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FcγRIIB Prevents Inflammatory Type I IFN
Production from Plasmacytoid Dendritic
Cells during a Viral Memory Response
This information is current as
of April 2, 2015.
Marcella Flores, Claude Chew, Kevin Tyan, Wu Qing
Huang, Aliasger Salem and Raphael Clynes
Supplementary
Material
http://www.jimmunol.org/content/suppl/2015/03/27/jimmunol.140129
6.DCSupplemental.html
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 27 March 2015
http://www.jimmunol.org/content/early/2015/03/27/jimmun
ol.1401296
Published March 27, 2015, doi:10.4049/jimmunol.1401296
The Journal of Immunology
FcgRIIB Prevents Inflammatory Type I IFN Production from
Plasmacytoid Dendritic Cells during a Viral Memory
Response
Marcella Flores,*,†,‡ Claude Chew,*,†,‡ Kevin Tyan,*,†,‡ Wu Qing Huang,*,†,‡
Aliasger Salem,x and Raphael Clynes*,†,‡
T
he type I IFN (IFN-a) response is crucial for viral
clearance during a primary viral infection, and it modulates both innate and adaptive components (1, 2). Although all cells can produce IFN-a, plasmacytoid dendritic cells
(pDCs) are thought to be important early responders during primary viral infections. Indeed, early studies using synthetic TLR
ligands, such as CpG, highlighted pDCs as the sole producer of
type I IFN after i.v. injections (3–5).
Results from recently developed pDC-deficient mice demonstrated that pDCs participate in the local and systemic production of
IFN-a after viral infections (6–8). However, these studies also
suggest that the role of pDCs as exclusive producers of IFN may
be virus specific (9–12) and route dependent (6, 13). When vesicular stomatitis virus or HSV was delivered i.v., pDC-derived
IFN-a was evident by 6 h (5, 6) in the circulation. The lack of
IFN-a production in pDC-deficient mice resulted in important
phenotypic consequences, with increased viral titers and impaired
adaptive immune responses. However, when mouse CMV or
mouse hepatitis virus was injected in the i.p. cavity, a pDC*Department of Pathology and Cell Biology, Columbia University Medical Center,
New York, NY 10032; †Department of Medicine, Columbia University Medical
Center, New York, NY 10032; ‡Department of Dermatology, Columbia University
Medical Center, New York, NY 10032; and xDivision of Pharmaceuticals, College of
Pharmacy, University of Iowa, Iowa City, IA 52242
Received for publication May 23, 2014. Accepted for publication February 9, 2015.
This work was supported in part by National Institutes of Health Grant 1R01CA164309
(to R.C.) and by National Institutes of Health Minority Supplemental Grant 2/06-2/08
(to M.F.) for National Institutes of Health/National Institute of Diabetes and Digestive
and Kidney Diseases Grant R01 DK70999 (to R.C.).
Address correspondence and reprint requests to Dr. Raphael Clynes, Columbia University, Departments of Pathology, Medicine, and Dermatology, 1150 St. Nicholas
Avenue, Russ Berrie Pavilion, Room 303B, New York, NY 10032. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BM, bone marrow; cDC, conventional DC;
h, human; IC, immune complex; KO, knockout; pDC, plasmacytoid dendritic cell;
ROI, region of interest; SeV, Sendai virus; WT, wild-type.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401296
specific IFN-a response was delayed and less pronounced (6, 7).
Taken together, these studies support a role for pDCs as important
producers of type I IFN and suggest that the route of viral inoculation may dictate when and to what degree pDCs engage the
immune response.
Traditionally, IFN-a has been thought to be less critical for viral
clearance during secondary viral responses, because other mechanisms provided by the adaptive memory immune response are
operative and available for rapid deployment (i.e., CTL- and/or
Ab-mediated clearance). Indeed, early studies found that clearance of secondary pox virus infections occurs comparably between wild-type (WT) and IFNAR-knockout (KO) mice (14).
However, in mouse models of secondary Sendai virus (SeV)
respiratory infections, IFN-a was shown more recently to be
crucial in activating memory CD8+ T cells to produce granzyme B
and contain viral spread (15). Yet, it is clear too that IFN-a may
be injurious to the host (16–18). pDC-derived IFN was shown to
have direct negative effects on pDCs themselves, resulting in
apoptosis (19) and a state of overactivation (20). Therefore, it
would be advantageous for pDCs to distinguish between a primary
and a secondary response and appropriately regulate their IFN
production to prevent unnecessary and untoward inflammatory
responses.
We previously described the dominant expression of the inhibitory FcgRIIB on murine pDCs and posited that it may have
important implications for IFN regulation during viral infections
when circulating, virus-specific Abs are present. In the current
study, we find that pDCs are the exclusive producers of IFN-a
during a primary systemic SeV inoculation. However, IFN production is absent during a memory response, despite the presence
of virus and pDCs together in the spleen. The absence of IFN was
dependent on circulating virus-specific Ab and was reversed by
the transgenic expression of the activating FcgRIIA receptor on
pDCs. This Ab-dependent IFN-a regulation may be an important
mechanism by which the potential deleterious effects of IFN-a are
prevented during a secondary infection.
