of TLR2 and NOD2 mRNA expression were equal in IL-21R-deficient mice
and wild-types, and also the upregulation of these receptors four days after the injection of
SCW fragments was comparable (data not shown). Since triggering of TLR2/NOD2 not only
results in induction of proinflammatory cytokines, but also in negative feedback via
Suppressor of Cytokine Signaling (SOCS), the regulation of SOCS expression was also studied.
QPCR analysis of synovial tissue demonstrated that despite comparable basal levels of
Dual role of IL-21 in experimental arthritis | 101
A
Histological analysis (0-3)
SOCS1 and SOCS3, IL-21R-deficient mice had a clearly impaired upregulation of SOCS1/3
during arthritis (Figure 5A). The decreased expression of SOCS3 at day 4 was confirmed on
protein by immunohistochemical staining on the sections of the knee joints of IL-21R-/- mice
(Figure 5B). SOCS3 expression at day 1 was not altered compared to the WT mice. In addition,
SOCS1 expression was hardly detectable (data not shown).
This IL-21-dependent upregulation of SOCS1 and SOCS3 was confirmed in subsequent
in vitro experiments, where we cultured splenocytes from IL-21R-/- mice and wild-types
with or without recombinant IL-21 for 24 hours. As demonstrated in Figure 5C, WT cells
have enhanced SOCS1/3 mRNA levels upon IL-21 stimulation, whereas IL-21R-deficient cells
do not respond. Interestingly, cells stimulated with SCW fragments after pre-exposure to
IL-21 produce less IL-6 and CXCL-1 than cells in the absence of IL-21 (Figure 5D), suggesting
that the IL-21-mediated SOCS upregulation can inhibit responses to TLR2/NOD triggering.
This lack of SOCS upregulation may contribute to the enhanced local activation in IL-21Rdeficient mice during SCW-induced arthritis.
**
WT
IL-21R-/-
3
2
*
*
1
0
Inflammation
B
WT
PG depletion
Chondrocyte death Cartilage erosion
IL-21R-/-
C
Figure 3 | Histopathological effects of IL-21R deficiency during chronic phase of SCW arthritis.
IL-21R deficiency during chronic SCW arthritis a suppressed influx of pro-inflammatory cells, cartilage
proteoglycan (PG) depletion and chondrocyte death at day 28 (A). Total knee joints were isolated
for histopathological analysis (n=8 mice/ group). Representative histological sections for infiltrate of
inflammatory cells (original magnification 50x) (B) and chondrocyte death (original magnification
200x) (C) stained with H&E in frontal knee sections (original magnification 100x).Values are the mean
+/- SEM from 6 mice per group; * P < 0.05, ** P < 0.01, by Student’s t-test.
102 | Chapter 5
A
0.65
WT
OD/50
IL-21R-/-
*
0.55
0.45
*
0.35
0.25
0.15
0.05
IgG1
B
20000
IgG2b
WT
IL-21R-/-
16000
*
12000
Netto counts (cpm)
IgG3
WT netto cpm
*
P = 0.07
*
2000
1000
00
A
Co
n
00
0.2
0.1
00
0.4
00
00
0.8
1.6
3.1
25
50
6.2
.50
12
25
.00
0
0
0
SCW antigen concentration (µg/ml)
WT
IL-21R-/-
IL-17
C
IFNγ
50
10
Precentage of CD4+ (%)
Precentage of CD4+ (%)
12
40
30
20
10
0
WT
IL-21R-/-
IL-17- IFNγ+
IL-17+ IFNγ+
60
20
*
Precentage of CD4+ (%)
D
IL-17+ IFNγ-
8
6
4
2
0
WT
IL-21R-/-
15
10
5
0
WT
IL-21R-/-
Figure 4 | Decreased adaptive immune responses in IL-21R deficient mice during chronic
SCW arthritis. Serum levels of SCW specific IgG1 and IgG3 antibodies were decreased at day 28
(A). Non-adherent spleen cells were stimulated with various concentrations of SCW or 1 µg/ml
Concanavalin A (Con A) for 72 hours, followed by 18 hours incubation with 0.25 µCi 3H-thymidine
(n=7-8 mice/ group). Results are shown as netto counts (cpm stimulation – cpm background) (B).
Representative dot plots of the isolated cells gated on CD4+ are shown (C). The mean percentage of
isolated IFN-γ and IL-17 producing T cells (CD4+ population) are shown (n = 6 mice/group) (D). Values
are the mean ±SEM ; * P < 0.05 by Student’s t-test.
Dual role of IL-21 in experimental arthritis | 103
A
WT
IL -21R- /-
*
ns
*
10
ns
5
ns
ns
0
naive
day 1
B
day 1
naive
day 4
SOCS1
day 4
SOCS3
WT
SOCS1
*
2.0
1.5
1.0
0.5
15
10
5
100
IL- SC S 21
W CW
+I
L-2
1
IL- SC S 21
W CW
+I
L-2
1
0
-
IL30
P = 0.06
100
50
0
20
10
0
IL- SC S 21
W CW
+I
L-2
1
IL- SC S 21
W CW
+I
L-2
1
200
Cytokine production (pg/ml)
*
IL-10
CXCL1 (KC)
150
IL- SC S 21
W CW
+I
L-2
1
300
21
-
IL-
IL-6
WT
IL-21R-/-
IL- SC S 21
W CW
+I
L-2
1
-
21
IL-
21
IL-
-
0
400
Cytokine production (pg/ml)
WT
IL-21R-/-
***
20
0.0
D
SOCS3
25
Cytokine production (pg/ml)
Relative mRNA expression
2.5
Relative mRNA expression
C
IL-21R-/-
21
Relative mRNA expession
Synovial SOCS regulation
250
200
150
100
50
Figure 5 | IL-21R deficient mice show impaired upregulation of SOCS1 and 3 during arthritis.
IL-21R deficiency results in an decreased upregulation of SOCS1 and SOCS3 during SCW arthritis on
day 4 (A). Representative histological sections of immunohistochemical staining of SOCS3 on day 4
(original magnification 50x and 200x)(B). Spleen cells from WT mice stimulated with IL-21 upregulate
SOCS 1 and SOCS 3 (C). Spleen cells from IL-21R deficient mice fail to decrease IL-6 and CXCL1 (KC)
levels after IL-21exposure (2h) following 24 hours of stimulation with SCW (n=6) (D). Results are mean
±SEM; ** = P < 0.01, *** = P < 0.001, ns = none significant by Student’s t-test.
104 | Chapter 5
Discussion
In the present study, we demonstrated that IL-21R-deficiency during experimental AIA
causes clearly reduced inflammation and proteoglycan depletion, and protection against
severe destruction of cartilage and bone. However, IL-21R-/- mice show enhanced local
activation after the induction of SCW arthritis, accompanied by an impaired upregulation
of SOCS1 and SOCS3. Therefore, this is the first study to describe a dual role for IL-21 in
experimental arthritis, on the one hand driving adaptive immunity via proinflammatory
IFNγ+IL-17+ T helper cells, while on the other hand suppressing innate responses via SOCS
signaling.
CD4+ T cells play an important role in the arthritis process. Where genetic studies
revealed an important contribution for genes related to T cell activation such as the
HLA-DR4 allele, PTPN22, and CTLA4 (18), clinical evidence for the T cell contribution was
obtained by the successful treatment of RA patients with CTLA4-Ig (Abatacept) (19). A new
subset of T cells, the Th17 cell, has recently emerged as a major pathogenic T cell involved
in the RA pathogenesis. While the role of IL-17, the main cytokine produced by Th17 cells,
has already been studied extensively in experimental arthritis (reviewed by 20) and other
autoimmune and inflammation models (reviewed by 21), the role of the other Th17
cytokines IL-21 and IL-22 remains to be further elucidated.
To investigate the role of IL-21 in experimental arthritis, we used IL-21R-deficient mice
during antigen-induced arthritis. In this chronic destructive model, IL-17 was previously
demonstrated to be expressed in the synovial tissue (22), and RAG-1-/- and CD4-deficient
mice showed clear protection from arthritis pathology (23). In our current study, joint
swelling and histological inflammation during AIA were significantly reduced in IL-21R
knockout mice. In addition, lack of IL-21R expression protected against proteoglycan
depletion and chondrocyte death as parameters of cartilage destruction, and also bone
erosions were clearly less prominent in IL-21R-deficient mice, indicating a clear proinflammatory role for IL-21 in arthritis. This was in line with previous studies of IL-21 blocking in
CIA (16) and the observation that IL-21R deficiency completely blocks arthritis development
in K/BxN mice (24).
IL-21 has a broad spectrum of responsive cell types, including cells from the innate as
well as the adaptive immune system (4). The role of IL-21 has been intensively investigated
in B cell responses (5-7), and in our study we confirmed that IL-21 is an important player in
antigen-specific antibody responses. IL-21R-deficiency resulted in significant inhibition of
the Th2-related IgG1 and Th1-related IgG3 antibody levels in the serum of arthritic mice.
Since the SCW-specific IgG2b subclass was not affected by IL-21R-deficiency, and Ozaki et
al previously reported a general suppression of IgG1 ànd IgG2b basal levels in IL-21R-/mice, we conclude that the suppressed IgG1 levels are not merely due to a generic
difference between WT and KO mice, but that this reduced antibody response is a specific
IL-21-dependent effect. While antigen-specific T cell responses were highly suppressed in
Dual role of IL-21 in experimental arthritis | 105
the absence of IL-21 signaling, an inhibitory effect on T cell cytokines in the arthritic joint
could only be found for IL-17 levels in synovial washouts, while no differences were
observed for the Th1 cytokine IFNγ.
We attempted to confirm our findings of IL-21 as proinflammatory cytokine in
experimental arthritis by inducing chronic SCW arthritis in IL-21R-/- mice. By local intra-articular injection of TLR2/NOD2 ligands (25), this acute macrophage-driven arthritis
becomes T cell dependent after repeated weekly exposure to the antigen (26). In line with
our findings in the AIA model, IL-21R-deficiency during SCW arthritis resulted in reduced
antigen-specific T and B cell responses. To study the role of IL-21 on T helper cells in closer
detail, flowcytometry was performed on lymphocytes isolated from the synovial tissue.
This analysis showed comparable percentages of CD4+IL-17+ Th17 cells in IL-21R-/- mice
and wild-type controls, but a remarkable effect of IL-21R-deficiency on CD4+ T cells that
produce both IFNγ as well as IL-17.
From this additional arthritis model we might conclude that IL-21R-deficiency indeed
suppresses adaptive immunity and in particular double-positive Th1/17 cells, although the
protective effects on histological scores of inflammation and destruction were less
pronounced than in the chronic AIA model. However, of great interest, throughout the
SCW model a continuously enhanced joint swelling was observed in IL-21R-/- mice
compared to the wild-type controls. Also in the synovial washouts of the arthritic joint, no
suppression of cytokines and chemokines could be found as was previously reported for
the AIA model. On the contrary, enhanced local activation was observed with a trend for
increased levels of IL-6 and CXCL1. The aggravated response to SCW fragments in IL-21Rdeficient mice could not be explained by a difference in expression levels of TLR2 and
NOD2, the receptors responsible for a reaction to the SCW fragments (25), since equal
mRNA levels were found in synovial biopsies of both mice strains. Since IL-21R-deficiency
does not interfere with KxB/N serum-induced arthritis (24), acting through immune-complexes, Fcγ receptors and complement activation, the aggravated response of IL-21R-/mice to SCW fragments cannot be generalized to all innate immune responses.
In vitro stimulation assays showed that IL-21 can induce SOCS expression. These
Suppressor of Cytokine Signaling proteins act as negative regulators and inhibit cytokine
pathways as well as TLR signaling (27). In our IL-21R-deficient mice, basal expression levels
of SOCS1 and SOCS3 levels were similar to WT controls, but remarkably IL-21/IL-21R
signaling seems to be essential for SOCS upregulation during experimental arthritis: while
control mice demonstrated a massive increase of SOCS expression, the upregulation of
SOCS1/3, was clearly impaired in IL-21R-/- mice. We assume that in vivo, early after the
injection of SCW fragments, infiltrating lymphocytes e.g. γδ-T cells and NKT cells contribute
to increased IL-21 levels. In addition, in vitro responses to SCW fragments were significantly
suppressed in the presence of IL-21, suggesting that the IL-21-induced SOCS expression
affects this innate immune response. IL-21-driven SOCS-dependent inhibition of LPS
signaling was previously described for monocyte-derived DCs in vitro (28), and our
106 | Chapter 5
findings suggest that this mechanism might well account for the enhanced joint swelling
in IL-21R-/- mice during SCW arthritis.
In summary, studying the role of IL-21 in arthritis we have seen two independent
mechanisms that determine the final outcome on joint inflammation and destruction: (1)
IL-21 as proinflammatory cytokine driving adaptive immunity, with a specific effect on
double positive IFNγ+IL-17+ T helper cells, and (2) IL-21 as negative regulator of innate
immune responses via induction of SOCS1/3 expression as demonstrated in
TLR2-dependent SCW arthritis. The net effects of inhibiting IL-21 will be dependent on the
environment: IL-21 will act more proinflammatory in T cell-driven arthritis like our AIA
model in which the memory response is independent of TLR-pathways (van den Brand,
manuscript in preparation), whereas in inflammation that is still partly driven through the
innate system, like during SCW-arthritis, IL-21 can also demonstrate its anti-inflammatory
capacity.
IL-21 seems to have a dual role in immune responses during arthritis, turning off an
initial innate response (mediated through TLRs) and upregulated in adaptive immune
responses to continue to drive a chronic inflammatory response. Understanding this dual
role of IL-21 in various immune processes will be important in human disease and could
make IL-21 a difficult therapeutic target in RA.
A
WT
IL-21R-/-
B
Supplemental figure S1 | Histopathological effects of IL-21R deficiency during antigen
induced arthritis (AIA). Total knee joints were isolated for histopathological analysis (n=8 mice/
group). Representative H&E histological sections for infiltrate of inflammatory cells (original
magnification 50x) (A) and representative Safranin O staining for PG depletion (original magnification
200x) (B).
Dual role of IL-21 in experimental arthritis | 107
Acknowledgements
We thank the personnel of the animal facility for taking good care of our mice, and the
research technicians Birgitte Walgreen, Monique Helsen and Liduine van den Bersselaar
for their excellent technical assistance throughout the experimental arthritis studies.
108 | Chapter 5
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interleukin-21 receptor pathway ameliorates disease in animal models of rheumatoid arthritis. Arthritis
Rheum 2007;56:1152-63.
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arthritis and flare-up reaction in mice induced by homologous or heterologous cell-walls. Am J Pathol
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McInnes IB, Schett G. Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol 2007;7:429-442
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Kremer JM, Westhovens R, Leon M, Di Giorgio E, Alten R, Steinfeld S, et al. Treatment of rheumatoid arthritis
by selective inhibition of T-cell activation with fusion protein CTLA4Ig. N Engl J Med 2003;349:1907-15.
Koenders MI, Joosten LA, van den Berg WB. Potential new targets in arthritis therapy: interleukin (IL)-17 and
its relation to tumour necrosis factor and IL-1 in experimental arthritis. Ann Rheum Dis 2006;65 Suppl
3:iii29-33 (Review).
Iwakura Y, Ishigame H, Saijo S, Nakae S. Functional specialization of interleukin-17 family members.
Immunity 2011;34:149-62.
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22. Koenders MI, Lubberts E, Oppers-Walgreen B, van den Bersselaar L, Helsen MM, Di Padova FE, et al. Blocking
of interleukin-17 during reactivation of experimental arthritis prevents joint inflammation and bone erosion
by decreasing RANKL and interleukin-1. Am J Pathol 2005;167:141-9.
23. Wong PK, Quinn JM, Sims NA, van Nieuwenhuijze A, Campbell IK, Wicks IP. Interleukin-6 modulates
production of T lymphocyte-derived cytokines in antigen-induced arthritis and drives inflammation-induced osteoclastogenesis. Arthritis Rheum 2006;54:158-68.
24. Jang E, Cho SH, Park H, Paik DJ, Kim JM, Youn J. A positive feedback loop of IL-21 signaling provoked by
homeostatic CD4+CD25- T cell expansion is essential for the development of arthritis in autoimmune K/
BxN mice. J Immunol 2009;182:4649-56.
25. Joosten LA, Koenders MI, Smeets RL, Heuvelmans-Jacobs M, Helsen MM, Takeda K, et al. Toll-like receptor 2
pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation
factor 88. J Immunol 2003;171:6145-53.
26. Joosten LA, Abdollahi-Roodsaz S, Heuvelmans-Jacobs M, Helsen MM, van den Bersselaar LA, Oppers-Walgreen B, et al. T cell dependence of chronic destructive murine arthritis induced by repeated local activation
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Chapter 6
Increased expression of interleukin-22
by synovial Th17 cells during late stages
of murine experimental arthritis is
controlled by interleukin-1 and enhances
bone degradation
Renoud J. Marijnissen1, Marije I. Koenders1, Ruben L. Smeets2,
Mark H.T. Stappers1,3, Cheryl Nickerson-Nutter4, Leo A.B. Joosten3,
Annemieke M.H. Boots2,5, Wim B. van den Berg1
adboud University Nijmegen Medical Centre, Rheumatology Research and Advanced Therapeutics,
R
Department of Rheumatology, Nijmegen, The Netherlands
2
Merck Research Laboratories (MSD), Department of Immune Therapeutics, Oss , The Netherlands
3
Radboud University Nijmegen Medical Centre, Department of Medicine and Nijmegen Institute for Infection,
Inflammation & Immunity (N4i), Nijmegen, The Netherlands
4
Pfizer Inc, Cambridge, Massachusetts, USA
5
University Medical Center Groningen, Department of Rheumatology and Clinical Immunology,
University of Groningen, The Netherlands
1
112 | Chapter 6
Abstract
Objective
Interleukin-22 (IL-22) is a mediator in antimicrobial responses and inflammatory autoimmune
diseases. Although IL-22 and its receptor, IL-22R, have been identified in the synovium of
rheumatoid arthritis patients, the source of IL-22 and its contribution to disease pathogenicity
remain to be established. This study was undertaken to investigate the regulation of IL-22
by Th17 cells in vitro and to evaluate the potential for IL-22 depletion in an experimental
arthritis model using mice deficient in the IL-1 receptor antagonist (IL-1Ra−/−).
Methods
Naive murine T cells were cultured under conditions leading to polarization of the cells
into subsets of Th1, Th2, induced Treg, and Th17. Cytokines were measured in the culture
supernatants, and the cells were analyzed by fluorescence-activated cell sorting. Tissue
samples from the inflamed ankle synovium of IL-1Ra−/− mice were isolated, and messenger
RNA levels of marker genes were quantified. IL-1Ra−/− mice were treated with neutralizing
anti–IL-22 antibodies. Synovial cells were isolated from the inflamed tissue and sorted into
fractions for analysis of cytokine production.
Results
In vitro tests showed that Th17 cells produced high levels of IL-22 after stimulation with IL-1
or IL-23. Interestingly, a synergistic increase in the production of IL-22 was observed after
combining IL-1 and IL-23. In vivo, IL-1Ra−/− mice displayed a progressive erosive arthritis,
characterized by up-regulation of IL-17 in mildly and severely inflamed tissue, whereas the
levels of IL-22 and IL-22R were increased only in severely inflamed synovia. Anti–IL-22
treatment of IL-1Ra−/− mice significantly reduced the inflammation and bone erosion.
Analysis of isolated single cells from the inflamed synovia revealed that IL-22 was mainly
produced by IL-17–expressing T cells.
Conclusion
These findings suggest that IL-22 plays an important role in IL-1–driven chronic joint
destruction.
IL-1–driven regulation of IL-22 in chronic arthritis | 113
Introduction
Rheumatoid arthritis (RA) is a systemic joint disease with an unknown etiology,
characterized by a chronic inflammatory infiltration of the synovial membrane and
associated with destruction of cartilage and bone. Among the T cells residing in the
inflammatory infiltrates of the synovium of RA patients, CD4+ T helper cells are dominant
(1–3). Depending on the cytokine environment, activated T helper cells may differentiate
toward a Th1, Th2, or Th17 cell subset, each of which has different functionalities. In
addition, CD4+ T cells can differentiate into regulatory T cell subsets (Treg cells) that
control effector T cell responses and prevent autoimmunity (4). In the past, autoimmune
diseases, such as RA, multiple sclerosis (MS), psoriasis, and inflammatory bowel disease
(IBD), were considered to be Th1-driven diseases. After the discovery of the Th17 cell
subset, that concept has changed. Blocking antibodies against interleukin-17 (IL-17) reduce
inflammation, and mice deficient in the IL-17 receptor (IL-17R−/−) show reduced sensitivity
to inflammation induction (5). Moreover, in experimental models of arthritis, blocking of
IL-17 significantly reduces bone erosions (6, 7).
Th17 cells are characterized by expression of CCR6 and IL-23R and by production of IL-17A,
IL-17F, IL-21, IL-22, and CCL20. In mice, activation of naive CD4+ T cells in the presence of IL-6
and transforming growth factor β (TGFβ) is sufficient to induce high levels of IL-17 production.
In addition, IL-1 has been shown to drive early Th17 cell differentiation (8, 9), whereas IL-23
is required for the maintenance/survival and expansion of Th17 cells in vivo (10, 11).
The pathogenic effects of IL-17 are well explored, but the contribution of the other
effector cytokines, such as IL-22, is not yet fully understood. Understanding their functions
will advance the knowledge on the pathogenicity of these Th17 cells. Although IL-22
production is not restricted to CD4+ T cells, adaptive T cells are still considered to be the
main producers (12, 13).
