KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 264
KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 264 KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 264 GILLES LAVERNY Identification of a potent and safe vitamin D receptor agonist for the treatment of inflammatory bowel disease Doctoral Dissertation To be presented by permission of the Faculty of Natural and Environmental Sciences of the University of Kuopio for public examination in Auditorium L21,Snellmania Building, University of Kuopio,on Thursday 10th December, at 3 p.m. Department of Biosciences University of Kuopio KUOPIO YLIOPISTO KUOPIO 2009 Distributor Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 207 87 2200 Fax +358 17 163 410 http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.shtml Series Editors: Professor Pertti Pasanen, Ph.D. Department of Environmental Science Author’s address: Department of Biosciences University of Kuopio P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 40 355 3084 E-mail: [email protected] Supervisors: Professor Carsten Carlberg, Ph.D. Department of Biosciences University of Kuopio Luciano Adorini, M.D. Intercept Pharmaceuticals Corciano (Perugia), Italy Reviewers: Professor Annemieke Verstuyf, Ph.D. Laboratorium voor Experimentele Geneeskunde en endocrinologie (Legendo) Leuven Belgium Professor Alberto Muñoz, Ph.D. Instituto de Investigaciones Biomédicas "Alberto Sols" Madrid, Spain Opponent: Professor Hans van Leeuwen, Ph.D. Department of Internal Medicine Erasmus MC Rotterdam, Netherlands ISBN 978-951-27-1402-5 ISBN 978-951-27-1297-7 (PDF) ISSN 1235-0486 Kopijyvä Kuopio 2009 Finland Laverny, Gilles. Identification of a potent and safe VDR agonist for the treatment of IBD. Kuopio university publications C. natural and environmental sciences 264. 2009. 135 p. ISBN 978-951-27-1402-5 ISBN 978-951-27-1297-7 (PDF) ISSN 1235-0486 Abstract The bioactive form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), is a secosteroid hormone that binds to the vitamin D receptor (VDR), a member of the nuclear receptor superfamily expressed in many cell types, and modulates a variety of biological functions. 1α,25(OH)2D3 is essential for bone and mineral homeostasis but also regulates growth and differentiation of multiple cell types, and displays immunoregulatory and anti-inflammatory activities. The anti-proliferative, pro-differentiative, anti-bacterial, immunomodulatory and anti-inflammatory properties of synthetic VDR agonists could be exploited to treat a variety of chronic inflammatory and autoimmune diseases, such as benign prostatic hyperplasia (BPH) and inflammatory bowel diseases (IBDs). We have analyzed the capacity of VDR agonists to treat BPH, a complex syndrome characterized by a static component related to prostate overgrowth, a dynamic component responsible for urinary irritative symptoms, and an inflammatory component. Data in this thesis demonstrate that VDR agonists, and notably elocalcitol, reduce the static component of BPH by inhibiting the activity of intraprostatic growth factors downstream of the androgen receptor, the dynamic component by targeting the RhoA/ROCKpathway in prostate and bladder cells, and the inflammatory component by targeting the NF-κB pathway. Inflammatory bowel diseases (IBDs) comprise Crohn’s disease (CD) and ulcerative colitis (UC), both characterized by relapsing inflammation of the gastrointestinal tract due to unbalanced activation of the mucosal immune system in genetically predisposed individuals. In addition to genetic factors, including VDR gene polymorphisms, environmental factors are also implicated in IBD development and notably vitamin D deficiency, suggesting VDR agonists as potential therapeutic agents. 1α,25(OH)2D3 efficacy has been shown in different models of experimental colitis; but its calcemic liability could pose safety issues in patients and limit its clinical use. Inhibition of TLR4-mediated TNF-α secretion in PBMCs from healthy donors was used to identify the less calcemic 1α,25(OH)216-ene-20-cyclopropyl-vitamin-D3 (BXL-62) as a VDR agonist with superior TNF-α inhibitory activity (IC50:1.5x10-15M), compared to 1α,25(OH)2D3 (IC50:8x10-9M). This higher anti-inflammatory potency from BXL-62 compared to 1α,25(OH)2D3 was confirmed in PBMCs from IBD patients. In addition, PBMCs from CD and UC patients activated by different TLR agonists are equally sensitive to the anti-inflammatory properties of BXL-62. Other pro-inflammatory cytokines, such as IL12/23p40 and IL-6, were also inhibited both at mRNA and protein levels. BXL-62 induced VDR primary response genes, like CYP24 and CAMP, at lower concentrations than 1α,25(OH)2D3, indicating VDR-mediated effects. The strong potency of BXL-62 can be explained by a different metabolism resulting in the accumulation of its stable 24-oxo metabolite, which shows antiinflammatory properties superimposable to the parent compound. The efficacy of BXL-62 in experimental IBD was shown in DSS-induced colitis. Intrarectal administration of BXL-62 induced faster recovery of clinical symptoms of colitis at normocalcemic doses, and its beneficial effects were significantly superior to 1α,25(OH)2D3. The results obtained in this thesis confirm the beneficial effects of VDR agonists in chronic inflammatory and autoimmune disorders like IBD and BPH and suggest BXL-62 as a potentially promising compound for IBD treatment. Medical subject headings: autoimmunity, benign prostate hyperplasia, toll like receptor, antigenpresenting cell, colitis, inflammatory response, elocalcitol, proinflammatory cytokines, metabolite, lamina propria mononuclear cells, peripheral blood mononuclear cells. Acknowledgements I would like to express my sincere thanks to all people who supported me professionally and privately during the last years to achieve this milestone in my life and create this thesis. I acknowledge the department of Biosciences from the University of Kuopio, BioXell S.p.a. and Intercept Pharmaceuticals from Milan for the friendly and inspiring environment where it is pleasure to study and to do research. I am grateful to the European Union Marie Curie Research Trainings Network “NucSys” which supported this work. I would like to express my deepest gratitude to my principal supervisors Prof. Carsten Carlberg and Luciano Adorini, M.D. for the possibility to do my doctoral studies under their supervision. Thank you for your support, advices and time that lead to the success of this work. I acknowledge Giuseppe Penna for his technical and scientific guidance as well as his friendship. Prof. Alberto Muñoz and Prof. Annemieke Verstuyf, the official pre-examiners of this thesis, are acknowledged for their valuable comments and advices, which helped to improve this thesis. I would like to thank Dr. Arja Hirvonen and Taru Nylund who helped me a lot for all the administrative tasks to get the thesis accepted. I would like to thank all my co-authors because without them, none of this work would have been achieved. Dr. Milan Uskokovic, Dr. Hubert Maehr and Prof. Satya Reddy for sharing with me their knowledge in chemistry and metabolism of VDR agonists, Elisa Corsiero, Laura Giudici and Thomas Lemeur for their help in performing the experiments and their daily good mood and Silvio Danese, M.D. and its group, who introduced me to the gastrointestinal field and shared their knowledge. I would like to acknowledge all the members of NucSys for enjoyable and productive meetings, lots of helpful discussions and all the support, especially Fabio, Carole, Pedro(s), Marcin, Claudia and Thomas. A special acknowledgement to Tatjana who hosted me in Kuopio and always support me during these three years, and to Tom for our lovely dinners and discussions. I would like to thank people in the Laboratory of Computational Biology from the University of Luxembourg, specially Aleksandra, Anna and Janine for the kind atmosphere in the lab. A special thanks to the personnel from BioXell and the Humanitas Institute from Milan for creating a friendly and productive working environment. To all my friends scattered around the world who were always present despite the distance, especially Julien, Bertrand, Manu, Pierre and Béatrice. I dedicate my doctoral thesis to my parents and grandmother, for their eternal support during the totality of my studies and this even in times of trouble and difficulties. To my grandfather, who is certainly taking care of me. Finally to you, that shared my life, for your love and unlimited support. Abbreviations 1α,25(OH)2D3 1α,25-dihydroxy-vitamin D3 25(OH)D3 25-hydroxy-vitamin D3 AF activation function APC antigen presenting cell BcR B cell receptor BPH benign prostatic hyperplasia BW body weight BXL-143 1α,25(OH)2-16-ene-20-cyclopropyl-24-oxo-vitamin D3 BXL-62 1α,25(OH)2-16-ene-20-cyclopropyl-vitamin D3 CAMP cathelicidin anti-microbial peptide CARD caspase recruitement domain CD Crohn's disease CD cluster of differentiation CDK cyclin dependent kinase CoA coactivator CoR corepressor COX cyclooxygenase CpG 5' cytosine-phospho-guanine CTL cytotoxic T lymphocyte CYP24A1 24-hydroxylase CYP27B1 1α-hydroxylase CYP450 cytochrome P450 DAI disease activity index DBD DNA-binding domain DC dendritic cell DNA desoxyribonucleic acid DSS dextran sodium sulfate DTT 1,4-dithiothreitol EAP experimental autoimmune prostatitis FBS fetal bovine serum FC-1 fetal clone 1 FcR fragment crystalisable receptor FGF fibroblast growth factor GM-CSF granulocyte macrophage colony stimulating factor GWA genome wide association HLA human leukocytes antigen IBD inflammatory bowel disease iDC immature dendritic cell IFN interferon Ig immunoglobulin IL interleukin ILT immunoglobulin-like transcripts iNOS inducible nitric oxide synthase ITAM immunoreceptor tyrosine-based activation motifs JNK jun N-terminal Kinase KH1060 20-epi-22-oxa-24α-homo-26,27-dimethyl-1α,25(OH)2D3 LBD ligand-binding domain LPMC lamina propria mononuclear cell LPS lipopolysaccharide LRR leucine-reach repeat domain LUTS lower urinary tract symptoms MAP mitogen activated protein MCP monocyte chemotactic protein mDC mature dendritic cell M-DC myeloid dendritic cell MDP muramyl dipeptide MHC major histocompatibility complex MLR mixed lymphocyte reaction MTD maximal tolerated dose MW molecular weight NFAT nuclear factor of activated T cells NF-kB nuclear factor κB NLR nucleotide binding site (NBS)–leucine-rich repeats NLS nuclear localisation signal NO nitric oxyde NOD non obese diabetic Nod nucleotide oligomerisation domain NR nuclear receptor PAMP pathogen-associated molecular pattern PBMC peripheral blood mononuclear cell PBS phosphate-buffered saline P-DC plasmacytoid dendritic cell PGE2 prostaglandin E2 PGN peptidoglycan PMN polymorphonuclear leukocytes PRR pathogen recognition pattern PTH parathyroid hormone RNA ribonucleic acid ROR RAR-related orphan receptor RT reverse transcription RXR retinoid X receptor STAT signal transducer and activator of transcription T1D type 1 diabetes TcR T cell receptor TGF transforming growth factor Th T helper cell TLR toll like receptor TNBS 2,4,6-trinitrobenzene sulfonic acid TNF tumor necrosis factor Treg regulatory T cell TRPV transient receptor potential vanilloid TX-527 19-nor-14,20-bisepi-23-yne-1α,25(OH)2D3 UC ulcerative colitis VDR vitamin D receptor VDRE vitamin D response element ZK156979 22-ene-25-oxa-1α,25(OH)2D3 List of original publications This thesis is based on the following publications referred to in the text by roman numerals (IV): I. Penna G, Fibbi B, Amuchastegui S, Cossetti C, Aquilano F, Laverny G, Gacci M, Crescioli C, Maggi M, Adorini L Human Benign Prostatic Hyperplasia Stromal Cells as Inducers and Targets of Chronic Immuno-mediated Inflammation. (2009) Journal of Immunology 182(7):4056-64 II. Penna G, Fibbi B, Amuchastegui S, Corsiero E, Laverny G, Silvestrini E, Chavalmane A, Morelli A, Sarchielli E, Vannelli GB, Gacci M, Colli E, Maggi M, Adorini L. The vitamin D receptor agonist elocalcitol inhibits IL-8-dependent benign prostatic hyperplasia stromal cell proliferation and inflammatory response by targeting the RhoA/Rho kinase and NF-kB pathways. (2009) Prostate 69(5):480-93 III. Laverny G, Penna G, Uskokovic M, Marczak S, Maehr H, Jankowski P, Ceailles C, Vouros P, Smith B, Robinson M, Reddy GS, Adorini L. Synthesis and Antiinflammatory Properties of 1alpha,25-Dihydroxy-16-ene-20-cyclopropyl-24-oxovitamin D(3), a Hypocalcemic, Stable Metabolite of 1alpha,25-Dihydroxy-16-ene-20cyclopropyl-vitamin D(3). (2009) Journal of Medicinal Chemistry. 52(8):2204-13 IV. Laverny G, Penna G, Vetrano S, Correale C, Danese S, Adorini L Identification of a potent and safe VDR agonist for the treatment of inflammatory bowel disease. (2009) submitted V. Laverny G, Penna G, Vetrano S, Correale C, Danese S, Adorini L Toll like receptor4-dependent selective defect in IL-10 production by blood leukocytes from inflammatory bowel disease patients. (2009) Table of content ABSTRACT ........................................................................................................................................................... 3 ACKNOWLEDGEMENTS .................................................................................................................................. 5 ABBREVIATIONS ............................................................................................................................................... 7 LIST OF ORIGINAL PUBLICATIONS........................................................................................................... 11 TABLE OF CONTENT ...................................................................................................................................... 13 1. INTRODUCTION ........................................................................................................................................... 17 1.1 Overview of the immune system ................................................................................................................ 19 1.1.1 Components of the innate immunity .................................................................................................... 19 1.1.1.1 Toll like receptors..........................................................................................................................................19 1.1.1.2 Nucleotide oligomerisation domain-Like Receptors (NLR) ..........................................................................22 1.1.1.3 Complement ..................................................................................................................................................23 1.1.1.4 Fragment crystallizable Receptor (FcR) ........................................................................................................24 1.1.2 Cells of the innate immunity................................................................................................................ 25 1.1.2.1 Mucosal epithelia ..........................................................................................................................................25 1.1.2.2 Phagocytes.....................................................................................................................................................25 1.1.3 Components of the adaptive immunity ................................................................................................ 27 1.1.3.1 Major histocompatibility complex.................................................................................................................27 1.1.3.2 Major histocompatibility complex class I molecules .....................................................................................27 1.1.3.3 Major histocompatibility complex class II molecules ...................................................................................28 1.1.3.4 B cell receptor ...............................................................................................................................................28 1.1.3.5 T cell receptor................................................................................................................................................29 1.1.4 Cells of the adaptive immunity, the lymphocytes ................................................................................ 29 1.1.4.1 B cells ............................................................................................................................................................30 1.1.4.2 Effector T cells ..............................................................................................................................................31 1.1.4.3 Regulatory T cells .........................................................................................................................................35 1.1.5 Dendritic cells, a key role in innate and adaptive immunity ............................................................... 36 1.1.5.1 Inflammatory dendritic cells..........................................................................................................................37 1.1.5.2 Myeloid dendritic cells ..................................................................................................................................37 1.1.5.3 Plasmacytoid dendritic cells ..........................................................................................................................38 1.1.5.4 Tolerogenic dendritic cells ............................................................................................................................38 1.2 VDR and 1α,25(OH)2D3 ............................................................................................................................. 40 1.2.1 A brief history ..................................................................................................................................... 40 1.2.2 1α,25(OH)2D3 ..................................................................................................................................... 41 1.2.2.1 Synthesis of vitamin D ..................................................................................................................................41 1.2.2.2 Catabolism of vitamin D ...............................................................................................................................41 1.2.2.3 Vitamin D analogs .........................................................................................................................................43 1.2.3 VDR..................................................................................................................................................... 45 1.2.3.1 NR superfamily .............................................................................................................................................45 1.2.3.2 Classification .................................................................................................................................................45 1.2.3.3 Structural features..........................................................................................................................................46 1.2.3.4 Structure and functions of VDR ....................................................................................................................47 1.2.4 Target genes and biological role ........................................................................................................ 50 1.2.4.1 Calcium and phosphate homeostasis .............................................................................................................50 1.2.4.2 1α,25(OH)2D3 metabolism and catabolism.................................................................................................... 51 1.2.4.3 Vitamin D deficiency ....................................................................................................................................51 1.2.5 VDR-mediated non-calcemic activities ............................................................................................... 53 1.2.5.1 Regulation of cell proliferation and tumorigenesis ........................................................................................53 1.2.5.2 Regulation of the immune system .................................................................................................................54 1.2.6 Anti-inflammatory properties of VDR agonists ................................................................................... 55 1.2.6.1 Dendritic cells ...............................................................................................................................................55 1.2.6.2 T cells ............................................................................................................................................................58 1.2.6.3 Regulatory T cells .........................................................................................................................................60 1.2.6.4 Treatment of autoimmune diseases ...............................................................................................................62 1.3 Begnin prostate hyperplasia ........................................................................................................................ 64 1.3.1 Definition ............................................................................................................................................ 64 1.3.2 VDR agonists in BPH treatment ......................................................................................................... 64 1.3.2.1 Elocalcitol ameliorates experimental autoimmune prostatitis .......................................................................65 1.3.2.2 VDR agonists treat BPH-associated LUTS ...................................................................................................66 1.4 Inflammatory bowel disease ....................................................................................................................... 68 1.4.1 Diagnosis and clinical features........................................................................................................... 69 1.4.1.1 Epidemiology ................................................................................................................................................70 1.4.2 Etiology ............................................................................................................................................... 71 1.4.2.1 Environmental factors ...................................................................................................................................71 1.4.2.2 Genetic factors...............................................................................................................................................71 1.4.2.3 Immunological factors ...................................................................................................................................72 1.4.3 Current treatments .............................................................................................................................. 75 1.5 Vitamin D and inflammatory bowel disease ............................................................................................... 77 1.5.1 Vitamin D deficiency and VDR polymorphisms in IBD patients ......................................................... 77 1.5.2 VDR agonists in IBD treatment .......................................................................................................... 77 1.5.2.1 In vitro activity ..............................................................................................................................................77 1.5.2.2 In vivo activity ..............................................................................................................................................78 2. AIMS OF THE STUDY .................................................................................................................................. 80 3. MATERIALS AND METHODS .................................................................................................................... 