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The type I IFN (IFN-a) response is crucial for viral clearance during primary viral infections. Plasmacytoid dendritic cells (pDCs)
are important early responders during systemic viral infections and, in some cases, are the sole producers of IFN-a. However,
their role in IFN-a production during memory responses is unclear. We found that IFN-a production is absent during a murine
viral memory response, despite colocalization of virus and pDCs to the splenic marginal zone. The absence of IFN was dependent
on circulating Ab and was reversed by the transgenic expression of the activating human FcgRIIA receptor on pDCs. Furthermore, FcgRIIB was required for Sendai virus immune complex uptake by splenic pDCs in vitro, and internalization via FcgRIIb
prevented cargo from accessing TLR signaling endosomes. Thus, pDCs bind viral immune complexes via FcgRIIB and prevent
IFN-a production in vivo during viral memory responses. This Ab-dependent IFN-a regulation may be an important mechanism
by which the potentially deleterious effects of IFN-a are prevented during a secondary infection. The Journal of Immunology,
2015, 194: 000–000.
2
FcgRIIB PREVENTS pDC IFN-a PRODUCTION IN VIRAL MEMORY RESPONSE
Materials and Methods
Mice
C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor,
ME). Fcgr2b (FcgRIIB2/2), Fcer1g (FcgRg2/2), and double-KO Fcgr2b
(FcgRIIB2/2) Fcer1g (FcgRg2/2) mice, referred to in this article as
FcgR-null mice, were purchased from Taconic Farms (Germantown, NY).
Human (h)FcgRIIA transgenic mice were generated as described (21) and
obtained from S. McKenzie (Department of Medicine, Cardeza Foundation
for Hematological Research, Thomas Jefferson University). YFP-IFNb
mice were obtained from B. Reizis (Department of Microbiology and
Immunology, Columbia University) with permission from R. Locksley
(University of California, San Francisco). YFP-IFNb hFcgRIIA mice were
generated in the animal facility in the Institute of Comparative Medicine at
the Columbia University Medical Center. All animal experiments were
performed in compliance with institutional guidelines and approved by
Columbia University’s Institutional Animal Care and Use Committee.
Intracellular IC trafficking. Splenic pDCs were isolated from spleens by
depleting CD19 + cells and then selecting for BST2 + cells using
MicroBeads (Miltenyi Biotec, San Diego, CA). Images were acquired by
confocal microscopy using a Nikon A1 confocal microscope with a 1003
objective and analyzed by ImageJ. Integrated density above a certain
threshold of Lamp1, LC3, and LysoTracker was measured at specific
regions of interest (ROIs). Measurements at random ROIs were taken on
images pivoted 90˚, as described (23). Manders coefficients were determined using the ImageJ plugin JACoP (Just Another Colocalization Plugin) developed by Fabrice P. Cordelieres (Institut Curie, Paris, France) and
Susanne Bolte (Sorbonne Universite´, Paris, France).
Statistical analysis
Differences between two groups were evaluated using the Student t test.
ANOVA was used to compare three or more groups with the Tukey
multiple-comparison posttest.
Results
Bone marrow (BM) cells (2 3 106 cells/ml) were cultured for 7–9 d in
RPMI 1640/10% FCS supplemented with 100 ng/ml murine Flt3 ligand
(R&D Systems, Minneapolis, MN). It typically consisted of 80% conventional DCs (cDCs) and 20% pDCs.
IFN-a production is absent during secondary exposure to SeV
Virus
SeV-Cantell strain was purchased from Charles River Labs and obtained
from Vincent Racaniello (Columbia University).
Murine immune sera and immune complex formation
SeV antisera. Mice were immunized with either live or heat-killed SeVCantell by tail vein injection. Four to six weeks postinjection, mice were
boosted, and sera were collected from mice 1 wk postboost. The blood of
five C57BL/6 mice was pooled, and sera were frozen in aliquots.
For intracellular immune complex (IC)-trafficking studies, IgG was
purified with protein A/G columns (Thermo Scientific, Rockford, IL). An
ELISA was developed in-house to determine the presence of mouse antiSeV IgG using SeV-coated plates.
For in vitro functional assays, SeV ICs were made using 5 ml live virus
stock incubated with 5 ml antisera and 0.5 ml RPMI 1640 for $15 min in
a 37˚C water bath. SeV ICs or SeV alone was incubated overnight with
purified BM pDCs, and IFN-a was read by ELISA.
For in vivo experiments, 20 ml anti-SeV immune sera was injected i.v.
before, after, or concurrently with 50 ml virus (i.v.), both into the tail vein.
OVA was purchased from Worthington Biochemical Industries
(Lakewood, NJ). CpGB–OVA fusion molecules were kindly provided by
Aliasger Salem and George Weiner (University of Iowa); they resulted in
IFN-a production as expected from previous published data (22) and our
own data (data not shown). Rabbit anti-OVA was prepared by Protein
G chromatography (Repligen, Waltham, MA) from the sera of OVAimmunized rabbits (contracted at Covance, Princeton, NJ). OVA ICs
were made at Ag/Ab (mg/ml) ratios of 1:10 or 1:5.