IL-22, belonging to the IL-10 superfamily, has been demonstrated to promote innate
immunity (14, 15). It signals via the IL-22R (a complex consisting of IL-22 receptor α1
[IL-22Rα1] and IL-10R2) and induces high phosphorylation of STAT-3, but can also activate
STAT-1, STAT-5, and several MAP kinases (15, 16). Interestingly, the receptor is only present
on resident tissue cells and not on hematopoietic immune cells. Furthermore, the IL-22
binding protein (IL-22BP), a secreted soluble receptor that binds IL-22 with high affinity,
acts as a natural antagonist of IL-22 signaling (14). Until now, most studies have focused on
the effect of IL-22 on hepatocytes and keratinocytes, inducing a wide range of antimicrobial
proteins, such as β-defensins, S100 proteins, serum amyloid A proteins, and matrix metalloproteinases (12, 15).
IL-22 plays a dual role in animal autoimmune models of psoriasis, IBD, and MS
(experimental autoimmune encephalomyelitis [EAE]), which is not yet fully understood.
While blocking of IL-22 may be beneficial in skin disease models, it aggravates disease in IBD
models (17, 18). In addition, IL-22−/− mice are fully susceptible to EAE (19). The presence of
114 | Chapter 6
IL-22–producing T cells in the inflamed tissue of animals in these models implies that the
expression of IL-22R on the tissue-resident cells determines the function and response to IL-22.
In a recent study, the susceptibility to and development of collagen-induced arthritis
(CIA) was evaluated in IL-22−/− C57BL/6 mice (20). Although these IL-22−/− mice showed
a reduced disease incidence and less pannus formation, it is currently not known whether
therapeutic blocking of IL-22 is beneficial. Interestingly, IL-22 and its receptor are expressed
in the synovial lining of RA patients (21). In the present study, we investigated the
production of IL-22 and IL-17 by murine CD4+ T cells under Th17-skewing conditions.
Furthermore, we assessed the role of IL-22, in comparison with that of IL-17, in an
experimental model of spontaneous arthritis in IL-1Ra−/− mice. The findings presented in
this study reveal an important role for IL-22 in IL-1–mediated joint destruction.
Material en methods
Animals. Male C57BL/6 mice were obtained from Janvier. IL-1Ra−/− mice on the BALB/c
background were kindly provided by Dr. M. Nicklin (Sheffield, UK) (22). All mice were
housed in filter-topped cages under specific pathogen–free conditions, and a standard
diet and water were provided ad libitum. The mice used for antibody treatment were
between 10 weeks and 14 weeks of age. All animal procedures were approved by the
institutional ethics committee.
T cell differentiation. The protocol used for T cell differentiation was kindly provided by
Prof. Brigitta Stockinger, with some modification. In short, naive CD4+CD62+ T cells were
isolated from the spleens of naive C57BL/6 mice (Miltenyi Biotec) and were activated with
plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (2.5 μg/ml). These cells were then
cultured in the presence of various cytokines and anticytokine antibodies to skew T cell
development. For Th1 cell differentiation, 10 ng/ml recombinant murine IL-12 (rmIL-12;
eBioscience) and 10 μg/ml anti–IL-4 (eBioscience) were used. For Th2 cell differentiation,
10 ng/ml rmIL-4 (R&D Systems) with 10 μg/ml anti–interferon-γ (anti-IFNγ; eBioscience)
were used. For Th17 cell differentiation, 50 ng/ml rmIL-6 (Sigma-Aldrich) and 1 ng/ml
recombinant human TGFβ1 (rhTGFβ1; R&D Systems) with 10 μg/ml anti–IL-2 were used. For
induced Treg cell differentiation, 1 ng/ml rhTGFβ1 (R&D Systems) and 830 CU/ml rhIL-2
(R&D Systems) were used. In additional experiments, 10 ng/ml rmIL-1β (R&D Systems), 10
ng/ml rmIL-23 (R&D Systems), or 1 μg/ml rmIL-1Ra (R&D Systems) was used.
After 4 days of activation, the supernatant was collected for cytokine measurement,
and the cells were prepared for flow cytometric analysis of intracellular cytokines. In
additional experiments, various numbers of cells were collected after 4 days, washed, and
transferred to a fresh plate with plate-bound anti-CD3/anti-CD28 and stimulated for
another 4 days in the presence of various cytokines and anticytokine antibodies.
IL-1–driven regulation of IL-22 in chronic arthritis | 115
Intracellular flow cytometry. Cells were stimulated for 4–6 hours (10 hours for the ex
vivo IL-22 staining) with phorbol myristate acetate (50 ng/ml; Sigma-Aldrich) and
ionomycin (1 μg/ml; Sigma-Aldrich) and GolgiPlug (1 μl/ml; BD Biosciences). For intracellular
detection of IFNγ, IL-17, and FoxP3, cells were washed, fixed, and permeabilized according
to the manufacturer’s protocol (eBioscience), and incubated with anti-IFNγ (BD Biosciences),
anti–IL-17 (BD Biosciences), anti-FoxP3 (eBioscience), and anti–IL-22-02 (Pfizer).
Cytokine detection. To determine the levels of IL-17, IFNγ, IL-10, and tumor necrosis factor
α (TNFα), Luminex multianalyte technology was used, in combination with Milliplex
cytokine kits (Millipore). IL-22 was measured with Flowcytomix kits from Bender
Medsystems. All assays were performed according to the manufacturer’s instructions.
Quantitative polymerase chain reaction (PCR). RNA was isolated from synovial ankle
biopsy tissue using TRI Reagent (Sigma) and treated with DNase to remove genomic DNA.
The RNA was subsequently reverse transcribed with oligo(dT) primers in a reverse
transcriptase procedure. Quantitative real-time PCR was performed, using primer pairs
and SYBR Green PCR Master Mix from Applied Biosystems. The ΔCt method was used to
normalize transcript levels to those for GAPDH and to calculate (using the 2 method) the
relative expression of messenger RNA (mRNA) for Th17 cell marker genes. All primers were
developed using Primer Express version 2.0 (Applied Biosystems) and validated according
to the recommended protocol. In order to allow for statistical analysis, the ΔCt value in
samples showing no detectable marker mRNA during the quantitative PCR analysis of
IL-22, IL-22Rα1, and IL-22BP was set at 21 cycles (representing the detection limit).
Experimental arthritis treatment protocol. IL-1Ra−/− mice with early arthritis (defined
as an arthritis score of 0.75–1.0 within a score range of 0–4) were treated with anti–IL-17
(MAB421; R&D Systems), anti–IL-22 (anti–IL-22-01; Pfizer), or control antibodies (rat IgG).
Mice received anticytokine treatment (8 mg/kg) intraperitoneally 3 times every week. The
clinical severity of arthritis (arthritis score) was macroscopically graded, 3 times per week,
on a scale of 0–2 for each paw, with higher scores indicating increasing severity of redness
and swelling of the paws. After 4 weeks of treatment, the mice were killed and the ankle
joints were isolated for histopathologic analysis.
Histopathology. For standard histologic assessment, isolated joints were fixed for 4 days
in 10% formalin and decalcified in 5% formic acid, and the specimens were then processed
for paraffin embedding (8). Tissue sections were stained with hematoxylin and eosin
(H&E). The severity of inflammation in the joints was scored on a scale of 0–3 (0 = no cells,
1 = mild cellularity, 2 = moderate cellularity, and 3 = maximal cellularity). In addition, bone
destruction was graded on a scale of 0–3, ranging from no damage to the complete loss
of bone structure. Proteoglycan (PG) depletion was determined using Safranin O staining.
116 | Chapter 6
Loss of PGs was scored on a scale of 0–3, ranging from fully stained (normal) cartilage to
destained (fully depleted of PG) cartilage. The scoring of the sections was performed in a
blinded manner.
Cathepsin K staining. The presence of active osteoclasts was visualized by immunohistochemical staining for cathepsin K. Joint sections (7 μm) were deparaffinized, rehydrated,
and incubated with anti–cathepsin K (ab19027; Abcam) or with rabbit Ig (Ab-105-C; R&D
Systems). Subsequently, the sections were incubated with biotinylated goat anti-rabbit
IgG (PK-6101; Vector), followed by peroxidase-labeled avidin–biotin complex (PK-6101;
Vector). Peroxidase was developed with diaminobenzidine, and tissues were counterstained
with hematoxylin. Cathepsin K–positive cells were quantified along the bone surface. The
mean number of positive cells in 2 sections per mouse was determined in a blinded manner.
Isolation, culture, and stimulation of synovial cells. The protocol for stimulation of
synovial cells was kindly provided by Prof. Shimon Sakaguchi, with some modification. In
short, after the mice were killed, the ankle joint synovium was dissected, and synovial
biopsy specimens were incubated with enzymatic digestion buffers (Liberase; Roche) for
30 minutes at 37°C. A 70-μm nylon cell strainer (BD Falcon) was then used to process the
digested tissue. The cell preparation was collected in RPMI with 10% fetal calf serum (FCS).
To isolate the single mononuclear cells, Lympholyte-M (Cederlane) was used, according
to the manufacturer’s protocol. All cells were cultured in RPMI 1640 (Gibco-Invitrogen)
supplemented with 10% FCS. In addition, the cells were prepared for intracellular
flow cytometry using magnetic-activated cell sorting for positive selection of CD11b+,
CD19+, and CD3+ cells with magnetic-labeled beads (Miltenyi Biotec), according to the
manufacturer’s protocol.
Statistical analysis. Results are expressed as the mean ± SEM. Differences between
experimental groups were tested using the Mann-Whitney U test or one-way analysis of
variance with Dunnett’s test for multiple comparisons, as appropriate. P values less than
0.05 were considered significant.
Results
Regulation of IL-22 expression by Th17 cells driven by IL-23 and IL-1
Th17 cells were reported to coexpress IL-17 and IL-22 (23). To validate this, we differentiated
naive CD4+ T cells into Th1, Th2, Th17, and induced Treg cell subsets. As expected, high
expression of IFNy was detected in the Th1 cell culture, while high expression levels of IL-5
and IL-10 were observed in the Th2 cell culture. High levels of IL-17 were a hallmark of
Th17-polarized cultures (Figure 1A).
IL-1–driven regulation of IL-22 in chronic arthritis | 117
A
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4500
**
**
***
IL-22 (pg/ml)
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IL-17 (pg/ml)
diu
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eg
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Me
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IL-22 (pg/ml)
Me
diu
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17 7
Th + IL
17 -1
+I
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iTr
eg
IL-17 (pg/ml)
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B
0
0
0
3500
3000
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ns
100
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diu
m
Th Th Th17
17 17
+I +I
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+
Th Th IL-1r
17 17 a
+I +I
L-2 L-2
3+ 3
IL1ra
Me
Me
diu
m
Th Th Th17
17 17
+I +I
L-1 L-1
+
Th Th IL-1r
17 17 a
+I +I
L-2 L-2
3+ 3
IL1ra
0
Figure 1 | Interleukin-22 (IL-22) is mainly produced by Th17 cells after coculture with IL-1 or
IL-23. Naive CD4+CD62L+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28
antibodies and differentiated for 4 days under specific culture conditions, to achieve differentiation
of Th1 cells (using recombinant murine IL-12 [rmIL-12] with anti–IL-4), Th2 cells (using rmIL-4 with
anti–interferon-γ [anti-IFNγ]), Th17 cells (using recombinant human transforming growth factor
β [rhTGFβ] and rmIL-6 with anti–IL-2), and induced Treg (iTreg) cells (using rhTGFβ1 and rhIL-2). A,
Production of IFNγ, IL-5, IL-10, IL-17, and IL-22 was measured in each cell culture on day 4. B, Th17
cells were generated from naive cells in the same manner as described above and cultured with
or without IL-1, IL-23, and IL-1 receptor antagonist (IL-1Ra). Thereafter, production of IL-17 and IL-22
was measured. Bars show the mean ± SEM representative results from 1 of 3 experiments, each
performed in triplicate. ** = P < 0.01; *** = P < 0.001, by Student’s t-test. NS = not significant.
118 | Chapter 6
Surprisingly, under standard Th17 cell culture conditions, we detected only low levels
of IL-22, whereas IL-17 was readily detected (Figure 1A). IL-23 has been shown to amplify
the IL-22 production by activated CD4+ T cells (19, 23). When IL-23 was added to our Th17
cell culture, we observed a significant induction of IL-22 production (Figure 1A). Moreover,
since IL-1 has been described as an important factor for the early differentiation of Th17
cells (9), we assessed whether the addition of IL-1 would also potentiate IL-22 production.
Indeed, IL-1 significantly increased the expression of IL-22, revealing that the Th17-polarizing culture condition is, by itself, a mediocre inducer of IL-22 production, whereas culture
conditions including both IL-1 and IL-23 can drive IL-22 production.
We next used IL-1Ra, the natural receptor antagonist of IL-1, to confirm a role of IL-1 in
Th17 cell generation. As expected, the addition of IL-1Ra abrogated the IL-1–enhanced
production of IL-22 (Figure 1B). Remarkably, the addition of IL-1Ra during IL-23–mediated
Th17 cell differentiation resulted in a significant reduction in the production of IL-17,
whereas no effect was seen on the production of IL-22, thereby suggesting that IL-23–
mediated potentiation of IL-22 production by Th17 cells is independent of (endogenous)
IL-1. These findings combined imply a role for both IL-23 and IL-1 in IL-22 production by
Th17 cells.
Synergistic effects of IL-1 and IL-23 on IL-22 production during Th17 cell
differentiation
Because IL-1 and IL-23 both drive IL-22 production by Th17 cells, we assessed their
synergistic potential during Th17 cell differentiation. The addition of both IL-1 and IL-23
significantly amplified the production of IL-22, whereas no added or synergistic effect was
seen on the production of IL-17 (Figure 2A). Interestingly, fluorescence-activated cell
sorting (FACS) analysis of the expression of IL-17 and IL-22 in Th17 cells revealed that these
Th17 cytokines have different kinetic expression profiles (Figure 2B). IL-17 expression was
already induced after 2 days, whereas IL-22 expression was nearly undetectable. However,
after 4 days of culture, 4–5% of the IL-1/IL-23–cultured Th17 cells produced both IL-17 and
IL-22. Thus, IL-17 production clearly precedes IL-22 production in Th17 cell differentiation.
Since IL-23 has been shown to maintain the IL-22 production by CD4+ cells (24), we
investigated whether long-time culture and restimulation of the IL-22–producing Th17
cells could affect the observed synergy between IL-1 and IL-23. Restimulated differentiated
Th17 cells still produced high levels of IL-17, which was not significantly influenced by
cytokine addition (Figure 2C). Cell cultures with IL-23 alone maintained the expression of
IL-22 by Th17 cells, in contrast to that with IL-1 alone. Interestingly, although a clear synergy
between IL-1 and IL-23 was observed in the maintenance of IL-22 production, the balance
shifted toward a more prominent role for IL-23. These findings are consistent with the
notion that IL-1 potentiates the generation of Th17 cells, and that IL-23 maintains the Th17
cell phenotype at the level of both IL-17 production and IL-22 production.
IL-1–driven regulation of IL-22 in chronic arthritis | 119
Expression of synovial IL-22 and IL-22R during chronic T cell–driven arthritis
Our results demonstrate that IL-1 potentiates IL-22 expression by Th17 cells in vitro. To
study the potential role of IL-22 in vivo, we tested the expression of IL-22 and IL-22R in
IL-1Ra−/− mice, an animal model in which IL-1/Th17–driven arthritis spontaneously
1200
5000
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IL-22 (pg/ml)
6000
4000
3000
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+
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+ I IL-23
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A
Th17+IL-1+IL-23
Th17
Medium
Day 2
IL-22
Day 4
IL-17
***
1250
5500
Figure 2 | Interleukin-1
(IL-1) and IL-23 have synergistic effects on the expression of IL-22 by
C
***
L-1
+I
23
1
23
IL-
IL-
IL-
Th
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diu
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23
+
1
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IL-
Th
17
IL-
IL-
Me
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m
IL-17 (pg/ml)
IL-22 (pg/ml)
Th17 cells. Naive CD4+CD62L+
T cells were activated with plate-bound anti-CD3 and soluble anti4500
1000
CD28 antibodies and differentiated for 4 days under Th17-polarizing conditions (using recombinant
human transforming growth
IL-6 with anti–IL-2), and then cultured
3500 factor β and recombinant murine
750
with IL-23 and/or IL-1. A, Concentrations of IL-17 and IL-22 were measured in each
*** cell culture on day
4. B, On day 2 and day 4,2500
cells were cultured in the same manner
500 as described above and stimulated
with phorbol myristate acetate, ionomycin, and GolgiPlug. Thereafter, the cells were stained for
1500
intracellular IL-17 and IL-22. C, On day 4, the T cells that differentiated
in the presence of IL-1 and IL-23
250
were washed and restimulated
for another 4 days, and production of IL-17 and IL-22 was measured
500
0
in each cell culture. Bars show the mean ± SEM of triplicate experiments.
** = P < 0.01; *** = P < 0.001
versus the other culture conditions.
120 | Chapter 6
IL-22
Day 4
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IL-17 (pg/ml)
C
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ILIL- 23
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ILIL- 23
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0
Figure 2 | Continued.
develops (8, 25). Serum, draining lymph node cells, and inflamed synovial biopsy tissue
from 10–12-week-old arthritic IL-1Ra−/− mice were isolated.
As shown in Figure 3A, IL-6 was detected at clearly increased levels in the serum,
whereas IL-10 levels were very low. Interestingly, the Th17 cytokines IL-17 and IL-22 were
also detectable in the serum of arthritic animals.
Rechallenge of the draining lymph node cells via anti-CD3/anti-CD28 stimulation for
48 hours (Figure 3B) revealed a clearly notable production of Th17 cytokines (IL-22 and
IL-17), as well as that of IL-10 and IFNγ (a Th1 cytokine). The severity of arthritis was not
correlated with increasing cytokine production per cell (results not shown). Although the
IFNγ levels were high, we have previously shown that the quantity of Th1 cells in this
model does not differ from that found in naive wild-type mice (8).
Synovial biopsy tissue samples obtained from 10–12-week-old IL-1Ra−/− mice with
various degrees of arthritis severity (mild, moderate, and severe) were analyzed for
differential expression of Th17 cell marker genes (Figure 3C). When compared to the
synovium of the mildly inflamed ankle joints, the moderately and severely inflamed
synovia displayed high expression of mRNA for IL-17 and the lineage transcription factor
retinoic acid–related orphan receptor γT. In contrast, the expression of IL-22 mRNA was
low, but significantly higher in mice with severe arthritis. Concomitantly, the expression of
the IL-22BP was also increased in severely inflamed ankle synovia. Messenger RNA for the
IL-22Rα1 subunit was only detected in the severely inflamed ankle joints. These findings
emphasize that IL-22 and its receptor are up-regulated mainly in chronically inflamed
synovial tissue of IL-1Ra−/− mice.
IL-1–driven regulation of IL-22 in chronic arthritis | 121
IFN γ (pg/ml)
30
20
200
1500
1000
500
5
0
250
250
200
200
150
100
IL-10 IL-17 IL-6 IL-22
C
IL-22
8
100
50
0
0
IL-17A
**
RE
3
**
30
100
*
RE
5
***
40
125
4
RORyT
50
150
6
75
20
50
IL-10R2
75
*
50
5
***
rat
e
de
25
RE
RE
3
**
30
6
4
IL-22Bp
35
*
7
25
Se
ve
re
ld
Mo
de
Mi
ld
0
Mo
Mo
de
Mi
IL-22Rα1
8
10
Mi
0
rat
e
0
Se
ve
re
25
ld
1
rat
e
2
20
15
10
2
5
1
0
Se
ve
re
rat
e
ld
Mo
de
Se
ve
re
rat
e
de
Mi
ld
0
Mo
Se
ve
re
rat
e
Mo
de
Mi
ld
0
Mi
RE
150
50
175
*
7
100
50
IL-22 (pg/ml)
IL-17 (pg/ml)
10
150
0
15
0
RE
250
2000
Se
ve
re
Serum levels (pg/ml)
25
2500
IL-10 (pg/ml)
B
A
Figure 3 | Expression of Th17 and interleukin-22 (IL-22)–related genes and proteins in
the serum (A), draining lymph nodes (B), and ankle synovium (C) of 10–12-week-old IL-1
receptor antagonist–deficient (IL-1Ra−/−) mice. A, Concentrations of IL-10, IL-17, IL-6, and IL-22
were measured in the sera (n = 8). B, Draining lymph node cells (1 × 105/well) were stimulated
with anti-CD3/anti-CD28 antibodies for 48 hours, and production of interferon-γ (IFNγ), IL-10, IL-17,
and IL-22 was measured by Luminex assay (n = 17–25). Circles represent individual samples, and the
horizontal line indicates the mean. C, IL-1Ra−/− mice were macroscopically scored for arthritis and
divided into 3 groups according to the degree of inflammation (mild, moderate, or severe) (n =
7–10 mice per group). Expression of mRNA for IL-22, IL-17A, retinoic acid–related orphan receptor γT
(RORγT), IL-22 receptor α1 (IL-22Rα1), IL-10R2, and IL-22 binding protein (IL-22BP) in the synovial biopsy
tissue from the ankle joints was measured by quantitative real-time polymerase chain reaction, with
results shown as the relative expression (RE) compared to that of GAPDH. Bars in A and C show the
mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 versus the mild arthritis group.
122 | Chapter 6
Suppression of arthritis by blockade of endogenous IL-22 in arthritic
IL-1Ra−/− mice
To explore the role of IL-22 during the progression of arthritis, IL-1Ra−/− mice with early
(initiating) arthritis (arthritis score of 0.75–1.0) were treated with blocking antibodies
against IL-22, or isotype control antibodies, for 4 weeks. In addition, anti–IL-17 treatment
was included as a positive control.