81 3.1 VDR agonists .............................................................................................................................................. 81 3.2 Cell cultures ................................................................................................................................................ 83 3.2.1 Primary prostate cell lines .................................................................................................................. 83 3.2.2 Immortal cell lines .............................................................................................................................. 83 3.2.3 Peripheral blood mononuclear cells ................................................................................................... 84 3.2.4 Lamina propria mononuclear cells ..................................................................................................... 84 3.3 In vitro experiments .................................................................................................................................... 85 3.3.1 Mixed lymphocyte reaction ................................................................................................................. 85 3.3.2 TLR-activated PBMCs or LPMCs....................................................................................................... 85 3.3.3 BPH cell activation ............................................................................................................................. 86 3.3.4 Enzyme-linked immunosorbent assay (ELISA) ................................................................................... 86 3.3.5 Total RNA purification ........................................................................................................................ 87 3.3.6 cDNA synthesis ................................................................................................................................... 87 3.3.7 Real time PCR ..................................................................................................................................... 87 3.4 In vivo experiments .................................................................................................................................... 88 3.4.1 Mice .................................................................................................................................................... 88 3.4.2 Assessment of the MTD ....................................................................................................................... 88 3.4.3 Induction of experimental colitis......................................................................................................... 89 3.4.4 Administration of VDR agonists ......................................................................................................... 89 3.4.5 Assessment of inflammation ................................................................................................................ 89 3.4.6 Histology ............................................................................................................................................. 90 3.5 Statistical analysis....................................................................................................................................... 91 4. RESULTS ......................................................................................................................................................... 92 4.1 BPH cells can act as non-professional APCs to induce chronic prostate inflammation ............................. 92 4.2 VDR agonist elocalcitol inhibits IL-8-dependent BPH cell proliferation and inflammatory response ....... 93 4.3 Potent anti-inflammatory properties of 1α,25(OH)2-16-ene-20-cyclopropyl-vitamin D3 (BXL-62) in inflammatory bowel disease models ................................................................................................................. 95 4.4 24-oxo BXL-62 metabolite exerts biological activities similar to its parent compound ............................. 98 4.5 Specific IL-10 production deficiency in inflammatory bowel disease patients compared to healthy controls .......................................................................................................................................................................... 99 5. DISCUSSION................................................................................................................................................. 102 5.1 Prostatic cells as inducers and targets of chronic inflammation ............................................................... 102 5.2 VDR agonists inhibit intraprostatic inflammatory responses ................................................................... 103 5.3 TLR specific deficiency for IL-10 production .......................................................................................... 104 5.4 BXL-62 ameliorates symptoms in experimental model of colitis............................................................. 106 5.5 24-oxo metabolite accumulation, a key event for BXL-62 potency ......................................................... 108 6. SUMMARY AND CONCLUSIONS ............................................................................................................ 110 7. FUTURE ASPECTS...................................................................................................................................... 112 8. REFERENCES .............................................................................................................................................. 113 APPENDIX: ORIGINAL PUBLICATIONS .................................................................................................. 136 1. Introduction The Roman numerals (I-V) refer to the manuscripts included in the thesis, as classified in the list of original publications. Despite all the efforts for the improvement of sanitary conditions and vector control, infections remain the leading cause of morbidity and mortality worldwide and represent a major challenge for the biomedical sciences. The development of vaccines and therapeutics is absolutely required, and these implicate a deeper understanding of the host immune system. The mammalian immune system is divided between innate immunity and adaptive immunity, which cooperate to protect the host against microbial and viral infections. The innate immune system is the phylogenetically older system to control microbe invasion and represents an immediate and direct immune response, induced after recognition of specific composite of bacteria, called pathogen-associated molecular pattern (PAMP). Conversely, the adaptive immune system, evolutionary more recent, is a later addition to the immune system, mediated by antibodies (humoral immunity) or by T and B lymphocytes (cell-mediated immunity). Such host-pathogen discrimination is essential for the host capacity to eliminate the pathogen without excessive damage to its own tissues. This avoidance of destruction of self-tissues is referred to as self-tolerance. Environmental factors, in genetic predisposed individuals could lead to failure of this control system, leading to autoimmune diseases. Vitamin D is produced in the skin by enzymatic modifications of cholesterol after exposure to ultraviolet B (UVB) radiation. 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the active form of vitamin D, is produced in the kidney by hydroxylation of its precursor, 25hydroxyvitamin D3 (25(OH)D3), and plays a central role in calcium homeostasis and bone remodelling. 1α,25(OH)2D3 effects are mediated by its receptor, the vitamin D receptor (VDR), a member of the superfamily of nuclear receptors (NRs). Since the discovery of VDR expression in cells regulating the immune response, 1α,25(OH)2D3 was shown to have benefits in various models of autoimmune and chronic inflammatory diseases. In addition, recent epidemiological studies correlate auto-immune disorders with low 25(OH)D3 serum levels. Since the supra-physiologic doses of 1α,25(OH)2D3 required to show robust antiinflammatory effects induce hypercalcemia, vitamin D analogues were synthesized in order to potentiate anti-inflammatory properties of VDR agonists without inducing hypercalcemic side effects. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 17 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Inflammatory bowel diseases (IBD) are chronic, relapsing inflammatory disorders of the gastrointestinal tract, most commonly the terminal ileum and colon, with two major forms recognized: Crohn’s disease (CD) and ulcerative colitis (UC). Whether or not these IBD subtypes fully share pathogenic mechanisms, the underlying factors are similar. Their current pathogenesis is focussed on deregulated mucosal immune response against intestinal bacterial flora in genetically predisposed individuals. In addition to genetic factors, including VDR gene polymorphisms, many environmental factors are also implicated in IBD development, and in this context vitamin D deficiency, especially observed in Northern latitudes, is now well documented as a high-risk factor for IBD pathogenesis. VDR expression is required to control inflammation of spontaneous and induced colitis models, as demonstrated by exacerbation of symptoms in Vdr-deficient mice. In addition, 1α,25(OH)2D3 has been shown to ameliorate spontaneous colitis in mice fed with a low calcium diet. This thesis extends the research on the potential used of VDR agonists in autoimmune disorder by identifying a potent anti-inflammatory VDR agonist as potential treatment for IBD and in addition, through the understanding of the mechanisms of action of this analog, contributes to the identification of events involved in the pathogenesis of experimental colitis. 18 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction 1.1 Overview of the immune system 1.1.1 Components of the innate immunity The innate immune system includes defence mechanisms that are encoded in the host germline. These include the epithelial barriers and the mucociliary blanket that sweeps away inhaled or ingested particles. They also include soluble proteins and bioactive small molecules that are either constitutively present in biologic fluids (for example, the complement proteins and defensins) or that are released from cells as they are activated (including cytokines that regulate the function of other cells, chemokines that attract inflammatory leukocytes, lipid mediators of inflammation, and bioactive amines and enzymes). Lastly, the innate immune system includes pattern recognition receptors (PRR) that bind PAMP expressed on the surfaces of invading microbes or viruses. 1.1.1.1 Toll like receptors The Toll like receptor (TLR) family has been extremely conserved during evolution. This family is the homolog of the Drosophila protein called Toll that was identified as a maternaleffect gene (Hashimoto, Hudson et al. 1988). TLRs are type I transmembrane proteins characterized by an extracellular leucine-rich repeat domain (LRR) coupled to an intracellular Toll/IL-1 receptor (TIR) with homology to the cytoplasmic domain of the IL-1 receptor (Hashimoto, Hudson et al. 1988; Medzhitov, Preston-Hurlburt et al. 1997; Medzhitov 2001; Athman and Philpott 2004). So far, 13 members have been identified in mammals, 11 in humans (TLR1-11) and 12 in mice (TLR1-9 and TLR11-13). For most of them, a specific ligand has been identified (Athman and Philpott 2004). These ligands are lipid, carbohydrate, peptide and nucleic acid structures representing common structural features of microorganisms, known as PAMP (Table 1). Subcellular localisation of the TLR is associated to its type of ligand. TLR1, 2, 4, 5 and 6, which recognize microbial specific components, are expressed on the cell surface, while TLR3, 7, 8 and 9, which recognize amino acids, are localized in endosomes or lysosomes (Athman and Philpott 2004). The primary function of TLRs is to signal that microbes have breached the body’s barrier defenses. TLRs are highly expressed on macrophages and dendritic cells (DCs) but are also expressed on neutrophils, eosinophils, epithelial cells and keratinocytes. Activation of most TLR induces cellular responses associated with acute and chronic inflammation Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 19 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD (Medzhitov 2001). When TLR ligands interact with their specific TLRs, intracellular adaptor proteins transduce signals that lead to enhanced expression of genes encoding proinflammatory cytokines and other inflammatory mediators. For example, tissue-resident macrophages stimulated by TLR agonists produce pro-inflammatory cytokines, including tumor-necrosis factor α (TNF-α), interleukin-1β (IL-1β), IL-12 and IL-6, which coordinate local and systemic inflammatory responses (Medzhitov, Preston-Hurlburt et al. 1997; Athman and Philpott 2004). In addition, TLR activation is the main signal for dendritic cell (DC) maturation (Steinman, Hawiger et al. 2003). Molecular mechanisms of TLR activation involve numbers of proteins enrolled in two main pathways depending on the adaptor protein Myd88 implication. For example, binding of bacterial lipopolysaccharide (LPS) to TLR4 in association with the MD2 and CD14 coreceptors elicits signaling through both the MyD88 pathway (using the adaptor TIR-domain-containing adaptor protein (TIRAP), also known as MyD88-adaptor-like protein, MAL) to activate nuclear factor κB (NF-κB) and proinflammatory responses, and also through an MyD88-independent pathway, including the adaptors TRIF (TIR domain–containing adaptor inducing IFN-β), TRAM (TRIF-related adaptor molecule) and TBK1 (TRAF family member-associated NF-κB activator-binding kinase) leading to the phosphorylation and nuclear translocation of IFN-regulatory factor 3 (IRF3) and expression of IFN-β and induction of anti-inflammatory responses (Athman and Philpott 2004). TLRs, in addition to their role in driving the innate immune system, are able to shape the adaptive immune system and especially the Th1/Th2 balance. Activation of nearly all TLRs programs T helper cell type 1 (Th1) by TLR-induced IL-12 production, as shown by the defect of Th1 response in Myd88-deficient mice upon immunization with ovalbumin in Freund’s complete adjuvant. However, a specific TLR2 ligand suppresses IL-12 and enhances IL-10 production, favoring a T helper cell type 2 (Th2) response via an ERK/MAPKdependent mechanism (Medzhitov, Preston-Hurlburt et al. 1997). Furthermore, TLRs that induce strong production of TGF-β, IL-6 and IL-23 promote IL-17 producing T helper cell type 17 (Th17) (Manicassamy and Pulendran 2009). 20 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction Table 1. Human TLRs and their respective ligands. Eleven TLR were identified in the human genome and only for nine of them the ligands have been characterized. The ligand is representated of the subcellular localization, since the cellular TLR1, 2, 4, 5 and 6 are expressed on the cell membrane and recognize specific surface microbial components, while TLR3, 7, 8 and 9 are localized in endosomes or lysosomes and recognize amino acids (Akira and Takeda 2004). Receptor TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 TLR11 Ligand Triacyl lipopeptides Soluble factors Lipoprotein/lipopeptides Peptidoglycan Lipoteichoic acid Lipoarabinomannan Phenol-soluble modulin Glycoinositolphospholipids Glycolipids Porins Atypical lipopolysaccharide Atypical lipopolysaccharide Zymosan Double-stranded RNA Lipopolysaccharide Taxol Fusion protein Envelope protein Heat-shock protein 60 Flagellin Diacyl lipopeptides Lipoteichoic acid Zymosan Imidazoquinoline Loxoribine Bropirimine Single-stranded RNA Imidazoquinoline Single-stranded RNA CpG-containing DNA N.D. N.D. Origin of ligand Bacteria and mycobacteria Neisseria meningitidis Various pathogens Gram-positive bacteria Gram-positive bacteria Mycobacteria Staphylococcus epidermidis Trypanosoma cruzi Treponema maltophilum Neisseria Leptospira interrogans Porphyromonas gingivalis Fungi Viruses Gram-negative bacteria Plants Respiratory syncytial virus Mouse mammary-tumor virus Chlamydia pneumoniae Bacteria Mycoplasma Gram-positive bacteria Fungi Synthetic compounds Synthetic compounds Synthetic compounds Viruses Synthetic compounds Viruses Bacteria and viruses N.D. Uropathogenic bacteria Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 21 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 1.1.1.2 Nucleotide oligomerisation domain-Like Receptors (NLR) As discussed in the previous section, detection of invaders by the host is mediated by the recognition of PAMPs by specific PRRs. While TLR recognition involves mainly membrane receptors, in the cytoplasm are present PRR, termed nucleotide binding site (NBS)–leucinerich repeats (NLR), able to sense cytosolic invasion. NLRs homologues are found in plants and are involved in the hypersensitive response against virulent plant pathogens (Athman and Philpott 2004). Bioinformatics approaches revealed the presence in the human genome of 23 NLRs genes, whereas about 34 genes are found in the mouse genome. General domain organisation of NLRs include a variable N‑terminal effector binding domain, a central nucleotide-binding domain (NBD) and C‑terminal leucine-rich repeats detecting PAMPs (Kanneganti, Lamkanfi et al. 2007). Based on the structure of the N terminal domains, NLRs contain 3 sub-families:, the caspase recruitement domain (CARD)-containing nucleotide oligomerization domain (Nods), the pyrin (PYD) and baculovirus inhibitor repeat (BIR). Nod1 and Nod2 (CARD15) sense bacterial molecules resulting from the synthesis and/or degradation of peptidoglycan (PGN). Nod1 recognizes the dipeptide-γ-D-glutamyl-meso-diaminopimelic produced by Gramnegative bacteria and specific Gram-positive bacteria. Nod2 is mainly expressed in cells of the myeloid lineage and is sensing the muramyl dipeptide (MDP), present on all types of PGN. Upon ligand binding, conformation changes lead to the recruitment of the serinethreonine kinase RICK (also called RIP2) through the CARD domain. This dimerization results in the degradation of NF-κB inhibitor IκBα, allowing the nuclear translocation of active NF-κB. In addition, Nod1 and Nod2 activation could also activate mitogen activated protein (MAP) kinases such as p38 or JNK (Jun N-terminal kinase). Finally, NF-κB and MAP kinases activation induce production of pro-inflammatory cytokines and promote the recruitment of neutrophils to the site of infection (Inohara, Chamaillard et al. 2005). Furthermore, Nod2 is essential in the production of anti-microbial peptides, such as defensins in Paneth cells (Lala, Ogura et al. 2003). The importance of NLRs is highlighted by their genetic variation, which correlates with disease susceptibility. Mutations in Nod2 were recently correlated with an increased susceptibility to the chronic intestinal inflammatory disease, Crohn’s disease. One of the most common mutation associated with this disease leads to the inability of the mutant protein to respond to MDP and to its incapacity to activate NF-κB. However, it is still not understood, 22 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction how the inability to sense bacterial products might lead to the aberrant inflammation that characterizes Crohn’s disease (Cho and Abraham 2007). 1.1.1.3 Complement The complement system is part of the innate immune response and underlies one of the main effector mechanisms of antibody-mediated immunity. The main role of complement is to defend the organism against bacterial infections, bridging innate and adaptive immunity and disposing of immune complexes and the products of inflammatory injury (Walport 2001). The complement system is composed of more than 30 plasma and cell surface proteins and 3 different pathways have been described, the classical pathway, the alternative pathway and the mannose-binding lectin pathway (Walport 2001; Chaplin 2003; Chaplin 2006). The first pathway discovered, and then called classical pathway, is initiated by the binding of the C1 complex (which consists of a C1q molecule, two molecules of C1r, and two molecules of C1s) to antibodies bound to an antigen on the surface of a bacterial cell via fragment crystalisable receptor (FcR). C1s first cleaves C4, which binds covalently to the bacterial surface, and then cleaves C2, leading to the formation of a C4b2a enzyme complex, the C3 convertase of the classical pathway (Walport 2001; Walport 2001; Chaplin 2003; Chaplin 2006). The mannose-binding lectin pathway is triggered by microbial cell wall components containing mannans and is called the lectin pathway of complement activation. Interaction between mannan-containing microbes and mannose-binding lectin–associated proteases 1 and 2 (MASP1 and MASP2, respectively) result to the lise of the mannose groups on the surface of a bacterial cell. These form a protease analogous to the activated C1 of the classic pathway that then goes on to activate C4, C2, and the remainder of the pathway (Walport 2001; Walport 2001; Chaplin 2003; Chaplin 2006). The alternative pathway is antibody-independent and is initiated by the covalent binding of a small amount of C3b to hydroxyl groups on cell-surface carbohydrates and proteins and is activated by low-grade cleavage of C3 in plasma. This C3b binds factor B, a protein homolog to C2, to form a C3bB complex. Factor D cleaves factor B bound to C3b to form the alternative pathway C3 complex C3bBb (Walport 2001; Walport 2001; Chaplin 2003; Chaplin 2006). The C3 convertase enzyme is really efficient to cleave C3 in C3b. Then C3b binds covalently around the site of complement activation. Some C3b binds to the C4b and C3b in Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 23 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD the classical and alternative pathways, respectively, forming C5 convertase enzymes. This C3b acts as an acceptor site for C5, which is cleaved to form C5a and C5b, and initiates the formation of the membrane-attack complex, a complex of the complement proteins C6, C7, C8, and C9, which are assembled into a membrane pore that causes lysis of the cells where the complement is activated (Chaplin 2003; Chaplin 2006). 1.1.1.4 Fragment crystallizable Receptor (FcR) In contrast to B Cell Receptors (BCRs) and T Cell Receptors (TCRs), receptors for the fragment crystallizable (Fc) domain of the immunoglobulins (Ig) do not recognize antigens but the Fc portion of antibodies. FcR play an important role in immune defense. FcRs for IgG and IgE are present on the surface of several cell types of the immune system. These receptors, designated FcγRs for those that bind IgG, and FcεRs for those that bind IgE, interact with antibody-antigen complexes to activate various biological responses. The biological responses elicited include antibody-dependent cell-mediated cytotoxicity, phagocytosis, release of inflammatory mediators and regulation of lymphocyte proliferation and differentiation (Daeron 1997). Affinities and genes encoding FcR present heterogenity within the family. FcγR is divided in three major classes (FcγRI, FcγRII, FcγRIII) and FcεRs in two (FcεRI and FcεRII). The form I recognizes with high affinity the Fc, while forms II and III bind Fc with a lower affinity. The Ig-binding portions of FcγRI, FcγRII, FcγRIII and FcγRI (the γ chains) are members of the Ig gene superfamily, all type I transmembrane proteins containing an extracellular region with two or more Ig-like domains and a polypeptide or lipid anchor in the membrane. The extracellular regions of the FcεR and FcγRI receptors show significant sequence similarity to each other: 70-98% sequence identity within the FcγRs and about 40% sequence identity between FcεRs and FcγRI (Raghavan and Bjorkman 1996). FcRs capable of triggering cell activation possess one or several intracytoplasmic activation motifs designated immunoreceptor tyrosine-based activation motifs (ITAMs), which resemble to those of the BCR and TCR signal transduction subunits. This signal activates sequentially src family tyrosine kinases and syk family tyrosine kinases that connect transduced signals to common activation pathways shared with other receptors (Ravetch and Bolland 2001). 