Cytokine assays
IFN-a was measured by ELISA (PBL, Piscataway, NJ). A BD CBA Mouse
Inflammatory Cytokine kit was used to measure other cytokines (BD
Biosciences, San Jose, CA).
Abs
Anti-CD11c (HL3), anti-CD19 (1D3), anti-B220 (RA3-6B2), and antiCD11b (M1/70) mAbs were obtained from BD Pharmingen (San Diego
CA). Anti-PDCA1 (120G8) was a gift of Georgio Trinchieri (formerly of
Schering-Plough). Chicken anti-Sendai, anti-chicken DyLight 488 or 594,
and sheep anti-LC3 were purchased from Abcam (Cambridge MA). Antichicken HRP was purchased from Thermo Scientific (Waltham, MA).
Donkey anti-rabbit Alexa Fluor 568 and donkey anti-sheep Alexa Fluor 647
were purchased from Life Technologies (Eugene, OR). Anti–MOMA-1 and
anti-MARCO were purchased from AbD Serotec (Kidlington, U.K.).
Immunofluorescence and microscopy
Detecting SeV in the spleen. Spleens were harvested at various times
postinjection and analyzed as single-cell suspensions or placed into OCT
compound and frozen at 280˚C prior to sectioning (6–8 mm continuous
sections) and staining, as indicated. Thawed tissue sections were stained
with the indicated Abs prior to fixing with 4% paraformaldehyde. SeV was
stained using chicken anti-SeV Ab, followed by an anti-chicken secondary
Ab, as described above.
Type I IFN’s potent innate antiviral properties also have potentially deleterious local and systemic toxic effects that could cause
unnecessary cellular activation and immunopathology if left unbridled during resolving or secondary infections. This suggests
that a host would benefit by restricting IFN production to a first
line of defense (i.e., before an effective adaptive immune response
develops).
To investigate whether IFN-a is produced during a memory
response to virus delivered i.v., a cohort of mice was immunized
with SeV and then rechallenged 14–21 d later (memory response).
A similar cohort of mice received only a single viral immunization
(primary response). Both groups of mice were analyzed for circulating IFN-a levels in the sera over the next 2 d. During a primary challenge, IFN-a was detected in the sera as early as 5 h
postinjection and began to wane by 24 h. In contrast, the SeV
memory response failed to produce any detectable IFN throughout
the 48-h period (Fig. 1A).
Given the importance of pDCs in the production of type I IFN
during certain viral infections, we sought to determine whether
pDCs were involved in IFN-a production during a naive response
to SeV. We used a YFP-IFNb reporter mouse to indirectly measure IFN-b production. YFP-IFNb reporter mice were injected via
the tail vein with live SeV, and spleens were harvested 3 h postinjection. Beyond 3 h, our ability to accurately gate on pDCs was
compromised as the result of IFN-dependent induction of the pDC
lineage marker BST2 on other cells (24). In this analysis of
splenocyte populations, we found that, in a naive setting, IFN
production was limited to pDCs; cDCs (CD11c+ BST22), macrophages (CD11b+ CD11c2), B cells (CD19+), and cells negative
for all markers (the vast majority of which are T cells) did not
produce detectable YFP in response to SeV (Fig. 1B). In contrast,
by backgating on YFP+ cells, CD11cmid, BST2+ pDCs were
identified as the sole producers of IFN-b during a naive viral
challenge at 3 and 4 h (Fig. 1C). Interestingly, only ∼10–20% of
total pDCs in the spleen responded to the viral inoculation to
produce YFP. This limited percentage was not dose dependent,
because the percentage of YFP+ pDCs could not be increased by
increasing the dose of virus injected (data not shown).
The induction of other inflammatory cytokines by SeV also was
differentially regulated between a primary and memory response.
In particular, when measured in the sera 3 h after exposure, IL-6,
like IFN-a, was limited to the primary response, whereas MCP1
and IFN-g were significantly induced in both the primary and
memory responses (Fig. 1D).
A lack of type I IFN during a viral memory challenge might be
explained by an efficient and rapid clearance of virus before it is
detected by pDCs. Yet Western blot analysis of splenocytes shows
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FLT3L bone marrow cultures
The Journal of Immunology
3
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FIGURE 1. IFN-a production is absent during secondary exposure to SeV. (A) WT mice, previously immunized with SeV, were rechallenged with SeV
(memory) and compared with mice receiving a single injection (naive). IFN-a in the sera was measured at the indicated time points by ELISA. Repeated at
least four times with a total of n = 12 mice in each group. (B) YFP-IFNb mice were injected as in (A); at 3 h post-SeV challenge, spleens were analyzed by
FACS for YFP+ cells in pDCs (CD11cmid BST2+), cDCs (CD11c+ CD11b+), and B cells (CD19+ CD11b2). Each symbol (square, triangle, or circle) is
a mouse obtained from pooled experiments. The experiments were repeated five times, with seven to nine mice total/group. (C) FACS analysis of YFP+
versus ungated cells. Representative sample from (B). (D) Cytokine bead assay analysis of sera 5 h post-SeV challenge, as in (A). The experiment was
repeated twice with four to six mice in each group. (E) Western blot analysis of total splenocytes of primary and memory mice, as in (A). Representative of
one mouse, as in (A). *p , 0.05, **p , 0.01, *** p , 0.001. ns, not significant.
that the presence of SeV is remarkably similar between a primary
and memory response. Although about half of the initial inoculum
is cleared in a memory versus a primary setting, the kinetics of the
virus are similar in that they peak at 1 h and persist through 48 h
compared with no treatment (Fig. 1E).