As shown in Figure 4A, the clinical scores of the inflamed ankles from the mice in the
control group steadily increased over time. Consistent with earlier findings (8), treatment with
anti–IL-17 significantly blocked the progression of arthritis. In anti–IL-22–treated mice, there was
a trend toward reduction of the clinical score when compared to the control group.
Similar to the effects on macroscopic arthritis scores, anti–IL-17 treatment significantly
reduced the influx of inflammatory cells in the arthritic joint tissue (Figure 4B). Cartilage PG
depletion was not significantly suppressed by blockade of IL-17, but anti–IL-17 treatment
did clearly reduce bone erosion (Figure 4B). However, we found no clear reduction in the
number of osteoclasts in the anti–IL-17–treated group (Figure 4B). Figures 4C and D show
the results from histologic assessment of representative H&E-stained sections from each
group.
Although the anti–IL-22 treatment did not affect the macroscopic arthritis scores
significantly, the histopathologic analysis revealed a significant suppression of the amount
of inflammatory cells after treatment (Figures 4A–C). Interestingly, the reduction in PG
depletion in anti–IL-22–treated mice nearly reached significance when compared with
controls (P = 0.07) (Figure 4B). Unlike the reduction in bone erosions, we found no
reduction in the number of osteoclasts with anti–IL-22 treatment (Figure 4B). These results
show that endogenous IL-22 may not affect edema, but does play a role in the joint
inflammation seen in this model. In addition, IL-22 may partially contribute to osteoclast
activity, but has a less substantial effect on osteoclast differentiation during arthritis
progression.
Role of T cells as the main local producers of IL-22 and IL-17 in the
inflamed synovium
Because anti–IL-22 treatment was found to prevent structural joint damage in this IL-1–
driven murine model, and the levels of IL-22 mRNA clearly correlated with arthritis severity,
we next investigated whether resident T cells are the main IL-22–producing cells within
the inflamed synovium. We therefore isolated and stimulated the CD11b+, CD19+, and
CD3+ cell subsets, as well as the negatively selected remaining cells, from the synovial
tissue of arthritic IL-1Ra−/− mice. As expected, production of IL-10 and TNFα was mainly
found in the CD11b+ (macrophages and granulocytes) and CD19+ (B cell) compartments.
Most of the IL-6 production was detected in the B cell fraction and in the remainder of the
cells (Figure 5A). The main population residing in the inflamed synovial tissue responsible
for the production of IL-17 and IL-22 was the CD3+ T cell fraction.
IL-1–driven regulation of IL-22 in chronic arthritis | 123
Inflammation (0-3)
1.75
1.50
1.25
0.0
4
Control
Control
A
10
anti-IL-17
anti-IL-17
22
IL-
An
ti-
22
IL-
An
ti-
IL-
17
A
l
0
An
ti-
22
IL-
An
ti-
An
ti-
IL-
17
A
l
0.0
Co
nt
ro
D
**
0.5
20
A
0.5
**
1.0
17
1.0
1.5
IL-
Bone erosion (0-3)
1.5
0.0
C
30
2.0
P = 0.07
Co
nt
ro
PG depletion (0-3)
2.0
An
ti-
Time (weeks)
IL22
3
l
2
0.5
l
1
1.0
Co
nt
ro
0
*
*
1.5
An
ti-
**
**
2.0
An
ti-
0.75
***
2.5
IL17
1.00
3.0
Co
nt
ro
Macroscopic score (0-4)
B
Control
anti-IL-17A
anti-IL-22
2.00
# Osteoclasts/ slide
A
anti-IL-22
anti-IL-22
Figure 4 | Treatment of interleukin-1 receptor antagonist–deficient (IL-1Ra−/−) arthritic mice
with anti–IL-22 antibodies reduces joint inflammation and destruction. IL-1Ra−/− mice with
early arthritis were treated with anti–IL-17A, anti–IL-22, or control rat IgG antibodies (n = 6–7 mice per
group). A and B, In each group, the severity of arthritis in the ankle joints was scored macroscopically
(A), and histopathologic scores for inflammation, cartilage proteoglycan (PG) depletion, bone
erosion, and number of osteoclasts were assessed 4 weeks after the start of treatment (B). Results
are the mean ± SEM of at least 8 mice per group. * = P < 0.05; ** = P < 0.01 versus control antibody–
treated group. C and D, Representative slides show hematoxylin and eosin–stained arthritic ankle
joint sections from mice in each treatment group. Original magnification × 50 in C; × 100 in D.
Arrows in D indicate a site of bone erosion.
124 | Chapter 6
70
IL-10 (pg/ml)
CD
Moderate
res
t
t
3
rat
e
Se
ve
re
de
rat
e
Se
ve
re
ld
Mi
Mo
Se
ve
re
rat
e
ld
Mi
de
Mo
5000
0
0
Mild
*
10000
ld
0
15000
Mi
100
*
1000
**
20000
Mo
200
CD
3
b
CD
19
IL-17+ IL-22−
**
2000
ND
ND
25000
Number of cells
ns
ND
11
b
CD
19
0.0
IL-17+ IL-22+
Number of cells
Number of cells
CD
11
0.5
0
*
300
1.0
100
3000
400
1.5
res
200
2.0
CD
300
IL-17− IL-22+
500
C
res
t
2.5
400
11
b
CD
19
t
3
res
CD
CD
11
b
CD
19
0.0
3.0
500
IL-22 (pg/ml)
0.5
2.5
CD
IL-17 (pg/ml)
1.0
5.0
600
t
res
t
CD
11
1.5
7.5
0.0
CD
3
0
b
CD
19
0
CD
3
250
b
CD
19
10
2.0
IFNγ (pg/ml)
500
3
20
750
res
30
1000
CD
IL-6 (pg/ml)
40
CD
11
TNF-α (pg/ml)
1250
50
B
10.0
1500
60
de
A
Severe
Figure 5 | Production of interleukin-22 (IL-22) by synovial cells is mainly derived from
IL-22
CD3+IL-17+ T cells. Ankle joints of interleukin-1 receptor antagonist–deficient (IL-1Ra−/−) mice were
scored macroscopically for the severity of arthritis (mild, moderate, or severe), and the synovium was
dissected and further processed. A, Synovial biopsy tissue samples were pooled in 4 groups (n = 4–6
mice per group), and after enzymatic digestion, the CD11b+, CD19+, or CD3+ cells were positively
selected using magnetic-labeled beads. The negatively selected cell population (designated
“rest”) was also assayed. TheIL-17cells (1 × 105 cells/well) were then stimulated for 4 hours with phorbol
myristate acetate (PMA) and ionomycin, and cytokine production (tumor necrosis factor α [TNFα],
IL-6, IL-10, interferon-γ [IFNγ], IL-17, and IL-22) was measured in each cell fraction supernatant. B
and C, In addition, after enzymatic digestion, the cells were incubated with PMA/ionomycin and
GolgiPlug for 10 hours before flow cytometric analysis (n = 6–14 mice per group from 3 independent
experiments). The cells were counted and stained for CD3, IL-17, and IL-22. Results are expressed as
the mean total number of isolated cells per synovial biopsy tissue group (B) or as dot plots of the
isolated cells gated on the CD3+ cell population (C) (representative results are shown). Bars show the
mean ± SEM. * = P < 0.01; ** = P < 0.001. ND = not detectable; NS = not significant.
100
*
15000
10000
*
5000
Moderate
Se
ve
re
Se
ve
re
Mi
ld
Mo
de
rat
e
Mi
ld
Mo
de
rat
e
Severe
IL-22
Mild
Se
ve
re
IL-1–driven regulation of
IL-22 in chronic arthritis | 125
0
0
0
C
1000
Number of c
ns
Mi
ld
Mo
de
rat
e
200
Number of c
Number of c
300
IL-17
Figure 5 | Continued.
In order to determine the percentage of CD3+ cells producing IL-22 or IL-17 and the
amount of double-positive cells present in the inflamed synovium, single cells were
isolated from the synovial tissue of IL-1Ra−/− mice with different arthritis severity scores.
FACS analysis of the expression of IL-17 and IL-22 revealed that most of the IL-22+ cells were
positive for IL-17 (Figures 5B and C), and the presence of this cell population in the
synovium increased with the severity of arthritis. Of interest, a large population of the
IL-17+ T cells was found to be IL-22 negative, possibly due to the differential kinetic
expression profiles of IL-17 and IL-22.
Discussion
In this study, we demonstrated that both IL-23 and IL-1 are important for the regulation of
IL-22 during Th17 cell differentiation. Strikingly, IL-1 and IL-23 synergistically enhanced the
production of IL-22 during Th17 cell differentiation. Furthermore, the results of this study
clearly showed that there are IL-22–producing T cells within the joints of arthritic IL-1Ra−/−
mice, and that neutralizing the production of IL-22 modulates joint inflammation and
bone destruction.
Th17 cells are considered to be the main IL-22 producers, in an IL-23–dependent
manner (23). Surprisingly, in our experiments, the addition of not only IL-23 but also IL-1 to
the Th17 cell differentiation culture clearly enhanced the IL-22–productive capacity of the
cells. Remarkably, combining IL-1 and IL-23 synergistically increased the production of
IL-22. To assess the role of IL-1 and IL-23 on polarized Th17 cells, we restimulated the cells
producing high amounts of IL-22 (in a Th17 cocktail culture with IL-23 and IL-1) with
combinations of IL-23 and IL-1. As a result, the synergy between IL-1 and IL-23 in the
maintenance of IL-22 production was still visible; nevertheless, culture with IL-23 alone
could already maintain high production of IL-22. Of interest, IL-1 alone was unable to
maintain the IL-22–producing capability of the Th17 cells. In contrast, the production of
IL-17 was induced earlier during the differentiation of Th17 cells and proved stable over a
126 | Chapter 6
longer culture period. Generally, it is therefore concluded that the IL-23–dependent
production of IL-22 seems more important in a later phase of Th17 cell differentiation.
To explore the role of IL-22 during arthritis, we investigated its role in IL-1Ra−/− mice,
which develop spontaneous arthritis as a result of excess IL-1 signaling due to the absence
of the natural inhibitor IL-1Ra (26). Because IL-17 and Th17 cells are important for the
development of arthritis in these animals (8, 25, 27), we selected this model to validate the
therapeutic potential of anti–IL-22. First, we were able to detect IL-22 systemically in the
sera of mice with established arthritis. Moreover, the draining lymph node cells produced
comparably high levels of IL-17 and IL-22 after anti-CD3/anti-CD28 stimulation, indicating
that in this model, T cells produce high levels of IL-22. To assess the presence of IL-22 and
its receptor during arthritis, mRNA levels were determined in the synovial biopsy tissue
from the mice, which confirmed that IL-22 and IL-22Rα1 are highly expressed within the
synovia of severely inflamed arthritic murine joints. In addition, the mRNA levels of IL-22BP
were also increased, indicating the presence of a possible local modulator of IL-22
signaling. Taken together, these findings indicate that IL-22 is produced locally within the
synovia of inflamed joints, which reflects the possibility that the functionality of IL-22 can
be attributed to the presence of its receptor.
We have previously observed that IL-17 contributes to the progression of inflammation
and joint destruction in this model (28), and as expected, the administration of anti–IL-17
ameliorated the macroscopic inflammation in IL-1Ra−/− mice. As described previously (8),
the results of histopathologic analysis clearly revealed a significant reduction in the influx
of inflammatory cells after treatment with anti–IL-17. Furthermore, anti–IL-17 treatment did
not significantly reduce the depletion of cartilage PG in this model (8), whereas bone
erosions decreased significantly. Thus, these findings show that even after long-term
therapeutic intervention against IL-17, the disease activity remains suppressed.
Because IL-1 and Th17 cells play important roles in this model, we hypothesized a role
for IL-22 in the progression of the disease. Although the reduction in macroscopic swelling
was less pronounced in the anti–IL-22–treated mice compared to that in the anti–IL-17–
treated mice, a significant reduction in the influx of inflammatory cells after anti–IL-22
treatment was clearly observed, as confirmed on histopathologic analysis. This reduction
of infiltrating cells is consistent with the findings from the study by Geboes et al, in which
a decrease in pannus cells was observed in IL-22−/− mice in the CIA model, confirming the
proinflammatory character of IL-22 (20). The minor decrease in macroscopic joint swelling
seen with anti–IL-22 suggests that IL-17 facilitates extravasation and edema formation
more prominently than does IL-22. Since IL-22 is mainly increased in highly inflamed
synovia in the later phases of arthritic disease, longer treatment with anti–IL-22 may lead
to more pronounced effects on joint edema.
Interestingly, therapeutic neutralization of IL-22 and that of IL-17 both resulted in a
reduction in the influx of proinflammatory cells and had similar effects on bone erosion.
This is remarkable, in view of the fact that most T cells produced IL-17 alone, and only
IL-1–driven regulation of IL-22 in chronic arthritis | 127
10–15% of these T cells coexpressed IL-22. Notably, only very low numbers of IL-22+IL-17− T
cells were found. Moreover, expression of IL-22 and IL-22R in the synovia coincided with
late-stage destructive disease. These findings are consistent with the hypothesis that Th17
cells are pathogenic and destructive by virtue of IL-22 production. In addition, in airway
inflammation, it has recently been shown that IL-17 may govern the proinflammatory and
pathologic properties of IL-22 (29). For further exploration of this notion, combination
blocking of both IL-17 and IL-22 should provide more insights.
The mechanism by which IL-22 specifically contributes to disease pathogenicity
remains unclear. Although it has been shown that blood immune cells neither respond to
IL-22 nor express the IL-22R (15), Ikeuchi et al demonstrated that IL-22 and IL-22R are
expressed within the synovium of RA patients, and that this expression was mainly
attributed to fibroblasts that produced monocyte chemotactic protein 1 upon stimulation
of IL-22 production (21). Recent data from our laboratory confirm the expression of the
IL-22R specifically on fibroblast-like cells isolated from inflamed human synovium (results
not shown).
In addition, Geboes et al showed that IL-22 stimulation of spleen cells from immunized
mice with CIA contributes to osteoclastogenesis (20). In the present study, we described a
role for IL-22 in bone degradation that was not associated with a decrease in the number
of residing osteoclasts at the end of the treatment period. Recently, it was shown that
bone erosion at end point is the cumulative result of dynamic osteoclast activity (i.e.,
numbers at a given phase in the process) over the course of arthritis progression (30).
However, in our experiments, we started with mice that had an early arthritis (defined as
an arthritis score of 0.75–1.0 within a score range of 0–4), a stage at which osteoclasts and
osteoclast progenitors are likely to be present within the joint. Moreover, proinflammatory
cytokines, especially IL-1, are very potent stimuli of osteoclast formation and survival (31,
32). In addition, we have observed that the levels of IL-22 are mainly increased in the highly
inflamed synovia and are less affected during the early stages of arthritis. Thus, although
IL-22 might be able to accelerate osteoclast formation in vitro, during the early initiating
arthritis in this model, the lower IL-22 levels might be secondary to the high IL-1 levels.
Since we observed no differences in the number of osteoclasts after 4 weeks of therapy,
whereas we did observe a significant reduction in bone erosion after IL-22 neutralization,
we conclude that IL-22 may exert its effect via the bone-resorbing capacity of the
osteoclasts.
This study thus demonstrates that IL-1 and IL-23 are important for maximal induction
of IL-22 production by murine Th17 cells. IL-17 production was found to precede IL-22
production. Furthermore, IL-22 production was maintained by IL-23, independent of IL-1. In
addition, we demonstrated that both systemic IL-22 and synovial IL-22 are mainly produced
by T cells that also produce IL-17. Finally, in a Th17-driven experimental arthritis model, IL-22
contributes to joint inflammation and structural damage. Our combined results imply that
IL-22 is responsible for Th17-mediated arthritis at the level of the joints. Since IL-22 and its
128 | Chapter 6
receptor have been detected in the synovium of RA patients, these novel insights may
contribute to the design of novel therapeutic strategies for the management and control
of structural damage in RA.
Acknowledgements
We thank Birgitte Walgreen, Monique Helsen, and Liduine van den Bersselaar for excellent
technical assistance.
IL-1–driven regulation of IL-22 in chronic arthritis | 129
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destruction in arthritis. Arthritis Rheum 2011; 63: 1365–75.
(31) Wei S, Kitaura H, Zhou P, Ross FP, Teitelbaum SL. IL-1 mediates TNF-induced osteoclastogenesis. J Clin Invest
2005; 115: 282–90.
(32) Jimi E, Nakamura I, Duong LT, Ikebe T, Takahashi N, Rodan GA, et al. Interleukin 1 induces multinucleation
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247: 84–93.
IL-1–driven regulation of IL-22 in chronic arthritis | 131
Chapter 7
T cell lessons from the rheumatoid arthritis
synovium SCID mouse model: CD3-rich
synovium lacks response to CTLA-4Ig
but is successfully treated by interleukin-17
neutralization
Marije I. Koenders1, Renoud J. Marijnissen1, Leo A. B. Joosten1, Shahla Abdollahi-Roodsaz1,
Franco E. Di Padova2, Fons A. van de Loo1, John Dulos3, Wim B. van den Berg1,*,
Annemieke M. H. Boots3,†
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
Novartis Pharma, Basel, Switzerland
3
Organon NV, Oss, The Netherlands
†
U
niversity Medical Center Groningen, Groningen, The Netherlands
1
2
134 | Chapter 7
Abstract
Objective
To provide an intermediate step between classic arthritis models and clinical trials, the
rheumatoid arthritis (RA) synovium SCID mouse model is a valuable tool for use during
preclinical research. We undertook this study to investigate the validity of this humanized
mouse model using anti–tumor necrosis factor (anti-TNF) and anti–interleukin-1 (anti–IL-1)
treatment and to investigate the direct effect of T cells– and B cell–related therapies on
the transplanted RA synovial tissue.
Methods
CB17/SCID mice were engrafted with human RA synovial tissue and systemically treated
with anti-TNF, anti–IL-1, anti–IL-17, CTLA-4Ig, anti-CD20, or isotype control antibodies.
Results
Validation of the model with anti-TNF treatment significantly reduced serum cytokine
levels and decreased histologic inflammation, whereas anti–IL-1 therapy did not show any
effect on the RA synovial grafts. In mice engrafted with B cell–rich synovial tissue,
anti-CD20 treatment showed clear therapeutic effects. Surprisingly, CTLA-4Ig treatment
did not show any effects in this transplantation model, despite prescreening of the
synovial tissue for the presence of CD3+ T cells and the costimulatory molecules CD80
and CD86. In contrast, great therapeutic potential was observed for anti–IL-17 treatment,
but only when CD3+ T cells were abundantly present in the RA synovial tissue.
Conclusion
This human RA synovium SCID mouse model enabled us to show that CTLA-4Ig lacks
direct effects on T cell activation processes in the synovial tissue. Further evidence was
obtained that IL-17 might indeed be an interesting therapeutic target in RA patients with
CD3-rich synovial tissue. Further characterization of the RA patients’ individual synovial
profiles is of great importance for achieving tailored therapy.
T cell targeting in the RA synovium SCID mouse model | 135
Introduction
Rheumatoid arthritis (RA) is a systemic autoimmune disease that is characterized by
chronic inflammation and destruction of synovial joints. The etiology of RA is still not clear,
but its pathogenesis has been studied extensively both in patients and in experimental
animal models. During the process of joint inflammation, the synovium is thickened by
hyperplasia of the synovial lining as well as by the influx of proinflammatory cells into the
joint. Besides the resident synovial fibroblasts and tissue macrophages, immune cells such
as neutrophils, natural killer cells, and T and B lymphocytes, can be found in the arthritic
joint, and all contribute to the final destruction of cartilage and bone matrix.
Animal models of arthritis are widely used to study the potential key players in the
process of RA (1). Blocking of proinflammatory cytokines or depletion of certain immune
cells might improve the outcome of the disease in experimental arthritis models in rats
and mice, and thereby help to unravel new therapeutic targets for RA. Unfortunately, not
all aspects of animal models can be translated directly to the human situation. The relative
roles of tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) in the pathogenesis of
arthritis are a striking example in the field of RA. From in vitro studies and murine arthritis
models, IL-1 was found to be a major cytokine in joint pathology, especially driving
cartilage destruction, in contrast to the less potent cytokine TNF (2–4). However, clinical
trials resulted in the opposite finding: anti-TNF treatment is now the most frequently
prescribed biologic agent for RA patients, while blocking IL-1 turned out to have only
relatively modest therapeutic effects in this disease (5).
To enhance the predictive value of our research findings in animal models, the use of
so-called humanized arthritis models might be a valuable additional tool that can be used
during preclinical research. SCID mice are widely used as host for the transplantation of
various tissues, since their impaired T and B cell function prevents them from rejecting
grafts. Transplantation of human cells or tissue from RA patients into immunodeficient
mice enables us to study and target the human homologs of various proteins, such as
cytokines, receptors, and growth factors, thereby providing an important step between
our standard animal models and clinical trials.
The RA SCID mouse model is used in 2 variants, each with its own advantages and
limitations. First described by Geiler et al and Pap et al, cotransplantation of RA synovial
tissue or, as more frequently applied, of RA synovial fibroblasts (RASFs) with pieces of
cartilage into SCID mice is used to study the capacity of the fibroblast-like synoviocytes to
degrade matrix and invade the cartilage (6, 7). A recent elegant article reported the
migratory and destructive characteristics of RASFs traveling from the transplantation site
to healthy cartilage in the contralateral flank (8). Obviously, this cartilage invasion model is
limited to one specific cell type as a major key player in the arthritis process, focusing
completely on the fibroblast and its destructive mediators and excluding the potential
contribution of other cell types to the degradation of cartilage.