24 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction 1.1.2 Cells of the innate immunity Cells involved in innate immune response are essentially phagocytes differentiated from the same myeloid progenitor. These cells do not need a somatic rearrangement, because most of the genes involved in the innate immune response are encoded in the genome. The old concept that cells of the innate immune system have as only role the engulfement of pathogens and then its destruction by apoptosis is incorrect. A wealth of evidence shows that engagement of innate immune mechanisms is required for shaping the adaptive immune response, such as the maturation of DCs, enhancing major histocompatibility complex (MHC) class II expression on their surface and promoting antigen presentation to T cells. 1.1.2.1 Mucosal epithelia The mucosal epithelia is one of the most ancient and universal modules of innate immunity. The mucosal epithelia from the skin, airways, reproductive tract and intestine are the main interface between the host and the microbial world (including both pathogenic and symbiotic microorganisms) and therefore are susceptible to colonization and invasion of pathogens, such as viruses, bacteria, fungi or parasites. Maintaining homeostasis with symbiotic bacteria while protecting the host from pathogen invasion represents a hard challenge for the mucosal epithelia (Artis 2008). During evolution, mammals have developed a mucosa associated lymphoid tissues, such as the Peyer’s patches in lamina propria in the gut, which are rich in cells involved in innate or adaptive immune responses, in order to lighten the mucosal challenge. Epithelia present the complete array of PRRs and in addition to the soluble factors produced after activation; they are able to produce anti-microbial peptides, like defensins or cathelicidin antimicrobial peptide (CAMP), which are potent immuno-regulators. In addition, epithelial cells at the mucosal surface can produce mucins inhibiting the attachment and entry of pathogens (Medzhitov 2007; Artis 2008). 1.1.2.2 Phagocytes Phagocytes are white blood cells, deriving from stem cell and having a common myeloid precursor. The primary function of phagocytes is to identify and engulf microbes. Phagocytosis is a very complex process having as main role the production of molecules required for efficient antigen presentation to the adaptive immune system after pathogen recognition. This is accompanied by intracellular signals that trigger cellular processes as Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 25 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD diverse as cytoskeletal rearrangement, alterations in membrane trafficking, activation of microbial killing mechanisms, production of pro- and anti-inflammatory cytokines and chemokines and activation of apoptosis (Underhill and Ozinsky 2002). The two most important members of this family are macrophages and neutrophils that share the same initial stage, the monocyte. Circulating monocytes represent 5-10% of circulating leukocytes, leave the bone marrow incompletely differentiated and then give rise to a variety of tissue-resident neutrophils and macrophages throughout the body, as well as to specialized cells, such as DCs (Volkman and Gowans 1965). In order to discriminate between infectious agents and self components, phagocytes have evolved a restricted number of phagocytic receptors, such as mannose and lectin receptors, PRR, FcR and the broad class of complement proteins. In addition, they express MHC class I and II molecules, but they represent poorly efficient antigen-presenting cells (APC) (Jensen 2007). In addition to their crucial role in the embryogenesis, the main functions of macrophages in the immune system are recognition and phagocytosis of pathogens, antigen presentation, production of superoxide, cytokines and chemokines for the recruitement of effector cells to the site of inflammation (Artis 2008). Macrophages are pleiomorphic in different tissues, defined as Langerhans cells in the epidermis, Kuppfer cells in the liver, microglia in the central nervous system, osteoclast in the bone and alveolar macrophages in the lung. In the gut, macrophages resides in the lamina propria but are not specifically named. The important step in the functional maturation and inflammation of macrophages is the conversion from a resting to an activated macrophage, meaning that they have increased their capacity to kill microbes (Medzhitov 2007). Polymorphonuclear leukocytes (PMNs or neutrophils) are the most important population of leukocytes. Neutrophils play an essential role in the human innate immune system because they are usually the first cell type recruited to sites of infection or areas of inflammation. Then, neutrophils are able to orchestrate the inflammatory response by recruiting, activating and programming APCs. As macrophages, neutrophils destroy microorganisms by phagocytosis. Interestingly, neutrophils present a short half-life, a mechanism of resolution of the inflammatory response (Theilgaard-Monch, Porse et al. 2006). 26 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction 1.1.3 Components of the adaptive immunity In contrast with innate immunity, mechanisms for generating receptors in the adaptive immune system involve great variability and rearrangement of receptor gene segments. The adaptive immune system can provide specific recognition of foreign antigens, immunological memory of infection and pathogen-specific adaptor proteins. However, the adaptive immune response is also responsible for allergy, autoimmunity and the rejection of tissue grafts (Janeway and Medzhitov 2002). Two main pathways encompass the adaptive immune system, humoral and cellmediated immunity where Ig secreted by B cells, and the TCR are the proteins presenting the somatic rearrangements. Cell-mediated immunity, orchestrated by T cells, serves as a defense mechanism against microbes that survive within phagocytes or infect nonphagocytic cells, while humoral immunity, mediated by secreted antibodies, protects mostly against extracellular microbes and microbial toxins (Janeway and Medzhitov 2002). 1.1.3.1 Major histocompatibility complex T cell-mediated immune response recognize antigen present only on infected cells, but not free antigen in solution. This mechanism is possible because T cells, in addition to the microbial antigen, have to recognize self structures. These self structures are the antigenic peptide-binding major histocompatibility complex (MHC) molecules, also called human leukocytes antigen (HLA) in humans. 1.1.3.2 Major histocompatibility complex class I molecules MHC class I molecules are cell surface heterodimers, consisting of a polymorphic transmembrane 44 kDa α-chain (named also class I heavy chain) and a 12 kDa nonpolymorphic β2 microglobulin protein, both non covalently linked. Three distinct classes of MHC class I molecules have been defined in humans, called HLA-A, HLA-B and HLA-C determined by α-chain genes encoded within the same chromosome. The fully assembled class I molecule is a heterotrimer consisting of a β2-microglobulin chain, a bound antigenic peptide, and the α chain stably expressed on cell surfaces (Chaplin 2003). Binding of antigen to the MHC class I pocket results in a structure that is the molecular target for TCR. Antigens presented by MHC class I molecules are described as “endogenous peptides” or self antigens due to the fact that they derive from proteins produced Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 27 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD within the cells and processed by the proteasome and the endoplasmic reticulum. Antigen can be recognized by the TCR only in combination with the MHC molecule and this phenomenon is known as “MHC restriction”. The simultaneous recognition of antigenic peptide bound to MHC molecules by the TCR ensures efficacy and specificity of T cell responses, allowing T cells to ignore free extracellular antigen and to focus on infected cells. MHC class I extracellular domains interact only with CD8+ cytotoxic T lymphocytes (CTLs) resulting in the lysis of the target cell expressing the appropriate peptide-MHC combination (Jensen 2007). 1.1.3.3 Major histocompatibility complex class II molecules As the MHC class I molecules, MHC class II molecules are formed by two polypeptide chains, but both are MHC-encoded transmembrane proteins, called α and β chains. The three major human MHC class II molecules are designated as HLA-DR, HLA-DP and HLA-DQ. Each MHC class II chain contains a cytoplasmic anchor, a transmembrane domain and two extracellular domains. The α2 and β2 domains provide a unique support for CD4 binding (Chaplin 2003). In contrast to MHC class I molecules, which are expressed on all cell types, MHC class II molecules are expressed only on particular cells called APCs. Professional APCs are DCs and macrophages. As non-professional APCs are considered neutrophils, basophils and B cells (Jensen 2007). This list could be extended, because many cell types are able to present MHC class II molecules on their surface. Antigenic peptides presented by MHC class II molecules result from the lysosomal and endosomal degradation of phagosized products, before transport into a specialized MHC class II loading compartment. Thus, MHC class II molecules present exogenous antigens to CD4+ T cells, alerting them about the presence of intracellular invaders (Watts 2004). 1.1.3.4 B cell receptor As previously mentioned, the BCR is a member of the multichain immune recognition receptor family that includes the TCR and FcεR1. The BCR, expressed only in mature B cells, is composed of a membrane-anchored specific immunoglobulin associated with a 32kDa phosphoprotein Ig-α and Ig-β. The latter, also defined as CD79α and CD79β, form a dimer through a disulfide bridge and show homology with CD3α and β from the TCR. They present 28 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction a 26 aa intra-cytoplasmic region called immunoreceptor tyrosine-based activation motif (ITAM) (Chaplin 2003). Once activated, ITAM is phosphorylated by the Src family kinase Lyn, providing a binding site for the SH2-domain-containing kinase Syk, triggering the signalling cascades. These phosphorylations events require co-stimulatory molecule such CD19 and CD45 that once activated, induce the dephosphorylation of the negative regulatory site of src family kinases. Antigen recogntion result in its internalisation and its processing in the MHC-II complex, where the peptide takes place (Kurosaki 1997). 1.1.3.5 T cell receptor The TCR, expressed on the surface of T lymphocytes, is a transmembrane protein consisting of a heterodimer formed by α/β chains or by γ/δ chains, resulting in the subclassification of T cells in α/β or γ/δ T cells. As in antibodies, each chain of the TCR heterodimer contains a variable and a constant region, where the variable region possesses 3 complementarity determining regions allowing the recognition of peptides presented by MHC molecules. TCRs, as BCRs, are associated with transmembrane molecules that allow signal transduction. For the TCR, these proteins form the CD3 complex, constituted by the transmembrane accessory molecules CD3γ, CD3δ, CD3ε, and a CD3ζ intracytoplasmic homodimer (Zidovetzki, Rost et al. 1998). As discussed for B cells, the cytoplasmic domain of CD3 contains an ITAM domain, that once phosphorylated by receptor-associated kinases Lck and Fyn, initiates an activation cascade involving the proteins ZAP-70, LAT, and SLP-76. These phosphorylation events result in the stimulation of phospholipase C, activation of the G proteins Ras and Rac and both protein kinase C and the MAP kinases. Activation of this pathway controls T cell activation and proliferation (Smith-Garvin, Koretzky et al. 2009). 1.1.4 Cells of the adaptive immunity, the lymphocytes Phagocytes and lymphocytes share the same precursors, the hematopoietic stem cells. However, this common precursor gives rise to two main subclasses, myeloid stem cells, from which phagocytes originate and lymphoid stem cells generating T and B cells and plasmacytoid DCs (Fig. 1). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 29 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Fig. 1. Major haematopoietic stem cell maturation pathways. Within the haematopoietic system, the haematopoietic stem cell (HSC) has self-renewal potential and multipotent differentiation potential. Its progeny include multipotent common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). In turn, these progenitors give rise to progenitors that have more limited differentiation potential, Pro-B and Pro-T cells, megakaryocyte erythroid progenitors (MEPs) and granulocyte monocyte progenitors (GMPs). In the myeloid lineage, MEPs give rise to mature erythrocytes and platelets in the peripheral blood. GMPs give rise to monocytes and the various granulocyte lineages. In the lymphoid lineages Pro-B and Pro-T cells give rise to mature (naive) B and T lymphocytes and then stimulated B and T cells, respectively, following exposure to antigen. Adapted from (Huntly and Gilliland 2005). 1.1.4.1 B cells B cells are lymphocytes that play a large role in the humoral immune response, as opposed to the cell-mediated immune response, which is governed by T cells. Their definition comes from the bursa of Fabricius in birds, where they mature. In mammals, immature B cells are formed in the bone marrow (Raff 1973). B cells differentiate from hematopoetic stem cells in the bone marrow under the control of IL-7 produced by stromal cells (Burrows and Cooper 1997). Their maturation takes subsequently place in lymph node follicles. B cells represent about 15% of the total leukocytes (Chaplin 2003). On their surface they express co-stimulatory molecules like CD19, CD81 or CD21, but also MHC-II molecules (Burrows and Cooper 1997). The main function of B cells is the production of Igs, antigen-binding proteins also known as antibodies 30 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction (LeBien and Tedder 2008). Antibodies are polypeptides formed by two heavy and two light chains linked by disulfide bonds. Antigen specificity is provided by the diversity of Nterminal regions (called variable regions), while the conserved C-terminal part is defined Fc (Chaplin 2003). An antibody is composed of two identical light (L) and two identical heavy (H) chains, and the genes specifying them are found in the 'V' (Variable) region and the 'C' (Constant) region. In the heavy chain 'V' region there are three segments; V, D and J, which recombine randomly, in a process called VDJ recombination, to produce a unique variable domain in the immunoglobulin of each individual B cell (Chaplin 2003). Similar rearrangements occur for light-chain 'V' region except there are only two segments involved; V and J. The amino terminal portion of each heavy chain is created by somatic joining of genes encoding a variable (VH), diversity (DH), and joining (JH) region (Chaplin 2003). The VH-JH and VL-JL light chain junctions formed by this recombination make up the third hypervariable region that contributes to the antigen-binding site. B cell development occurs through several stages, each stage representing a change in the genome content at the antibody loci (Schatz, Oettinger et al. 1992). B cells are the key cell of the humoral immunity, representing an essential component of the adaptive immune system, but can also secrete a number of cytokines and chemokines. In addition, B cells express MHC class II molecules and can act as non-professional APCs, thus playing a role also in immunoregulation. They eventually develop into memory B cells after activation by antigen interaction B cells. A critical difference between B cells and T cells is based on their mode of antigen recognition. B cells recognize their cognate antigen in its native form (Batista and Harwood 2009). They recognize free (soluble) antigen in the blood or lymph using their BCR or membrane-bound Ig. In contrast, T cells recognize their cognate antigen in a processed form, as a peptide fragment presented to the TCR by MHC molecules of APCs (Smith-Garvin, Koretzky et al. 2009). 1.1.4.2 Effector T cells All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors derived from hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes (Schwarz and Bhandoola 2006). The earliest thymocytes express neither CD4 nor CD8, and are therefore classified as double-negative (CD4-CD8-) cells. As they progress through their development they become double-positive thymocytes (CD4+CD8+), and finally mature to single-positive (CD4+CD8- or Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 31 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD CD4-CD8+) thymocytes that are then released from the thymus to peripheral tissues (Ellmeier, Sawada et al. 1999). About 98% of thymocytes die during the development processes in the thymus, which involves positive and negative selection (Delves and Roitt 2000). Positive selection "selects for" T-cells capable of interacting with MHC molecules (Delves and Roitt 2000). Double-positive thymocytes (CD4+/CD8+) move deep into the thymic cortex where they are presented with self-antigens (i.e., antigens that are derived from molecules belonging to the host of the T cell) complexed with MHC molecules on the surface of cortical epithelial cells (Robey and Fowlkes 1994). Only those thymocytes that bind the MHC/antigen complex with adequate affinity will receive a "survival signal". Developing thymocytes that do not have adequate affinity cannot serve useful functions in the body (i.e. the cells must be able to interact with MHC molecules and peptide complexes in order to affect immune responses) (Marrack and Kappler 2004). Because of this, the thymocytes with low affinity die by apoptosis and are engulfed by macrophages (Cohen, Duke et al. 1992) . A thymocyte's fate is also determined during positive selection (Starr, Jameson et al. 2003). Double-positive cells (CD4+/CD8+) that are positively selected on MHC-II molecules will eventually become CD4+ cells, while cells positively selected on MHC-I molecules mature into CD8+ ells (Ellmeier, Sawada et al. 1999). A T cell becomes a CD4+ cell by downregulating expression of its CD8 cell surface receptors (Delves and Roitt 2000). If the cell does not lose its signal through the ITAM pathway, it will continue down-regulating CD8 and become a CD4+, single positive cell. But if there is signal drop, the cell stops down-regulating CD8 and switches over to down-regulating CD4 molecules instead, eventually becoming a CD8+, single positive cell (Starr, Jameson et al. 2003). Negative selection removes thymocytes that are capable of strongly binding with "self" peptides presented by the MHC complex (Starr, Jameson et al. 2003). Thymocytes that survive positive selection migrate towards the boundary of the thymic cortex and thymic medulla. While in the medulla, they are again presented with self-antigen in complex with MHC molecules on APCs, such as DCs and macrophages (Anderson, Moore et al. 1996). Thymocytes that interact too strongly with the antigen receive an apoptotic signal that leads to cell death. The vast majority of all thymocytes end up dying during this process. The remaining cells exit the thymus as mature naive T cells. This process is an important component of immunological tolerance and serves to prevent the formation of self-reactive T cells that are capable of generating autoimmune diseases in the host (Starr, Jameson et al. 2003). 32 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction Although the specific mechanisms of activation vary slightly between different types of T cells, the "two-signal model" in CD4+ T cells holds true for most. Activation of CD4+ T cells occurs through the engagement of both the TCR and CD28 on the T cell by the MHCpeptide complex and B7 family members on the APC, respectively. Both are required for production of an effective immune response; in the absence of CD28 co-stimulation, TCR signaling alone results in anergy (Schwartz 1997). The signaling pathways downstream from both CD28 and the TCR involve many proteins (Smith-Garvin, Koretzky et al. 2009). The first signal is provided by binding of the TCR to a short peptide presented by the MHC molecule. This ensures that only a T cell with a TCR specific to that peptide is activated. The partner cell is usually a professional APC, a dendritic cell in the case of naïve responses, although B cells and macrophages can be important APCs (Itano and Jenkins 2003). The peptides presented to CD8+ T cells by MHC-I molecules are 8-9 amino acids in length; the peptides presented to CD4+ cells by MHC-II molecules are longer, as the ends of the binding cleft of the MHC-II molecule are open (Jensen 2007). The second signal comes from co-stimulation, in which surface receptors on the APC are induced by a relatively small number of stimuli, usually products of pathogens, but sometimes breakdown products of cells, such as necrotic-bodies or heat-shock proteins (Akira, Takeda et al. 2001). The only co-stimulatory receptor expressed constitutively by naïve T cells is CD28, so co-stimulation for these cells comes from the CD80 and CD86 proteins on the APC (Adorini, Penna et al. 2004). Other receptors are expressed upon activation of the T cell, such as OX40 and ICOS, but these largely depend upon CD28 for their expression (Smith-Garvin, Koretzky et al. 2009). The second signal licenses the T cell to respond to an antigen. Without it, the T cell becomes anergic, and it becomes more difficult for it to activate in future. This mechanism prevents inappropriate responses to self, as selfpeptides will not usually be presented with suitable co-stimulation (Schwartz, Mueller et al. 1989). As the Th cells continue to respond to the activating signal, they progress toward polar extremes of differentiation designated Th1 and Th2, depending on the nature of the cytokines present at the site of activation (Zenewicz, Antov et al. 2009) (Fig. 2). Naïve T cells can be differentiated in vitro into Th1 cells by culturing with IL-12, an innate-system-derived cytokine that is highly expressed by activated macrophages and DCs (Hsieh, Macatonia et al. 1993). IL-12 activates signal transducer and activator of transcription 4 (STAT4) signaling pathways, resulting in activation of the genes encoding the cytokine Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 33 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD IFN-γ and the T-box family transcription factor T-bet essential for Th1 development (TBX21) (Kaplan, Sun et al. 1996). Additionally, expression of the IL-12 receptor is up-regulated by Tcells and thus makes Th1 cells even more sensitive to this polarizing signal, thereby enabling their expansion (Mullen, High et al. 2001). In combating infectious diseases, Th1 cells are especially useful at eliminating intracellular pathogens, including viruses and intracellular bacteria. These cells secrete high levels of IFN-γ, which is important for macrophage activation, and IL-2, which is important for directing cytotoxic CD8+ T cell responses. In contrast, aberrant Th1 responses are thought to be important for driving autoimmune diseases and chronic inflammation (O'Garra and Arai 2000). IL-4 is the signature molecule of Th2 cells; the role of this cytokine is not only to promote auto-stimulation, but also to trigger isotype switching towards IgE, an antibody isotype necessary for combating extracellular parasites, in B cells. IL-4 stimulation leads to activation of STAT6 pathways, which are necessary for GATA-3 expression, a master transcription factor for the Th2 regulation. In addition, GATA-3 drives expression of IL-4, creating a positive feedback loop and inducing expression of other cytokines (O'Garra and Arai 2000). Th2 cells direct the immune response against extracellular parasites, including helminths. However, in some circumstances, they cause asthma and allergies. Th2 cells produce a myriad of cytokines with distinct functions such as IL-4, IL-5 for eosinophil recruitment, IL-9 important for mast cells and T cells and mucin production by epithelial cells during allergies or the potent anti-inflammatory cytokine IL-10 (Zenewicz, Antov et al. 2009). Recently, the Th1/Th2 paradigm has been expanded; following the discovery of a third subset of effector Th cells that produce IL-17 and exhibits effector functions distinct from Th1 and Th2 cells. Development of Th17 cells can be divided into three stages: differentiation (driven by TGFβ and IL-6), amplification (triggered by IL-21) and, lastly, stabilization (maintained by IL-23). Naïve T cells can be differentiated into Th17 cells in vitro by activation in the presence of TGF-β, which drives Smad signaling, and a secondary inflammatory stimulus, driven by signaling molecules such as IL-6 or IL-21 that activates the transcription factor STAT3 (Zenewicz, Antov et al. 2009). Activated STAT3 drives expression of two transcription factors essential for shaping Th17, retinoid-acid-receptorrelated orphan receptor (ROR) γ and RORα. IL-21 is induced by IL-6 and leads to activation of RORγ, driving expression of IL-17A and other cytokines. Thus, this IL-21 loop represents an important autocrine factor for amplification of Th17 cells (Nurieva, Yang et al. 2007). IL23 was originally thought to be the cytokine driving Th17 differentiation. However, as shown 34 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction by the unresponsiveness of naïve T cells for IL-23, this cytokine is not needed for Th17 differentiation, but is instead important for maintenance of these cells. At the initial stage of Th17 differentiation, TGF-β-mediated activation leads to the increased expression of the receptor IL-23R, enabling the cells to become responsive to IL-23 (Mangan, Harrington et al. 2006; Zhou, Lopes et al. 2008). Signature cytokines secreted by Th17 include IL-17A, IL17F, IL-21 and IL-22 (Korn, Bettelli et al. 2009). Th17 cells have been implicated in the progression of inflammatory diseases, but it seems that they could act also as anti-inflammatory and protect against certain conditions, mainly through production of IL-22, that activates anti-apoptotic and proliferative responses (Zenewicz, Antov et al. 2009). 