The failure of pDCs to respond to SeV during a memory response
also may be explained by a lack of access to the virus. We and other
investigators found pDCs in the red and white pulp in spleens of
naive mice. In the white pulp, pDCs exist largely at the marginal
zone and within the T cell area. More detailed analysis of pDCs at
4
FcgRIIB PREVENTS pDC IFN-a PRODUCTION IN VIRAL MEMORY RESPONSE
Virus-specific Ab is required for IFN suppression during
a memory response
We found that, at 14–21 d postimmunization, mouse sera contained SeV-specific Ab, as determined by hemagglutination inhibition and an SeV-specific ELISA (data not shown). To investigate
whether Ab was necessary for IFN suppression during a memory
response, we repeated the primary and memory viral challenge
using mice unable to produce virus-specific Ab. HELmMT mice
express a transgenic BCR specific for hen egg lysozyme on an
IgM-deficient background. Interestingly, and in complete contrast
to WT mice, HELmMT mice did not suppress the IFN-a response
during a memory rechallenge (Fig. 3A).
To more directly determine whether virus-specific Ab was required for IFN-a suppression, a cohort of naive mice was passively
immunized by transfer of immune sera and then challenged with
SeV as before; IFN-a was measured in the serum as described
earlier. We found that immune sera was sufficient to prevent any
type I IFN production in response to SeV (Fig. 3B) and flu
(Supplemental Fig. 1). Lastly, colocalization of SeV and pDCs in
the spleens of mice 30 min following injection of preformed SeV
ICs suggests again that the inhibition of IFN by pDCs is not due to
a lack of access by pDCs (Fig. 3C). Combined, these results
suggest that inhibition of IFN production by pDCs during a viral
memory response requires the presence of virus-specific Ab.
Transgenic expression of hFcgRIIA on pDCs permits IFN-a
production during a memory response
We previously described the dominant expression of FcgRIIB on
murine pDCs and that the limited expression of FcgRs prevented
Ag presentation of OVA ICs to T cells (25). We designed a series
of experiments to directly examine a role for FcgRIIB in the inhibition of IFN-a through the use of the KO mouse. Although we
show that, in both a memory response and by directly injecting
SeV ICs, there is an increase in IFN-a in the sera in the absence of
FcgRIIB, we could not determine whether pDCs were responsible
for this effect because the FcgRIIB-KO mouse is not pDC specific
(Supplemental Fig. 2A, 2B). In vitro experiments coculturing WT
and FcgRIIB-KO pDCs with SeV ICs showed that both WT and
FcgRIIB-KO pDCs fail to produce IFN-a; however, the latter
is due to the inability of FcgRIIB-KO pDCs to acquire ICs
(Supplemental Fig. 2C, 2D).
Being unable to determine the direct requirement for FcgRIIB
in vivo, we determined whether expressing the activating FcgRIIA
would reverse the IFN suppression seen in WT responses.
Transgenic mice expressing the hFcgRIIA receptor, an activating
Fc receptor present only in primates, were compared with WT
mice in a memory SeV response. Analysis of the pDCs from these
transgenic mice showed the transgene expressed on pDCs (25). In
contrast to WT mice, FcgRIIA-transgenic mice responded with
limited, although significant, IFN-a concentrations in the sera (Fig
4A). To determine whether pDCs were responsible for producing
the IFN-a measured in sera, YFP-IFNb mice were crossed to
FcgRIIA-transgenic mice, and the memory experiment was repeated. Importantly, we found that, endowed with the presence of
an activating FcgR, pDCs are capable of inducing an IFN response
in a memory setting and, again, were the only cell involved in the
response at 3 h (Fig. 4B). Furthermore, direct injection of SeV ICs
into hFcgRIIA-transgenic mice resulted in IFN-a production,
contrasting the suppressed response in WT mice (Fig. 4C).
To directly examine the interaction between SeV ICs and pDCs,
we turned to an in vitro system. BM-expanded and isolated pDCs
were enriched and incubated with SeV ICs overnight and sampled
for IFN-a production. Although both WT and hFcgRIIA pDCs
responded equally to virus alone (Fig. 4D), only the transgenic,
activating FcgR bearing pDCs produced IFN in response to viral
ICs (Fig. 4E). This was not the result of an increased IC binding
potential by the transgenic pDCs over the WT, because both bound
SeV ICs equally (Fig. 4F, 4G).
Thus, taken together, the data suggest that the prevention of an
IFN-a response to SeV ICs is not because neutralizing Ab renders
the complex inert; rather, the dominant expression of FcgRIIB
on murine splenic pDCs prevents IFN-a production during viral
memory responses.