136 | Chapter 7
The second humanized arthritis model is the RA synovium SCID mouse model. In this
model, standardized pieces of synovial tissue from RA patients are transplanted
subcutaneously on the back or under the renal capsule of immunodeficient mice (9, 10).
This model is more complex than the RASF model, since it uses whole RA synovial tissue
with a mixture of cell types including various immune cells, preferably freshly isolated
during joint surgery. Although this model lacks the possibility of studying destruction of
cartilage and/or bone, it is a great model with which to investigate multiple cell types and
their interactions within the inflamed synovium, to study the migration of lymphocytes
into this tissue (9–11), and to test new therapeutic strategies that target various
immunologic aspects of the synovial inflammation. Methotrexate (MTX) treatment in the
RA synovium SCID mouse model decreased the inflammatory cells in the graft by inducing
apoptosis (12), and previous anti-TNF treatment in the RA synovium SCID mouse model
demonstrated a reduced amount of synovial inflammatory cells in these chimeric mice
(13). Elegant studies by Klimiuk et al and Takemura et al in which human T cells and B cells
were depleted from the RA synovial grafts showed a crucial role for B cells in T cell
activation, whereas T cells drive synovial cytokine and matrix metalloproteinase expression
(14, 15). Adoptive T cell transfer into the RA synovium SCID mouse model augmented this
cytokine production (14).
In our study, we systematically investigated the research value of the RA synovium
SCID mouse model using a selection of the most widely prescribed biologic agents that
are used in the treatment of RA. First, the validity of this humanized mouse model was
studied using anti-TNF and anti–IL-1 treatment. In addition, the direct effect of T cell– and
B cell–related therapies on the transplanted RA synovial tissue was investigated with
CTLA-4Ig, anti-CD20, and anti–IL-17 antibodies, with specific focus on the responsiveness
to anti–IL-17 treatment. In our animal models, we have seen great efficacy of anti–IL-17
treatment, particularly during antigen-specific reactivation of the arthritis and in Th17
cell–driven arthritis models (16, 17). Since the presence of T cells seems to be a requirement
for successful treatment with IL-17–neutralizing antibodies, we investigated the
preselection of synovial tissue to provide tailored treatment for potential anti–IL-17–
responsive patients.
Materials and methods
Mice. Female CB17/SCID mice were obtained from Charles River. Because of their immune
deficiency, all mice were housed in individually ventilated cages, and a standard diet and
water were provided ad libitum. The mice were used between ages 6 and 18 weeks. All
animal procedures were approved by the institutional ethics committee.
T cell targeting in the RA synovium SCID mouse model | 137
Patients and tissues. Synovial tissue was obtained from 26 RA patients who underwent total
joint replacement surgery or synovectomy of the hip, knee, ankle, shoulder, elbow, or wrist
joint. The patients were 80.8% female, with a mean age of 64.7 years (range 39–84 years) and
a mean disease duration of 18.6 years (range 1–36 years). The RA patients took a variety of
antirheumatic drugs: 38.5% were treated with anti-TNF biologic agents (adalimumab,
infliximab, etanercept) with or without additional MTX or prednisone, 19.2% were receiving
MTX, and the remaining 42.3% took only prednisone, disease-modifying antirheumatic drugs,
and/or nonsteroidal antiinflammatory drugs. All patients fulfilled the American College of
Rheumatology (ACR) 1987 revised criteria for the classification of RA (18). Procedures were
performed after informed consent approved by the hospital ethics committee.
Study protocol. Synovial tissue was collected in culture medium with antibiotics and
freshly processed into standardized biopsy samples with a diameter of 6 mm using
disposable skin biopsy punches (Staffel). Part of the tissue was directly embedded in OCT
compound (Tissue-Tek) and snap-frozen in liquid nitrogen, and cryosections were stained
with hematoxylin and eosin (H&E) to study the quality and cellularity of the synovial tissue.
The rest of the biopsy samples were transplanted to the CB17/SCID mice. CB17/SCID mice,
maintained under pathogen-free conditions, were anesthetized by isoflurane inhalation.
After swabbing the skin in the neck region with 70% ethanol, a small incision was made in
the dorsal skin behind the ear of each SCID mouse, the tissue was inserted subcutaneously,
and the wound was closed with suture clips.
Treatment protocol. After an engraftment period of 1 week, serum was collected by
orbital puncture to screen for the expression of human cytokines and chemokines by
Luminex analysis. Mice were randomly assigned to the various treatment groups and
treated on days 7 and 10 with intraperitoneal injections of antibodies at a fixed dose of 10
mg/kg. On day 14 after transplantation, the mice were killed by exsanguination, and the
grafts were isolated for histologic analysis. For antibody treatment, anti-TNF (adalimumab;
Abbott), anti–IL-1 (ACZ885; Novartis), CTLA-4Ig (abatacept; Bristol-Myers Squibb), anti-CD20
(rituximab; Genentech), anti–IL-17 (AIN457; Novartis), or isotype control human IgG1
(Sigma) antibodies were used. All antibodies were specific for their human targets and did
not cross-react with the murine homologs.
Luminex analysis. To determine levels of the cytokines and chemokines in serum
samples, Luminex multianalyte technology in combination with multiplex cytokine kits
(Milliplex; Millipore) was used. Cytokines were measured in 25 μl of serum diluted 1:3 in
assay buffer. The sensitivity of the multiplex kit was <1 pg/ml. Samples that showed
cytokine levels below the detection limit were set at a value of 0.01 pg/ml to include them
in further statistical analysis.
138 | Chapter 7
Histologic analysis. Synovial grafts were isolated on day 14 after transplantation. For
standard histology, tissue was embedded in OCT compound, and 6 μm cryosections were
subsequently prepared. H&E staining was performed to study synovial inflammation. The
severity of inflammation of the graft was scored on an arbitrary scale of 0–2 (0 = no cells,
0.25–0.75 = mild cellularity, 1.0–1.5 = moderate cellularity, and 1.75–2.0 = maximal
cellularity).
Immunohistochemistry. Cryosections were fixed in 4% paraformaldehyde and incubated
with 1% H2O2 in methanol. Next, sections were stained with antibodies against human
IL-1β (GF12; Calbiochem), TNFα (AB1793; Abcam), CD3 (MCA1477; Serotec), CD80 (AF140;
R&D Systems), CD86 (PN IM2728; Beckman Coulter), and CD20 (CMC302; Cell Marque).
Subsequently, the sections were incubated with biotinylated secondary antibodies (P0260
and P0162; Dako) (BA-5000; Vector) followed by labeling with streptavidin–horseradish
peroxidase (P0397; Dako). Peroxidase was developed with diaminobenzidine as substrate.
Sections were counterstained with hematoxylin for 1 minute.
Statistical analysis. Differences between experimental groups were tested using the
Mann-Whitney U test or Kruskal-Wallis test, unless stated otherwise. Results are expressed
as the mean ± SEM, unless stated otherwise. P values less than 0.05 were considered
significant.
Results
Successful validation of the RA synovium SCID mouse model using
anti-TNF treatment
One week after transplantation of synovial tissue into SCID mice, serum was collected and
Luminex analysis was performed to screen for human and murine cytokine and chemokine
expression in the circulation. Although murine mediators were hardly detected (data not
shown), human IL-6 and to a lesser extent human IL-8 and human TNF were clearly present
in the serum of the engrafted mice (Figure 1A). Simultaneously with the serum samples,
the synovial grafts were collected and scored for histologic cellularity as a measure of
inflammation. This analysis demonstrated that serum human IL-6 levels correlated
significantly with the histologic inflammation of the synovial grafts (Figure 1B). To validate
our model, we used anti-TNF treatment as the most successful biologic agent in the
treatment of RA. After an engraftment period of 1 week, mice were injected on days 7 and
10 with adalimumab in an excess amount of 10 mg/kg. On day 14 after transplantation,
the mice were killed, and serum and histologic analysis showed a significant reduction of
both serum human IL-6 levels and the cellularity of the grafts in mice treated with anti-TNF
antibodies compared to the control group (Figures 2A and B). In addition, immunohisto-
T cell targeting in the RA synovium SCID mouse model | 139
chemical staining for human TNFα and IL-1β demonstrated a clear reduction in the
synovial expression of these 2 cytokines after anti-TNF treatment (Figures 2C and D).
A
B
Figure 1 | A and B, In the rheumatoid arthritis synovium SCID mouse model 1 week after
transplantation, human interleukin-6 (hIL-6) is highly expressed in serum (A), and the serum levels
of human IL-6 correlate significantly with histologic inflammation of the graft as scored on a scale of
0–2 (B). Horizontal lines in A represent the mean. hTNFα = human tumor necrosis factor α.
140 | Chapter 7
Human RA synovial tissue does not respond to anti–IL-1 treatment
In our classic experimental arthritis models in mice, IL-1 seemed a promising candidate for
successful therapeutic targeting in RA. However, IL-1– blocking therapy in clinical trials
turned out to be not as effective in RA as expected (5, 19). In our humanized mouse model,
we tested the effect of anti–IL-1 antibodies in comparison with anti-TNF treatment. In
Figure 1A, we had already demonstrated that serum levels of IL-1 in the engrafted SCID
mice were much lower than the levels of TNF. Consistent with this finding, the RA synovium
SCID mice treated with anti–IL-1 antibodies did not show a significant reduction in serum
A
B
C
D
Figure 2 | A and B, Anti-TNF treatment significantly suppresses serum human IL-6 levels (horizontal
lines represent the mean; *** = P < 0.001, by Mann-Whitney U test) (A) and histologic scores for
cellularity of the synovial grafts in the chimeric mice (B), determined by Luminex analysis 14 days
after transplantation (A; 75–77 mice/group) and on hematoxylin and eosin–stained cryosections
(B; 36–39 grafts/group). C and D, Anti-TNF treatment also significantly reduces synovial staining with
IL-1β (C) and TNFα (D) by immunohistochemistry. Values in B–D are the mean ± SEM. * = P < 0.05
versus isotype control treatment, by Mann-Whitney U test. See Figure 1 for definitions.
T cell targeting in the RA synovium SCID mouse model | 141
cytokines and histologic cellularity, whereas the mice engrafted with synovium from the
same RA donors did respond significantly to anti-TNF treatment (Figures 3A and B). This
result suggests that our humanized mouse model of arthritis reflects the modest
responsiveness of RA patients to IL-1 blocking therapy, while neutralizing TNF serves as a
positive reference control.
B cell depletion using anti-CD20 antibodies is effective in the RA
synovium SCID mouse model
Selective depletion of CD20+ B cells has shown good results in the treatment of RA. In our
model, we investigated whether the transplanted synovial tissue of RA patients can be
responsive to such a B cell therapy by treating the mice with anti-CD20 antibodies. Since
not all arthritic tissue obtained during surgery contains B cells, the synovial biopsy samples
were first screened for CD20+ B cells by immunohistochemistry before the start of
treatment (Figure 4A). As shown in Figure 4B, targeting CD20+ B cells in the RA synovium
SCID mouse model significantly reduced the serum levels of human IL-6 as a systemic
marker of activation of the synovial graft, to the same extent as our positive control
treatment with anti-TNF antibodies. This indicates that the B cell–positive arthritic
synovium, despite being isolated from the joint and its supporting immune system,
remains directly sensitive to the therapeutic efficacy of anti-CD20 treatment.
A
B
Figure 3 | A and B, Anti–IL-1 treatment in the human rheumatoid arthritis synovium SCID mouse
model does not suppress serum human IL-6 levels (A) or histologic scores for cellularity of the synovial
grafts in the chimeric mice (B), determined by Luminex analysis 14 days after transplantation (A; 12
mice/group) and on hematoxylin and eosin–stained cryosections (B; 18–20 grafts/group). Horizontal
lines in A represent the mean. *** = P < 0.001 versus isotype control treatment, by Kruskal-Wallis and
Dunn’s multiple comparison tests. Values in B are the mean ± SEM. See Figure 1 for definitions.
142 | Chapter 7
A
B
Figure 4 | A, Human rheumatoid arthritis (RA) synovium was stained for CD20+ B cells before
treatment of RA synovium SCID mice with depleting anti-CD20 antibodies. Original magnification ×
100. B, Anti-CD20 treatment in the human RA synovium SCID mouse model significantly suppressed
human IL-6 levels in the serum of the engrafted mice, determined by Luminex analysis 14 days
after transplantation (7–10 mice/group). Horizontal lines represent the mean. *** = P < 0.001 versus
isotype control treatment, by Kruskal-Wallis and Dunn’s multiple comparison test. See Figure 1 for
other definitions.
CTLA-4Ig treatment lacks direct therapeutic effects on synovial grafts
in the RA synovium SCID mouse model
In contrast to the positive effect of anti-CD20 antibodies in the RA synovium SCID mouse
model, treatment with CTLA-4Ig did not show any effect. Since the therapeutic effect of
CTLA-4Ig is based on binding to the CD80 and CD86 proteins on antigen-presenting cells
(APCs), thereby preventing costimulation and activation of T cells, the synovial tissue was
first screened for the presence of CD3+ T cells and CD80+ and CD86+ APCs (Figures 5A
and B). However, despite this selection the synovial grafts did not respond to CTLA-4Ig
treatment either with changes in human IL-6 expression in serum or with changes in
histologic inflammation (Figures 5C and D), suggesting that CTLA-4Ig might execute most
of its effect not locally in the synovial tissue, but mainly systemically in lymphoid structures
like spleen and lymph nodes to reach clinical efficacy in RA patients.
Responsiveness to anti–IL-17 treatment is determined by CD3+ T cells
in synovium
Although CTLA-4Ig, inhibiting the T cell costimulation process, was not effective in our RA
synovium SCID mouse model, this does not necessarily mean that the RA synovium SCID
mouse model is not sensitive to any T cell–based therapy. Therefore, we investigated the
effect of blocking the T cell effector cytokine IL-17 in the RA synovium SCID mice. Before
the start of treatment, human RA synovial tissue was stained for the presence of CD3+ T
cells, the major source of IL-17 production. As shown in Figures 6A and B, two groups could
T cell targeting in the RA synovium SCID mouse model | 143
be clearly distinguished: synovium with low numbers of CD3+ T cells diffusely spread or
clustered around small blood vessels (CD3low), and tissue with high amounts of CD3+ T
cells, mainly aggregated in large lymphocyte clusters (CD3high). Initially it seemed that
despite the presence of CD3+ T cells in the synovial tissue, the engrafted SCID mice did
not show a response to anti–IL-17, as demonstrated by levels of human IL-6 in serum
comparable to those in control-treated mice (Figure 6C). Interestingly, after stratification
for CD3low and CD3high synovial tissue, only the mice transplanted with the synovium rich
in CD3+ T cells showed a significant response to anti–IL-17 treatment (Figure 6C),
A
C
B
D
Figure 5 | A and B, Human rheumatoid arthritis (RA) synovium was stained for CD80+ (A) and
CD86+ (B) antigen-presenting cells before treatment of RA synovium SCID mice with CTLA-4Ig.
Original magnification × 100. C and D, CTLA-4Ig treatment in the human RA synovium SCID mouse
model did not suppress human IL-6 serum levels (C) or histologic scores for cellularity of the synovial
grafts in the chimeric mice (D), determined by Luminex analysis 14 days after transplantation (C; 13–
14 mice/group) and on hematoxylin and eosin–stained cryosections (D; 13 grafts/group). Horizontal
lines in C represent the mean. *** = P < 0.001 versus isotype control treatment, by Kruskal-Wallis and
Dunn’s multiple comparison tests. Values in D are the mean ± SEM. See Figure 1 for other definitions.
144 | Chapter 7
A
C
B
Figure 6 | A and B, Human rheumatoid arthritis (RA) synovium was stained for CD3+ T cells to
discriminate between RA tissues with low amounts of CD3+ T cells (A) and high amounts of CD3+ T
cells (B). Original magnification × 100. C, Anti–interleukin-17 (anti–IL-17) treatment in the human RA
synovium SCID mouse model significantly suppressed human IL-6 (hIL-6) levels in the serum of the
mice engrafted with CD3-rich synovial tissue, but not with synovial tissue with low amounts of CD3,
determined by Luminex analysis 14 days after transplantation (9–11 mice/group). Horizontal lines
represent the mean. * = P < 0.05 versus isotype control treatment, by Mann-Whitney U test.
suggesting that the relative presence of CD3+ T cells in the arthritic joint is a good marker
for responsiveness to this type of treatment.
The first clinical trials with anti–IL-17 treatment in RA patients showed promising
numbers of patients meeting the ACR 20% improvement criteria (20, 21). However, in our
opinion, preselection of the patients before treatment, based on the presence of CD3+ T
cells in the synovial tissue, might further improve clinical responses to blocking IL-17.
Discussion
This is the first study in the human RA synovium SCID mouse model that has systematically
validated and screened the predictive value of such a humanized arthritis model by
testing the therapeutic effectiveness of widely accepted biologic treatments for RA. In
addition, based on the findings of this study, we would like to propose a selection method
based on CD3+ T cell screening to enhance the therapeutic responsiveness of RA patients
to anti–IL-17 treatment.
The RA synovium SCID mouse model seems to be a very suitable tool for screening
the therapeutic efficacy of new biologic agents and promising chemical compounds on
T cell targeting in the RA synovium SCID mouse model | 145
inflamed RA synovial tissue. In several respects, the RA synovium SCID mouse model is
superior to in vitro synovium explant studies. Although the in vivo model requires far
more synovial tissue than is needed for in vitro studies, the engraftment of SCID mice
results in a dynamic model with good vascularization of the tissue within 1 week after
engraftment. In vitro synovial explant cultures, mainly used for stimulation assays, can also
be used to study the antiinflammatory effect of various inhibitors on the spontaneous
secretion of cytokines and chemokines, but readout is often limited to 24–48 hours of
culture (22, 23). The good vascularity of the synovium in the RA synovium SCID mouse
model enables prolonged cell survival up to weeks after engraftment (10, 11), and also
improves targeting of the tissue with potential new therapeutics.
Initially we performed several RA synovium SCID mouse model experiments to
investigate the therapeutic window of this arthritis model using anti-TNF as a widely
accepted biologic agent. For this purpose, adalimumab was selected because of its fully
humanized structure, which does not cross-react with murine TNFα. Anti-TNF treatment
in this model significantly and consistently reduced serum human IL-6 levels, and
suppressed inflammation scores of the synovial grafts being analyzed for cellularity as well
as proinflammatory cytokine staining by immunohistochemistry. In the set-up phase of
the model, various serologic markers were determined in the engrafted SCID mice using
Luminex and enzyme-linked immunosorbent assay technique. Human IL-6 was most
abundantly present in the serum of the RA synovium SCID mice, but human IL-8 levels,
although 10-fold lower, showed a pattern similar to that of human IL-6, correlating with
the inflammatory status of the grafts and responding to anti-TNF treatment. Consistent
with earlier findings (11), human IgG and, to a lesser extent, human IgM were detected in
the serum of our SCID mice engrafted with RA synovial tissue. Because of the various IgG1
antibody treatments in this study, human IgG were not used as a readout in our SCID
mouse experiments, but human IgG levels might be a very useful additional readout
when testing new chemical entities in this model.
After successful validation of the model with anti-TNF antibodies, the effect of IL-1
blocking was investigated in this RA synovium SCID mouse model. Although a trend
toward therapeutic efficacy was observed, no significant suppressive effect of anti–IL-1
treatment was found on both serum IL-6 levels and histologic scores. This result is in line
with the clinical finding that blocking IL-1 in RA patients resulted in quite modest responses
(5). Two important remarks must be made concerning the potential underestimation of
the effect of anti–IL-1 treatment. First, the followup of the RA synovium SCID mice injected
with anti–IL-1 antibodies was restricted to 1 week of treatment, while the first clinical
effects of IL-1 blockers in RA patients were only reported after 24 weeks of treatment (24).
In addition, during the validation of the RA synovium SCID mouse model with anti-TNF
and anti–IL-1 treatment, we deliberately chose not to preselect the RA synovial tissue for
the presence of certain target cells or proteins. However, since IL-1 plays a crucial role in T
cell activation and Th17 cell differentiation, screening for and selection of CD3-rich synovial
146 | Chapter 7
tissue might increase the responsiveness to anti–IL-1 treatment. Further RA synovium SCID
mouse studies are needed to investigate the potential of such selection criteria, which
might result in an interesting change of practice for the treatment of RA patients with IL-1
blockers.
While B cell targeting in the RA synovium SCID mouse model using anti-CD20
antibodies resulted in clear therapeutic effects, the lack of any response to CTLA-4Ig
treatment was quite surprising. After engraftment of SCID mice with RA synovial tissue,
low levels of human IL-17 and interferon-γ could be detected in the serum of some of the
mice (data not shown), indicating the presence of activated Th17 and Th1 cells in the
synovial grafts. Despite optimal selection of potentially responsive synovial tissues after
immunohistochemistry for various markers involved in APC–T cell interaction, CTLA-4Ig
did not reduce serum cytokine levels or histologic scores. Based on these results, we could
speculate that abatacept treatment does not act directly on the synovium, but more likely
prevents T cell activation at a systemic level of the immune system. More complicated
humanized arthritis models integrating RA synovial tissue and human peripheral blood
mononuclear cells need to be developed to test this hypothesis.
Although CTLA-4Ig treatment in our RA synovium SCID mouse model failed,
neutralizing IL-17 as another target involving T cells did show significant therapeutic
effects. Interestingly, the relative presence of CD3+ T cells clearly determined the
responsiveness of the engrafted mice to the anti–IL-17 antibodies. Stratification of the RA
synovial tissue by expression of high and low amounts of CD3+ T cells resulted both in
good responders and in mice that did not respond to anti–IL-17 treatment, respectively.