1.1.4.3 Regulatory T cells Among the various populations of regulatory and suppressor T cells described, naturally occurring thymic and peripheral CD4+ T cells that co-express CD25 are currently most actively investigated (Shevach, DiPaolo et al. 2006). CD4+CD25+ Treg cells prevent the activation and proliferation of potentially autoreactive T cells that have escaped thymic deletion. They fail to proliferate and secrete cytokines in response to polyclonal or antigenspecific stimulation, and are not only anergic but also inhibit the activation of responsive T cells. Although CD25, CD152, and glucocorticoid-induced TNF-related protein (GITR) are markers of CD4+ CD25+ Treg cells, they are also expressed by activated T cells (Shevach and Stephens 2006). A more faithful marker distinguishing CD4+CD25+ Treg cells from recently activated CD4+ T cells is Foxp3, a member forkhead family of transcription factors that is required for CD25+ Treg cell development and is sufficient for their suppressive function (Sakaguchi, Wing et al. 2007) (Fig. 2). Foxp3+ CD4+ CD25+ Treg cells play an important role in preventing the induction of several autoimmune diseases, such as the autoimmune syndrome induced by day 3 thymectomy in genetically susceptible mice, IBD, type 1 diabetes (T1D) in thymectomized rats and in non obese diabetic (NOD) mice. A defect in peripheral regulatory cells affecting both CD25+ Treg cells and natural killer cells has been described also in T1D patients, and autoreactive T cells in diabetics are skewed to a pro-inflammatory Th1 phenotype lacking the IL-10-secreting T cells found in non-diabetic, HLA-matched controls. The clinical relevance of CD4+ CD25+ Treg cells has also been shown in patients affected by rheumatoid arthritis and multiple sclerosis (Baecher-Allan and Hafler 2006). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 35 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD CD4- Naïve CD8- T cell CTL CD8+ CD4+/CD25+ CD4+ IL-12 IFNγ STAT4 Th0 TGF-β IL-6 RORγ FOXP3 IL-4 GATA3 Th17 Treg Th2 IL-10 TGF-β IL-4 IL-13 Th1 IFN-γ IL-2 IL-23 IL-17 Fig. 2. T cell differentiation. T-cell differentiation is tightly controlled by cytokines and transcription factors that determine the type of inflammatory response. CD4+ T cells differentiate in T helper cells (Th) or T regulatory (Treg), while CD8+ differentiates in cytotoxic T lymphocytes (CTL). Adapted from (Korzenik and Podolsky 2006). 1.1.5 Dendritic cells, a key role in innate and adaptive immunity DCs derive, as phagocytes, from monocytes, and after stimulation with IL-4 and granulocyte macrophage colony stimulating factor (GM-CSF) differentiate into immature DCs (iDCs). iDCs are continuously produced from hematopoietic stem cells in the bone marrow and are widely distributed in lymphoid and nonlymphoid tissues. Mature DCs are professional APCs, which are strategically positioned at the boundaries between the inner and the outside world, thus bridging innate and adaptive immunity. DCs, including epidermal Langerhan’s cells, splenic marginal zone DCs and interstitial DCs within nonlymphoid tissues, continuously sample self-antigen to maintain T cell self-tolerance (Banchereau and Steinman 1998). At the immature stage, iDCs express PRRs and cytokine receptors, allowing them to sense pathogens and contribute to the innate immune response and induced DCs maturation. DCs are known as the most efficient APC to activate naïve T cells. However, they are able to do more than just efficiently present antigen to T cells. They are key modulators of the immune response that can influence Th cell differentiation by preferentially inducing Th1 or 36 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction Th2 cell responses, and the differential polarization of CD4+ T cells appears to be mediated by discrete dendritic cell subsets (Steinman, Hawiger et al. 2003). 1.1.5.1 Inflammatory dendritic cells In most tissues, DCs are present in an immature stage, lacking the requisite accessory signal for T cells activation. In contrast, they express the complete panel of receptors involved in sensing pathogen invasion such as TLRs or NLRs and many antigen-capturing Fcγ and Fcε receptors. Antigen uptake by phagocytosis or recognition of PAMPs by its receptor transform immature DCs into mature DCs, showing now a reduced capacity for antigen uptake but an exceptional capacity for T cell stimulation. Thus, mature DCs express T cells co-stimulatory molecules, such as CD40, CD86, CD80 and MHC-II. This maturation process induces DC migration from the periphery to lymphoid organs, where antigen presentation to T cells could occur (Itano and Jenkins 2003). DCs are heterogeneous not only in terms of maturation stage, but also of origin, morphology, phenotype and function. Two distinct DC subpopulations were originally defined in the human blood based on the expression of CD11c, and they have been subsequently characterized as belonging to the myeloid or lymphoid lineage, and defined as myeloid (M-DCs) and plasmacytoid (P-DCs) (Colonna, Trinchieri et al. 2004). 1.1.5.2 Myeloid dendritic cells M-DCs are characterized by a monocytic morphology; express myeloid markers like CD13 and CD33, the β2 integrin CD11c, the activatory receptor Ig-like transcripts 1 (ILT1), and low levels of the IL-3 receptor α chain CD123 and at a high level the complete TLRs familly (Steinman and Banchereau 2007). M-DCs are the most efficient APCs directly able to prime naïve T cells and can become, under different conditions, immunogenic or tolerogenic (Steinman and Banchereau 2007). As already mentioned, MHC present on the M-DCs surface is recognized by TCR and with engagement of co-receptors, such as CD86, CD80 or CD40-CD40L. This lead to the production of IL-12 by M-DCs driving the CD4+ naïve T cells to a Th1 phenotype (Banchereau, Briere et al. 2000). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 37 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 1.1.5.3 Plasmacytoid dendritic cells Conversely, P-DCs have a morphology resembling plasma cells, are devoid of myeloid markers, express high levels of CD4, CD62L and CD123 and ILT3. Oppositely to M-DCs, PDCs express high level of TLRs involved in virus recognition, such as TLR3, 7 and 9, but not those TLRs that are involved in bacteria component sensing (Colonna, Trinchieri et al. 2004). P-DCs produce high levels of IFN-α (Colonna, Trinchieri et al. 2004), cytokines with clearly distinct effects on T cell activation and differentiation due to its IL-12 inhibiting properties. PDCs are poor APCs due to its low MHC-II surface expression. However even if P-DCs are less efficient than M-DCs as APCs, P-DCs are able to drive Th1-mediated response after virus infection and to activate CTLs (Banchereau, Briere et al. 2000). Interestingly, P-DCs primed CTLs present a poor proliferation capacity due to substantial production of IL-10. In addition, a very important aspect of P-DC-mediated regulation of adaptive immunity is the ability, through the production of both type I interferons and IL-6, to induce human B cells to differentiate into plasma cells and produce immunoglobulin. These observations, coupled to the high expression of ILT3 suggest a role for P-DCs, under steady-state conditions, in the maintainance of peripheral immune tolerance as naturally occurring tolerogenic DCs (Penna, Roncari et al. 2005). 1.1.5.4 Tolerogenic dendritic cells It is now clear that DCs can be not only immunogenic but also tolerogenic, both intrathymically and in the periphery, and they can modulate T cell development (Steinman and Banchereau 2007). Tolerogenic DCs are characterized by reduced expression of costimulatory molecules, in particular CD40, CD80, CD86, although this is not an absolute requirement. In addition, they usually show reduced IL-12 and increased IL-10 production, and often an early stage of maturation (Steinman, Hawiger et al. 2003). While these well-established phenotypic and functional properties of tolerogenic DCs can easily explain their propensity to induce regulatory rather than effector T cells, several other mechanisms may play a role in favoring Treg cell induction by tolerogenic DCs. However, the simplistic concept that iDCs are intrinsically and uniquely able to induce regulatory/suppressor T cells has been dispelled by the observation that mature DCs can also be very efficient inducers of Treg cells (Yamazaki, Iyoda et al. 2003), a property already noted for semi-mature DCs (Lutz and Schuler 2002). 38 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction Fig. 3. Dendritic cell subsets. DCs are heterogeneous not only in terms of maturation stage, but also of origin, morphology, phenotype and function. Two distinct DC subpopulations were originally defined in the human blood based on the expression of CD11c, and they have been subsequently characterized as belonging to the myeloid or lymphoid lineage, and defined as myeloid (M-DCs) and plasmacytoid (P-DCs). M-DCs could differentitated in tolerogenic or inflammatory DCs, resulting in a phenotype and cytokine production different, whileP-DCs differentitate and express BDCA2, TLR3-7-9 and produce high amont of IFN-α (Colonna, Trinchieri et al. 2004). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 39 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 1.2 VDR and 1α,25(OH)2D3 1.2.1 A brief history The first scientific description of a vitamin D deficiency, namely rickets, was provided in the 17th century by both Dr. Daniel Whistler (1645) and Professor Francis Glisson (1650). In 1824, German scientists found that cod-liver oil have excellent anti-rickets properties, and in 1861, Trousseau in France proposed that rickets was induced by lack of sun exposure and a faulty diet, where a cure was as simple as cod-liver oil ingestion. Before the scientific agreement to define the biologically active form of vitamin D as a secosteroid hormone, it was accidentally classified as vitamin (vital-amine). Around 1920, Sir Edward Mellanby was working with dogs raised exclusively in the absence of UVB. He developed a diet that allowed him to unequivocally establish that the bone disease, rickets, was caused by a deficiency of a trace component present in the diet (Mellanby 1976). In 1921 he wrote "The action of fats in rickets is due to a vitamin or accessory food factor which they contain, probably identical with the fat-soluble vitamin." Furthermore, he established that cod-liver oil was an excellent anti-rachitic agent. Shortly thereafter, McCollum and associates observed that oxidized cod-liver oil still retained its calcium-depositing properties. Based on this, they concluded that the anti-rachitic substance found in certain fats was distinct from fat-soluble vitamin A and its “specific property was to regulate the metabolism of the bones.” In the sequence of discovery of vitamins, the newly discovered anti-rachitic substance was the fourth; hence it was called vitamin D (McCollum, Simmonds et al. 1995). The chemical structures of the vitamins D were determined in the 1930s in the laboratory of Professor A. Windaus at the University of Göttingen in Germany. Vitamin D3 was not chemically characterized until 1936, when it was shown to result from the UVB irradiation of 7-dehydrocholesterol (Windaus A 1936). Virtually simultaneously, the elusive anti-rachitic component of cod-liver oil was shown to be identical to the newly characterized vitamin D3. These results clearly established that the anti-rachitic substance vitamin D was chemically a steroid, more specifically a secosteroid. 40 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction 1.2.2 1α,25(OH)2D3 21 18 12 11 Fig. 4. Structure of vitamin D3 (cholecalciferol) and its numbering system. 13 20 23 17 8 24 25 16 14 9 22 26 27 15 7 6 5 4 3 HO 19 10 1 2 1.2.2.1 Synthesis of vitamin D Vitamin D is composed of 3 rings named A, C and D, of a seco-B ring and 1 side chain for a total of 27 carbons (Fig. 4). 1α,25(OH)2D3, the bioactive hormone, is synthesized from vitamin D3 in a highly regulated multistep process. The initial step is the transformation by UVB light of the 7-dehydrocholesterol circulating in the skin, in an unstable precursor, the pre-vitamin D3 (Fig. 5). This precursor is transformed in vitamin D3 in a heat dependent process. In the liver, vitamin D3 is hydroxylated to 25(OH)D3, by a mitochondrial cytochrome P450 (CYP450) enzyme, the 25-hydroxylase (encoded by the gene CYP27A1). Next, in the proximal renal tubule, another enzyme belonging to the CYP450 family, the 1α-hydroxylase (encoded by the gene CYP27B1), transforms the precursor in the bioactive form, the 1α,25(OH)2D3 (Bell 1998; Deeb, Trump et al. 2007). CYP27B1 is not expressed exclusively in the proximal renal tubule, but extra renal sites of 1α,25(OH)2D3 synthesis have been found in cells of the immune system, as well as in breast, prostate and gut cells (Bell 1998). The mechanisms leading to less active or completely inactive metabolites are now fully characterized. 1.2.2.2 Catabolism of vitamin D The limiting rate from the bioactive hormone turnover is the product of its hydroxylation by 24-hydoxylase-1α,25(OH)2D3 (CYP24A1) to form 1α,24,25(OH)3D3 (Haussler, Whitfield et al. 1998). Interestingly, CYP24A1 is directly up-regulated by 1α,25(OH)2D3 via a VDRdependent manner, confirming a negative feedback induced by the hormone to control its concentration (Ohyama, Ozono et al. 1994). The principal pathway leading to the elimination of 1α,25(OH)2D3 is mediated by the hydroxylation on carbon 24 (C24) (1α,24,25(OH)2D3), then this hydroxyl group is reduced in a keto group leading to 24-oxo-1α,25(OH)2D3. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 41 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Interestingly, the oxydation of C24-hydroxy group in C24-keto group is also catalyzed by CYP24A1 (Uskokovic, Norman et al. 2001). After a second hydroxylation step, always catalysed by CYP24A1, 24-oxo-1α,25(OH)2D3 is transformed in C23 (24-oxo- 1α,23,25(OH)3D3) and finally oxidized into calcitroic acid, the final water soluble metabolite excreted by the kidneys (Reddy and Tserng 1989) (Fig. 5). In some tissues, a secondary pathway was described using successively an hydroxylation at carbons C23 and C26 resulting in the calcitriol lactone (Reddy and Tserng 1989). More recently, an alternative pathway was discovered in some tissues or malignant cell lines (Uskokovic, Norman et al. 2001), This alternative pathway, named C3 epimerization pathway, induces a stereochemical modification on the A ring resulting to the 3-epi-1α,25(OH)2D3 (Siu-Caldera, Sekimoto et al. 1999). Next, elimination of 3-epi-1α,25(OH)2D3 is following the classical pathway by CYP24A1 hydroxylation (Kamao, Tatematsu et al. 2004) (Fig. 5). This metabolite has been shown to be an inactive metabolite, while C24 and C23 metabolites were able to show, in some aspects, activities similar to the parent compound (Lemire, Archer et al. 1994; Siu-Caldera, Sekimoto et al. 1999). 42 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction Fig. 5. Metabolism and catabolism of 1α,25(OH)2D3. After UV exposure, the 7-dehydrocholesterol is transformed into vitamin D3, that after two successive hydroxylations in the liver (CYP27A1) and the kidney (CYP27B1) is converted into the active metabolite, 1α,25(OH)2D3. The hormone is catabolized into calcitroic acid, the final water soluble metabolite excreted by the kidneys. All these elimination steps involve the same enzyme CYP24A1 and could occur in different tissues (Haussler, Whitfield et al. 1998). 1.2.2.3 Vitamin D analogs The discovery of the immunomodulatory properties of VDR prompted the study of 1α,25(OH)2D3 as a therapeutic agent for immuno-mediated diseases (DeLuca 2004). Unfortunately, the dominant role of 1α,25(OH)2D3 is to adjust serum calcium and phosphorus concentrations, and its in vivo immunomodulatory properties are mostly achieved at hypercalcemic doses. These observations have opened up a new research area, where the design of 1α,25(OH)2D3 analogs with stronger anti-inflammatory properties but lower calcium-increasing capacity has generated interesting compounds (Adorini 2002). Two classes of 1α,25(OH)2D3 analogs containing 16-ene or 20-cyclopropyl moieties were intensively studied because of their unique biological activity (Uskokovic, Manchand et al. 2006). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 43 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD In the early 90s, the first report of 16-ene VDR agonist demonstrated potent leukemic cell growth inhibition without modification of intestinal calcium absorption (Norman, Zhou et al. 1990). Since then, many different analogs were described containing this 16-ene modification and their peculiar metabolism was shown to be responsible for their increased potency (IV) (Lemire, Archer et al. 1994). As described previously, CYP24A1 is the main enzyme catalysing 1α,25(OH)2D3 degradation, leading by various steps to calcitroic acid. While 24-oxo-1α,25(OH)2D3 is rapidly converted in 24-oxo-1α,23,25(OH)3D3, it appears that analogs containing 16-ene are protected from the C23-hydroxylation step, leading to an accumulation of C24-oxo. This metabolite is a non-calcemic metabolite, which presents similar activity than its parent compound, both in vitro and in vivo (Lemire, Archer et al. 1994; Uskokovic, Norman et al. 2001). Elocalcitol (BXL-628, 1α-fluoro-25-hydroxy-16,23Ediene-26,27-bishomo-20-epi-vitamin D3) (Fig. 12 and table 4) is an example of a 16-ene modified compound, and it was proposed as a treatment for benign prostatic hyperplasia (BPH) (II) (Maggi, Crescioli et al. 2006). The second well-described family includes the 20-epi analogs. The 20-epi modification leads to a conformational change, where the hydrogen at C20 is converted from R to S (Binderup, Latini et al. 1991; Sicinska and Rotkiewicz 2009). This modification leads to a stabilization of the VDR-retinoid X receptor (RXR) complex and a modification of the coactivator (CoA) or corepressor (CoR) recruitment (Schwinn and DeLuca 2007). An example of a potent 20-epi compound is KH1060 (1α,25(OH)2-20-epi-22-oxa-24,26,27trishomo-vitamin D3) that was shown to have enhanced anti-proliferative and antiinflammatory properties in many in vitro and in vivo models, such as T1D or IBD (Mathieu, Waer et al. 1995; Stio, Treves et al. 2002; Penna, Amuchastegui et al. 2006). Molecular mechanisms induced by this compound were extensively studied, and the crystal structure of KH1060-VDR-RXR complex was solved, showing higher stability and longer half-life compared to the natural hormone (Tocchini-Valentini, Rochel et al. 2001). A similar group of analogs, based on similar stereochemistry, was synthesized later on, the 20-cyclopropyl. These family of compounds show higher potency than the natural hormone in inhibiting the proliferation of cancer cell lines, in inhibiting production of pro-inflammatory cytokines, such as IFN-γ or TNF-α, and a stronger potency in primary VDR target gene induction with a controlled calcemic activity (III) (Uskokovic, Manchand et al. 2006). In addition, introduction of 16-ene moiety in 20-cyclopropyl analogs has been shown to increase anti-proliferative and 44 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction anti-inflammatory properties of VDR agonists without enhancing calcemic activity (III) (Uskokovic, Manchand et al. 2006). 1.2.3 VDR The biological effects of 1α,25(OH)2D3 are mediated by the VDR, a member of the superfamily of NRs. The VDR was discovered as a NR in 1975, but it was cloned a decade later (Baker, McDonnell et al. 1988); two years after the first genomic identification of a steroid receptor (glucocorticoid receptor) (Weinberger, Hollenberg et al. 1985). The sequencing of other receptors highlighted the presence of many more NRs genes than previously expected (Chawla, Repa et al. 2001). The VDR is now classified as member of the endocrine receptor subfamily together with the glucocorticoid receptor. Its natural ligand, 1α,25(OH)2D3, presents an affinity constant for the VDR in the nanomolar range (Haussler, Whitfield et al. 1998). Recently, a novel classification based on mouse tissue expression and function, considered the VDR as a NRs also involved in bile acid and xenobiotic metabolism based on its high expression in gastroenteric tissues (Bookout, Jeong et al. 2006). However, the main physiological process regulated by the VDR is the calcium and the phosphate homeostasis. 1.2.3.1 NR superfamily 1.2.3.2 Classification NRs belong to a large superfamily of transcription factors comprising 48 members in the human genome. These transcription factors regulate the expression of target genes to affect processes as diverse and important as reproduction, development and metabolism (Chawla, Repa et al. 2001; Novac and Heinzel 2004). They are classified based on ligand-binding affinity, but more recently a new classification based on the interpretation of physiological role from tissue-specific expression patterns has been proposed (Chawla, Repa et al. 2001; Bookout, Jeong et al. 2006). Based on the ligand sensitivity, NRs could be divided in three subgroups. The first class called “endocrine receptors” presents a high affinity for hormonal lipids (at the nanomolar range). The second group is called “sensors” and presents a lower affinity in the micromolar range. This class senses xenobiotic or nutritional components, such as cholesterol, lipids or fatty acids. The third group contains NR, for which no ligand has yet been identified. More recently, a novel classification, based on expression levels and tissue distribution of NRs in mice, classified NRs by their physiological shared functions. This Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 45 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD classification highlights the important role of NRs in the regulation of inflammation, cell growth and differentiation, development and reproduction and nutrient use and storage (Bookout, Jeong et al. 2006). Fig. 6. Circular dendrogram representing the relationship between NR expression and their physiological functions. The relationship between NR expression, function and physiology is depicted as a circular dendrogram using the hierarchical, unsupervised clustering of NR tissue expression distribution. The analysis reveals the existence of a higher order network tying NR function to reproduction, development, central, and basal metabolic functions, dietary-lipid metabolism and energy homeostasis. Adapted from (Bookout, Jeong et al. 2006). 