Splenic pDC IC binding occurs via FcgRIIB and prevents
trafficking to TLR9 signaling compartments
SeV ICs did not induce IRF-7 translocation to the nuclei in WT
pDCs (data not shown), suggesting that the block to IFN-a production occurred upstream, potentially by derailing trafficking of
the IC cargo to the canonical TLR signaling endolysosome. We
reported previously that murine pDCs bind and internalize OVA
ICs via FcgRIIB exclusively (25) and fail to reach the proteolytic
endosome required for Ag processing. Therefore, we similarly
assessed the intracellular trafficking of OVA ICs and TLR ligand–
containing ICs (OVA-CpGB ICs), which may interact directly
with endosomal TLRs in pDCs and be internalized via an
FcgRIIB-independent manner.
First, to assess the FcgR dependence of OVA-CpG IC binding,
we compared the IC binding potential of WT, FcgRIIB-deficient,
and FcgR-null splenic pDCs by confocal microscopy. We found
that, in the absence of FcgRIIB, OVA IC binding was significantly
reduced on splenic pDCs and equaled that of the FcgR-null pDCs
(Fig. 5A, 5B) confirming previous experiments (Supplemental
Fig. 2D). The addition of a TLR9 moiety (CpG B) on the Ag
did not alter the FcgRIIB dependence (Fig. 5B, right panel),
strongly suggesting that WT pDCs exclusively bind ICs through
the inhibitory FcgRIIB.
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the marginal zone showed that pDCs were interweaved between
Marco+ and Moma-1+ cells, clearly delineating the zone by
forming short chains of pDCs (Fig. 2).
To address spatiotemporal questions and pDC accessibility to
injected virus during a memory response, previously immunized
mice were rechallenged with SeV, and cross-sections of spleens
were stained at various time points. We found that, during a memory
response, SeV was largely present at the marginal zone associated
equally between Marco+, Moma-1+ macrophages and marginal
zone B cells at 20 and 35 min, moving eventually to follicular
B cells at 50 and 75 min (Fig. 2A, 2C, left panel). En route to
follicular B cells, SeV colocalized with pDCs at the marginal zone
between 20 and 50 min. Surprisingly, this analysis indicated that
only ∼10% of SeV is present on pDCs during a memory response.
We wondered how this compared with a primary response where
the interaction between pDCs and SeV is known to result in a strong
IFN-a response. SeV was injected as above into the tail veins of
naive mice, and spleens were harvested at the indicated time points.
Interestingly, we found that, at early time points (20 min), SeV in
a primary setting followed nearly the same route as in a memory
setting, colocalizing with Marco+ and Moma-1+ cells. However,
SeV trafficking differences between the memory and primary
responses became evident by 40 min and more apparent by 75 min.
SeV traveled further into the B cell area than in a memory setting
and did not persist at the marginal zone (Fig. 2B, 2C, right panel).
Interestingly, the early access to SeV by pDCs was nearly equivalent
between the memory and primary settings (Fig. 2D).
Taken together, these data suggest that, unlike in a primary response, the interaction between SeV and pDCs during a memory
response at the splenic marginal zone fails to induce an IFN response.
The Journal of Immunology
5
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FIGURE 2. Splenic pDCs interact with SeV at the marginal zone. Memory (A) and primary (B) mice were compared for SeV (green) colocalization at the
indicated time points with various cellular populations (red). Sendai virus was detected using chicken anti-SeV followed by anti-chicken Alexa 488 or
Alexa 594. Cellular subsets were detected by using anti–moma-1 biotin with streptavidin Alexa 488, anti-Marco PE, anti-BST2 Alexa 488, and CD45R
Alexa 647. All images are pseudocolored with SeV in green and cell subset in red. Original magnification 340. One representative experiment from two
experiments is shown. (C and D) Quantification of (A) and (B), respectively, for each cell population. Between 5 and 10 sections were quantified for each
time point and cell population. One representative experiment of two is shown with similar patterns. (D) Expansion of the white box in (A) (left panel).
Quantification of 20 and 35 min (right panel). SeV was detected using chicken anti-SeV followed by anti-chicken Alexa 594 and anti-BST2 Alexa 488.
Original magnification 386.6 was achieved with an optical zoom. n.s., not significant.
6
FcgRIIB PREVENTS pDC IFN-a PRODUCTION IN VIRAL MEMORY RESPONSE
We next sought to examine the intracellular trafficking pathways
that may prevent viral ICs from reaching a TLR signaling com-
Discussion
In the current study, we described one mechanism by which the
innate IFN-a response is averted upon secondary exposure to virus. We determined that the presence of circulating virus-specific
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FIGURE 3. Virus-specific Ab is required for IFN-a suppression. (A) WT
mice were compared with HELmMT mice for IFN-a secretion in the sera by
ELISA after i.v. re-exposure to SeV in a memory response. (B) WT naive
mice were injected with SeV or preformed SeV ICs, and IFN-a concentrations were measured in the sera by ELISA. Experiment was repeated twice
with a total of six mice. (C) As in (B), sections of spleens were stained for SeV
and BST2+ cells at 30 min postinjection. Sendai virus was detected using
chicken anti-SeV followed by anti-chicken Alexa 594. BST2 was detected
using anti-BST2 Alexa 488. Images are pseudocolored with BST2 shown in
red and SeV in green. Original magnification 360. Quantification of pooled
experiments from four and five mice (SeV and SeV IC, respectively) shown
from two experiments. **p , 0.01, *** p , 0.001. n.s., not significant.
partment in pDCs. OVA ICs and OVA-CpG ICs were incubated
with splenic pDCs, and colocalization with various intracellular
markers was assessed by immunofluorescence and confocal microscopy.