These findings suggest that a selection method based on CD3+ T cell screening might be
very useful in daily practice to provide tailored therapy to RA patients, and to enhance the
therapeutic responsiveness of these patients to anti–IL-17 treatment. Despite successful
validation of the RA synovium SCID mouse model with various biologic treatments, in this
study we could not yet link the responsiveness of the synovial grafts to the clinical
responses of the patients, except for anti-TNF treatment, because these patients were not
treated with biologic agents other than TNF blockers. Future studies in the RA synovium
SCID mouse model might provide such a correlation of efficacy for various specific
biotherapies.
In conclusion, our studies show that the RA synovium SCID mouse model mirrors
most clinical observations with biologic treatments in RA patients. Consequently, the
predictive value of this model for evaluating novel treatments is expected to be high.
Moreover, the model allows for studies of RA heterogeneity, which can be exploited to
optimize protocols for targeted treatment in preselected patient groups.
T cell targeting in the RA synovium SCID mouse model | 147
Acknowledgements
We thank the patients, rheumatologists, and orthopedic teams at Radboud University
Nijmegen Medical Centre and St. Maartenskliniek (Nijmegen, The Netherlands) for their
cooperation in obtaining the synovial tissue; in particular, we would like to thank Saskia
Susan, Petra Heesterbeek, and Lenny Geurts-van Bon. In addition, we thank the personnel
of the animal facility for taking good care of our mice and the research technicians Birgitte
Walgreen, Monique Helsen, and Liduine van den Bersselaar for their excellent technical
assistance throughout the RA synovium SCID mouse studies.
148 | Chapter 7
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Chapter 8
The functional role of the Th17 cytokines
in Rheumatoid Arthritis
R.J. Marijnissen, M.I. Koenders, L.A.B. Joosten, W.B. van den Berg
152 | Chapter 8
Abstract
The utility of cytokines as therapeutic targets in Rheumatoid Arthritis (RA) has been
demonstrated with the introduction of tumor necrosis factor (TNF) blockade in clinical
practice. And although very effective in combination with methotrexate, anti-TNF-α
agents are certainly not sufficient in all RA patients, so there is a need for broader coverage.
In this current update we focus upon the recent developments regarding Th17 cytokines
as therapeutic targets in RA. Since the discovery in 2003, IL-17 producing T-cells have been
shown to play an important role during chronic (auto-) immune disorders like RA. Until
now anti-IL-17 therapy has only shown mild efficacy compared to the anti-TNF agents.
Continuing efforts are ongoing to target other Th17 cytokines like IL-17, IL-21, IL-22 and
GM-CSF with promising data emerging. This article reviews the current understanding
about the role of IL-17, IL-21, IL-22 and GM-CSF in relation to Th17 cells and their contribution
in the pathogenesis of Rheumatoid Arthritis and experimental arthritis models.
The functional role of the Th17 cytokines in Rheumatoid Arthritis | 153
Characteristics of Th17 biology
IL-17 producing CD4+ T cells (Th17) are characterized by the expression of CCR6 and the
IL-23R and production of IL-17A (IL-17), IL-17F, IL-21, CCL20, IL-22, IL-26 (only humans) and
more recently GM-CSF. In 2003, the discovery of the IL-23/IL-17 axis led to the recognition
of Th17 cells, a third effector CD4+ T cell lineage, producing mainly IL-17 and being distinct
from classic Th1 and Th2 lineages (1;2). This discovery led to a paradigm shift, showing that
autoimmune diseases are predominantly driven by autoreactive Th17 cells and not Th1
cells (3). The biology of IL-17 induction by these Th17 cells differs slightly between mice
and humans. In mice, exposure to IL-6 in combination with TGF-beta to activated naïve
CD4+ T-cells is sufficient to induce IL-17 production (4), by which TGF-beta blocks the Th1
and Th2 pathways. (5). On the contrary, in humans TGF-beta seems to play a modest role
(6;7). TGF-beta does seem to block the human Th1 pathway (8;9), thereby favoring Th17,
but IL-1β and IL-23 are the driving factor with IL-6 or IL-21. In vivo, IL-23 is necessary to
maintain the IL-17 production (10). Although IL-23 itself cannot drive de novo generation
of Th17 (in mice), exposure to IL-23 during the differentiation induces an increased
pathogenic cytokine profile; a decreased IL-10 production versus an increased IL-22
expression (11). Th17 cells turned out to produce IL-21, that creates an amplification loop
that further expands these cells (12;13). Finally, IL-1 has been shown to drive Th17 cell differentiation in vitro and in vivo (14;15).
Generally, still not much is known about the temporal expression of the Th17
cytokines and their exact role in the pathogenesis of Rheumatoid Arthritis. This variable
cytokine expression during the course of the disease has major consequences for the
success of pioneering new therapies targeting Th17 cells and their cytokines. Here we give
an update on the Th17 cytokines IL-17, IL-21, IL-22 and GM-CSF and their role in Rheumatoid
Arthritis.
IL-17A
Interleukin-17A (IL-17) was first described in 1993 as CTLA-8, produced by human T cells
(16). It is part of the IL-17 family, consisting of 6 members (A-F), and signals via the IL-17RA/
RC complex. IL-17F shares 50% homology with IL-17A (IL-17), highest of all IL-17 family
members and has similar effects as IL-17A (17). The signaling cascade mediated by IL-17 is
still not fully known, but it activates PI3-kinase/Akt and the classical NFκB pathway(18).
Numerous studies have shown a role for IL-17 in the immunity against extracellular
pathogens, especially the host defense against fungi (19).
Despite the beneficial role of IL-17 in host defense, it is also associated with
autoimmune diseases such as Crohn’s disease, multiple sclerosis, systemic sclerosis,
systemic lupus erythematosus, and RA.
154 | Chapter 8
IL-17 is spontaneously produced by RA synovial membrane cultures (17;20) and elevated
IL-17 levels and IL-17 producing T cells have been detected in the synovial fluid correlating
with joint destruction (21-24). In experimental arthritis models, depletion or neutralization
of IL-17 (signaling) strongly reduces disease, with major effects on joint damage (25;26).
The IL-17 receptor is widely expressed in all tissues studied to date. On stromal cells,
IL-17 induces IL-6, G-CSF and CXCL-8/IL-8 (CXCL-1/KC in mice) that induces infiltration of
neutrophils (27;28). In addition, it attracts monocytes partly via the induction of CCL2
(MIP-1α) (29;30). Furthermore, direct activation on chondrocytes induces matrix metalloproteinases (MMPs) that degrade cartilage matrix, in addition, it inhibits matrix deposition
(31;32). Finally, IL-17 directly potentiates osteoclastogenesis (33). Taken into account that
Th17 cells themselves also express RANKL, makes them a good candidate for inducing
osteoclatogenenesis directly (34). This shows that IL-17 plays a major role in inducing
cartilage and bone destruction.
Although there is evidence of other sources of IL-17 besides Th17 cells, including γδ T
cells (gamma delta T cells), CD8+ T-cells (Tc17), neutrophils and mast cells, CD4+ T cells are
considered to be the main producers. In patients with established arthritis, synovial IL-17
has been shown to be associated with mast cells (35). It might have been taken up by
these cells since IL-17 mRNA could not be found in mast cells, not excluding a subsequent
release and activity of mast-cell derived IL-17. In our hands, in the SCID transfer model,
using biopsies from RA patients with an established arthritis, anti-IL-17 therapy is only
effective when high numbers of CD3+ cells are present (36), demonstrating the significance
between IL-17 and T cells during chronic joint disease in humans.
The first result of anti-IL-17 as a monotherapy besides methotrexate showed variable
responses (37). The potency of several cytokines can be markedly increased through
synergism with IL-17, of which TNF-α is probably the most important. TNF-α is a pro-inflammatory cytokine that plays a dominant role in inflammatory cascades, and is known
to be produced in considerable concentrations in the joints of RA patients (38). To date,
anti-TNF-α (39) induces low disease activity or remission in most patients, of note still
thirty percent of the patients do not respond to anti-TNF-α treatment (40). After single
blocking of TNF-α in CIA, draining lymph nodes contain an increase in cells producing
both IL-17 and IFN-y (41). Peripheral increase in IL-17/Th17 after TNF-α blockade is also
observed in patients (42;43), suggesting the therapeutic potential of additional anti-IL-17
treatment. In vitro, IL-17 in combination with TNF-α induces a tremendous synergistic
upregulation of pro-inflammatory cytokines and chemokines in RA synovial fibroblast-like
cells (unpublished observation and (44)), synovial tissue and osteoclasts (45;46). The
combined over expression of IL-17 and TNF results in considerably increased synovial
mRNA levels of the alarmin S100A8, the cytokine IL-1β and several matrix metalloproteinase’s (MMP’s). This effect is partly explained by the observation that IL-17 can stabilize TNF
induced mRNA expression, thereby enhancing cytokine and chemokine expression
(47;48). Our group has shown that in CIA, combination therapy of anti-TNF-α and anti-IL-17
The functional role of the Th17 cytokines in Rheumatoid Arthritis | 155
is very potent in reducing inflammation and joint destruction, even during late stages of
disease (39), when TNF-α dependency declines (49).
Overall, IL-17 is an important cytokine in the induction of cartilage destruction and
bone erosions during the course of disease. Most importantly, since IL-17 cooperates and/
or synergizes with TNF-α during arthritis, exploring combination therapy will be crucial.
IL-21
IL-21 was first described as a product of activated CD4+ T cells in 2000 (50). IL-21 is a
pleiotropic cytokine belonging to the IL-2 cytokine family, closely related to IL-2, IL-4 and
IL-15 and acts on a variety of hematopoietic immune cells, including T, B, DC and NK cells
as well as on tissue cells including endothelial cells and fibroblasts (51). IL-21 signals via the
IL-21 receptor complex, consisting of the IL-21R with the common γc receptor (52;53). IL-21
primarily activates JAK1 and JAK3 resulting in STAT3 and STAT1 activation, with weaker
activation of STAT5 phosphorylation (54;55).
Although discovered by production of activated CD4+ T cells, γδ T cells and NK(T)
cells can also produce high levels of IL-21 (56-59). Human memory CD4+ T cells produce
high levels of IL-21 after activation, with most cells being double positive for IFN-γ and IL-17
and few also producing Th2 cytokines. Furthermore, it has become clear that T follicular
helper cells are high producers of IL-21. This IL-21 contributes to the formation of lymph
nodes and is crucial for the development of plasma cells (57;60). Synovial fibroblasts show
increased expression of various matrix metalloproteinases (MMPs) as well as RANKL upon
IL-21 stimulation (61). Depending on the cellular environment and cellular activation, IL-21
induces the differentiation, proliferation and cytokine induction of NK, NKT, Th17 and
CD8+ T cells, and supports the differentiation of cells of the myeloid lineage (62;63).
In 2007, the research on IL-21 was boosted when IL-21 was shown to be an autocrine
factor in Th17 differentiation, amplifying the Th17 differentiation and expansion (12;13;64).
IL-21 is induced after stimulation with IL-6, positioned between STAT3 phosphorylation
and RORγT upregulation, since STAT3-/-mice do not induce IL-21, while RORγT-/- mice do
(64). In mice, direct stimulation with IL-21 results in the induction of IL-17, but less potent as
compared to IL-6 and no synergy observed when combining the two cytokines (64). In
mice, IL-6 signaling is sufficient to drive Th17 and autoimmunity in the absence of IL-21
(65;66). On the contrary, in humans IL-21 seems to play a more prominent role in driving
Th17 responses in combination with TGF-β (67).
Recent genome-wide association studies have provided convincing evidence for the
chromosomal 4q27 region, harboring the IL-2 and IL-21 genes, as a risk factor for chronic
inflammatory disorders, including SLE, psoriasis and RA (68;69). In RA synovial tissue IL-21
mRNA has been detected in tissues and cells (70). In addition, increased IL-21 levels were
found in RA sera and synovial fluid compared to Osteoarthritis (OA) and healthy volunteers
156 | Chapter 8
(61;71). Furthermore, these levels correlate with IL-17 levels and numbers of CD4+ T-cells. In
animals models, neutralizing IL-21 in the collagen induced arthritis and antigen induced
arthritis results in an decreased inflammation and destruction ((72) and unpublished own
observation). Most interesting, neutralization of IL-21 lowers IL-6 serum levels, which might
provide an indirect effect on lowering Th17 induction. Overall, these observations suggest
a direct role for IL-21 in the pathogenesis of RA.
Most interesting IL-21 affects B-cells and might be directly involved in auto-antibody
production (73). Additionally, in early RA patients only serum IL-6 and IL-21 are associated
with increased IgM-RF, ANCA titers and general B cell activation (74). Since autoantibodies
are increased years before clinical onset, this points towards the involvement of IL-21 early
in the disease process. Besides this direct role on B-cell development, IL-21 has major
effects on dendritic cell function (75). IL-21 increases SOCS proteins in DC’s and
macrophages, resulting in lowered TLR responses in vitro and in vivo ((76) and unpublished
own observation). Most interesting, in a model for atopic dermatitis, the defective response
of IL-21R signaling results in an impaired migration of skin dendritic cells towards draining
lymph nodes after antigen capture (77;78). A process that seems to be partly dependent
on the regulation of CCR7 by IL-21. These results show, that IL-21 not only induces
proliferation and activation of lymphocytes, but also regulates a negative feedback loop
between DC’s and T-cells.
Taken together, besides the early activation of Th17 cells, IL-21 shows to play a crucial
role in the initiation of the adaptive immune response. Therefore, targeting IL-21 for the
treatment of RA patients might be most beneficial during the early course of disease.
IL-22
IL-22 (IL-TIF) was first described in 2000 and belongs to the IL-10 family of cytokines, closely
related to IL-10, IL-19, IL-20, IL-24 and IL-26 (79). IL-22 is produced by various types of
hematopoietic cells, including T helper cells, gamma delta T cells, NK and NKT cells.
However, soon after the characterization of Th17 cells it became clear that this T cell
subtype is the dominant IL-22 producer (80). IL-22 signals through a complex of the IL-22R1
and IL-10R2. The IL-22R1 subunit is shared with IL-20 and IL-24, the IL-20R2 is shared with
IL-10, IL-28 and IL-29 (81;82). Interestingly, the IL-22R is only present on resident tissue cells
and not on hematopoietic immune cells (83). Expression has been shown on hepatocytes,
keratinocytes and epithelial cells. IL-22 signaling results in the activation of JAK1 and TyK2
tyrosine kinases, primarily leading to the phosphorylation of STAT-3, but can also activate
STAT-1, STAT-5, and ERK, JNK and p38 MAP kinases (83;84). A soluble receptor, IL-22 binding
protein (IL-22Bp) has been described as a natural inhibitor of IL-22 signaling (85).
Classically, Th17 cells produce both IL-17 and IL-22. During the differentiation of Th17
cells, IL-6 together with TGF-β induce optimal IL-17 expression, but not IL-22 expression,
The functional role of the Th17 cytokines in Rheumatoid Arthritis | 157
mainly because TGF- β inhibits IL-22 expression (9). IL-23 is essential for inducing maximal
IL-22 production in vitro and in vivo during experimental arthritis (86;87). In our hands, IL-1
and IL-23 synergize for an optimal IL-22 production during Th17 differentiation (88).
Furthermore, activation of the aryl hydrocarbon receptor (AhR), a receptor which ligands
include environmental toxins, during Th17 differentiation not only increases the amount
of cells, but more specifically induces IL-22 production (89). There is evidence that mature
Th17 retain their IL-17 producing capacity, but lose their IL-22 producing capacity (90). On
the contrary, Th22 cells have been described, CD4+ T helper cells producing IL-22 without
IL-17 (91;92). Possibly, this temporal expression of IL-17 and IL-22 reflects different stages of
the differentiation/ maturation or destabilization of Th17 cells.
IL-22 plays a dual role in animal autoimmune models of psoriasis, IBD, RA, and MS
(EAE), which is not yet fully understood (86;93-96). While blocking of IL-22 may be beneficial
in skin disease models (93), blocking IL-22 in IBD models aggravates disease (94;96). In
addition, IL-22-/- mice are fully susceptible to EAE (86). The presence of IL-22-producing T
cells in the inflamed tissue during chronic inflammation implies that the expression of
IL-22R on the tissue-resident cells determines the function and response to IL-22. Until
now, most studies have focused on the pro-inflammatory effect of IL-22. On hepatocytes
and keratinocytes IL-22 induces a wide range of antimicrobial proteins, such as betadefensins, S100 proteins, serum amyloid A proteins, Reg family members, and matrix metalloproteinases (83;84;97). In contrast to the strong pro-inflammatory effects, IL-22 has a
protective and regenerative effect on epithelial cells. IL-22 enhances the migration and
proliferation of epithelial cells in skin and lung tissue, but the exact mechanisms still need
to be elucidated (98-100).
Understanding the role of IL-22 in Rheumatoid Arthritis is still limited. The expression
of IL-22 and the IL-22R within the synovium has been shown (101). The expression of the
IL-22R was mainly attributed to fibroblasts that proliferated and produced MCP-1 upon
IL-22 stimulation (101). Recent unpublished data from our department confirms the
expression of the IL-22R specifically on fibroblast-like cells and endothelial cells isolated
from human inflamed synovium (data not shown). In addition, Geboes et al, showed that
IL-22 stimulation of spleen cells from immunized CIA mice contributes to osteoclastogenesis via a RANKL dependent mechanism (95). Since IL-22R expression on leukocytes has
not been described, this effect of IL-22 might have occurred via the stromal cells found in
raw spleen cultures. Indeed, it has been shown that IL-22 induces RANKL by fibroblast-like
cells, promoting osteoclastogenesis in monocytes co-cultures (102). Most important,
development of collagen-induced arthritis (CIA) in IL-22-/- C57BL/6 mice showed reduced
disease incidence and less pannus formation (95). In addition, our group has shown that
progression of inflammation in IL-1ra deficient mice is halted after therapeutic blocking
with antibodies against IL-22 (88). Moreover, neutralization of IL-22 specifically showed a
decrease in bone erosions. In RA patients, IL-22 can only be picked up in a subpopulation
of RA patients ((103) and own unpublished observation). Remarkably, patients having high
158 | Chapter 8
IL-22 levels show a more destructive and more progressive phenotype (103). Therefore
IL-22 seems be a good biomarkers to distinguish patients with erosive RA which may
require a more aggressive therapy.
Since Th17 cells can both express IL-17 and IL-22 and combining cytokines can have
synergistic or antagonistic effects, it would be interesting to evaluate the combined
exposure. Until now, additional effects of combining the two cytokines has not been
studied extensively. On keratinocytes exposure to both IL-17 and IL-22 results in a
synergistic increase in S100 and defensins, directing towards an enhanced pro-inflammatory response (80). Furthermore, Sonnenberg et al have shown that the presence or
absence of IL-17A is decisive for the pro-inflammatory or protective properties of IL-22 in a
model of airway inflammation (104). This implies that IL-22 on its own might have a
protective function, but in an inflamed environment has pro-inflammatory actions.
Further work will be needed to elucidate the exact role of IL-22 in the pathogenesis of
Rheumatic diseases like RA.
GM-CSF
Granulocyte macrophage-colony stimulating factor (GM-CSF) is a major driver of the
maturation of macrophages and granulocytes. GM-CSF is most known for its potency to
induce DC development in vitro and in vivo (105;106). It is mainly produced by T-cells and
stromal cells (107), although there are also reports showing GM-CSF production by
monocytes, endothelial cells and chondrocytes, especially in response to IL-1 (108-110).
GM-CSF binds to a heterodimeric complex consisting of 2 distinct subunits, the GM-CSF–
specific α-chain and the common β-receptor which is shared between the GM-CSFR, the
IL-3 receptor, and the IL-5 receptor (111;112). Activation of the GM-CSFR results in JAK/STAT,
MAPK, PI3K, and canonical NF-κB pathway activation (112).
Th17 derived GM-CSF has drawn great attention in the recent literature (113). Two
independent groups have shown that in EAE models Th17 cells are the main producers of
GM-CSF (114;115) Furthermore, this GM-CSF production by Th17 cells was dependent on
IL-23 via the induction of the transcription factor RORyT (114). Interestingly, over expression
of GM-CSF leads to increased IL-23 and IL-6 production; two key-cytokines for the Th17
polarization, revealing a possible loop that can perturbate chronic inflammation (115).
GM-CSF can be detected in the synovial fluid of RA patients, as well as in the arthritic
joints in experimental models (116-120). The importance of GM-CSF in the induction and
perturbation of arthritis has been shown in T-cell dependent and independent
experimental models (121). Direct injection of GM-CSF in the joints of mice leads to an
increased influx of granulocytes and macrophages that aggravate the disease severity
(122). Neutralization of GM-CSF in arthritis models decreases disease severity (107;123).
Depletion of GM-CSF during LPS exposure or deficiency of GM-CSF in CIA lowers TNF-α
The functional role of the Th17 cytokines in Rheumatoid Arthritis | 159
levels. In addition, case reports from patients suffering from arthritis and receiving GM-CSF
after chemotherapy or for treating Felty’s syndrome, showed an exacerbation of the
arthritis (118). Again pointing out the pro-inflammatory character of the cytokine. The fact
that GM-CSF deficiency or neutralization fully blocks the disease in the EAE model has
given extra interest in the current phase 1 and 2 trials with GM-CSF targeting in MS and RA
(124). Of note, these trials will not directly answer the question whether the source of
GM-CSF is indeed T cell derived which needs further clarification.