1.2.3.3 Structural features The structural and functional organisation within the NRs is highly conserved. NRs genes present five distinct domains, named A to E. The N-terminal region, containing domains A/B, is highly variable and contains at least one constitutively active transactivation function-1 (AF-1). This domain is implicated in transactivation and acts in a ligand-independent manner outside of the receptor context. In addition, the C-terminal part of the A/B domain, due to its proximity with C domain (DNA-binding domain) may play a role in the interaction with DNA 46 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction by regulating the ability of the receptor to interact with other members of the family or by altering the choice of the DNA target sequence (Robinson-Rechavi, Escriva Garcia et al. 2003). The DNA-binding domain is the most conserved region and contains the P-box, responsible for the DNA-binding specificity and two zinc-binding motifs that maintain the domain architecture. The variability of this domain correlates with the DNA-binding preferences of the NRs. Two NRs, the small heterodimerizing partner 1 (SHP1) and dosagesensitive sex reversal congenital adrenal hypoplasia critical region on the X chromosome (DAX1) lack a DBD and function principally as dominant negative repressors for other NRs (Nagy and Schwabe 2004). Between the DNA-binding and the ligand-binding domain (E domain) is a less conserved region (D domain) that behaves as a flexible hinge between the C and E domains, and contains the nuclear localization signal (NLS) (Robinson-Rechavi, Escriva Garcia et al. 2003). Finally, the large E domain, ligand-binding domain, is moderately conserved as sequence but well conserved as 3D structure. The ligand-binding domain is responsible for many functions, mostly ligand induced, notably the AF-2 (helix 12) transactivation function, a strong dimerization interface, another NLS, and often a repression function (RobinsonRechavi, Escriva Garcia et al. 2003). Fig. 7. Schematic representation of NR domains. Most of the NR exhibit five specific domains, the N terminal containing AF-1 involved in transactivation, followed by the DNA-binding domain (DBD). The hinge is a poorly conserved region presenting the nuclear localisation signal (NLS). The C terminal portion contains the ligand-binding domain (LBD) and the helix 12 (AF-2), crucial for the ligand-induced activity (Robinson-Rechavi, Escriva Garcia et al. 2003). 1.2.3.4 Structure and functions of VDR The human VDR gene, located on chromosome 12q, is composed of a promoter and a regulatory region and 8 exons encoding the 427 amino acid of the VDR protein (MW 48 kDa) (Fig. 8) (Deeb, Trump et al. 2007). The VDR is composed of domains that allow translocation to the nucleus, ligandbinding, heterodimerization with its partner RXR, DNA-binding and the co-factor interactions (Fig. 8) (Carlberg 2003). In the absence of ligand, VDR is partitioned between the cytoplasm Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 47 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD and the nucleus. The ligand induces interaction of VDR with importin β, via its nuclear localization signal regions, increasing nuclear translocation of VDR, as well as the translocation of the VDR-RXR heterodimer (Yasmin, Williams et al. 2005). The DNA binding domain (DBD) recognizes a specific DNA sequence present in regulatory regions of primary 1α,25(OH)2D3 responding genes, which is referred to as a vitamin D response element (VDRE). VDREs are hexameric DNA sequence composed of the consensus sequence RGKTSA (R=A or G, K=G or T, S=C or G) separated by three or four spacing nucleotides (Carlberg and Seuter 2009). VDR-RXR heterodimers bind to VDREs form by a direct repeat (DR) of two hexameric core binding motifs with 3 intervening nucleotides (DR3-type) (Carlberg, Bendik et al. 1993), but also to DR4-type REs along with other members of the nuclear receptor superfamily (Quack and Carlberg 2000). It should be noted that effective VDR binding has also been observed on everted repeat (ER)-type REs with 6 to 9 spacing nucleotides (ER6, ER7, ER8, ER9) (Schrader, Muller et al. 1994; Schrader, Nayeri et al. 1995) The ligand-binding domain contains the 1α,25(OH)2D3-binding pocket and the transactivation domain called AF-2 (Carlberg 2003). This last domain is essential for the ability of NRs to activate gene transcription, as the change of positioning of helix 12, upon ligand binding, creates a binding surface that favors the interaction with CoAs instead of CoRs (Nagy and Schwabe 2004). CoRs suppress the expression of responsive genes, while CoAs favor transcription and act as a bridge between the VDR-RXR heterodimer and the basal transcription machinery (Nagpal, Na et al. 2005). CoRs recruit histone deacetylase involved in chromatin condensation that wrap VDREs, which silences gene expression. CoAs recruit histone acetyltransferases, destabilize the nucleosome core and unravel DNA for transcription (Nagpal, Na et al. 2005). Passive diffusion across cell membrane allows 1α,25(OH)2D3 to bind to its receptor and induce VDR phosphorylation at serines 51 and 208. Phosphorylations have been proposed to induce conformational changes in ligand- and DNA-binding domains that allow heterodimerization of VDR with RXR (Arriagada, Paredes et al. 2007). As a consequence, CoRs (such as NR co-repressors and the silencing mediator for retinoid and thyroid hormone receptors) are released. Next, the CoA complexes (steroid receptor co-activators, nuclear coactivator 62 kDa–SKI-interacting protein, chromatin modifiers CREB binding protein–p300, polybromo and SWI‑2 related gene 1 associated factor) are recruited to VDRE region to initiate the transcription with the help of transcription factor 2B and RNA polymerase II 48 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction (Haussler, Whitfield et al. 1998). VDR binding does not necessarily induce transcript upregulation, but it can result in direct or indirect inhibition of gene expression (White 2004; Carlberg and Seuter 2009). VDR expression is regulated by positive feedback, whereby VDR activation increases its mRNA synthesis, while increased expression of PTH inhibits VDR synthesis (Brown, Zhong et al. 1995). As observed in various cell line, VDR can be down-regulated due to the inhibitory effect of SNAIL 1 or 2 (zinc-finger transcription factors involved in cell movement) but it could be up-regulated in colon and breast cancer cells after estradiol treatment in a ERK dependent manner (Palmer, Larriba et al. 2004; Gilad, Bresler et al. 2005; Larriba, Martin-Villar et al. 2009). Interestingly, in inflammatory conditions, VDR expression could be up-regulated as shown in IL-8 stimulated BPH cells (II). Fig. 8. Chromosomic and protein domains of the Vitamin D receptor. The human VDR gene located on chromosome 12q, is composed of non translated exons (1a–1f) and exons 2–9, which encode 6 domains (A–F) of the full-length VDR protein. VDR nuclear localization signals (blue) direct the receptor into the nucleus. VDR associates with RXR through the dimerization domains (yellow). The 1α,25(OH)2D3–VDR–RXR complex binds to VDREs through the DNA-binding domain in the regulatory region of target genes. Conformational changes in the VDR result in the dissociation of the CoR, silencing mediator for retinoid and thyroid hormone receptors (SMRT), and allows interaction of the transactivation domain AF-2 (light grey) with stimulatory CoAs (Haussler, Whitfield et al. 1998) that mediate transcriptional activation. Non-synonymous (FokI) and synonymous (BsmI, ApaI, TaqI and Tru9I) single-nucleotide polymorphisms (SNPs) have been identified in VDR (defined by restriction enzymes, polymorphisms are indicated in parentheses). FokI polymorphism at translation initiation codon results in a smaller VDR that interacts with transcription factor 2B (TF2B) more efficiently and has greater transcriptional activity than the full length VDR. Adapted from (Deeb, Trump et al. 2007) Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 49 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 1.2.4 Target genes and biological role About 1000 different genes are believed to be under the direct control of 1α,25(OH)2D3. As revealed by rickets development caused by VDR loss of function, most of the VDR-regulated genes are involved in the calcium/phosphate homeostasis and in the maintenance of bone content. However, VDR expression in cells not involved in these processes, especially actors of the immune system, led to the recognition of non-calcemic action of 1α,25(OH)2D3 (Nagpal, Na et al. 2005). 1.2.4.1 Calcium and phosphate homeostasis Biological functions of 1α,25(OH)2D3 are the regulation of calcium and phosphate homeostasis and maintenance of bone integrity. These activities are achieved through direct actions of the hormone on the intestine, kidney or bone and through feedback inhibition of parathyroid hormone (PTH) production at the parathyroid glands. However, bone deficiency in children’s severe rickets could be rescued by intra-venous calcium administration, indicating an important role of vitamin D-dependent calcium regulation in the intestine rather than in bone, kidney or parathyroid gland (Bouillon, Van Cromphaut et al. 2003). Calcium ion channel transient receptor potential vanilloid type 5 and 6 (TRPV5-6 also known as epithelial calcium channel 1 and 2) present VDREs in their regulatory regions and are up-regulated after 1α,25(OH)2D3 treatment. TRPV5 and 6 are respectively involved in transepithelial uptake of calcium by the kidney and in the absorption of calcium from the intestinal lumen, and were considered as the “gatekeepers” of epithelial calcium transport (Pike, Zella et al. 2007). Interestingly, Trpv6 and CalbindinD9K deficient mice present a physiological intestinal calcium absorption and 1α,25(OH)2D3 treatment in Trpv6 deficient mice respond equally well for the intestinal calcium regulation compared to the wild type (Benn, Ajibade et al. 2008; Kutuzova, Sundersingh et al. 2008). Thus, the mechanisms underlying the 1α,25(OH)2D3 intestinal calcium regulation appears to be still incompletely defined. Moreover, up-regulation after 1α,25(OH)2D3 treatment and substantial reduction in both Cyp27b1-null and VDR-null mice can explain the important role of 1α,25(OH)2D3 in calcium homeostasis (Van Cromphaut, Dewerchin et al. 2001; van Abel, Hoenderop et al. 2003). Phosphate homeostasis involves a phosphaturic hormone, fibroblast growth factor 23 (FGF23), which is secreted by osteoblasts and functions as a suppressor of phosphate reabsorption from the kidney filtrate and represses 1α,25(OH)2D3 synthesis, closing this 50 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction endocrine regulatory loop (Haussler, Whitfield et al. 1998). In addition, 1α,25(OH)2D3 upregulates also receptor activator of NF-κB ligand (RANKL), an osteoblast cell surface protein that stimulates osteoclastogenesis and bone resorption (Boyce and Xing 2008). Interestingly, 1α,25(OH)2D3 acts as a direct repressor of PTH in contrast with its positive effect on calcium regulation. 1.2.4.2 1α,25(OH)2D3 metabolism and catabolism 1α,25(OH)2D3 has a direct regulatory effect on its metabolic and catabolic enzymes. First, CYP27B1 is up-regulated by PTH and down-regulated by 1α,25(OH)2D3, highlighting a direct negative control of the bioactive hormone on its actual synthesis (Brenza and DeLuca 2000). Similarly, CYP24A1, encoding for the principal enzyme involved in 1α,25(OH)2D3 catabolism, is the most important positively regulated gene by 1α,25(OH)2D3 (Ohyama, Ozono et al. 1994). 1.2.4.3 Vitamin D deficiency As previously discussed, vitamin D discovery was directly linked to the rickets, a disease caused by vitamin D deficiency. The main source of vitamin D precursor is the UVBirradiated skin, while limited quantities are contained in the diary nutriments, except oily fish and fish liver oil (Hollis 2005). This implicates sun exposure as a critical step in vitamin D synthesis, since the main catalyzer of its synthesis is UVB. Then, it became obvious that living in higher latitudes, lack of sunlight represents as an important environmental factor for vitamin D deficiency (Cantorna and Mahon 2004; Holick 2007). Vitamin D deficiency was also shown to be correlated with the incidence and the severity of osteoporosis, a bone disorder (Lips 1996). The discovery of the non-calcemic effects of 1α,25(OH)2D3 and the symptoms amelioration in autoimmune animal models after 1α,25(OH)2D3 treatment confirmed vitamin D deficiency as a potential environmental factor in autoimmune diseases (Cantorna and Mahon 2004). Nowadays, it is evident that low 25(OH)D3 serum levels are linked to the development and the severity of many disorders from autoimmune disease to cancer (Brenza and DeLuca 2000; Jurutka, Bartik et al. 2007). Lack of sun exposure does not represent the sole cause of vitamin D deficiency, but skin absorption and loss of function of VDR or enzymes responsible for its synthesis are equally important (Holick 2007). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 51 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Fig. 9. Physiological functions of 1〈,25(OH)2D3. Vitamin D3 made in the skin or ingested in the diet can be stored in and then released from fat cells. Vitamin D in the circulation is bound to the vitamin D–binding protein, which transports it to the liver, where vitamin D is converted by CYP27A1. This is the major circulating form of vitamin D that is used by clinicians to determine vitamin D status. This form of vitamin D is biologically inactive and must be converted in the kidneys by CYP27B1 to the biologically active form 1α,25(OH)2D3. Serum phosphorus, calcium, FGF-23, and other factors can either increase (+) or decrease (–) the renal production of 1α,25(OH)2D3 that decreases its own synthesis through negative feedback and decreases the synthesis and secretion of PTH by the parathyroid glands. 1α,25(OH)2D3 increases the expression of CYP24A1 to catabolize 1α,25(OH)2D3 to the water-soluble, biologically inactive calcitroic acid, which is excreted in the bile. 1α,25(OH)2D3 enhances intestinal calcium absorption in the small intestine by interacting with the VDR-RXR to enhance the expression of the epithelial calcium channel TRPV6 and calbindin D9K, a calcium-binding protein (CaBP). 1α,25(OH)2D3 is recognized by its receptor in osteoblasts, causing an increase in the expression of RANKL, which induces preosteoclasts to become mature osteoclasts. Mature osteoclasts remove calcium and phosphorus from the bone, maintaining calcium and phosphorus levels in the blood. Adapted from (Deeb, Trump et al. 2007) and (Holick 2007). 52 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction 1.2.5 VDR-mediated non-calcemic activities Epidemiological data on increased susceptibility to various diseases in people suffering of 25(OH)D3 deficiency, and the observation that VDR is also present in cells other than those of the intestine, bone, kidney and parathyroid gland led to the recognition of non-calcemic actions of VDR ligands. Discovery of VDR expression in lymphocytes and its antiproliferative properties were the first evidence for the potential role of 1α,25(OH)2D3 as antiproliferative and anti-inflammatory agent (Abe, Miyaura et al. 1981; Colston, Colston et al. 1982; Provvedini, Tsoukas et al. 1983). The immunomodulatory and anti-inflammatory properties of VDR agonists will be described in detail below. 1.2.5.1 Regulation of cell proliferation and tumorigenesis The important role of 1α,25(OH)2D3 in the regulation of cell proliferation and tumorigenesis is reflected by the phenotype observed in Vdr deficient mice. These mice present numerous tumors after exposure to oncogene or carcinogen, compared to wild-type mice, and colorectal hyper-proliferation. However, they do not present spontaneous tumors despite precancerous lesions in mammary glands (Kallay, Pietschmann et al. 2001; Bouillon, Eelen et al. 2006). As previously mentioned, vitamin D deficiency is directly correlated with cancer, but also genes coding the metabolic enzymes, CYP24A1 and CYP27B1, are found to be down-regulated in many cancers, such as in breast or prostate tumors (Palmer, Gonzalez-Sancho et al. 2001). Mechanisms underlying 1α,25(OH)2D3 anti-proliferative effects are principally mediated by its capacity to perturbate the cell cycle (Deeb, Trump et al. 2007). E-cadherin, a transmembrane linker of the intercellular adherens junctions, is a membrane protein classified as tumor suppressor gene. E-cadherin binds to β-catenin, inhibiting its nuclear translocation. In the nucleus, β-catenin binds to T cell transcription factor/lymphoid enhancer-binding factor 1 (TCF/LEF1), a transcription factor involved in the cell proliferation control. 1α,25(OH)2D3 treatment increases the level of E-cadherin, resulting in a sequestration of β-catenin in the cytoplasm and blocking the TCF/LEF1 gene regulation (Palmer, Gonzalez-Sancho et al. 2001). Transition from G0/G1 to S phase is directed by cyclin dependent kinases (CDKs) or CDK inhibitors (CDKIs) causing phosphorylation /dephosphorylation events of the tumor suppressor retinoblastoma protein. VDREs were found in regulatory regions of some CDK and CDKI, such as GADD45A or p21waf1, demonstrating the direct regulatory process of 1α,25(OH)2D3 by arrest of G1 cycle (Deeb, Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 53 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Trump et al. 2007). An alternative anti-tumor mechanism of 1α,25(OH)2D3 is to promote cell apoptosis. Key mediators of apoptosis such the anti-apoptotic, pro-survival proteins BCL2 and BCL-XL are repressed by 1α,25(OH)2D3, while it induces expression of pro-apoptotic proteins, such as BAX, BAK and BAD (Ylikomi, Laaksi et al. 2002). Recent evidence involves 1α,25(OH)2D3 also in angiogenesis inhibition, as demonstrated by inhibition of vascular endothelial growth factor (VEGF) and up-regulation of the potent anti-angiogenic factor thrombospondin 1 (THBS1) in SW480-ADH human colon tumor cells (FernandezGarcia, Palmer et al. 2005). However, tumors exhibit many mechanisms to escape the VDR-dependent antiproliferative activity. As previously mentioned, VDR expression in late tumor phases is down-regulated. In addition, in colorectal cancer, SNAIL1 and 2 repress VDR gene promoter, inducing the inhibition of 1α,25(OH)2D3 dependent E-cadherin up-regulation, resulting in the abolition of its anti-proliferative activity (Palmer, Gonzalez-Sancho et al. 2001; Palmer, Larriba et al. 2004; Larriba, Martin-Villar et al. 2009). 1.2.5.2 Regulation of the immune system VDR is expressed in most cell types of the immune system, in particular in APCs, such as macrophages and DCs, as well as in both CD4+ and CD8+ T cells (Veldman, Cantorna et al. 2000). Moreover, macrophages and DCs express, under the control of pro-inflammatory signals, such as IFN-γ or NF-κB, the functional enzymatic machinery to synthesize and metabolize the active hormone (van Etten and Mathieu 2005). From these observations, it is conceivable that 1α,25(OH)2D3 could contribute to physiological regulation of the innate and adaptive immune responses; thus VDR agonists could represent valuable anti-inflammatory agents (Adorini and Penna 2008). Data accumulated in the last few years clearly demonstrate that, in addition to exert direct effects on T-cell activation, VDR agonists markedly modulate the phenotype and functions of APCs, in particular DCs. It is also possible that 1α,25(OH)2D3 may contribute to the physiological control of immune responses, and possibly be also involved in maintaining tolerance to self antigens, as suggested by the enlarged lymph nodes containing a higher frequency of mature DCs in Vdr-deficient mice (Griffin, Lutz et al. 2001). Recently, novel regulatory roles of 1α,25(OH)2D3 were highlighted in wound repair enabling keratinocytes to recognize and respond to microbes and to protect wounds against infection, as well as its role as key component of innate immunity in microbial recognition and anti-microbial response during injury (Liu, Stenger et al. 2006). This appealing concept 54 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction has emerged by the observation that vitamin D3 induced by sunlight in the skin is hydroxylated by local DCs into the active hormone, which in turn up-regulates on activated T cells expression of the epidermiotropic chemokine receptor CCR10, a primary VDRresponsive gene, enabling them to migrate in response to the epidermal chemokine CCL27 (Sigmundsdottir, Pan et al. 2007). Thus, the autocrine production of 1α,25(OH)2D3 by DCs can program the homing of skin-associated T cells, which could include Treg cells able to counteract the pro-inflammatory effects induced in the skin by sun exposure. In addition, high CYP27B1 expression was found in wounds that were induced in keratinocytes in response to TGF-1ß, triggering production of 1α,25(OH)2D3 by keratinocytes, which in turn increased expression of CAMP and induced TLR2 and CD14 expression (Schauber, Dorschner et al. 2007). Thus, VDR agonists possess not only anti-inflammatory but also anti-infective properties, which could provide additional clinical benefits in different inflammatory conditions. 1.2.6 Anti-inflammatory properties of VDR agonists 1.2.6.1 Dendritic cells Earlier indications for the capacity of VDR agonists to target APCs were corroborated by their ability to inhibit the production of IL-12. More recent work has demonstrated that 1α,25(OH)2D3 and its analogs have profound effects on the phenotype and function of mDCs. VDR agonists arrest the differentiation and maturation of DCs, maintaining them in an immature state, as shown by decreased expression of maturation markers and increased antigen uptake (Penna and Adorini 2000; Ferreira, van Etten et al. 2009). Studies performed either on monocyte-derived DCs from human peripheral blood or on bone marrow-derived mouse DCs have consistently shown that in vitro treatment of DCs with VDR agonists leads to down-regulated expression of the co-stimulatory molecules CD40, CD80, CD86, to markedly decreased IL-12, to enhance IL-10 production, and the modulation of chemokine production; resulting in inhibition of T-cell activation. The near abrogation of IL-12 production and the strongly enhanced secretion of IL-10 highlight the important functional effects of 1α,25(OH)2D3 and its analogs on DCs and are, at least in part, responsible for the induction of DCs with tolerogenic properties (Penna and Adorini 2000). In addition, DCs treated with VDR agonists up-regulate the expression of ILT3, an inhibitory molecule associated with tolerance induction, although ILT3 expression is dispensable for the capacity of 1α,25(OH)2D3-treated DCs to induce regulatory T cells (Penna, Roncari et al. 2005). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 55 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 1α,25(OH)2D3 utilizes different mechanisms to regulate cytokine production by DCs. IL-12 secretion is inhibited by targeting the NF-κB pathway, via NF-κB proteins, such as Vrel reticuloendotheliosis viral oncogene homolog B (RelB) and c-Rel. RelB is essential in differentiation and maturation of DCs, and exhibits an active VDRE that could explain is strongly down-regulation in mice splenocytes after 1α,25(OH)2D3 intra-peritoneal (IP) administration (Griffin, Dong et al. 2007). RelB inhibition is directly mediated by binding of liganded-VDR-RXR complex in VDREs present in its promoter. This induces recruitment of CoR complexes containing HDAC1 and 3 and SMRT. Direct RelB inhibition seems to occur selectively in APCs, confirming predominant immunomodulation of 1α,25(OH)2D3 on this subtypes (Griffin, Dong et al. 2007). Interestingly, antigen-exposed DCs, in which RelB function is inhibited induce a population of antigen-specific CD4+ cells that regulate immune responses in an IL-10-dependent manner (Griffin, Lutz et al. 2001). Suppression of the monocyte recruited GM-CSF is achieved by interaction of ligand-VDR monomers with functional repressive complexes in the promoter region of the cytokine (Towers and Freedman 1998). In this case, the ligand-VDR complex acts selectively on the two components required for activation of this promoter/enhancer: it competes with the transcription factor NFAT1 for binding to the composite site and positioning itself adjacent to Jun–Fos on the DNA. Co-occupancy apparently leads to an inhibitory effect on c-Jun transactivation function. These two events mediated by VDR effectively block the NFAT1AP-1 activation complex, resulting in an attenuation of GM-CSF transcription (Towers and Freedman 1998; Towers, Staeva et al. 1999). The prevention of DC differentiation and maturation, as well as the modulation of their activation and survival, which leads to DCs with tolerogenic phenotype and function that result in T-cell hyporesponsiveness, are not limited to in vitro activity. 