IFN-a production by TLR9 ligands requires engagement of
IRF7 in Lamp2+, LysoTracker+ endosomes (26). We examined the
trafficking of ICs in splenic pDCs and found that OVA ICs incubated with WT splenic pDCs were not colocalized with LysoTracker, as was expected from previous work (25). However, the
transgenic expression of activating FcgRIIA significantly increased colocalization of ICs and LysoTracker (Fig. 5C, 5D), and
the results were confirmed with studies using Lamp1 (Fig. 5E, 5F).
Interestingly, when CpG was present in the cargo, WT pDCs
delivered more ICs to LysoTracker. Furthermore, the FcgRIIAdependent increase in the colocalization of ICs to acidic compartments was supported by Lamp1 (Fig. 5E, 5F). Thus, FcgRIIBinternalized ICs access a spatiotemporal endosomal pathway distinct
from activating FcgRs that inefficiently access the acidic LAMP1+
IRF7+ IFN-a signalosome, thus preventing IFN-a production.
Recently published work identified a noncanonical IFNogenic
pathway involving the LC3-associated phagosome. In these studies
using BM-derived murine pDCs, the investigators found that Abcoated beads were internalized via LC3, an autophagy-related
protein, and that the recruitment of LC3 was Fc g-chain dependent (27); both were functionally required for IFN-a production.
We performed a similar experiment using splenic pDCs and soluble ICs. Recruitment of LC3 was measured by calculating the
integrated density of LC3 fluorescence at each IC particle (ROI).
We controlled for background fluorescence by measuring the same
parameters and ROIs on images pivoted 90˚. Our experiments
yielded results consistent with an LC3-mediated internalization
through activating FcgRs (Fig. 5G, 5H). However, we found that
FcgRIIB did not recruit LC3 above background when using either
WT or g-chain–deficient pDCs. Indeed, only when an activating
FcgR was transgenically expressed did we detect LC3 recruitment
on splenic pDCs. Furthermore, the presence of a TLR moiety
coupled to OVA did not change LC3 recruitment in the WT pDCs,
supporting the conclusions of Henault et al. (27) that LC3 recruitment is initiated by the IgG component of the IC and not by
its cargo. Our results show that ICs internalized through the inhibitory FcgRIIB do not lead to LC3 recruitment.
Taken together, our data demonstrate that FcgRIIB on splenic
murine pDCs is required to internalize ICs and that this internalization occurs outside of LC3+ compartments and results in IC
trafficking away from the LysoTracker+ endolysosomes where
IRF7 and TLR9 signal. In contrast, ICs internalized through activating FcgRs access an LC3-mediated internalization pathway
that leads to LAMP1+, LysoTracker+ endolysosomes, enabling
MyD88 signaling and IRF7 nuclear translocation. In human pDCs,
in which both activating and inhibitory FcgRs are coexpressed, as
modeled in this study, the amplitude of the IFN-a signal would be
balanced by the relative functional expression of these opposing
receptors. This situation may be relevant to primates, including
humans, in which the activating FcgRIIA is dominant. In mice, the
exclusive expression of FcgRIIB should prevent untoward IFN-a
production during recurrent or chronic exposure to a virus, in
which the presence of adequate functional viral-specific neutralizing Abs enable benign clearance and limited viral replication
and systemic spread.
The Journal of Immunology
7
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FIGURE 4. IFN production in response to ICs is rescued by transgenic expression of FcgRIIA. (A) IFN-a concentrations in the sera of WT and
hFcgRIIA-transgenic mice were compared during a memory response, using ELISA, at the indicated time points. Pooled experiments of 6–12 mice/group
are shown. (B) Dual transgenic FcgRIIA+ YFP-IFNb reporter mice were compared with WT YFP-IFNb reporter mice at 3 h postrechallenge. The percentage of YFP+ pDCs (CD11c+ BST2+) was determined by FACS (n = 4–8 mice/group). (C) WT or hFcgRIIA-transgenic mice were injected with
preformed SeV ICs. Sera were analyzed for IFN-a, using ELISA, at the indicated time points. Pooled experiments of at least six mice are shown. (D) BMexpanded WT or hFcgRIIA-expressing pDCs were incubated overnight with SeV, and the supernatant was analyzed for IFN-a. Repeated twice in duplicate
and triplicate. (E) As in (D), except that enriched BM pDCs were incubated with SeV ICs. (F) Splenic pDCs from WT or hFcgRIIA mice were incubated
with preformed SeV ICs for 30 min and analyzed by confocal microscopy. Sendai virus was detected using chicken anti-SeV followed by anti-chicken
Alexa 594. B220 was detected with anti-B220 488. Image is pseudocolored with red for SeV, DAPI in blue, and B220 in white. (Figure legend continues)
8
FcgRIIB PREVENTS pDC IFN-a PRODUCTION IN VIRAL MEMORY RESPONSE
Ab and the dominance of the inhibitory FcgR on pDCs are required for preventing the potentially toxic side effects of systemic
IFN. Thus, the humoral immune memory response functions to
temper the innate response upon re-exposure to virus.