Recruitment
-Neutrophils, macropages
Chondrocyte
Osteoblast
Cytokines/chemokine, recruitment
-IL-8, IL-1, IL-6, TNF-α, MIP-1α, G-CSF
Fibroblast
Destructive mediators, remodeling
-MMPs, NO, S100A8
-RANKL
IL-17
Macrophage
Differentiation, proliferation, migration
-NK(T), CD4 (Th17), CD8
-DC
CCR6
IL-23R
IL-21
B cell
Dendritic cell
Cytokines/Chemokines
-IL-6, SOCS↓
Destructive mediators
-MMPs
Fibroblast
Th17 cell
Th17
Proliferation
-Endothelial cells, fibroblasts
IL-22
Anti-microbial proteins
Fibroblast
Endothelial cell
GM-CSF
Destructive mediators, remodeling
-RANKL, MCP-1
Differentiation
-DC, Macrophage
Dendritic cell
Macrophage
Cytokines/Chemokines
-IL-6, IL-23, TNF-α
Figure 1 | The functional role of the Th17 cytokines in Rheumatoid Arthritis. Th17 cells are
characterized by the expression of CCR6 and the IL-23R and the production of several cytokines and
chemokines, including IL-17A (IL-17), IL-21, IL-22 and GM-CSF. These cytokines induce proinflammatory
and destructive mediators that contribute to the arthritic process.
160 | Chapter 8
Concluding remarks
The knowledge from experimental arthritis models clearly points towards a central role for
IL-23, IL-17 and Th17 cells in the pathogenesis of rheumatic diseases (1;14;25). Furthermore,
the preceding literature shows that the Th17 cytokines IL-21, IL-22 and GM-CSF are
important in fine-tuning the inflammatory responses in the early and chronic phase of
arthritis (figure 1). However, the exact importance of Th17 cells and the Th17 cytokines in
heterogeneous diseases like RA is still under debate.
Increased IL-17 producing CCR6+ memory T cells have been found in the peripheral
blood of patients with early RA (125). On the contrary, in the synovium of patients with
established RA most likely IFN-γ producing T helper cells are found rather than IL-17
producing T helper cells (126). These observations already point to an altered expression
of IL-17 during the course of disease. Similarly, isolated IL-17 producing T-cell clones can
also express features of the other effector subsets. While in vitro generated Th17 cells by no
means produce IFN-γ, IL-4 or express FoxP3, isolated Th17 cells from the joints of mice
suffering from experimental arthritis can co-express IFN-y (88;127). Comparing the Th17
phenotype during infection, expanded C. albicans and S. aureus specific memory T-cells
isolated from human PBMC’s also produce IL-17 in combination with IFN-γ (128;129). Similar,
in peripheral blood from RA patients, an increase is found in effector Th cells producing
combinations of IL-22, IFN-γ and IL-17 (103). Finally, isolated regulatory T cells form healthy
subject start to produce IL-17 under the influence of IL-1 (130). Whether or not these cells
arise from or are closely related to Th17 cells is a big debate in current literature.
T helper cells recovered from inflamed joints in Juvenile idiopathic arthritis (JIA) that
produced IFN-γ, express chemokine receptors that are intermediate in phenotype
between Th1 and Th17 cells (131). Supporting this, in experimental autoimmune encephalomyelitis (EAE), the IFN-γ producing T helper cells, isolated from the inflamed tissue,
exclusively arose from IL-17 producing T-cells, and still possess certain Th17 specific cell
markers that are not present on ‘classical’ Th1 cells (5). These findings suggest that there is
a close relationship between T helper cell subsets and their differentiation in a later stage.
In summary, increasing number of reports have shown the involvement of CD4+ T
cells in the initiation and perturbation of Rheumatoid Arthritis. Th17 cells are crucial for the
induction of autoimmune disorders in mice. In human disease, Th17 cells and Th17
cytokines have been demonstrated at the site of inflammation and are correlated with the
extent of joint destruction. Important advances have been made in understanding how
and when the different Th17 cytokines play a role in the disease pathogenesis.
Heterogeneity between patients, plasticity of cytokine expression during the course of
disease and effects of therapy on the phenotype of immune cells, make it difficult to
predict the success rate of targeting Th17 cytokines in RA patients. Advancing knowledge
will help to design the best therapy for each individual patient.
The functional role of the Th17 cytokines in Rheumatoid Arthritis | 161
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The functional role of the Th17 cytokines in Rheumatoid Arthritis | 167
Chapter 9
Summary and final considerations
170 | Chapter 9
Summary and final considerations | 171
Summary
Th17 cells have been implicated in the pathogenesis of chronic destructive arthritis. In
physiological conditions these cells give protection against extracellular bacteria and
fungi. Especially C. albicans is known for its potent induction of Th17 cells. First, we tried to
induce pathogenic Th17 cells in vivo and study their effects on joint pathology. We used
the chronic SCW-induced arthritis and investigated whether co-exposure to a small
amount of yeast particles on top of the repetitive local exposure to TLR2/NOD2 agonist
could specifically enhance the Th17 cell expansion and polarize the arthritis towards a
more destructive phenotype (chapter 2). The i.a. injections with C. albicans resulted in a
significant increase in joint inflammation, which was accompanied by enhanced cartilage
erosion. In summary, we found an increase in IL-17, IFN-γ and IL-21 together with more IL-17
producing T-cells. Previous studies in Chrohn’s patients have demonstrated increased
levels of antibodies directed against C. albicans, which imply a direct role for C. albicans in
the disease. Furthermore, colonization of C. albicans in the gut of immunized collagen-induced arthritis mice increases disease severity. Since the gut is also implied to be involved
in RA, investigating whether or not C. albicans is directly involved in other autoimmune
disorders like RA is of great importance.
To understand the mechanism by which C. albicans induces the Th17 response, we
explored the role the specific pattern recognition receptors (chapter 3). Freshly isolated
PBMCs were stimulated with several pathogen-associated molecular patterns (PAMPs)
and whole (heat-killed) microorganisms in the presence of human serum, without
additional costimulatory factors. The fungal pathogen C. albicans was found to induce
potent IL-17 responses, even more potently than several Gram-negative bacteria.
Furthermore, several pathogens were able to induce IL-1 and/or IL-6, but only C. albicans
could induce a clear IL-17 response. This Candida-induced IL-17 response turned out to be
dependent on the mannose receptor (MR; CD206) expressed by monocytes/macrophages.
Finally, this MR-induced IL-17 production was augmented by the TLR2/dectin-1 pathway.
This study identified a new role for the MR being involved in triggering Th17 cells in
response to C. albicans.
Previous studies from our department have clearly shown the potency of anti-IL-17
therapy in reducing cartilage destruction and bone erosions in several experimental
arthritis models (1). TNF-α is the most commonly targeted cytokine in RA, but still
approximately 30% of the patients do not respond to anti-TNF-α therapy. Because of the
heterogeneity of the disease, more specific cytokine targeting or combination therapies
might be beneficial for big cohorts of patients. IL-17 has been shown to synergize with
several other cytokines in vitro, including TNF-α (2). Because of this synergy and since
synovial explants of patients that did not respond to anti-TNF-α treatment (TNF-non-re-
172 | Chapter 9
sponders) still respond to anti-TNF-α treatment (unpublished observation) ex vivo, we
explored the interaction between TNF-α and IL-17 in more detail. (chapter 4) First, by over
expressing IL-17 and TNF-α in vivo, we observed that synovial IL-17 expression promotes
TNF-α-induced joint pathology. Further analysis revealed that exposure to both IL-17 and
TNF-α synergistically enhanced up-regulation of S100A8, IL-1β, and matrix metalloproteinases (MMPs). Additional in vitro experiments revealed that S100A9 deficient mice (which
also lack functional S100A8 protein) were protected against IL-17/TNF-α-induced
expression of cartilage NITEGE, a neoepitope that reflects ADAMTS-mediated proteoglycan
cleavage. Both in vivo and in vitro experiments clearly demonstrate an important role for
S100A8/S100A9 in the IL-17/TNF-α-induced cartilage destruction. Finally, neutralization of
IL-17 and TNF-α signaling during established arthritis was more effective than single
anti-cytokine treatments, and significantly inhibited further joint inflammation and
cartilage destruction. In summary, this study showed that neutralizing IL-17 in addition to
TNF-α was still effective in the late stages of arthritis, even under conditions in which
blocking of IL-17 or TNF-α alone was no longer effective. Therefore, combination therapy
might show great potential, especially in RA patients that do not sufficiently respond to
anti-TNF-α therapy alone.
Besides the growing knowledge about the induction and expansion of Th17 cells, it
became clear that IL-17 is not the only cytokine produced by Th17 cells, but also IL-21 and
IL-22 might have some immunogenic and pathogenic properties. In chapter 5 and 6, we
sought to explore the role of both cytokines during experimental arthritis, specifically
focusing on their expression and role in T-cell driven joint pathology. IL-21 is one of the
Th17 cytokines that acts on the T cells themselves via an autocrine loop, and contributes
to their formation and expansion. An earlier study had shown that IL-21 was involved in
the development of the collagen induced arthritis model (3). The purpose of our study
(chapter 5) was to investigate the effect of IL-21R-deficiency on joint pathology in relation
to Th17 cells during chronic experimental arthritis. We investigated the inflammation and
joint destruction during the chronic T-cell driven stages of the antigen-induced arthritis
(AIA) and the chronic Streptococcal Cell Wall (SCW) arthritis. In both models IL-21R-/- mice
were protected against severe arthritis. This reduced pathology was accompanied by a
decrease in serum IgG1 levels and suppressed antigen-specific T cells responses. IL-17
levels were reduced during AIA and in the SCW arthritis we observed a decline in the
IL-17+ IFN-γ+ T-cells isolated from the arthritic knee joints. Both observations confirm the
importance of IL-21 on the Th17/IL-17 induction. In contrast, clearly enhanced local
activation and joint inflammation were observed in IL-21R-deficient mice shortly after
each SCW injection. Previously, it has been shown that IL-21 could restrain TLR4 signaling
in dendritic cells via a SOCS (suppressor of cytokine signaling) dependent mechanism (4).
Consequently, we performed in vitro assays to explore the involvement of SOCS in the
enhanced SCW response in the IL-21R-/- mice. Exposure to SCW together with IL-21
Summary and final considerations | 173
stimulation did result in a lower inflammatory response due to the upregulation of SOCS1
and SOCS3, a mechanism lacking in the IL-21R-/- mice. Nonetheless, IL-21 has a more
dominant pro-destructive role driving Th1/17 cells and joint pathology during chronic
experimental arthritis, even despite the suppressive role of IL-21 via SOCS during acute
inflammatory responses. The data from chapter 5 suggests that anti-IL-21 therapy might
have a potential value as a therapeutic in the course of RA.
Recently it was discovered that IL-22 is mainly produced by Th17 cells, but the exact role in
the pathogenesis of rheumatoid arthritis was still unclear. In chapter 6, we investigated
the regulation of IL-22 by Th17 cells and evaluated the potential for therapeutic blocking
of IL-22 in an in vivo model. In vitro tests showed that Th17 cells produce high levels of IL-22
after exposure to IL-1 or IL-23. This production of IL-22 could be further increased after
combining the two cytokines. Because of the observed role for IL-1 on Th17 cells in vitro
and during the IL-1Ra model in vivo (5), we decided to select this IL-1-dependent model to
test the potential of the anti-IL-22 antibody. Interleukin-1 receptor antagonist-deficient
(IL-1Ra-/-) mice spontaneously develop an inflammatory and destructive arthritis, which is
IL-1, IL-17 and T-cell dependent. Investigating the progression of the erosive arthritis in
these mice, we only detected increased IL-22 and IL-22R in the severely and chronically
inflamed synovial tissues, whereas IL-17 was already upregulated during early inflammation.
Analysis of isolated single cells from the inflamed synovia revealed that IL-22 was mainly
produced by IL-17-expressing T cells. Because of this late expression of IL-22, we decided to
start the anti-IL-22 treatment with mice having active arthritis. Anti-IL-22 treatment
significantly reduced the inflammation and bone erosion, verifying the pro-inflammatory
role of IL-22 during arthritis.
Recent reports have shown that RA patients with high serum levels of IL-22 represent
a group of patients suffering from a more destructive and more progressive phenotype.
These findings suggest that IL-22 differentiates the more progressive RA patients, which
might require a more aggressive therapy. Whether IL-22 will be a good target for treatment
in RA will depend on its relationship with other cytokines. It has been described that IL-17
can promote the expression and pro-inflammatory properties of IL-22, while it impedes its
regenerative properties (6). Therefore, it might be more reasonable to block the IL-17
pathway instead of the IL-22 pathway, which will decline inflammation and at the same
time restore the regenerative capacities of IL-22.
Murine experimental arthritis models are widely used to study inflammatory processes,
but not all of these findings can be translated directly to the patients. The best example
might be the discrepancy of the role of IL-1 and TNF-α between mice and humans. The
classical murine arthritis models have shown a central role for IL-1β in driving joint
pathology, with a lesser potential for TNF-α (7). On the contrary, anti-TNF-α treatment is
now the most frequently prescribed biologic approach for RA patients, while blocking of
174 | Chapter 9
IL-1β only shows modest therapeutic effects (8). In the preceding years, our group has put
a lot of effort in setting up a humanized arthritis model, with the expectation to make
better predictions for new therapeutics that will proceed to clinical trials. In chapter 7, we
describe the validation of this model, using SCID-CB17 mice that were engrafted with
human RA synovial tissue. We validated the model by treatment with anti-TNF-α antibodies
and comparing anti-TNF-α to anti-IL-1β treatment. In addition, the direct effect of new and
current T and B cell-related therapies were investigated. Anti-TNF treatment significantly
reduced serum cytokine levels and decreased histological inflammation, whereas anti-IL-1
therapy did not show any effect on the RA synovial grafts. Next, anti-CD20 treatment only
showed clear therapeutic effects in mice engrafted with B cell-rich synovial tissue.
Surprisingly, CTLA4-Ig treatment, which is effective in the clinics, did not show any effects
in this transplantation model. Pre-screening of the synovial tissue for the presence of
CD3+ T cells and the co-stimulatory molecules CD80/86 did not alter the outcome. One of
the reasons of this failure might be that CTLA4-Ig exerts its effects not locally within the
synovium. Finally, great therapeutic potential was observed for anti-IL-17 treatment,
however only when CD3+ T cells were abundantly present in the RA synovial tissue. In
conclusion, the humanized RA SCID model can be a valuable tool for predicting responses
of new therapeutics that act locally within the arthritic joint.
Conclusions and final considerations
In this thesis, we aimed to demonstrate the presence and function of Th17 cells and the
Th17 cytokines IL-21 and IL-22 in models of experimental arthritis. Secondly, we aimed to
characterize the (antigen-specific) induction of Th17 cells in vitro. We provided evidence
that C. albicans drives pathogenic Th17 cells via the macrophage mannose receptor. In
addition, we provided a rational for combination therapy of anti-IL-17 and anti-TNF-α
treatment for Rheumatoid Arthritis. Furthermore, we have confirmed that the Th17
cytokines IL-21 and IL-22 cytokines contribute to the joint pathology in arthritis. Finally, we
set-up and validated a humanized arthritis model and demonstrated the potential of
anti-IL-17 treatment in a subset of RA patients. The recent knowledge about the
involvement of the Th17 cytokines IL-17, IL-21, IL-22 and GM-CSF in Rheumatoid Arthritis is
discussed in chapter 8.
The cytokine network, ancestry and transcriptional control of Th17 cells during the
course of chronic inflammatory diseases, like RA is a complex field. Although clinicians and
immunologists do agree on the importance of IL-17 in driving chronic arthritic processes,
the response to anti-IL-17 therapy in RA patients has not been as impressive as compared
to the response achieved in animal models. One explanation offered is that the biology of
Th17 cells differs in important ways between mice and humans. Otherwise, we have
shown that IL-17 is important in a subset of RA patients that might benefit from anti-IL-17
Summary and final considerations | 175
therapy. The challenge will be to identify and characterize these possible anti-IL-17
responsive patients in the coming future. Alternatively, there might also be a prominent
role for the other Th17 cytokines IL-21, IL-22 and GM-CSF during the course of the disease.
Further understanding on how these Th17 cytokines are regulated in vivo, will aid in
understanding the processes that drive auto inflammatory/ autoimmune diseases and
determine the future direction in the development of new therapies.
176 | Chapter 9
Reference List
(1) Koenders MI, Lubberts E, Oppers-Walgreen B, van den BL, Helsen MM, di Padova FE et al. Blocking of
interleukin-17 during reactivation of experimental arthritis prevents joint inflammation and bone erosion
by decreasing RANKL and interleukin-1. Am J Pathol 2005; 167(1):141-149.
(2) Chabaud M, Page G, Miossec P. Enhancing effect of IL-1, IL-17, and TNF-alpha on macrophage inflammatory
protein-3alpha production in rheumatoid arthritis: regulation by soluble receptors and Th2 cytokines. J
Immunol 2001; 167(10):6015-6020.
(3) Young DA, Hegen M, Ma HL, Whitters MJ, Albert LM, Lowe L et al. Blockade of the interleukin-21/
interleukin-21 receptor pathway ameliorates disease in animal models of rheumatoid arthritis. Arthritis
Rheum 2007; 56(4):1152-1163.
(4) Strengell M, Lehtonen A, Matikainen S, Julkunen I. IL-21 enhances SOCS gene expression and inhibits
LPS-induced cytokine production in human monocyte-derived dendritic cells. J Leukoc Biol 2006;
79(6):1279-1285.
(5) Koenders MI, Devesa I, Marijnissen RJ, Abdollahi-Roodsaz S, Boots AM, Walgreen B et al. Interleukin-1 drives
pathogenic Th17 cells during spontaneous arthritis in interleukin-1 receptor antagonist-deficient mice.
Arthritis Rheum 2008; 58(11):3461-3470.
(6) Sonnenberg GF, Nair MG, Kirn TJ, Zaph C, Fouser LA, Artis D. Pathological versus protective functions of IL-22
in airway inflammation are regulated by IL-17A. J Exp Med 2010; 207(6):1293-1305.
(7) Joosten LA, Helsen MM, Saxne T, van de Loo FA, Heinegard D, van den Berg WB. IL-1 alpha beta blockade
prevents cartilage and bone destruction in murine type II collagen-induced arthritis, whereas TNF-alpha
blockade only ameliorates joint inflammation. J Immunol 1999; 163(9):5049-5055.
(8) Alten R, Gomez-Reino J, Durez P, Beaulieu A, Sebba A, Krammer G et al. Efficacy and safety of the human
anti-IL-1beta monoclonal antibody canakinumab in rheumatoid arthritis: results of a 12-week, Phase II,
dose-finding study. BMC Musculoskelet Disord 2011; 12:153.
Summary and final considerations | 177
Chapter 10
Nederlandse samenvatting
List of abbreviations
Dankwoord
List of publications
Curriculum vitae
Nederlandse samenvatting | 181
Nederlandse samenvatting
Reumatoïde artritis (RA) is een chronische gewrichtsontsteking die de gewrichten
beschadigt. Het is een auto-immuunziekte en treft ongeveer 1% van de wereld bevolking.
Vrouwen zijn vaker aangedaan dan mannen (ongeveer in de verhouding 3:1) en zowel
genetische als omgevingsfactoren spelen een rol bij de kans op het ontwikkelen van deze
ziekte. Met name roken en infecties worden vaak genoemd als risicofactoren.
RA wordt gekenmerkt door synoviale ontsteking en hyperplasie, de aanwezigheid
van auto-antilichamen (reumafactor en antilichamen tegen gecitrullineerde eiwitten
(ACPA)), kraakbeen- en bot destructie (“gewrichtsvervorming”) en een verhoogde kans op
hart- en vaatziekten. Het synoviale membraan van de gewrichten worden g
­ ekarakteriseerd
door toegenomen bloedvaten en een chronische infiltratie van ontstekingscellen. Met
name monocyten/ macrofagen, B- en T-cellen worden in hoge mate gevonden in het
ontstoken weefsel. De ontstekingscellen maken grote hoeveelheden pro-inflammatoire
cytokines waaronder TNF-α, IL-1β, IL-18, IL-6, IL-8, IL-12 en IL-23. Cytokines zijn signaaleiwitten waarmee cellen onderling communiceren. De genoemde cytokines spelen een
cruciale rol in de activatie, proliferatie, differentiatie en het rekruteren van de B- en T-cellen.
Bij progressie van de ziekte en door de chronische ontsteking ontstaat de destructie van
bot- en kraakbeenweefsel. De geactiveerde ontstekingscellen secreteren onder andere
matrix mettaloproteinases (MMP’s), die zorgen voor schade en gewrichtsvervorming.
Naast de introductie van methotrexaat als medicatie is een van de grootste vooruitgangen
van de laatste tien jaar de toepassing van TNF-α remmers. TNF-α is verhoogd in de
ontstoken synoviale membranen van de patiënten, tevens onderdrukt het remmen van
TNF-α ook de ontsteking in verschillende experimentele artritis modellen. Tegenovergesteld induceert de transgene overexpressie van TNF-α voor spontane artritis in muizen.
Aansluitend hebben verschillende studies laten zien dat TNF-α remmers de ziekteactiviteit verlagen en de kwaliteit van leven sterk verhogen. Dit succes wordt verder ondersteund
door het feit dat er nog steeds nieuwe TNF-α remmers op de markt komen. Therapeutische
remming van TNF-α heeft een klinische respons in ongeveer 70% van de patiënten en
induceert een snelle daling van de spiegels van acute fase eiwitten, IL-6 en herstelt de
functie van regulatoire T-cellen. Dit betekent toch dat bij 30% van de patiënten TNF-α
remmers niet voldoende zijn. Anderzijds, vanwege de veelzijdige effecten van TNF-α is er
ook een kans op bijwerkingen, waaronder een verhoogd risico op infecties. Het begrijpen
van de cytokine cascades die betrokken zijn in de pathogenese van RA kan nieuwe aangrijpingspunten opleveren die lager in de cascade voorkomen en daardoor specifieker
zijn en voor minder bijwerkingen zorgen. In dit proefschrift hebben we ons gericht op de
relatieve contributie van Th17 cellen (IL-17 producerende CD4+ T helper cellen) en
verschillende Th17 cytokines op de ontwikkeling van experimentele artritis modellen voor
RA.