1α,25(OH)2D3 and its analogs can also induce DCs with tolerogenic properties in vivo, as demonstrated in models of allograft rejection by oral administration directly to the recipient or by adoptive transfer of in vitro-treated DCs (Griffin, Lutz et al. 2001). Tolerogenic DCs induced by a short treatment with 1α,25(OH)2D3 are probably responsible for the capacity of this hormone to induce CD4+CD25+ Treg cells that are able to mediate transplantation tolerance (Adorini, Penna et al. 2003) (detailed in Regulatory T cells). Moreover, in autoimmune diabetic NOD-treated mice that exhibit a defect in Treg cells, VDR agonists restored the Treg cell population and arrest the development of autoimmune diabetes (Mathieu, Waer et al. 1995). 56 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction Although the immunomodulatory effects of VDR agonists on DCs are well established, the capacity of this hormone to modulate DCs subsets has been addressed only recently. Two DC subsets, M-DCs and P-DCs have been identified. These subsets are characterized by a distinct expression of PRRs and co-stimulatory molecules, and by the selective production of immunomodulatory cytokines. Analysis of immunomodulatory effects exerted by 1α,25(OH)2D3 on human blood M-DCs and P-DCs demonstrates a differential capacity of this hormone to modulate cytokines and chemokines production in DC subsets, showing marked effects in M-DCs and negligible ones in P-DCs (Penna, Amuchastegui et al. 2007). In addition, inhibition of Th1 development and enhancement of CD4+ suppressor Tcell activity are selectively induced by 1α,25(OH)2D3 in M-DCs but not P-DCs. This differential capacity of DC subsets to respond to 1α,25(OH)2D3 is not due to a diverse VDR expression or VDR-dependent signal transduction, but is associated with different effects of this hormone on NF-κB p65 phosphorylation and nuclear translocation in DC subsets (Penna, Amuchastegui et al. 2007). Thus, 1α,25(OH)2D3 appears to up-regulate tolerogenic properties in mDCs, down-regulating IL-12 and Th1 cell development, while promoting CD4+ suppressor T cell activity and enhancing the production of CCL22, a chemokine able to recruit Treg cells. By contrast, no immunomodulatory effects appear to be induced by 1α,25(OH)2D3 in P-DCs (Liu 2005). P-DCs, characterized by an intrinsic ability to prime naïve CD4+ T cells to differentiate into IL-10-producing T cells and CD4+CD25+ Treg cells, and to suppress immune responses, may represent naturally occurring regulatory DCs, and the lack of P-DCs modulation by 1α,25(OH)2D3 would thus leave this tolerogenic potential unmodified (Liu 2005). Innate immune regulation by 1α,25(OH)2D3 was recently emphasized by the discovery of a “nonclassical” mechanism mediated by the enhancement of anti-microbial peptide. Discovery of VDREs in CAMP gene encoding, a potent anti microbial peptide, proposes new functions for 1α,25(OH)2D3 in immunomodulation of innate immunity (Wang, Nestel et al. 2004). Protection by 1α,25(OH)2D3-induced CAMP expression was demonstrated in human PBMCs infected by tuberculosis, decreasing pro-inflammatory cytokines production and down-regulating HLA-DR expression (Schauber, Dorschner et al. 2007). Moreover, a clinical correlate of this important link is provided by the observation that sera from AfricanAmerican individuals, known to have increased susceptibility to Mycobacterium tuberculosis, have reduced levels of 25(OH)D3, the 1α,25(OH)2D3 precursor, and are inefficient in CAMP mRNA induction, suggesting that 1α,25(OH)2D3 sufficiency contributes to decreased Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 57 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD susceptibility to microbial infections (Schauber, Dorschner et al. 2007). Interestingly, in autoimmune disorders, especially psoriasis, CAMP overexpression in wound healing has been proposed as a pro-inflammatory signal for P-DCs (Lande, Gregorio et al. 2007). However, clear evidences for anti-inflammatory properties, such as arrest of DCs maturation, are also described. DCs maturation after TLR activation is inhibited by CAMP, as show by inhibition of maturation markers, such as CD86, CD80, CD83 and HLA-DR, as well as by inhibition of pro-inflammatory cytokines, via inhibition of NF-κB nuclear translocation (Kandler, Shaykhiev et al. 2006). Morevover, the immunomodulatory properties of CAMP could also affect T cells, as shown by the inhibition of IFN-γ and IL-2 production in DCs co-cultured with naive T cells in the presence of CAMP (Kang, Azad et al. 2005; Bandholtz, Ekman et al. 2006; Yu, Mookherjee et al. 2007). In vivo data on the relationship between 1α,25(OH)2D3 and CAMP, especially in mice models, are difficult due to the absence of VDREs in the promoter of the murine homolog, cramp (Gombart, Borregaard et al. 2005). However, cramptreated mice present amelioration of symptoms in chemically induced colitis or allergic contact sensitization, as also observed with 1α,25(OH)2D3 treatment (Di Nardo, Braff et al. 2007; Tai, Wu et al. 2007). 1.2.6.2 T cells Soon after the discovery of VDR expression in T cells, 1α,25(OH)2D3 was shown to inhibit antigen-induced T-cell proliferation and cytokine production. Later studies demonstrated selective inhibition of Th1 cell development, although it was not clarified how much of this effect could be accounted for by modulation of DC functions. Indeed, several key cytokines are direct targets for VDR agonists in T lymphocytes, in particular Th1-type cytokines, such as IL-2 and IFN-γ (Nagpal, Na et al. 2005). In activated T cells, inhibition of IL-2 transcription is mediated by antagonism of the ligand-VDR-RXR complex for the formation of the NFAT/AP-1 complex, resulting to a stable association of VDR to the NF-AT/AP-1 binding site on the IL-2 promoter (Bemiss, Mahon et al. 2002). On the other hand, IFN-γ transcription inhibition is directly mediated by interaction of the ligand–VDR–RXR complex via a negative VDRE in the promoter region of the gene (Cippitelli and Santoni 1998). 1α,25(OH)2D3 has been also shown to enhance the development of Th2 cells via a direct effect on naïve CD4+ cells. This could contribute to account for the beneficial effect of VDR agonists in the treatment of inflammatory conditions (Boonstra, Barrat et al. 2001). The capacity of 1α,25(OH)2D3 to skew T cells towards the Th2 pathway had been suggested 58 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction previously, but could not be confirmed by other studies. Th2 cells can be targets of VDR agonists but this depends on their activation and differentiation status (Mahon, Wittke et al. 2003). Thus, 1α,25(OH)2D3 can apparently up-regulate, down-regulate or have no effect on IL-4 production and, consequently, on Th2 cell development, illustrating the complexity of immunoregulatory pathways set in motion by 1α,25(OH)2D3. Treatment with VDR agonists also inhibits IL-17 production (Tang, Zhou et al. 2009), a proinflammatory cytokine shown recently to be produced by pathogenic T cells in various models of chronic inflammation and immune-mediated tissue injury, including organ-specific autoimmunity in the brain, heart, synovium and intestines, allergic disorders of the lung and skin, and microbial infections of the intestines and the nervous system (Steinman 2007). Interestingly, IL-17 production is induced by IL-23 where p40 chain is strongly inhibited by VDR agonists. As expected, 1α,25(OH)2D3 inhibits IL-17 produced by T cells, showing an amelioration of symptoms in different autoimmune diseases (Adorini and Penna 2008). Recent work on experimental autoimmune uveitis, a Th17 autoimmune visual disorder, show 1α,25(OH)2D3 potency in the inhibition of IL-17 leading to a suppression of Th17-mediated inflammation. This suppression involves the direct inhibition of IL-17 production by CD4+ T cells and indirect inhibition of IL-17 lineage commitment by down-regulation of the ability of DC to support priming of T cells toward the Th17 effectors pathway (Tang, Zhou et al. 2009). Thus, in addition to controlling Th1 and Th2 cells, 1α,25(OH)2D3 also modulates the Th17 lineage. Interestingly, 1α,25(OH)2D3 has also been shown to prevent and treat TNBS-induced colitis, by reducing Th1 and Th17 cells while up-regulating Foxp3+ Treg cells, associated with significant reduction of IL-12p75, IL-23p19, and IL-6 production by DCs (Daniel, Sartory et al. 2008). In conclusion, 1α,25(OH)2D3 in vivo appears primarily to inhibit pro-inflammatory, pathogenic T cells, such as Th1 and Th17, and, under appropriate conditions, may favor a deviation to the Th2 pathway. These effects could be, in part, a consequence of direct T-cell targeting by 1α,25(OH)2D3 and its analogs, but modulation of DC function by VDR agonists certainly plays an important role in shaping the development of T-cell responses. Thus, VDR agonists can target T cells both directly and indirectly, selectively inhibiting T-cell subsets able to mediate inflammation and tissue damage. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 59 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD CCL17 CCL22 IL12/23p40 IL-10 CD152 Inhibition CD80/CD86 CD28 TCR MHCII Activation Recognition DC Dendritic Cell CD11a/CD18 CD54 CD154 CD40 ILT3 CD4+ T cell Adhesion Activation Inhibition Fig. 10. Immunomodulatory effects of VDR agonists on myeloid DCs and CD4+ T cells. VDR agonists inhibit in M-DCs, but not in P-DCs, expression of surface co-stimulatory molecules, for example, CD40, CD80 and CD86, as well as MHC class II and CD54 molecules. Production of cytokines affecting T-cell differentiation into Th1 and Th17, IL-12 and IL-23, respectively, are also inhibited in M-DCs. Conversely, expression of surface inhibitory molecules, like ILT3, and of secreted inhibitory cytokines, such as IL-10, are up-regulated markedly. Chemokines potentially able to recruit CCR4+ regulatory T cells, such as CCL22 are also up-regulated, whereas the CCR4 ligand CCL17 is down-regulated. Upon interaction with M-DCs, CD4+ T cells up-regulate expression of the inhibitory molecule CD152 (CTLA-4). DCs expressing low levels of co-stimulatory molecules, secreting IL-10, and expressing high levels of inhibitory molecules (for example, ILT3) favor the induction and/or the enhancement of regulatory/suppressor T cells (Adorini 2002; Adorini and Penna 2008). 1.2.6.3 Regulatory T cells As discussed previously, induction of DCs with tolerogenic phenotype and function plays an important role in the immunoregulatory activity of VDR agonists. Tolerogenic DCs induced by a short treatment with 1α,25(OH)2D3 or its analogs are probably responsible for the capacity of the hormone to induce CD4+CD25+ suppressor T cells that are able to mediate transplantation tolerance (Adorini, Penna et al. 2003) and to arrest the development of autoimmune diabetes (Gregori, Giarratana et al. 2002). VDR agonists enhance CD4+CD25+ Treg cells and promote tolerance induction in transplantation and autoimmune disease models. A short treatment with 1α,25(OH)2D3 and mycophenolate mofetil, a selective inhibitor of T and B cell proliferation that also modulates APCs, induces tolerance to islet allografts 60 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction associated with an increased frequency of CD4+CD25+ Treg cells able to adoptively transfer transplantation tolerance (Gregori, Casorati et al. 2001). The induction of tolerogenic DCs could indeed represent a therapeutic strategy promoting tolerance to allografts (Adorini 2002) and the observation that immature mDCs can induce T cell tolerance to specific antigens in human volunteers represents an important proof of concept for this approach (Dhodapkar, Steinman et al. 2001). CD4+CD25+ Treg cells able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4+CD25- T cells are also induced by treatment of adult NOD mice with the VDR agonist BXL-219 (Gregori, Giarratana et al. 2002). This treatment arrests insulitis, blocks the progression of Th1 cell infiltration into the pancreatic islets and inhibits T1D development at non-hypercalcemic doses (Gregori, Giarratana et al. 2002). Although the T1D and islet transplantation models are quite different, in both cases administration of VDR agonists doubles the number of CD4+CD25+ Treg cells, in the spleen and pancreatic lymph nodes. However, tolerogenic DCs may not always be necessarily involved in the generation of Treg cells by VDR agonists. A combination of 1α,25(OH)2D3 and dexamethasone has been shown to induce human and mouse naïve CD4+ T cells to differentiate in vitro into Treg cells, even in the absence of APCs. Upon transfer, these IL-10 producing T cells could prevent CNS inflammation, indicating their capacity to exert a suppressive function in vivo (Barrat, Cua et al. 2002). VDR agonists not only favour induction of CD4+CD25+ Treg cells, but can also enhance their recruitment to inflammatory sites. Blood-borne M-DCs, in contrast to P-DCs, constitutively produce high levels of CCL17 and CCL22 ex vivo, which are enhanced further by CD40 stimulation (Penna, Amuchastegui et al. 2007). CCL22 and CCL27 are chemokines able to recruit activated T cells and, in particular, Th2 cells via CCR4. In addition, these chemokines can recruit CD4+CD25+ Treg cells (D'Ambrosio, Sinigaglia et al. 2003). CCL22, a chemokine mostly produced by DCs, has been found to selectively recruit, in ovarian carcinoma patients, Foxp3+ Treg cells able to suppress anti-tumor responses, leading to reduced patient survival (D'Ambrosio, Sinigaglia et al. 2003). Similarly, CCL22 secreted by lymphoma B cells attracts Foxp3+CCR4+CD4+CD25+ Treg cells able to suppress proliferation and cytokine production by tumor-infiltrating CD4+CD25- T cells (Yang, Novak et al. 2006). Thus, the high constitutive and inducible production of CCR4 agonists by immature mDCs could lead to the preferential attraction of CD4+CD25+ Treg cells. Intriguingly, the production Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 61 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD CCL22 is markedly enhanced by 1α,25(OH)2D3 in blood M-DCs (Penna, Amuchastegui et al. 2007), indicating that VDR agonists may favor the recruitment of Treg cells by this DC subset, a novel finding consistent with previous in vivo data (Gregori, Casorati et al. 2001; Gregori, Giarratana et al. 2002; Penna, Amuchastegui et al. 2007). 1.2.6.4 Treatment of autoimmune diseases A common pattern shared by most autoimmune diseases is the Th1-mediated inflammatory profile. Epidemiological analysis shows reasonably strong ecological and case-control evidence that vitamin D reduces the risk of autoimmune disease, including multiple sclerosis and T1D, and weaker evidence for rheumathoid arthritis (Grant 2006). The high prevalence of vitamin D inadequacy in the general population could indeed favor chronic inflammatory diseases, in addition to bone diseases and cancer (Holick 2007). The anti-inflammatory and immunoregulatory properties of VDR agonists have been studied in different models of autoimmune diseases. Notably, 1α,25(OH)2D3 and its analogs can prevent systemic lupus erythematosus in MRLlpr/lpr mice, experimental allergic encephalomyelitis (EAE), collagen-induced arthritis, Lyme arthritis, IBD, and autoimmune diabetes in NOD mice. VDR agonists are able not only to prevent but also to treat ongoing autoimmune disease, as demonstrated by their ability to inhibit T1D development in adult NOD mice, and the recurrence of autoimmune disease after islet transplantation in the NOD mouse, or to ameliorate significantly the chronic-relapsing EAE induced in Biozzi mice by spinal cord homogenate (Mattner, Smiroldo et al. 2000; Penna, Amuchastegui et al. 2006). As discussed previously, an important property of VDR agonists is their capacity to modulate both APCs and T cells. Distinct regulatory mechanism may predominate in different autoimmune disease models but a common pattern, characterized by inhibition of Th1 cell development, has been frequently observed. The induction of tolerogenic DCs, which leads to an enhanced number of CD4+CD25+ Treg cells renders them appealing for clinical use, especially for the prevention and treatment of autoimmune disease (Adorini, Giarratana et al. 2004; Hackstein and Thomson 2004). However, topical treatment of psoriasis is the only clinical application so far established for VDR agonists in the therapy of autoimmune diseases. The calcemic liability of systemically applied VDR agonists still hampers progress towards clinical applications, a situation that may be resolved by the ongoing development of more potent and less calcemic analogs. In addition, additive and even synergistic effects have been observed between VDR agonists and immunosuppressive agents, such as cyclosporine A 62 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction and sirolimus, in autoimmune diabetes and EAE models (van Etten and Mathieu 2005). In the following chapters, BPH and principally IBD will be extended. Peripheral lymphoid organs Target tissue IL-17 Treg Treg CD40 CTL Th17 IL-6 IL-23 IL-2 CD80 mDC CD86 IL-12 Th1 Th1 Th1 Th1 migration ILT3 Target cell Inflammatory chemokines NO TNF-α IFN-γ mφ PGE2 CCL22 Cox-2 iNOS Treg IL-10 Fig. 11. Immunomodulatory effects of VDR agonists in autoimmune diseases. VDR agonists can modulate the inflammatory response via several mechanisms in secondary lymphoid organs and in target tissues. In secondary lymphoid organs, VDR agonists inhibit IL-12 and IL-23 production and ILT3 expression. M-DC modulation by VDR agonists inhibit development of Th1 and Th17 cells, while inducing CD4+CD25+Foxp3+ regulatory T cells and, under certain conditions, Th2 cells. VDR agonists can also inhibit the migration of Th1 cells, and they up-regulate CCL22 production by M-DC, enhancing the recruitment of CD4+CD25+ regulatory T cells and of Th2 cells. In addition, VDR agonists exert direct effects on T cells by inhibiting IL-2 and IFN-γ production. In target tissue, pathogenic Th1 cells, that can damage target cells via induction of cytotoxic T cells and activated macrophages, are reduced in number and their activity is further inhibited by CD4+CD25+ Treg cells and by Th2 cells induced by VDR agonists. IL-17 production by Th17 cells is also inhibited. In macrophage, important inflammatory molecules like COX-2 and iNOS, are inhibited by VDR agonists, leading to decreased production of NO and prostaglandin E2 (PGE2). Macrophages, as well as DCs and T cells, can synthesize 1α,25(OH)2D3 and this may also contribute to the regulation of the local immune response (Adorini 2002; Adorini and Penna 2008). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 63 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 1.3 Begnin prostate hyperplasia 1.3.1 Definition BPH is the most frequent benign neoplasm in aging men and one of the most common chronic conditions in the male population, with a histological prevalence at autopsy of 50% in men aged 50 to 60 years and of 90% over 80 years (McVary 2006). Clinical BPH refers to the lower urinary tract symptoms (LUTS), associated with benign prostatic enlargement leading to bladder outlet obstruction. BPH is defined histologically by hyperproliferation of stromal and epithelial cells of the prostate, caused by complex cellular alterations including changes in proliferation, differentiation, apoptosis and senescence (Lee and Peehl 2004). Compared to normal prostate tissue, hyperplastic nodules are characterized by reduced epithelium-to-stroma ratio, determined by an imbalance between growth and death programs of stromal cells (Ishigooka, Hayami et al. 1996; Claus, Berges et al. 1997; Lin, Wang et al. 2000), leading to increased final stromal volume. Histological micronodular alterations appear early in young men, characterized by an immature mesenchyme displaying features of embryonic mesenchyme, able to differentiate into myofibroblasts and smooth muscle cells to generate a “reactive stroma” (Peehl and Sellers 1997; Rumpold, Untergasser et al. 2002; Lee and Peehl 2004; Untergasser, Madersbacher et al. 2005). These changes in stromal architecture and homeostasis, and in the microenvironment of prostatic stromal-epithelial cell interactions, induce subsequent epithelial rearrangements and BPH progression (McNeal 1990; Donjacour and Cunha 1991; Bierhoff, Walljasper et al. 1997). 1.3.2 VDR agonists in BPH treatment A link between the vitamin D system and the prostate was first established by epidemiological correlations of increased prostate cancer incidence and mortality rates in patients with vitamin D insufficiency(Schwartz 2005). The prostate was then recognized as an extrarenal site of vitamin D synthesis and action through the expression of 1α-hydroxylase and VDR, respectively (Ali and Vaidya 2007). VDR expression in tissues derived from the urogenital sinus, as prostate and bladder, is quantitatively similar to classic target organs for calcitriol, as liver, kidney, and bone, although lower than in the bowel and in malignant prostate or bladder cell lines (Maggi, Crescioli et al. 2006). Moreover, a growth-regulating role of calcitriol 64 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction synthetized by the prostate is suggested by the demonstration of in vitro and in vivo antiproliferative, prodifferentiative, proapoptotic and anti-invasive properties in several models of prostate cancer (Ali and Vaidya 2007), and by a marked decrease of 1α-hydroxylase activity in prostate cancer cell lines (Maggi, Crescioli et al. 2006). Based on these preclinical results, the chemotherapic efficacy of calcitriol, alone or in association with classical anti-tumoral drugs (dexamethasone, docetaxel, carboplatin, estramustine), was tested by several clinical trials in prostate cancer patients (Harzstark and Ryan 2008). Pharmacological management of BPH is a novel application of vitamin D analogs, prompted by the detection of VDR expression in cultured prostatic and bladder stromal cells derived from BPH patients (Maggi, Crescioli et al. 2006). Reducing prostate overgrowth by decreasing intra-prostatic androgen signalling, without directly interfering with systemic androgen action, would obviate the adverse systemic side effects of anti-androgens, such as 5α-reductase inhibitors. In addition, VDR agonists display marked anti-inflammatory properties and this class of agents could therefore represent an interesting therapeutic option for the pharmacological treatment of BPH (Maggi, Crescioli et al. 2006). 