Our work also describes a molecular pathway that differentiates
activating and inhibitory FcgRs in their ability to traffic ICs for
TLR sensing. We took several approaches, most of which were
technically challenging, including using TLR9 Abs to demonstrate
Original magnification 3100. (G) Quantification of images as in (F) were analyzed for the average number of SeV particles/cell/image. Pooled data from
two independent experiments, with .50 cells analyzed per group. *p , 0.05, **p , 0.01, *** p , 0.001. n.s., not significant.
Downloaded from http://www.jimmunol.org/ at The University of Iowa Libraries on April 2, 2015
FIGURE 5. Splenic pDCs internalize ICs via FcgRIIB and traffic particles to early endosomes. (A) A representative example of splenic pDCs binding OVA
ICs. (B) Indicated ICs were incubated with splenic pDCs for 30 min and analyzed for IC binding by confocal microscopy. OVA IC and OVA CpG IC graphs
were generated from at least two experiments. Each symbol represents the average number of IC particles/cell/image. (C) Representative example of WT and
hFcgRIIA-transgenic splenic pDCs incubated with OVA ICs and stained for LysoTracker (green) and anti-rabbit OVA ICs (red). (D) Quantification of experiments
provided in (C), showing integrated density of LysoTracker at IC+ ROI. Three pooled experiments are shown, with .150 IC particles analyzed. (E) Representative
example of WT or hFcgRIIA-transgenic splenic pDCs incubated with OVA ICs (red) and LAMP1 (green) and DAPI (blue). (F) Quantification of experiments as
in (E) for integrated density of LAMP1 at IC+ ROI. Two pooled experiments are shown. (G) Representative example of WT and hFcgRIIA-transgenic splenic
pDCs incubated with OVA ICs and stained for LC3 (green) and anti-rabbit OVA ICs (red). (A), (C), (E), and (G), original magnification 3100. (H) Quantification
of experiments as in (G) for integrated density of LC3 at IC+ ROI. Two pooled experiments are shown. *p , 0.05, **p , 0.01, *** p , 0.001.
The Journal of Immunology
covered by day 7 post-SeV injection, suggesting that they had
cleared the virus by days 14 and 21 (data not shown). However,
during the course of our studies, we found that there was a tendency for previously SeV-immunized mice to respond less robustly to unrelated stimuli (not statistically significant, data not
shown). This tendency may have contributed to the absence of
a response to SeV in memory mice. However, we also found that
passive immunization with antisera was sufficient to suppress the
IFN response in naive mice (Fig. 3B) and that the transgenic expression of activating FcgRs could reverse the IFN suppression
in a memory setting with presumably equally exhausted mice
(Fig. 4).
Our current study highlights the dominance of FcgRIIB on pDCs
as a mechanism to prevent IFN-a production by TLR-bearing ICs.
Systemic infections elicit IFN-a from pDCs and provide the host
with a protective, potent, and immediate source of IFN-a. In the
setting of a well-orchestrated adaptive memory response, this innate production of IFN-a is mitigated by circulating virus-specific
Abs and through inhibitory FcgRs on pDCs. If pDCs express an
FcgR repertoire equal to that of their cDC counterparts, secondary
infections would lead to unnecessary IFN-a production and elicit
an exacerbated immune response that could lead to untoward
consequences of injury and/or autoimmunity.
Alternatively, a study by Honke et al. (33) highlights another
possible mechanism for halting IFN-a production in a memory
setting. They found that IFN-a resistance by MOMA-1+ macrophages during primary viral responses is required for the mounting
of a protective adaptive immune response. By overexpressing
Usp18, an inhibitor of IFNAR signaling, MOMA-1+ macrophages
at the marginal zone capture virus and allow viral replication for
presentation to T and B cells. Thus, during memory responses,
a synergistic action to improve upon the memory response could
be aided by viral IC presentation and the viral replication allowed
by the absence of IFN.
The FcgRIIB-specific means of controlling pDC-derived IFN-a
also may be important during chronic infections when circulating
viral ICs may be present. In a classic model of chronic lymphocytic choriomeningitis virus infection, Zuniga et al. (34) found
that pDCs were prevented from making IFN in response to CpG
30 d postinfection with lymphocytic choriomeningitis virus. This
time point correlates with the presence of neutralizing Abs, as
gauged by Bergthaler et al. (35). It is possible then that, in this
model of chronic infection, IFN-a production is suppressed as
a result of circulating viral ICs being sensed through FcgRIIB on
pDCs.