182 | Chapter 10
CD4+ T cellen helpen andere ontstekingscellen in hun reactie tegen infecties en worden
daarom ’T helper’ of ‘Th’ cellen genoemd. T helper cellen worden verdeeld in drie effector
populaties; de Th1, Th2 en Th17 cellen, die elk op hun eigen manier het immuunsysteem
activeren en daarmee beschermen tegen pathogenen. Naast deze effector populaties
bestaan ook regulatoire T cellen, zogenaamde Tregs, die de immuunreactie onderdrukken.
De T helper cellen zijn niet alleen betrokken bij immuunreacties tegen infecties, maar
blijken ook betrokken te zijn bij het ontwikkelen van auto-inflammatoire aandoeningen.
In RA lijken T-cellen cruciaal te zijn in de initiatie en perturbatie van de chronische
ontstekingen. Ten eerste, zijn de meeste T-cellen die worden gevonden in de ontstoken
gewrichten CD4+. Dit wordt ondersteund door data van experimentele artritismodellen,
waarbij gewrichtsontsteking naar een naïeve muis kan worden overgedragen door het
injecteren van enkel de CD4+ T-cellen van een zieke muis. Voor lange tijd werden de
meeste T-cel gedreven immuunziekten, zoals multiple sclerose (MS), inflammatoire
darmziekten (IBD) en RA gezien als een IL-12 en Th1 gedreven ziekte. Na de ontdekking
van IL-23 zijn er aanwijzingen dat niet de IL-12/Th1 route, maar eerder de IL-23/Th17 route
dominant is voor het chronische inflammatoire proces, zoals wordt gezien in RA.
Th17 cellen zijn betrokken in de pathogenese van chronische destructieve artritis. Onder
fysiologische condities bieden deze cellen bescherming tegen extracellulaire bacteriën
en schimmels. Met name de gist C. albicans is bekend om zijn krachtige inductie van Th17
cellen. Eerst is geprobeerd om pathogene Th17 cellen in vivo te induceren en hebben we
de effecten op de gewrichtspathologie bestudeerd (hoofdstuk 2). We hebben daarvoor
het chronische SCW-artritis model gebruikt en onderzocht of blootstelling aan SCW in
combinatie met een kleine hoeveelheid gistdeeltjes de Th17 cellen specifiek kon induceren
die daardoor het model destructiever zouden maken. De gewrichtsinjecties met C.
albicans resulteerde in een significante toename van de lokale ontsteking, die werd
­gekarakteriseerd door verhoogde kraakbeenerosie. Aanvullend hebben we een toename
van IL-17, IFN-γ en IL-21 gevonden in combinatie met een toename in de IL-17 producerende
T-cellen. Om het mechanisme waarmee C. albicans Th17 cellen induceert beter te
begrijpen, onderzochten we de rol van de specifieke herkenningsreceptoren (hoofdstuk 3).
Vers geïsoleerde PBMCs werden gestimuleerd met verschillende pathogeen-geassocieerde
moleculaire patronen (PAMPs) en gehele micro-organismen zonder bijkomende co-­
stimulatoire factoren (anti-CD3/CD28). De inductie van IL-17 door C. albicans bleek potenter
dan verscheidene gramnegatieve bacteriën. Deze Candida-geïnduceerde IL-17 respons
bleek afhankelijk te zijn van de mannose receptor (MR, CD206) op de monocyten /
macrofagen. Vervolgens hebben we gevonden dat deze MR-geïnduceerde IL-17 productie
verhoogd wordt door TLR2 en Dectin-1 stimulatie. Dit onderzoek heeft een nieuwe rol
aangetoond van de MR bij het induceren ​​van Th17 cellen in reactie op C. albicans.
Nederlandse samenvatting | 183
Voorgaande proefdierstudies van onze afdeling hebben duidelijk aangetoond dat het
remmen van IL-17 tijdens (het ontstaan van) artritis, de kraakbeenschade en botschade
vermindert. Zoals eerder genoemd zijn meeste anti-cytokine therapieën gericht op het
blokkeren van TNF-α, waarbij 30% van de patiënten niet op de therapie reageert. Deze
patiënten noemen we TNF non-responders. Omdat de ziekte zo heterogeen is, niet iedere
patiënt reageert op de therapie en er een verhoogd risico is op infecties, is men steeds op
zoek naar nieuwere specifiekere therapieën of combinatietherapieën. Het is bekend dat
cytokines in synergie kunnen werken en daarmee hun effecten op cellen versterken.
Omdat dit ook voor IL-17 en TNF-α opgaat hebben we de interactie tussen TNF-α en IL-17
gedurende artritis in detail bestudeerd (hoofdstuk 4). Eerst hebben we IL-17 en TNF-α tot
overexpressie gebracht in de gewrichten van muizen. Daarbij hebben we gevonden dat
wanneer zowel IL-17 en TNF-α tot overexpressie worden gebracht er een synergetische
verhoging waarneembaar is van de eiwitten/ enzymen S100A8, IL-1β en verschillende
MMPs. Additionele in vitro experimenten op kraakbeen toonden aan dat muizen die deficiënt
zijn in S100A9 (en daarmee ook in S100A8) beschermd zijn tegen de gecombineerde
schade van IL-17 en TNF-α. Neutralisatie van zowel IL-17 als TNF-α na inductie van de artritis
was effectiever dan een monotherapie en remde de verdere ontwikkeling van gewrichtsontsteking en kraakbeenschade. Deze studie laat zien dat het remmen van IL-17 samen
met TNF-α nog steeds effectief is, zelfs onder condities waar monotherapie van anti-IL-17
of anti-TNF-α geen effect meer had. Combinatietherapie zou daarom mogelijk een goede
optie kunnen zijn voor RA patiënten die niet reageren op TNF-α remmers.
Naast de groeiende kennis over de inductie en expansie van Th17 cellen is duidelijk
geworden dat IL-17 niet het enige cytokine is dat geproduceerd wordt door deze T helper
cellen; ook IL-21 en IL-22 kunnen in grote hoeveelheden worden uitgescheiden. In
hoofdstuk 5 en 6, zochten we naar de rol van deze cytokines in experimentele artritis
modellen en keken daarbij specifiek naar de expressie en functie onder T-cel gedreven
gewrichtspathologie. IL-21 is een van de Th17 cytokines die via een autocriene lus bijdraagt​​
aan zijn eigen inductie en expansie. Een eerdere studie heeft aangetoond dat IL-21
betrokken is in de ontwikkeling van het collageen geïnduceerde artritis (CIA) model. Het
doel van deze studie (hoofdstuk 5) was om het effect van IL-21R-deficiëntie te
onderzoeken op de Th17 gerelateerde gewrichtspathologie in andere artritis modellen.
We onderzochten daarmee de rol van IL-21 op de ontsteking en gewrichtsschade in het
antigeen-geïnduceerde artritis (AIA) en het SCW-artritis model. In beide modellen werden
IL-21R-/- muizen beschermd tegen ernstige artritis. De verminderde pathologie ging
samen met een daling van serum IgG1 spiegels en onderdrukte antigeen-specifieke
T-cellen respons. De IL-17 niveaus waren verlaagd tijdens de AIA, daarnaast zagen we een
daling van het aantal IL-17 + IFN-γ + T-cellen geïsoleerd uit de ontstoken kniegewrichten
in de SCW-artritis. Beide observaties bevestigen het belang van IL-21 in de Th17/IL-17
inductie. Daarentegen vonden we een duidelijk toegenomen gewrichtszwelling bij IL-21R-
184 | Chapter 10
deficiënte muizen kort na iedere SCW injectie. Eerder is aangetoond dat IL-21 de activering
van dendritische cellen kan beperken via de inductie van SOCS (onderdrukker van
cytokine signalering) (4). Met opvolgende in vitro assays hebben we de laten zien dat de
blootstelling aan SCW samen met IL-21 stimulatie resulteert in een lagere ontstekingsreactie
door de inductie van SOCS1 en SOCS3, een mechanisme dat ontbreekt in de IL-21R-/muizen. Desalniettemin heeft IL-21 een dominante pro-destructieve rol op de gewrichtspathologie tijdens chronische experimentele artritis via de inductie van Th1/17 cellen.
Onze gegevens suggereren dat anti-IL-21 behandeling een potentiële therapeutische
waarde kan hebben voor RA.
Onlangs is ontdekt dat IL-22 geproduceerd wordt door Th17 cellen, maar de precieze rol in
de pathogenese van RA is niet helemaal duidelijk. In hoofdstuk 6 hebben we daarom de
regulatie van IL-22 door Th17 cellen bestudeerd en is tevens het potentieel van het
neutraliseren van IL-22 geëvalueerd in een in vivo model. De in vitro onderzoeken tonen
aan dat Th17 cellen hoge niveaus van IL-22 produceren na blootstelling aan IL-1 of IL-23.
Deze productie van IL-22 wordt verder vergroot na het combineren van de twee cytokines.
Vanwege de eerder beschreven rol van IL-1 op de Th17 cellen gedurende het IL-1Ra model
(5), besloten we dit IL-1-afhankelijke model te selecteren om het potentieel van de
anti-IL-22 therapie te testen. Interleukine-1 receptor antagonist-deficiënte (IL-1Ra-/-)
muizen ontwikkelen spontaan een inflammatoire en destructieve artritis die IL-1, IL-17 en
T-cel afhankelijk is. Onderzoek naar de expressie van IL-22 en de IL-22R toonde aan dat we
IL-22 alleen konden detecteren in ernstig en chronisch ontstoken synoviaal weefsel, terwijl
IL-17 reeds werd gedetecteerd tijdens vroege ontsteking. Analyse van geïsoleerde ontstekingscellen uit de ontstoken synovia lieten zien dat IL-22 voornamelijk wordt geproduceerd
door IL-17 producerende T-cellen. Door de late expressie van IL-22 besloten we de anti-IL-22
behandeling te starten in muizen met actieve artritis. Anti-IL-22 verminderde de ontsteking
en werkte met name beschermend op de boterosie.
Recente rapporten hebben aangetoond dat RA patiënten met hoge serumniveaus
van IL-22 een groep van patiënten vormt met een erg destructieve en progressieve ziekte.
Deze bevindingen suggereren dat IL-22 een biomarker kan zijn voor patiënten die een
agressieve behandeling nodig hebben. Of IL-22 zelf een goed doelwit is voor de
behandeling van RA is zal afhangen van zijn relatie met andere cytokines. Het is namelijk
bekend dat IL-17 de expressie en pro-inflammatoire eigenschappen van IL-22 kan
bevorderen, terwijl het de beschermende werking remt. Daarom is het mogelijk beter om
IL-17 te remmen in plaats van IL-22, zodat de ontsteking afneemt en tegelijkertijd de
regeneratieve capaciteit van IL-22 wordt hersteld.
Experimentele artritismodellen worden gebruikt om inflammatoire processen te
bestuderen, maar niet alle bevindingen kunnen direct worden vertaald naar de patiënt.
Het beste voorbeeld daarvoor is de verschillen in dominantie van IL-1 en TNF-α tussen
Nederlandse samenvatting | 185
muizen en mensen. De klassieke experimentele muismodellen laten een centrale rol zien
voor IL-1β op de gewrichtspathologie, met een kleinere rol voor TNF-α. Dit terwijl de
behandeling met anti-TNF-α medicijnen nu de meest voorgeschreven biological is voor
RA patiënten en het blokkeren van IL-1β slechts bescheiden therapeutische effecten
toont. In de voorgaande jaren heeft onze onderzoeksgroep daarom veel energie gestoken
in het opzetten van een gehumaniseerd artritis model met de verwachting betere
voorspellingen voor nieuwe therapieën in de kliniek te doen. In hoofdstuk 7 beschrijven
we de opzet en validatie van dit model, waarbij we SCID-CB17 muizen geïnoculeerd
hebben met humaan synoviaal weefsel van RA patiënten. Het model is vervolgens
gevalideerd middels behandeling met anti-TNF-α antilichamen en het vergelijken van
anti-TNF-α ten opzichte van anti-IL-1β behandeling. Bovendien werd de rechtstreekse
werking van nieuwe en lopende B- en T-cel-gerelateerde therapieën onderzocht.
Anti-TNF-α behandeling verlaagde de serum cytokine levels en liet een verlaging zien van
de histologische ontsteking, terwijl er op de anti-IL-1 therapie geen significante respons
waarneembaar was. Vervolgens liet de behandeling met een anti-B-cel therapie
(anti-CD20) alleen duidelijke therapeutische effecten zien in muizen die geïnoculeerd
waren met B-cel-rijk synoviaal weefsel. Verrassend genoeg vonden we geen effectief
verschil na CTLA4-Ig behandeling, terwijl deze therapie zijn effect in de kliniek heeft
bewezen. Dit zou kunnen wijzen op dat het effect van CTLA4-Ig in RA patiënten niet
lokaal in het synovium wordt bewerkstelligt. Tenslotte hebben we grote therapeutische
effecten waargenomen voor anti-IL-17 therapie wanneer CD3 + T cellen overvloedig
aanwezig waren in het RA synoviaal weefsel. Concluderend voorspelt pre-screening van
het synoviale weefsel op de aanwezigheid van CD3 + T-cellen en B-cellen de uitkomst van
specifieke behandelingen. Aansluitend kan het gehumaniseerde RA SCID model een
waardevol instrument zijn voor het voorspellen van de uitkomst van nieuwe therapieën
die hun werking lokaal in het gewricht hebben.
In dit proefschrift hebben we geprobeerd om de aanwezigheid en functie van Th17 cellen
en de Th17 cytokines IL-21 en IL-22 in modellen van experimentele artritis beter te
begrijpen. Ten tweede, hebben we de (antigeen-specifieke) inductie van Th17 cellen in
vitro gekarakteriseerd. We hebben aangetoond dat C. albicans pathogene Th17cellen
induceert via de macrofaag mannose receptor. Daarnaast is een rationeel geboden voor
de combinatietherapie behandeling met anti-IL-17 en anti-TNF-α voor patiënten met
Reumatoïde Artritis. Tevens hebben we bevestigd dat de Th17 cytokines IL-21 en IL-22
bijdragen aan de gewrichtspathologie in artritis. Ten slotte hebben we een gehumaniseerd
artritis model opgezet, gevalideerd en het potentieel van anti-IL-17 behandeling
aangetoond voor een subgroep van RA patiënten. De recente kennis over de betrokkenheid
van de Th17 cytokines IL-17, IL-21, IL-22 en GM-CSF bij RA is uiteengezet in hoofdstuk 8.
Het cytokine netwerk en de regulatie van Th17 cellen tijdens chronische inflammatoire
aandoeningen, zoals gezien wordt in RA, is een complex veld. Hoewel clinici en
186 | Chapter 10
immunologen het eens zijn over een rol voor IL-17 in chronische gewrichtsontstekingen,
is de respons op anti-IL-17 behandeling bij RA patiënten niet zo indrukwekkend vergeleken
met de respons verkregen in diermodellen. Een verklaring hiervoor kan worden gevonden
in het feit dat de inductie van Th17 cellen in belangrijke opzichten verschilt tussen muizen
en mensen. Anderzijds hebben we aan kunnen tonen dat IL-17 belangrijk is in een
subgroep van patiënten met RA die mogelijk kunnen profiteren van anti-IL-17 behandeling.
De uitdaging zal zijn om deze subgroep van patiënten in de komende toekomst verder
te karakteriseren. Beter inzicht in hoe Th17 cellen en gerelateerde cytokines in vivo
gereguleerd worden gedurende het ziektebeloop zal helpen bij het begrijpen van de
processen die een rol spelen in auto-immuunziekten en de toekomstige richting in de
ontwikkeling van nieuwe therapieën bepalen.
Nederlandse samenvatting | 187
188 | Chapter 10
List of abbreviations
-/ADAMTS-4
AdTNF
AIA
ANOVA
APC
APC
ATCC
CCR7
CD4
Ci
CIA
CII
CXCL-8
DAS28
DC
EAE
EDTA
ELISA
FACS
FCA
FCS
FcγR
FITC
FOXP3
FSC
GAPDH
G-CSF
GM-CSF
HE
HLA
HRP
IBD
IFN-γ
IgG
IL
IL-1Ra
IL-21R
JAK1
JIA
k.o.
KC
LPS
MBq
mBSA
MDP
MFI
MMP
MR
mRNA
MS
MTX
ND
NFκB
NK
knock-out
A disintegrin and metalloproteinase with thrombospondin motifs 4
adenoviral
antigen induced arthritis
analysis of variance
antigen presenting cell
allophycocyanin (dye protein)
American Type Culture Collection
chemokine receptor 7
cluster of diferentiation 4
Curie
collagen induced arthritis
collagen type 2
chemokine (C-X-C motif ) ligand 8 (also known as IL-8)
disease activity score for 28 joints
dendritic cell
experimental autoimmune encephalomyelitis
ethyl di-amine-tetra-acetate
enzyme-linked immunosorbent assay
fluorescence-activated cell sorting
Freund’s complete adjuvant,
fetal calf serum
Fc gamma receptor
fluorescein isothiocyanate (dye protein)
forkhead box P3
Forward Scatter
glyceraldehyde-3-phosphate dehydrogenase
granulocyte colony-stimulating factor
granulocyte macrophage colony stimulating factor
hematoxylin and eosin
human leukocyte antigen
horseradish peroxidase
inflammatory bowel disease
interferon gamma
immunoglobulin G
Interleukin
interleukin-1 receptor antagonist
IL-21 receptor
Janus kinase 1
Juvenile Idiopathic Arthritis
knock-out
keratinocyte chemoattractant (new nomenclature is CXCL-1)
lipopolysaccharide
megabecquerel
methylated bovine serum albumine
muramyl dipeptide
mean fluorescence intensity
matrix metalloproteinase
mannose receptor
messenger RNA
multiple sclerosis
metotrexate
not detectable
nuclear factor-kappaB
natural killer
List of abbreviations | 189
NKT
NLR
NOD2
ns
OCT
OPG
P
PAMP
PBMC
PBS
PCR
PE
PFA
PFU)
PG
PMA
PRR
QPCR
RA
RANKL
RE
RORγT
RPMI
RT-PCR
S100A8
SCID
SCW
SD
SEM
siRNA
SOCS
SSc
STAT
T-BET
Tc
TGF-β
Th
TLR
TNFBP
TNF-α
Treg
natural killer T
NOD-like receptors
nucleotide-binding oligomerization domain-containing protein 2
not significant
optimal cutting temperature compound
osteoprotegerin
p-value; probability (statistical significance)
pathogen-associated molecular pattern
peripheral blood mononuclear cell
phosfate buffered saline
polymerase chain reaction
phycoercythrin (dye protein)
paraformaldehyde
plaque-forming unit
proteoglycan
phorbol 12-myristate 13-acetate
pathogen recognition receptor
quantitative real time polymerase chain reaction
Rheumatoid Arthritis
receptor activato of nuclear factor kappa-B ligand
relative expression
Orphan Nuclear Receptor ROR-gammaT
Roswell Park Memorial Institute (culture medium)
reverse transcriptase polymerase chain reaction
S100 calcium binding protein A8
Severe Combined Immune Deficiency
streptococcal cell wall
standard deviation
standard error of the mean
small interfering RNA
suppressor of cytokine signalling
side Scatter
signal transducer and activator of transcription
T-box transcription factor
technetium
transforming growth factor alpha
T helper
toll-like receptor
TNF binding protein
tumor necrosis factor alpha
regulatory T cell
Dankwoord | 191
Dankwoord
Zo, de laatste woorden gaan op papier. Met ontzettend veel plezier heb ik op de afdeling
Reumatologie mogen werken aan mijn promotieonderzoek. Het project was uitdagend
en ik heb de ruimte gekregen om me te ontplooien in richtingen die niet direct met mijn
onderzoek te maken hadden. Voor iemand met een enorme regeldrang is het moeilijk om
toe te moeten geven dat er het afgelopen jaar veel op mijn bordje kwam; het afronden
van mijn promotie, het opzetten van een nieuwe onderzoeksafdeling binnen een
reorganiserend onderzoeksinstituut in Utrecht en uiteindelijk een nieuwe baan in Zeist. Ik
wil dan ook iedereen bedanken die me hierbij gesteund heeft. Zonder iemand tekort te
doen wil ik een aantal mensen graag persoonlijk bedanken.
Wim, je hebt mee de kans gegeven om te promoveren op jouw lab. Ik kon altijd bij je
terecht als ik je raad nodig had. Je hebt me ook erg gesteund in mijn (onderzoeks)plannen,
zolang ik maar met een goede onderbouwing kwam. Ik mocht naar alle (buitenlandse)
congressen/ cursussen en kreeg de ruimte om me ook naast het onderzoek te blijven
ontwikkelen. Dank daarvoor!
Mieke, ik kijk met erg veel plezier terug op mijn stage bij Organon en er was dan ook geen
twijfel om een project te accepteren waar we zouden blijven samenwerken. Bedankt voor
je steun de afgelopen jaren.
Marije, wat hebben we er een mooi project van gemaakt. Samen groeiden we in 2007 in
onze nieuwe rol, jij als begeleider, ik als promovendus. Ik heb veel kunnen leren van je
pragmatische aanpak en kritische blik op manuscripten en data. Je hebt me alle ruimte
gegeven om nieuwe onderzoekslijnen te ontwikkelen en daar heb ik veel van geleerd. Ik
kon altijd bij je terecht voor leuke discussies met een kop koffie. Bedankt!