1.3.2.1 Elocalcitol ameliorates experimental autoimmune prostatitis Based on marked inhibitory activity of the VDR agonist elocalcitol on basal and growth factor induced proliferation of human prostate cells (Crescioli, Ferruzzi et al. 2004), this compound was tested in experimental autoimmune prostatitis induced by injection of prostate homogenate-complete Freund’s adjuvant in NOD mice (Penna, Amuchastegui et al. 2006). Administration of elocalcitol, in normocalcemic doses for 2 weeks in already established experimental autoimmune prostatitis (EAP) inhibits significantly the intraprostatic cell infiltrate, leading to a profound reduction in the number of CD4+ and CD8+ T cells, B cells, macrophages, DCs and I-Ag7-positive cells. Immunohistological analysis demonstrates reduced cell proliferation and increased apoptosis of resident and infiltrating cells. Therapeutic administration of elocalcitol in NOD mice with established EAP decreases IFN-γ production by anti-TCR-stimulated lymph node cells, indicating inhibition of Th1 cell responses in prostate-draining periaortic lymph nodes. Treatment with elocalcitol also inhibits ex vivo production of IL-17, that could be relevant to the therapy of chronic prostate inflammation because this cytokine has been found elevated in situ in prostate specimens from patients affected by BPH, a condition characterized by prostate cell growth associated with an important inflammatory component (Hackstein and Thomson 2004). In Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 65 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD addition, a significantly decreased expression of inducible nitric oxide synthase (iNOS), a key enzyme required for the synthesis of the inflammatory agent nitric oxide (NO), and a markedly decreased production of NO itself is observed in peritoneal macrophages from elocalcitol-treated NOD mice with established EAP (Penna, Amuchastegui et al. 2006). A number of studies have associated excessive NO production with acute and chronic inflammation in model systems, and have also demonstrated that administration of NO synthase inhibitors can induce beneficial anti-inflammatory properties (Tinker and Wallace 2006). The capacity of elocalcitol to inhibit iNOS expression and NO production may thus represent an additional mechanism explaining its anti-inflammatory properties. 1.3.2.2 VDR agonists treat BPH-associated LUTS Inhibition of prostatic inflammation and proliferation has been also observed in human BPH cells. Elocalcitol treated human BPH cells inhibits significantly the inflammatory cytokine (IFN-γ, IL-17 and TNF-α)-stimulated production of IL-8 responsible for their proliferation via autocrine/paracrine mechanisms, but also inhibits the IL8-induced RhoA/ROCK pathway, known to be involved in contractile signaling in many tissues, accompanied by inhibition of cyclooxygenase 2 (COX-2) transcripts, prostaglandin E2 production and arrest of NF-κB p65 nuclear translocation (I and II). The capacity of elocalcitol to inhibit prostate inflammation could complement the anti-proliferative effects of this VDR agonist on prostate growth, providing a novel mechanism of action accounting for the arrest of prostate growth observed in BPH patients treated with elocalcitol (Penna, Amuchastegui et al. 2006). Interestingly, inhibition of prostatic inflammation by VDR agonists which is mediated by several different mechanisms including inhibition of COX-2 and prostaglandin E2, could contribute to the potential of these agents in prevention and treatment of prostate cancer (Krishnan, Moreno et al. 2007). Preclinical studies have shown reduced testosterone-induced BPH cell proliferation to a similar extent than finasteride and cyproterone acetate, prompting apoptosis even in presence of intraprostatic growth factors and completely antagonizing the effect of androgenic stimulation at subpicomolar concentrations in cells treated with VDR agonist elocalcitol, proposing this agonist as a potente regulator of the growth and survival of primary BPH stromal cells (Maggi, Crescioli et al. 2006). 66 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction Elocalcitol significantly decreased prostate growth both in unmanipulated and in castrated T-replaced rats, with an effect comparable to finasteride but without affecting testis androgenic secretion or pituitary function (Crescioli, Ferruzzi et al. 2004). Interestingly, both prostatic epithelial and stromal cells from elocalcitol-treated rats showed increased apoptotic rate and clusterin expression, confirming in vitro observations (Crescioli, Ferruzzi et al. 2004). Since rats do not develop BPH spontaneously, the anti-hyperplasic potential of elocalcitol was tested in male beagle dogs chronically treated for 9 months with 5μg/kg/die. In this model, reduction of prostate weight was observed, more evident after a 2-month recovery, suggesting a prolonged pharmacological activity of this compound, in the absence of side effects (Adorini, Penna et al. 2007). These preclinical data prompted a 12-week phase IIa, multicenter, double-blind, randomized, placebo-controlled clinical trial aimed at evaluating the efficacy and safety of elocalcitol administration (150 μg/die) in BPH patients (Colli, Rigatti et al. 2006). Elocalcitol exhibited a 7.2% reduction in prostate volume, measured by pelvic MRI, compared to placebo. Importantly, 92% of elocalcitol-treated patients did not experience a clinically significant growth in prostate volume compared with 48% in the placebo group. Thus, the reduction of prostate volume in elocalcitol-treated group against its marked increase in the placebo group indicates the ability of this VDR agonist to block the ongoing BPH process. During the trial, no difference was observed in symptom score or urodynamic parameters, probably because of the short duration of this proof-of-concept study and because patients were not screened for symptoms but only for prostatic volume (Colli, Rigatti et al. 2006). To elucidate this point, a 6-months phase IIb study was performed to measure maximum urinary flow rate and symptom severity as secondary end-points in patients with at least moderate symptomatology. Elocalcitol was effective in improving maximum urinary flow rate (Qmax) and ameliorating LUTS, as well as arresting prostate growth and preventing the risk of AUR and need for surgery (Fibbi, Penna et al. 2009), all key parameters of BPH progression. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 67 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 1.4 Inflammatory bowel disease The major forms of idiopathic IBD, UC and CD have been empirically defined by clinical, pathological, endoscopic and radiological features (Podolsky 2002). IBD are chronic inflammatory disorders of the gastrointestinal tract due to unbalanced activation of the mucosal immune system in response to luminal antigens in genetically predisposed individuals (Cho 2008). The onset of IBD typically occurs in the second and third decades of life and a majority of affected individuals progress to relapsing and chronic disease. Family aggregation has long been recognized. First-degree relatives of patients have a relative risk of five-fold or greater and the inheritable component seems stronger in CD than in UC (Tysk, Lindberg et al. 1988; Orholm, Munkholm et al. 1991). The remarkable increase in the incidence of IBD during the last half century implicates changes in the environment as a major cause for this evolution, since genetic variations are negligible in such a short period of time, while the ‘‘hygiene hypothesis’’ of allergic and autoimmune diseases has been invoked to explain the world-wide spreading of IBD (Danese, Sans et al. 2004). Whilst CD and UC both fall under the collective term IBD, these conditions can be quite distinct, with different pathogenesis, underlying inflammatory profiles, symptoms and treatment strategies. UC is a relapsing non-transmural inflammatory disease that is restricted to the colon, with characteristic histological findings such as acute and chronic inflammation of the mucosa by polymorphonuclear leukocytes and mononuclear cells, crypt abscesses, distortion of the mucosal glands and goblet cell depletion. In comparison, CD is a transmural disorder affecting the entire gastrointestinal tract from the mouth to the anus, including at the histological level small superficial ulcerations over a Peyer’s patch and focal chronic inflammation extending to the submucosa, sometimes accompanied by non-caseating granuloma formation (Baumgart and Carding 2007; Baumgart and Sandborn 2007; Xavier and Podolsky 2007). Both types of patients typically suffer from frequent and chronically relapsing flares, resulting in diarrhea, abdominal pain, rectal bleeding and malnutrition. UC and CD are associated with both intestinal and extraintestinal manifestations. Extraintestinal manifestations are usually related to intestinal disease activity and may precede or develop concurrently with intestinal symptoms (Danese, Semeraro et al. 2005). 68 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction 1.4.1 Diagnosis and clinical features UC and CD are generally diagnosed using clinical, endoscopic and histologic criteria. However, no single finding permits an absolute differential diagnosis. About 10-20% of patients have a clinical picture that falls between CD and UC; and they are diagnosed as affected by indeterminate colitis (Baumgart and Sandborn 2007). The most consistent feature of UC is the presence of blood and mucus in the stool with lower abdominal cramping, which is most intense during the passage of bowel movements. The location of abdominal pain depends on the extent of colonic involvement. Pain is present in the left lower quadrant with distal disease and extends to the entire abdomen with pancolitis. Pediatric patients have a higher frequency of pancolonic involvement, a higher likelihood of proximal extension of disease over time, and a higher risk of colectomy compared to adult patients (Cho 2008). In contrast to UC, the symptoms in CD could be subtle, leading to a delay in diagnosis. Gastrointestinal symptoms depend on the location, extent, and severity of involvement. In patients with ileocolonic involvement, abdominal pain is usually postprandial, usually in the periumbilical area, especially in children. Gastroduodenal CD presents with early satiety, nausea, emesis, epigastric pain or dysphagia. Due to postprandial pain and delay in gastric emptying, patients with gastroduodenal CD often limit their caloric intake to diminish their discomfort. Extensive small bowel disease causes diffuse abdominal pain, anorexia, diarrhea and weight loss and may result in lactose malabsorption (Cho 2008). While IBD can limit quality of life, due to pain, vomiting, diarrhea and rectal bleeding, it is rarely fatal on its own. In part because of recurrences after surgery performed, quality of life is lower in patients with CD than with UC. In case of IBD-dependent death, the most common causes are septic peritonitis, malignancy and surgery complications (Baumgart and Sandborn 2007). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 69 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Table 2. Differential diagnosis of UC and CD. UC and CD are associated with both intestinal and extraintestinal manifestations. Extraintestinal manifestations are usually related to intestinal disease activity and may precede or develop with intestinal symptoms. While UC and CD have a number of similarities in their clinical presentations, characteristic features highlight a number of diversities. UC is a relapsing non-transmural inflammatory disease that is restricted to the colon. CD is a relapsing, transmural inflammatory disease of the gastrointestinal mucosa that can affect the entire gastrointestinal tract from the mouth to the anus. Typical presentations include the discontinuous involvement of various portions of the gastrointestinal tract and the development of complications including strictures, abscesses, or fistulas (Cho 2008). Adapted from (Baumgart and Sandborn 2007). Clinical features Hematochezia Passage of mucus or pus Small-bowel disease Can affect upper-gastrointestinal tract Abdominal mass Extraintestinal manifestations Small-bowel obstruction Colonic obstruction Fistulas and perianal disease Ulcerative Colitis Crohn's disease Common Common No No Rare Common Rarely Rarely No Rare Rare Yes Yes Sometimes Common Common Common Common Biochemical features Anti-neutrophil cytoplasmic antibodies Common Anti-saccharomyces cerevisia antibodies Rarely Pathologival features Transmural mucosal inflammation Distorted crypt atchitecture Cryptitis and crypt abscesses Granulomas Fissures and skip lesions No Yes Yes No Rarely Rarely Common Yes Uncommon Yes Yes Common 1.4.1.1 Epidemiology IBD occurs most frequently in people in their late teens and twenties but also occurs in children. There have been cases in children as young as two years old and in older adults in their seventies and eighties. IBD is a disease of industrialized countries; the highest incidence rates and prevalence have been reported from Northern Europe, the UK and North America. Recently, it has been estimated to affect approximately four million people worldwide. In North America, prevalence rates of CD for Hispanic (4:1 per 100000) and Asian people (5:6 70 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction per 100000) is much lower than those for white individuals (43:6 per 100000) and AfricanAmerican people (29:8 per 100000) (Baumgart and Carding 2007). 1.4.2 Etiology 1.4.2.1 Environmental factors IBD‘s higher prevalence in more developed, or “Westernized” countries, could be explained by an increased sanitation and the lifestyles within these regions. This hypothesis is supported by large epidemiological studies in North America and Europe that shown a higher incidence in urban compared with rural communities. It has been proposed that the exposure to unhygienic conditions during development can prime the intestinal environment leading to optimal mucosal immune development and regulation, thus preventing a future inflammatory response. There are many environmental modifications that can be ascribed to the hygiene hypothesis, including better housing, safer food and water, improved hygiene and sanitation, vaccines, the widespread use of antibiotics, lack of parasites, fewer infections and better but selective nutrition (Danese, Sans et al. 2004; Geier, Butler et al. 2007). Some distinct environmental factors are equally considered as risk factors for IBD, such as smoking, diet, drugs, social status, stress, the enteric flora, altered intestinal permeability and appendectomy. Remarkably, smoking has a completely opposite effect on CD compared to UC, further indicating that distinct pathogenic mechanisms underlie each form of IBD (Danese, Sans et al. 2004). 1.4.2.2 Genetic factors A positive family history is still the largest independent risk factor for the disease, highlighting the transmission of genetic information as a key feature in disease onset. The frequency of familial occurrence among unselected individuals with IBD has been reported to be as high as 20-30% in referral-based studies, but has ranged between 5 and 10% in population-based surveys. Epidemiological studies have also shown that 75–80% of families with members affected by the disease present concordance for disease type. All family member affected by IBDs will be or CD or UCs but inside a family not both (Cho and Abraham 2007; Cho 2008). The strongest evidence of genetic factors contributing to susceptibility to IBD comes from concordance studies in twins. Monozygotic-twin concordance for CD has ranged Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 71 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD between 42 and 58% and between 6 and 17% for UC. This indicates that genetic factors are more significant in CD than in UC (Baumgart and Carding 2007). UC and CD are polygenic diseases: twelve genome-wide association (GWA) studies have identified susceptibility regions on chromosomes 16, 12, 6, 14, 5, 19, 1, 16, and 3. These regions have been renamed as IBD1–9, respectively (Baumgart and Carding 2007). Nod2 was the first specific gene on chromosome 16 associated with IBD (and so is also known as IBD1) and has been established as a susceptibility locus for CD but not UC. Because genetic factors are more significant in CD, most of the first studies were performed only on CD. However, recent studies extend analysis to UC and discover specific candidate genes like IL-10. Extensive GWA results open a window into the complex biology of IBD, revealing common candidate genes implicate in adaptive and innate immune system pathways, such as IL-23R, STAT3 or IL-12B (encoding IL12/23p40). In addition, these studies permit the identification of unexpected pathways, such as autophagy (especially ATG16L1 in CD) (Cho 2008; Budarf, Labbe et al. 2009). 1.4.2.3 Immunological factors The dominant hypothesis in IBD pathogenesis is the abnormal dynamic balance between microbes, particularly commensal flora, and host defensive responses at the mucosal frontier. As observed in conventional animal facilities, the Il-10 KO mice develop enterocolitis within 5–8 weeks of life. This is caused by uncontrolled immune responses to conventional microflora, since germ-free Il-10 KO mice do not develop the disease. In addition, mice raised in specific pathogen-free facilities develop a milder disease, which does not result in death (Kuhn, Lohler et al. 1993). Confirmation of the important role of the bacterial flora is also provided by treatment with antibiotics, exterting beneficial effects in both CD and UC patients (Baumgart and Sandborn 2007). However, precisely how commensal bacteria in the intestine interface locally with cells of the immune system to initiate and perpetuate intestinal inflammation remains unclear. The first line of defence of the mucosal immune system is the epithelial barrier. Per consequence, disturbances in epithelial barrier permeability represent the first signal required for an abnormal response to luminal antigens. The intestinal epithelium, which is considered to be a part of the innate immune system, plays an active role in the maintenance of mucosal homeostasis (Artis 2008). Epithelial cells form a tight, highly selective barrier between the body and the intraluminal microenvironment. Failure of this barrier may result in intestinal 72 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction inflammation, most likely through exposure to fecal antigens leading to inappropriate activation of the mucosal immune system. IBD patients present lowered epithelial resistance and increased permeability of the inflamed and non-inflamed mucosa (Soderholm, Olaison et al. 2002). Similar defect were shown to be involved in the pathogenesis in mice models. The classical model used to study experimental colitis is a dextran sodium sulphate (DSS)-induced colitis model. This chemical, once ingested, provokes severe bloody stool and a wasting disease by disruption of the intestine epithelial barrier. Genetically modified mice that express a dominant-negative N-cadherin transgene in epithelial cells present also a disruption of interepithelial cell adhesion resulting in modest inflammation, particularly in areas of the intestine that lie beneath areas of barrier disruption (Elson, Cong et al. 2005). Discovery of Nod2 mutations, a PRR recognizing the muramyl dipeptide, associated with a group of CD patients (those with small bowel and stricturing disease) suggests an important role of the innate immune system in disease pathogenesis (Danese and Fiocchi 2006). In the last few years, mutations in both TLRs and NLRs have been found to be associated with IBD, confirming their required dysfunctions for the pathology onset. Even if Nod2 mutations are the most important mutations observed in IBD patients, their exact role is still undefined. Nod2 silencing does not induce spontaneous colitis symptoms, but mice are more susceptible to intra-oral Listeria infection correlated to decreased expression of antimicrobial peptides (Kobayashi, Chamaillard et al. 2005). A recent study has highlighted a dysfunction in IL-10 production after TLR activation of PBMCs from IBD patients carrying a Nod2 mutation, by inhibiting MAP kinase phosphorylation (Noguchi, Homma et al. 2009). Thus, Nod2 seems to play a role in the regulation of the balance between pro- and antiinflammatory cytokine production. Nod2 is not the only PRR member involved in IBD. Most of the TLRs, especially TLR4, have been proposed in some GWA studies as possible hot spots, and hyporesponsiveness of certain TLR4 polymorphisms have been observed for CD patients in Northern Europe. In addition, colitis was exacerbated in mice treated with a TLR4 antagonist or in tlr4 KO mice (Fort, Mozaffarian et al. 2005; Fukata, Michelsen et al. 2005). Still, GWA studies could not confirm Nod2 mutations in all IBD patients. An important role for the adaptive immune system is clearly demonstrated by GWA studies showing IL-23R and IL-12B as hotspot mutations (Cho 2008). The two IBD subsets were thought to involve two different T helper cell-mediated responses. CD, characterized by elevated levels of IL-2 and IFN-γ, was considered as a Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 73 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD predominant Th1-mediated disease. Conversely UC, showing higher amount of IL-5 and IL13 was considered as Th2-mediated. Recently, this view was abandoned with the emergence of a new Th subset, the Th17 (Korzenik and Podolsky 2006). Mechanistic studies have highlighted the role of IL-23 in the Th17 lineage in IBD patients and in experimental models. Colitis initiation was originally correlated with IL-12 (Neurath, Fuss et al. 1995). However, recent findings on the shared p40 chain in both IL-12 and IL-23 highlight IL-23 as a required candidate for colitis development. Il-10 KO mice that were made Il-23 deficient do not develop colitis (Noguchi, Homma et al. 2009). These observations, confirmed by a GWA study, have highlighted the impact of the IL23/Th17 axis in IBD development. IL-23-dependent colitis does not require IL-17, but it seems to inhibit the frequency of Treg cells in the colon (Izcue, Hue et al. 2008). Although IL-23 has a pronounced inflammatory role in IBD, the individual Th17-associated cytokines have more complex roles. The role of IL-17 in colitis is controversial, because there are data suggesting that it is proinflammatory, protective, or that it has no observable role. IL-17 stimulation of colonic epithelial cells induces pro-inflammatory cytokines, such as IL-6 and IL-8, and chemokines like monocyte chemotactic protein-1 (MCP-1) (Andoh, Takaya et al. 2001). Accordingly, IL17 deficiency ameliorates colitis in mice (Zhang, Zheng et al. 2006). By contrast, neutralization of IL-17 using antibodies during acute colitis leads to more severe disease, suggesting a protective role of this cytokine (Ogawa, Andoh et al. 2004), while in a T-cellmediated model of colitis, IL-17 has been shown to be non-essential in disease development (Izcue, Hue et al. 2008). Each of these studies was performed using different colitis models and distinct ways of neutralizing IL-17 (anti-IL-17 antibody administration versus cytokine KO versus receptor KO). This might explain why differing roles for IL-17 have been described. Lastly, it is important to note that IL-17 is expressed by T cells in the healthy gastrointestinal tract and that this expression is dependent on the presence of commensal flora (Niess, Leithauser et al. 2008). IL-21 is also upregulated in lesions of IBD patients and seems to regulate the balance between Th17 and Treg cells in the gastrointestinal tract. Actually, IL-21 is required, in combination with TGF-β, to drive the Th17 lineage. In its absence, TGF-β stimulation leads to preferential induction of Treg cells (Fantini, Rizzo et al. 2007). In mice models, IL-21 deficiency leads to a reduced severity of chemically-induced colitis, and wild type mice present a significant increase of IL-21 levels (Fina, Sarra et al. 2008). Thus, IL-21-driven Th17 differentiation seems to actively contribute to the development of colitis. 74 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction Unlike other Th17 cytokines, IL-22 is protective in IBD. Mice deficient in Il-22, either genetically or through antibody depletion, display a more severe disease in models of acute and chronic colitis. In addition, gene delivery approaches have shown that IL-22 can be therapeutic, when directed to pre-existing inflammation (Sugimoto, Ogawa et al. 2008). The protective effects of IL-22 in the gastrointestinal tract are not limited to IBD, but IL-22 has also been shown to be protective to infection with the gastrointestinal pathogen Citrobacter rodentium (a cause of murine colonic mucosal hyperplasia) (Zheng, Valdez et al. 2008). IL-22 induces pro-survival and anti-apoptotic signaling pathways in the cells of the intestinal epithelium. IL-22 secretion by Th17 cell might provide protection through maintaining the integrity of the epithelial barrier and protect mucin secreting goblet cells (Sugimoto, Ogawa et al. 2008). Th17 pathways could represent novel candidates for the regulation of colitis, but their precise role in disease onset is still unresolved. Th1 pathways remain interesting targets, as shown by IL-12p40 antibody treatment in CD patients (Baumgart and Sandborn 2007). 