Human pDCs were shown to dominantly express the activating
receptor FcgRIIA (36). Nevertheless, evidence that human pDCs,
like mice, exhibit an Fc-dependent inhibition of IFN-a production
was described previously (37, 38), and it may have important
implications for vaccine development. In one prominent example, a Merck-designed adenovirus-based HIV vaccine (Ad5/HIV)
showed an increase in HIV susceptibility in patients with preexisting Abs to adenovirus (39). A separate study, using a systems analysis approach, identified the production of type I IFN as
the most significant difference between those with and without
pre-existing Abs to adenovirus (40). In another example, the
whole inactivated influenza vaccine elicits a strong IFN-a production and a potent adaptive immune response (41), whereas the
split vaccine mediates immunity through Fc receptors and prevents an IFN-a response (41, 42). Not surprisingly, the latter is far
less immunogenic, and children without pre-existing flu Abs require two vaccinations for efficacy. These studies point to the
perils involved in mounting an adaptive immune response in the
absence of an innate immune response. In the case of the Ad5/HIV
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that OVA-CpG ICs internalized by WT pDCs failed to colocalize
with TLR9, whereas hFcgRIIA-expressing pDCs showed remarkable colocalization (data not shown). However, staining with
TLR9-specific Abs was too weak for rigorous quantitative microscopy. In a second approach, we reasoned that pulsing pDCs
with CpG-FITC (InvivoGen, San Diego CA) would delineate
a pathway known to engage TLR9 and lead to IFN-a production.
However, these experiments were also characterized by unreliable
staining. Thus, we took advantage of well-described studies that
reveal the requirement of acidification for TLR9 activation and
IRF7 recruitment (26, 28) and capitalized on the highly reproducible staining that we achieved with LysoTracker, which reliably stains the acidic lysosomal organelles. Using this approach,
we found that FcgRIIB on splenic pDCs was required for binding
viral ICs and that subsequent internalization failed to deliver ICs
to LysoTracker+ compartments. These results were confirmed by
similar findings with Lamp1 and Lamp2. In contrast, the transgenic expression of an activating FcgR was sufficient to restore
IFN-a production and delivered ICs to LysoTracker+ Lamp1 and
Lamp2+ endolysosomes.
A recent study also described a noncanonical, autophagy-related
pathway that is alternatively used by activating FcgRs for IFN-a
induction by DNA-containing ICs (27). This pathway recruits LC3
to the IC in an IgG-dependent manner. Fig. 5 shows that LC3 was
not recruited by WT splenic pDCs or by g-chain–KO pDCs, but
only when the activating FcgR was transgenically expressed.
Taken together, these studies suggest that activating and inhibitory
FcgRs are discriminating pathways that modulate TLR sensing by
directing ICs either toward or away from TLR-sensing compartments. Furthermore, pDCs, by dominantly expressing the inhibitory FcgR, are prone to suppress the IFN-a response to ICs.
Our work supports the findings of Henault et al. (27) that LC3
is used by activating FcgRs to delivery cargo to TLR-sensing
compartments. However, they showed that BM-expanded WT
pDCs do recruit LC3 to internalized ICs in a g-chain–dependent
manner. Our studies consistently identified a lack of activating
FcgRs on murine splenic pDCs. This apparent conflict could be
due to the source of pDCs and contaminating cDCs. We found that
expanding BM with FLT3L to obtain pDCs inconsistently altered
FcgR expression compared with splenic pDCs. B220 and CD11b,
markers used in the literature to isolate pDCs and cDCs from
FLT3L cultures, were often not mutually exclusive or were suspected of containing a large percentage of cDC precursors, as has
been reported (29). By using primary splenic pDCs to investigate
the FcgRIIB-dependent requirement for binding, internalizing,
and intracellular trafficking of ICs, we circumvented the problems
that might arise by exposing dendritic cells to FCS and an inflammatory cytokine, such as FLT3L.
Our major finding that IFN-a production by splenic pDCs was
prevented during a memory response might have been explained
by a lack of access to the virus during transport through the spleen
in a memory setting. However, we found that, in both primary and
memory responses, pDCs had comparable access to the virus.
Indeed, it may be possible that the same molecular mechanisms
are directing traffic in both settings (i.e., complement by acting
with natural IgG and IgM Ab in the primary response and with
virus-specific IgG Ab in the memory) (30–33), which ultimately
delivers virus to follicular B cells for trafficking and presentation
by follicular dendritic cells.
Other mechanisms that may have contributed to the prevention
of an IFN response during a memory setting may include pDC
“exhaustion” due to low-level viral exposure (34). We found by
Western blot that SeV did not persist in the spleens of mice by day
14 (Fig. 1E), and in naive mice we found that weight was re-
9
10
FcgRIIB PREVENTS pDC IFN-a PRODUCTION IN VIRAL MEMORY RESPONSE
Acknowledgments
We thank the members of the Clynes laboratory, including Amy Bergtold,
Bitao Liang, and Dharmesh Desai, for advice and technical support. We
appreciate the support of Core Facilities at Columbia University Medical
Center: 1) The Columbia Center for Translational Immunology Flow Core
for both flow analysis (LSR II) and flow sorting [BD Influx II; (R. C.,
principal investigator)], and 2) We are very grateful for the tremendous
guidance that the staff of The Confocal and Specialized Microscopy Core
provided, specifically Theresa Swayne, Ph.D.; Adam White, Ph.D.; Jason
Vevea; and Cedric Espenel, Ph.D.
Disclosures
The authors have no financial conflicts of interest.
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