Leo, ik vond het erg jammer dat je kort na de start van mijn promotie aankondigde dat je
de afdeling ging verlaten en daarmee ook niet meer mijn dagelijkse begeleider kon zijn.
Toch was je er altijd voor me als het nodig was. Jouw overstap heeft geresulteerd in een
ontzettend leuke samenwerking met het lab interne. Wat hebben we mooie papers
neergezet! Bedankt.
Fons, ik kon de discussies die je tijdens de werkbesprekingen leidde altijd erg waarderen.
Soms als advocaat van de duivel, maar daarmee bleven we allemaal wel kritisch op onze
eigen bevindingen. Bedankt.
Lenny, waar zal ik beginnen? We kennen elkaar al zo lang. We begonnen samen aan de
medische faculteit (maandagclubje), werden vrienden en kregen ook nog de kans om
192 | Chapter 10
samen te werken. Ik mocht jullie huwelijk coördineren, vandaag sta je mij bij. Dankzij jouw
komst naar de afdeling had ik eindelijk iemand die mijn frustraties kon temmen. We
sloegen een mooie brug tussen de onderzoekslijnen. Je bent een erg goede vriendin en
het betekent voor mij dan ook veel om vandaag door jou als paranimf te worden
bijgestaan!
Ben, ik zal niet vergeten dat je me vanuit het buitenland mailde of ik nog wist of er posities
waren binnen de afdeling. Niet alleen heb ik erg leuke herinneringen aan onze studie
(altijd lopend met je bekertje koffie, soms samen als practicumduo), maar ook heb ik veel
steun van je gekregen in onze tijd samen op het lab. Ik heb erg veel geleerd van je
feedback op mijn presentaties en teksten. Jij hebt je (wilde) haren verloren en ik ben
gegroeid, ook in mijn vermogen om dingen wat meer los te laten. De leuke congressen en
middagen in de Aesculaaf werkten therapeutisch voor ons beiden. Ik ben erg blij met jou
aan mijn zijde als paranimf vandaag.
Ik wil natuurlijk iedereen van ‘groepje 1’ bedanken. Birgitte, niet alleen heb ik goede
herinneringen aan de gezellige roddelmomenten die we samen met Monique deelden
op het dierenlab, maar mede dankzij jou heb ik enorm veel geleerd over het muizenwerk
en de moeilijkheden van de verschillende artritismodellen. Ik denk ook met plezier terug
aan alle keren dat je Renske meenam naar het lab. Monique en Liduine, heel erg bedankt
voor jullie ondersteuning. Monique, jouw enorme kennis over de ‘oude’ experimenten uit
de beginjaren van het lab hebben mijn inzichten enorm vergroot en voorkwamen soms
dat we het wiel opnieuw probeerden uit te vinden. Liduine, je hebt me nog ontzettend
geholpen in de laatste fase. Monique en Liduine, we houden erg veel van koken en lekker
eten. Volgens mij moeten we nog steeds een keer bij ‘In geuren en kleuren’ gaan eten in
Berg en Dal. Shahla, bedankt voor je betrokkenheid en fijne discussies op de dinsdagen.
Ik heb veel bewondering voor de manier waarop je jouw eigen onderzoekslijn vorm hebt
gegeven. Bas aka ‘mister IL-32’. Hoewel ik het jammer vond dat het IL-32 project al was
vergeven toen ik mijn sollicitatiegesprek had, heb ik je daar later niet om benijd. Je hebt
dit moeilijke project toch maar even weten om te buigen naar een aantal mooie high
impact factor papers. Ik vond het altijd gezellig met je. Sabrina, it was fun having you as
a colleague, I learned a lot about Brazil and maybe even more about the Netherlands.
Isabel, we only worked together for a short period, but you were the one who thought
me the principles of T-cells and intracellular flow cytometry and I still benefit from that
today.
Ik wil ook alle studenten bedanken van de afdeling Reumatologie. Ik heb veel van jullie
mogen leren, in het bijzonder van de studenten die ik zelf begeleid heb. Jan Willem,
bedankt voor je inzet tijdens je stage. Volgens mij heb je vooral veel geleerd over het
invriezen en ontdooien van eiwitten en de activiteit ervan. Het deed me goed om van je
Dankwoord | 193
te horen dat je aan een promotietraject in Leiden bent begonnen. Thessa, hoewel we het
werk van je stage nog erg preliminair was, heeft het ons veel inzichten in de T-cel biologie
en TGF-b signalering gebracht. Het feit dat op basis van de uitkomsten van jouw studies
een promotietraject is gestart is een groot compliment. Mark, ik vind het nog steeds een
eer dat ik je toe mocht spreken tijdens je afstuderen. De enorme pipetteerschema’s en
stimulaties tijdens je stage kostten soms meer tijd dan het isoleren van de cellen. Ik vond
het erg leuk dat je uiteindelijk ook tijdens de opleiding koos voor een specialisatie
immunologie en daarbinnen ook een goede promotieplek hebt gevonden. Alsya, the
student I never had ;-), I am really happy that we finally got the chance to work together
on the same project.
Jeroen, ’you are me there one’! Ik weet niet of het verstandig was om twee ‘Huukske’members op het lab te hebben, maar heerlijk om af en toe eens alles eruit te gooien
zonder er teveel bij te moeten nadenken. Ik hecht erg veel waarde aan de lunches die we
in de prekliniek hadden. Niet alleen als vriend, maar ook als collega heb ik veel steun van
jou gehad. Je scherpte en kennis over genregulatie hebben menig onderzoek naar een
hoger niveau getild. Helaas ben je er vandaag waarschijnlijk niet bij, Janneke is namelijk
precies vandaag uitgerekend (timing). Gelukkig zien en spreken we elkaar nog vaak en kijk
ik ernaar uit om weer eens met Monica naar Basel af te reizen.
Sharon, je bent een voorbeeld voor me geweest in mijn tijd op de afdeling. Niet alleen
liet je zien dat gedegen onderzoek loont, maar ook dat de combinatie van immunologie
en pathologie wel degelijk kan resulteren in een mooi proefschrift. Ik vond de jaarlijkse
NVVI congressen, de zoektocht naar Schuitgraaf en de files heen en terug vanuit Noordwijkerhout altijd erg gezellig.
De werkgroepleiders, Peter en Peter, bedankt voor jullie behulpzaamheid, wetenschappelijke bijdrage en gezelligheid. Marianne, ik wil jou nog even speciaal noemen. Je staat
altijd voor iedereen klaar en dat waardeer ik heel erg. Ik bedank je voor je altijd oprechte
interesse en ik beloof vanaf nu echt eens (vaker) op vakantie te gaan. Arjen, kamergenoot
en een van de drijvende krachten achter de meest briljante sketches op de afdeling.
Buiten het feit dat je een supercollega bent, ben je ook een erg goede onderzoeker. De
aanhouder WNT! Richard, ik heb je altijd erg bewonderd om de energie waarmee jij
iedere dag aan het werk ging. Je bent iemand op wie iedereen altijd kan rekenen. Marta,
I always get inspired by your drive for perfection and good coffee. Rik, Marta, Kim, Mark,
Wouter, Ben, Jeroen en Menno. Bedankt voor de gezellige vrijdagmiddagborrels.
Heerlijk om de week af te sluiten en teleurstellingen van sommige experimenten aan de
kant te schuiven. Hoewel het waarschijnlijk beter is voor mijn postuur, mis ik deze
wekelijkse afsluitingen wel een beetje. Verder wil ik alle andere collega’s van het lab
Reumatologie bedanken: Onno, Miranda, Elly, Esmeralda, Eline, Genoveva, Arjan,
194 | Chapter 10
Dennis, Matthijs, Annet, Wouter, Debbie, Lilyanne, Mieke, Martijn, Henk, Isabel,
Nuria, Nozomi, Leif, Tamara en Calin. Dankzij jullie ging ik altijd met erg veel plezier naar
mijn werk. Bedankt voor de gezellige werkdagen, dagjes uit, sinterklaasvieringen,
lunchpauzes, borrels en kerstspel. Ook de collega’s in de kliniek: Piet, Roland, Mieke,
Lieke, Renske, Jaap, Elke, Yvonne, Kavish, Jos en Laura, erg bedankt voor jullie interesse
en support.
Ook dank aan de andere collega’s van het lab Interne, ik heb me altijd erg welkom gevoeld.
Frank, ik kijk met erg veel plezier terug op onze samenwerking. Niet alleen heb ik veel van
je geleerd, maar ik heb vooral ook goede herinneringen aan de avonden samen pipetteren
en FACSen. Ik zal nooit vergeten hoe de eerste HIES patiënt totaal geen IL-17 inductie liet
zien na Candida blootstelling. Beste Mihai bedankt voor de support, in het bijzonder voor
je steun bij de subsidieaanvraag. Louis and James , thanks for the nice collaboration. Cor,
Monique, Marije, Anne, Kathrin, Mark, Sanne, Theo en Duby, bedankt voor de leuke
discussies van tijd tot tijd.
De collega’s van Biochemie. Els bedankt voor je luisterend oor en de witte Twixen tegen
de suikerdipjes. Raymond, Sandy, Sander, Remon, Chantal, Joyce en Sanne bedankt
voor de gezelligheid op de retraites, in de wandelgangen en jullie inzichten in de MGP.
Matthijs en de collega’s van de IC, bedankt voor de feedback, de koffie en lunches tussen
de bedrijven door.
Alle medewerkers van het CDL, met name Kitty, Bianca, Daphne, Helma, Alex, Claudia
en Mieke. Hoewel er een hoge druk op jullie allemaal lag toen ik in 2007 begon met het
proefdieronderzoek, heb ik jullie inzet en interesse altijd erg kunnen waarderen.
Jasper, ik kan het altijd erg goed met je vinden en ben je sinds we als directe collega’s
samenwerken niet alleen gaan waarderen als collega, maar ook als vriend. Je kijk op de
medische wereld en de kansen die er liggen vind ik altijd erg verhelderend.
Tim, heel erg bedankt voor de kans die je me gegeven hebt en de support voor het
afmaken van mijn proefschrift. Ik heb in Utrecht met name mijn organisatorische
vaardigheden verder kunnen ontwikkelen. All colleagues from Utrecht, thanks for all of
your interest and your support to finish my thesis! Annelies en Rosalien, ik heb erg fijn
met jullie gewerkt, niet alleen vanwege jullie zorgvuldigheid, maar ook vanwege jullie
inspanning en doorzettingsvermogen. Ik zal de gezellige koffiemomentjes missen.
Sandra, Marzia, Wioleta, Sanne, without you the group would not have been so
successful.
Dankwoord | 195
Beste Frans, we kennen elkaar al sinds mijn tijd als student MLW. Ik heb altijd erg veel
waarde gehecht aan je adviezen als mentor en daar wil ik je voor bedanken.
Willemijn, niet alleen ben je een goede vriendin van Monica en mij, maar met jou heb ik
toch veel raakvlakken door de link met farm/tox, de open dagen en onze stages bij John.
Ik vind het prettig om af en toe met je te sparren over onderzoek.
John, ik heb veel van je geleerd de afgelopen jaren. Ik geniet nog steeds na van mijn tijd
bij Organon en kijk er daarom ook erg naar uit om weer met jou te gaan samenwerken bij
Triskelion.
Hanneke en Maaike , toch wel de twee maatjes gedurende mijn promotie! Samen
begonnen, samen in de NCMLS PhD commissie en samen geëindigd. Samen uit en samen
thuis. Bedankt voor de gezelligheid en ik hoop dat we elkaar niet uit het oog verliezen.
Zoals jullie weten zijn vrienden onmisbaar voor me, ook tijdens mijn promotieonderzoek.
Een aantal wil ik graag bij naam noemen. Dirk, natuurlijk geldt het voorgaande stukje over
Lenny ook voor jou. Je bent een Geurts en op een Geurts kun je bouwen. Ons beider
enthousiasme zorgt voor een synergie waarmee we een hoop mensen schrik aanjagen.
Het geduld dat jij opbrengt om naar iemand te luisteren maakt je een speciale vriend.
Bedankt. Lieve Berend, Dennis, Floor en Anke, bedankt voor de nodige afleiding en
vriendschap. Ondanks de verschillende carrières vinden we elkaar in de Bourgondische
levensstijl die we er op na houden. Ik hoop dat we nog lange tijd gaan genieten van de
lange zomeravonden onder het genot van een glas wijn. Igo, ik geniet erg van je nuchtere
kijk op situaties en tegelijk je enthousiasme in de avonduren. Martje en Gerrit, fijn dat bij
jullie de deur altijd voor me open staat en bedankt voor de support en interesse de
afgelopen jaren. Marnix en Laurens, twee extra broertjes, bedankt dat jullie er altijd zijn
als ik jullie nodig heb. Marnix, zoals je kunt lezen lukt het me (weer) niet om het kort te
houden. Erik en Marta, Linda en Julian, Berni en Marleen, Joris en Annelies, Ruud en
Rutger, Charlotte en Freek, Fred en Erwin, bedankt voor jullie interesse en adviezen de
afgelopen jaren.
Zonder de heren van A.H.G. Senex Captiosus had ik me nooit gerealiseerd dat er nog
zoveel te ontdekken valt buiten de medische wetenschappen.
Lieve Ineke, Rob, Ron, Nadine, Demi, Naomi, opa Dolf en oma Mia, lieve schoonfamilie,
nu ben ik dan echt student af. Ik ben erg blij met de enorme steun die ik van jullie heb
gekregen en krijg.
196 | Chapter 10
Lieve Jantien, Koenraad en Roeland, ik hoop dat jullie met dit boekje een beter idee
krijgen van wat jullie broer hier allemaal uitspookt in het Oosten. Het moge duidelijk zijn
dat ik mijn ‘verlichting’ hierin heb gezocht.
Liefste ome Jan en ome René, we weten nog even niet hoe dit avontuur af gaat lopen,
maar ik wil dat jullie weten dat ik me nooit zover zou hebben ontwikkeld zonder jullie
rolmodel en relativerende woorden.
Lieve Ronald en Loes, lieve pap en mam, bedankt voor alle steun en liefde die jullie me
gegeven hebben. Ik heb ontzettend veel van jullie geleerd, met name van jullie onder­
nemerschap en doorzettingsvermogen. Zonder jullie was ik nooit zo ver gekomen.
Monica, het allerlaatste woord is voor natuurlijk voor jou. Als geen ander begrijp je hoe ik
in elkaar zit en houd je me met twee benen op de grond. Het onderzoek heeft heel wat
van ‘onze’ tijd gekost. Vaker dan eens hebben we avonden en weekenden samen
doorgebracht op het lab. Jij maakt het thuiskomen na een lange werkdag fijn. Ik hoef dan
ook niemand uit te leggen hoe belangrijk je bent geweest voor dit project en hoe
belangrijk je voor mij bent………..want als je lacht…..
Dankwoord | 197
198 | Chapter 10
List of publications | 199
List of publications
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Marijnissen RJ, Koenders MI, Young D, Nickerson-Nutter C, van de Loo FAJ, Boots AMH, van den
Berg WB. A dual role of IL-21 in experimental arthritis: IL-21R-deficiency prevents adaptive
immunity mediated joint pathology, but may enhance the local innate inflammatory process.
Submitted for publication.
Marijnissen RJ, Koenders MI, van de Veerdonk FL, Dulos J, Boots AMH, Netea MG, Joosten LA, van
den Berg WB. C. Albicans polarizes a T-cell driven arthritis model towards Th17, resulting in a
more destructive arthritis. PLOS One, 2012;7(6):e38889.
Zwerina K, Koenders MI, Hueber A, Marijnissen RJ, Baum W, Heiland GR, Zaiss M, McInnes I, Joosten
LA, van den Berg WB, Zwerina J, Schett G. Anti-IL-17A therapy inhibits bone loss in TNF-α mediated
murine arthritis by modulation of T-cell balance. Eur J Immunol, 2012 Feb;42(2):413-23.
Koenders MI, Marijnissen RJ, Abdollahi-Roodsaz S, Di Padova FE, van de Loo FA, Dulos J, Joosten
LA, Boots AM and van den Berg WB. T cell lessons from the RA synovium SCID mouse model:
synovial tissue rich in CD3+ T cells lacks response to CTLA4-Ig, but is successfully treated with
anti-IL-17 antibodies. Arthritis & Rheumatism, 2012 Jun;64(6):1762-70.
van de Veerdonk FL, Lauwerys B, Marijnissen RJ, Timmermans K, Di Padova F, Koenders MI,
­Gutierrez-Roelens I, Durez P, Netea MG, van der Meer JW, van den Berg WB, Joosten LA. The
anti-CD20 antibody Rituximab reduces T helper 17 response. Arthritis & Rheumatism, 2011
Jun;63(6):1507-16.
Marijnissen RJ, Koenders MI, Smeets RL, Stappers MH, Nickerson-Nutter C, Joosten LA, Boots
AMH, van den Berg WB. Increased IL-22 expression by synovial Th17 cells during late stages of
arthritis is controlled by IL-1 and enhances bone degradation. Arthritis Rheum. 2011
Oct;63(10):2939-48.
Koenders MI, Marijnissen RJ, Devesa I, Lubberts E, Joosten LA, Roth J, van Lent PL, van de Loo FA,
van den Berg WB. TNF / IL-17 Interplay Induces S100A8, IL-1β, and MMPs, and Drives Irreversible
Cartilage Destruction In Vivo: Rationale for Combination Treatment During Arthritis. Arthritis &
Rheumatism, 2011 Aug;63(8):2329-39.
Chai LY, van de Veerdonk F, Marijnissen RJ, Cheng SC, Khoo AL, Hectors M, Lagrou K, Vonk AG,
Maertens J, Joosten LA, Kullberg BJ, Netea MG. Anti-Aspergillus Human Host Defense Relies on
Th1, Rather Than Th17, Cellular Immunity. Immunology. 2010 May;130(1):46-54.
van de Veerdonk FL, Marijnissen RJ, Joosten LA, Kullberg BJ, Drenth JP, Netea MG, van der Meer
JW. Milder clinical hyperimmunoglobulin E syndrome phenotype is associated with partial
interleukin-17 deficiency. Clinical Experimental Immunology, 2010 Jan;159(1):57-64.
van de Veerdonk FL, Marijnissen RJ, Kullberg BJ, Koenen HJ, Cheng SC, Joosten I, van den Berg
WB, Williams DL, van der Meer JW, Joosten LA, Netea MG. The macrophage mannose receptor
induces IL-17 and this pathway is amplified by dectin-1/TLR2. Cell Host & Microbe, 2009 Apr
23;5(4):329-40.
Koenders MI, Devesa I, Marijnissen RJ, Abdollahi-Roodsaz S, Boots AM, Walgreen B, di Padova FE,
Nicklin MJ, Joosten LA, van den Berg WB. Interleukin-1 drives pathogenic Th17 cells during
spontaneous arthritis in interleukin-1 receptor antagonist-deficient mice. Arthritis& Rheumatism,
2008 Nov;58(11):3461-70.
200 | Chapter 10
Curriculum Vitae
Renoud Johannes Marijnissen is geboren op 4 mei 1981 in Breda. Na het behalen van zijn
VWO diploma in 1999 begon hij de studie Scheikunde aan de Radboud Universiteit in
Nijmegen. Gedurende die periode merkte hij dat hij meer affiniteit had met het menselijk
lichaam en ziekteprocessen. In 2001 kon Renoud beginnen met de opleiding Biomedische
Wetenschappen en koos hij in zijn bachelor voor de hoofdrichting Toxicologie. In 2007
behaalde Renoud zijn master Biomedical Sciences
Tijdens zijn studiejaren was hij actief betrokken als voorlichter voor de Radboud Universiteit
en coördinator van de Nijmeegse Twee-daagse voor Biomedische wetenschappen. In 2003
werd hij penningmeester van het oprichtingsbestuur van 'de Aesculaaf', een uitdagend
project waarmee een ontmoetingsplek is gerealiseerd voor medewerkers en studenten
van het UMC St Radboud. In deze periode was hij betrokken als student-assistent bij het
onderwijs van de afdeling Toxicologie en lid van de opleidingscommissie van Biomedische
wetenschappen.
Van juni 2007 tot en met februari 2012 was hij werkzaam als junioronderzoeker binnen de
onderzoeksgroep van prof. Wim van den Berg binnen de afdeling Reumatische ziekten
van het UMC St Radboud. Hier heeft hij onderzoek gedaan naar de rol van Th17 cellen en
het effect van de Th17 gerelateerde cytokines op het destructieve proces in Reumatoïde
artritis. De resultaten van dit onderzoek, onder leiding van dr. Marije Koenders en prof.
Wim van de Berg, liggen ten grondslag aan dit proefschrift. Gedurende zijn promotie­
traject heeft hij zijn werk meerdere malen mogen presenteren op internationale congressen.
Naast zijn werk als junior onderzoeker was hij als lid van de NCMLS PhD commissie verantwoordelijk voor de organisatie van de PhD retraite in 2009 en 2010. Ook werd hij in 2010
als vertegenwoordiger van de promovendi en AIOS verkozen tot lid van de UMC-raad en
nam hij daarbinnen plaats in de agendacommissie.
Begin 2012 kreeg hij de kans om zich op de afdeling Translationele Immunologie van
Timothy Radstake in Utrecht verder te ontplooien. Als co-director was hij medeverantwoordelijk voor het opzetten en aansturen van deze nieuwe onderzoeksgroep. Onlangs
is hij vol enthousiasme begonnen als study director bij Triskelion (TNO) in Zeist.