1.4.3 Current treatments Glucocorticosteroids have been used in the treatment of active IBD for many decades and are effective in inducing clinical remission of CD and UC. However, corticosteroids are not effective for maintenance of remission and their long-term use is associated with sometimes severe and irreversible side effects (Faubion, Loftus et al. 2001). Within one year from the start of steroid therapy, most patients relapse or develop corticosteroid dependency. The introduction in 1998 of biologics, such as infliximab (Remicade; Centocor), a chimeric monoclonal antibody directed against TNF-α, for the treatment of CD, has changed the treatment of refractory IBD dramatically (Hanauer, Feagan et al. 2002). However, ideal therapeutic strategies for all IBD patients, inducing and maintaining long-term remission without steroid exposure or surgery, have yet to be developed. Epidemiological studies show that vitamin D deficiency correlates with IBD severity, leading to a growing interest on the role and action of VDR agonists in this pathology (Cantorna and Mahon 2004). VDR agonists show indeed potential therapeutic effects in IBD treatment. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 75 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Table 3. Novel agent currently under investigation for treatment of IBD. Recent advances in the understanding of UC and CD pathogenesis provide new biological candidates for therapeutic approach targeting diverse cellular pathways. Adapted from (Baumgart and Sandborn 2007). Therapeutic approach Agent ABT-974 Target IL-12 T-cell differentiation /subsets Atlizumab/MRA Daclizumab/basiliximab Visilizumab Inflammatory cytokines/pathways Selective adhesion molecules Innate immune stimulation Intestinal repair Other 76 IL-6 CD25 CD3 Certolizumab (CDP-870) TNF-α Adalizumab Fontolizumab TNF-α IFN-γ Semapimod RDP58 P38/JNK P38/JNK Rosiglitazone Alicaforsen Natalizumab MLN02 PPARγ ICAM1 α4 α4β7 GM-CSF EGF Growth hormone Trichuris suis ova Unknown Unknown Unknown Unknown Balance of gut flora Probiotics/prebiotics Autologous bone marrow transplant Leukopheresis Drug class mAb Receptor mAb mAb mAb PEG-Ab fragment Small molecule Peptide Small molecule Peptide Small molecule Antisense mAb mAb Disease Phase III CD UC UC I/II III III CD I/II CD CD III III CD UC II II UC UC CD UC II II III III Peptide Peptide Peptide Helminth CD UC CD CD/UC III III II I/II N/A CD/UC II/III T cells? N/A Leukocytes Device CD I CD/UC III Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction 1.5 Vitamin D and inflammatory bowel disease Immunomodulatory effects of VDR agonists are primarily observed on the inhibition of Th1mediated responses, but they can also inhibit Th17 responses (Penna and Adorini 2000; Tang, Zhou et al. 2009). In addition to these direct immunomodulatory effects, VDR agonists induce anti-microbial peptide expression, which shows benefits in colitis models (Tai, Wu et al. 2007). 1.5.1 Vitamin D deficiency and VDR polymorphisms in IBD patients IBD is more common in higher latitudes in Europe and North America, described as the ‘‘North–South’’ gradient in IBD incidence. A possible explanation for this higher prevalence could be vitamin D deficiency, since the Northern hemisphere receives less sunlight, especially during winter. Vitamin D deficiency has been observed as a common feature in IBD patients, and notably among pediatric one and in the Iranian population (Pappa, Grand et al. 2006; Naderi, Farnood et al. 2008). Furthermore, two studies have shown that fish oil, which is a rich source of vitamin D, decreases IBD severity (Cantorna and Mahon 2004),. Recently, a pilot clinical study was performed on IBD patients, resulting in short-term beneficial effect on bone metabolism and on disease activity after a one year administration of calcitriol (Miheller, Muzes et al. 2009). This study underlines the potential role of vitamin D in IBD treatment. In addition, the VDR maps to the IBD2 locus on chromosome 12, and VDR gene polymorphisms have been described in CD patients (Simmons, Mullighan et al. 2000; Gaya, Russell et al. 2006). These genetic observations coupled to the environmental hypothesis support the use of vitamin D analogs as therapeutic agents. 1.5.2 VDR agonists in IBD treatment 1.5.2.1 In vitro activity Lymphocytes represent key cells in IBD pathogenesis, and their recruitment in the gut promotes the inflammatory cascade (Korzenik and Podolsky 2006). Phytohaemagglutininactivated peripheral blood T lymphocytes from UC patients or healthy controls cultured in the presence of 1α,25(OH)2D3 or the VDR agonists EB1089 and KH1060 show significant and dose dependent inhibition of proliferation from day 3 of culture (Stio, Bonanomi et al. 2001). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 77 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD This anti-proliferative effect of VDR agonists is induced by increased apoptosis, as observed by higher protein levels of pro-apoptotic signals, such as Poly(ADP-ribose)-polymerase (PARP) cleavage or caspase-3 (Martinesi, Treves et al. 2008). In addition, VDR agonisttreated PBMCs activated by TNF-α or LPS and co-cultured with human umbilical vein endothelial cells (HUVEC) present an inhibition of ICAM-1, an adhesion molecule upregulated in IBD patients. This demonstrated the capacity of VDR agonists to inhibit cell–cell contacts as well as cell–cell interactions between endothelial cells and lymphocytes (Martinesi, Treves et al. 2008). Based on the key role of DCs in driving T cell responses, DC modulation would represent an important feature for VDR agonist activity in the context of IBD. Co-treatment with dexamethasone and 1α,25(OH)2D3 arrests the differentiation and the maturation of DCs activated with enteroantigen, as shown by decreased expression of maturation markers, such as CD40, CD80 and CD86 as well as MHC-II (Pedersen, Schmidt et al. 2009). These cotreated DCs are impaired in T-cell activation, as demonstrated by inhibition of T-cell dependent cytokine production, such as IL-4, IFN-γ, IL-2 or IL-17 (Pedersen, Schmidt et al. 2009). TNF-α represents a validated target in IBD, since this cytokine plays an important role in the initiation and perpetuation of intestinal inflammation in IBD and anti-TNF-α antibodies are approved therapies also for this indication. The VDR agonist TX-527 [19-nor-14,20bisepi-23-yne-1α,25(OH)2D3] inhibits proliferation and TNF-α production by PBMCs from CD patients (Stio, Martinesi et al. 2007). TNF-α inhibition in LPS-activated PBMCs is not restricted to CD, but also PBMCs from UC patient are responsive to VDR agonists (IV). Moreover, VDR agonists inhibit pro-inflammatory cytokines production such as IFN-γ, IL12p40, IL-6 or IL-1β in PBMCs from IBD patients activated by bacterial components or cytokine stimuli (IV, V) (Ardizzone, Cassinotti et al. 2009). After treatement with the VDR agonist TX-527, PBMC purified from CD patients stimulated by TNF-α present an inhibition of NF-κB nuclear translocation together with an inhibition of IκBα degradation (Stio, Martinesi et al. 2007). 1.5.2.2 In vivo activity Il-10 KO mice fed with a low calcemic diet develop diarrhea and a severe wasting disease earlier than mice fed with a normocalcemic diet or supplemented with vitamin D (Cantorna, Munsick et al. 2000). Moreover, administration of 1α,25(OH)2D3 significantly ameliorates 78 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Introduction IBD symptoms in Il-10 KO mice and treatment for as little as 2 weeks blocks the progression and ameliorates symptoms in mice with already established IBD (Cantorna, Munsick et al. 2000). As expected, VDR expression is required to control inflammation in the IL-10 KO mouse, since colitis is exacerbated in Il-10/Vdr double-deficient mice, associated with high local expression of IL-2, IFN-γ, IL-1β, TNF-α and IL-12 (Froicu, Weaver et al. 2003). Vdrdeficient mice are also extremely sensitive to DSS-induced colitis and then both dietary calcium and intrarectal administration of 1α,25(OH)2D3 directly and indirectly inhibits the TNF-α pathway, decreasing the severity and extent of DSS-induced colitis in wild-type mice. Moreover, treatment of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis with the VDR agonist 22-ene-25-oxa-1α,25(OH)2D3 (ZK156979) inhibits disease at normocalcemic doses, accompanied by down-regulation of myeloperoxidase activity, TNF-α, IFN-γ, and Tbet expression, whereas in local tissue IL-10 and IL-4 protein levels increased (Daniel, Radeke et al. 2006). The same group has recently proposed that 1α,25(OH)2D3, administered together with dexamethasone, inhibits IL-12, IL-23p19 and IL-17 linked with increased FoxP3 and TGF-β expression, hypothesizing a switch from Th1 and Th17 cells to Treg cells (Daniel, Sartory et al. 2008). Vdr-deficient mice undergoing DSS present impaired wound healing preceded by increased transepithelial electric resistance (Kong, Zhang et al. 2008). These observations suggest that VDR plays a critical role in mucosal barrier homeostasis by preserving the integrity of junction complexes and the healing capacity of the colonic epithelium. Therefore, vitamin D deficiency may compromise the mucosal barrier, leading to increased susceptibility to mucosal damage and increased risk of IBD. Vdr-deficient mice present mortality after intravenous LPS administration, likely due to a loss of negative control in the TLR activation pathway in absence of VDR (Froicu and Cantorna 2007). In addition, as already mentioned, 1α,25(OH)2D3 is a direct inducer of CAMP, shown to provide benefits in DSS-induced colitis. Moreover, 1α,25(OH)2D3 and other VDR agonists inhibit in vitro, in monocytes or PBMCs, TNF-α, IL-6, IL-12p40 and IL-1β after activation by any TLR human ligands (IV). These results highlight an important role for VDR and VDR agonists in the control of innate and adaptive immunity as well as in the epithelial membrane integrity in IBD, identifying the vitamin D system as a potential key regulator of gastrointestinal homeostasis. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 79 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 2. Aims of the study BPH and IBD are two chronic inflammatory disorders affecting the prostate and the gastrointestinal tract, respectively. A better understanding of the inflammatory component involved in their pathogenesis could support a potential therapeutic use of VDR agonists in these indications. Therefore, aims of the present thesis were: • To characterize the inflammatory component involved in BPH pathogenesis. • To evaluate in BPH cells the anti-inflammatory properties of elocalcitol, a VDR agonist proposed for the treatment of BPH. • To identify a potent and safe VDR agonist for a potential use in the treatment of IBD. • To characterize the selected compound in terms of metabolism and immunomodulatory properties • 80 To contribute to the understanding of IBD pathogenesis. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Materials and Methods 3. Materials and Methods The capital letters (I-V) refer to the manuscripts using the corresponding materials and methods. In this section, methods used by co-authors will not be described. 3.1 VDR agonists Crystalline 1α,25(OH)2D3 and its analogues (Fig. 12 and Table 4) were a gift of Dr. Milan Uskokovic (BioXell Inc. Nutley, NJ, USA). The compounds were reconstituted in 100 % ethanol, at the concentration of 1 mg/ml and stored in concentrated solutions at -80 °C under nitrogen atmosphere. 1α,25(OH)2D3 and its analogues were freshly diluted before each experiment, and the ethanol concentration in the test conditions did not exceed 0.00025%. Fig. 12. Chemical structures of 1α,25(OH)2D3 and key VDR agonists used in this thesis. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 81 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Table 4. Summary of VDR agonists used in this thesis. The maximal tolerated dose (MTD) is established as described below. The capital numbers refer to the text as numbered in the List of Publications (MW: molecular weight). Chemical name MW Generic MTD nomenclature Manuscript 1,25-dihydroxy-vitamin D3 417 Calcitriol 0.3 II-V 1,25-dihydroxy-16-ene-23-yne-20423 cyclopropyl-vitamin D3 BXL-36 10 III 1,25-dihydroxy-16-ene-23-yne-20411 cyclopropyl-19-nor-vitamin D3 BXL-37 10 III 1α-fluoro-25-hydroxy-16-ene-23yne-20-cyclopropyl-vitamin D3 425 BXL-38 100 III 407 BXL-39 100 III 519 BXL-47 0.3 III 531 BXL-48 0.3 III 533 BXL-49 0.3 III 521 BXL-50 0.01 III 533 BXL-51 0.3 III 535 BXL-52 3 III 415 BXL-60 1 III 427 BXL-62 3 III/IV 1,25-dihydroxy-24-oxo-16-ene-20441 cyclopropyl-vitamin D3 BXL-143 3 III 1α-fluoro-25-hydroxy-16,23Ediene-26,27-bishomo-20-epivitamin D3 BXL-628 / Elocalcitol 30/100 I/II 3-desoxy-1,25-dihydroxy-16-ene23-yne-20-cyclopropyl-vitamin D3 1,25-dihydroxy-16-ene-20cyclopropyl-23-yne-26,27hexafluoro-19-nor-vitamin D3 1,25-dihydroxy-16-ene-20cyclopropyl-23-yne-26,27hexafluoro-vitamin D3 1α-fluoro-25-hydroxy-16-ene-20cyclopropyl-23-yne-26,27hexafluoro-vitamin D3 1,25-dihydroxy-16,23E-diene-20cyclopropyl-26,27-hexafluoro-19nor-vitamin D3 1,25-dihydroxy-16,23E-diene-20cyclopropyl-26,27-hexafluorovitamin D3 1α-fluoro-25-hydroxy-16,23Ediene-20-cyclopropyl-26,27hexafluoro-vitamin D3 1,25-dihydroxy-16-ene-20cyclopropyl-19-nor-vitamin D3 1,25-dihydroxy-16-ene-20cyclopropyl-vitamin D3 82 443 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Materials and Methods 3.2 Cell cultures 3.2.1 Primary prostate cell lines Primary cell lines derived from BPH patients were used for work I and II. BPH cells were obtained from prostate tissues derived from three patients, who underwent transurethral resection of the prostate (TURP) for BPH, after informed consent and approval by the Local Ethical Committee. Patients did not receive any pharmacological treatment in the 3 months preceding surgery. The tissue was cut into small fragments and treated overnight with 2 mg/mL bacterial collagenase (700 U/ml). Fragments were than extensively washed in phosphate-buffered saline (PBS, 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4.2H2O) and cultured in DMEM-F12 1:1 supplemented with 10% heat-inactivated FBS, 2 mmol/l glutamine, 100 UI/ml penicillin and 100 mg/ml streptomycin. Cells began to emerge within 1 week and were used within the 15th passage. BPH cell cultures comprised fibroblasts (for the most part) and fibromuscular cells and were negative for endothelial and epithelial markers (I, Fig. 1). 3.2.2 Immortal cell lines Two cells lines, myelomonocytic THP-1 cells, originally derived form human peripheral blood from a patient suffering of acute monocytic leukaemia, and an ovarian cancer cell line (CAOV cells) derived from a human ovarian adenocarcinoma, were used in III. THP-1 cells were maintained in culture in phenol red-free RPMI 1640 with Glutamax and 10% (v/v) heatinactivated FBS supplemented with 0.1 mg/ml streptomicine and 100 UI/ml penicillin. 24 h prior the treatment THP-1 (106/ml) were grown overnight in phenol red-free RPMI 1640 with Glutamax and 5% (v/v) charcoal-treated heat-inactivated FBS with 0.1 mg/ml streptomicine and 100 UI/ml penicillin. After treatment with the corresponding concentration of VDR agonist, mRNA was extracted. CAOV cells were maintained in McCoy’s culture media supplemented with 10% FCS. For metabolism studies, 3x106 cells were seeded in T150 culture bottles and were grown to confluence. Confluent CAOV cells were incubated with 1 μM VDR agonists in 50 ml media containing 10% FCS. The incubations were carried out at 37 °C in a humidified atmosphere under 5% CO2 and were quenched at 24 h with 10 ml of methanol. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 83 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 3.2.3 Peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) purified from whole blood or from buffy coats were obtained by Ficoll gradient (III, IV and V). PBMCs were isolated from buffy coats of healthy subjects (through the courtesy of Centro Trasfusionale, Ospedale di Magenta, Milan, Italy) or blood from IBD patients (in collaboration with Dr. Danese from Istituto Clinico Humanitas, Milan, Italy) by Ficoll gradient (BioChrome AG, Berlin, Germany). Blood was collected from from 40 UC patients and 44 CD patients. Diagnosis was based on clinical, radiological, endoscopic and biopsy findings. Sometimes, PBMCs obtained from one patient were used only for a set of experiments, being insufficient to carry out all the programmed experiments. Informed consent for this study, carried out in vitro on PBMCs, was obtained from all patients. Briefly, buffy coats or blood were diluted with PBS supplemented with 2.5 mM EDTA (Sigma-Aldrich, USA) and loaded over a Ficoll-Paque. Density gradient was centrifuged for 30 min at 2000 rpm at room temperature (RT). About 95% of mononuclear cells at the interface containing PBMC were collected and washed twice with PBS. PBMC viability was determined by Trypan blue (Sigma-Aldrich) exclusion test. Cells were always >95% viable at culture initiation. 3.2.4 Lamina propria mononuclear cells Lamina propria mononuclear cells were purified from colon or ileum biospy from two CD and two UC patients (in collaboration with Dr. Danese from Istituto Clinico Humanitas, Milan, Italy) after informed consent and approval by the Local Ethical Committee. Briefly, the epithelium was removed from the lamina propria by incubation with 5 mM EDTA in HBSS (Gibco Invitrogen, Paisley, UK) for 20 min under gentle shaking. Then, the mucosal layer was cut in small pieces and digested for 30 min in 0.75 mg/ml collagenase type 2 and 20 µg/ml DNase type 1 (Sigma-Aldrich). Single cell suspensions were obtained by filtering with 100 µm and 70 µm cell strainers, followed by extensive washing in complete medium. About 50x106 cells were loaded over a Ficoll-Paque density gradient and centrifuged for 20 min at 690 g. The interface was collected, washed and then loaded on a Percoll density gradient (GE healthcare, Sweden) over 46% (v/v) and 100% and centrifuged 30 min at 2000 rpm. The interface containing mononuclear cells and the pellet containing leukocytes were recovered and washed twice with PBS. Viability was determined by Trypan blue (SigmaAldrich) exclusion test. Cells were always >95% viable at culture initiation. 84 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Materials and Methods 3.3 In vitro experiments 3.3.1 Mixed lymphocyte reaction After purification of PBMCs as previously described, the same number (3x105/200 μl/well) of allogeneic PBMCs from two different donors were cocultured in 96-well flat-bottom plates in RPMI 1640 with Glutamax and 5% (v/v) heat inactivated Fetal Clone-1 (FC-1, Charles River, Italy) with 1% non essential amino acids, 0.5 mg/ml gentamicin and 1 mM sodium pyruvate, in presence of the indicated concentrations of VDR agonists. Cultures were incubated at 37 °C in humidified atmosphere containing 5% CO2. After 5 days, proliferation and IFN-γ were measured. 3.3.2 TLR-activated PBMCs or LPMCs PBMCs (2x105/200 μl/well) were cultured in complete medium (RPMI 1640 with Glutamax and 10% (v/v) heat-inactivated FBS (Charles River, Italy) with 1% non essential amino acids, 0.5 mg/ml gentamicin and 1 mM of sodium pyruvate) in 96-well flat-bottom plates in presence of 100 ng/ml of LPS from Escherichia coli 0111:B4 (Sigma-Aldrich) (III) or with various TLR agonists (TLR agonist kit, InvivoGen, USA) (Table 5) (I, II, IV and V). In V, cells were treated in addition with an antibody targeting the IL-10 receptor (1 µg/ml, CDW210, BD biosciences, USA) or with its corresponding isotype control (rat IgG γ2a, BD biosciences) at similar concentrations. For mRNA quantification in IV, cells were washed after 6 h with PBS and lised with RLT buffer (Qiagen, Germany) following the protocol describer later. After 24 h, culture supernatants were harvested and stored at -80°C (III, IV, V). Lamina propria leukocytes (1x105/200 µL/well) were cultured in complete medium supplemented with 100 UI/ml penicillin, 0.1 mg/ml streptamicin and 0.5 µg/ml amphotericin B in 96-well round bottom plates in presence of 1 µg/ml coated anti-CD2 (BD biosciences) and 1 µg/ml soluble anti-CD28 (BD biosciences) in presence or absence of VDR agonists (IV). Mononuclear LPMCs (2x105/200 μl/well) were cultured as described for the PBMC with the supplemented medium. Cultures were incubating at 37 °C in humidified atmosphere containing 5% CO2. At the end of the experiment (24 or 72 h) the supernatants were harvested and stored at -80°C until analysis. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 85 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 3.3.3 BPH cell activation BPH cells at 70-85% confluence were cultured in DMEM-F12 1:1 supplemented with 10% heat-inactivated FBS, 2 mmol/l glutamine, 100 UI/ml penicillin and 100 mg/mLl streptomycin and stimulated with various TLR agonists (TLR agonist kit, InvivoGen) (Table 5) or with a cocktail of cytokine containing 10 ng/ml IFN-γ, IL-17 and/or TNF-α, (optimal cytokine concentrations to induce in BPH cells; IL-8 and IL-6 production as determined in separate experiments) or with the indicated concentrations of IL-8 (BD Biosciences). For mRNA expression, after 4 h stimulation cells were washed with PBS and then lised with RLT buffer (Qiagen). After 48 h, cell culture supernatants were analyzed for cytokine and chemokine production. Table 5. TLR ligands used for the studies. Source and nature of TLR agonists used and their specificities. Concentrations used for these studies are those recommended by the manufacturer. TLR Ligand Source TLR1/2 Pam3CSK4 synthetic TLR2 HKLM TLR3 poly(I:C) TLR4 LPS TLR5 Flagellin TLR6/2 FSL1 TLR7 Imiquimod synthetic TLR8 ssRNA40 synthetic TLR9 ODN2006 (type B) synthetic Listeria monocytogenes Synthetic Escherichia coli K12 Salmonella typhimurium Mycoplasma salivarium Nature tripalmitoylated lipopeptide heat-killed gram positive bacteria double-stranded RNA Concentration bacterial outer wall 100 ng/ml bacterial flagellar 100 ng/ml N-terminal part of lipoprotein LP44 imidazoquinoline amine analogue single stranded RNA GUrich sequence unmethylated CpG dinucleotides 0.5 µg/l 108 cells/ml 25 µg/l 100 ng/ml 500 ng/ml 500 ng/ml 5 µg/ml 3.3.4 Enzyme-linked immunosorbent assay (ELISA) ELISAs for human IFN-γ, TNF-α, IL-6, IL-1β, IL-10 and IL-8 were performed using mAb pairs and standards provided in the BD OptEIA™ Human ELISA set (BD Biosciences), according to the manufacturer’s procedures. ELISA for human IL-12/23p40 was performed using commercially available mAbs and standards (BD Biosciences) according to the manufacturer’s instructions. 86 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Materials and Methods 3.3.5 Total RNA purification Total RNA was extracted using the RNeasy Mini kit (Qiagen). Prior to collection, cells were washed with ice-cold PBS, after which cells were lysed using 350 μl of RLT buffer and RNA was extracted. The RNA lysates were collected and diluted with 70% ethanol (1:1 ratio) and loaded onto a silica membrane (RNeasy mini column, Qiagen). After washing, membranes were treated with DNase to exclude amplification of genomic DNA. Each step was preceded by a centrifugation of 15 s at 10000 rpm. Finally, RNAs were eluted with 14 μl of sterile H2O after a centrifugation at 14000 rpm for 1 min. Purities and RNA concentrations were measured with a NanoDrop ND-1000 (NanoDrop, USA). 3.3.6 cDNA synthesis To retrotranscribe 1 μg of total RNA to cDNA in III (Fig. 5), 100 pmol of oligodT18 primer, 20 nmol of dNTPs, 200 pmol of DTT, reverse transcriptase buffer (50 mM Tris–HCl, pH 8.3, 50 KCl, 4 mM MgCl2, 10 mM DTT), 40 U of reverse transcriptase and 40 U of RNAse Inhibitor (buffer and enzymes from Fermentas, Lithuania) were incubated for 1 h at 37 °C in 40 μl volume. Following synthesis the reverse transcriptase was inactivated for 5 min at 95 °C and cDNA was diluted 1:10 in sterile H2O. In I, II and IV, 1 µg of total RNAs were retrotranscribed using reverse transcriptase buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 30 mM MgCl2 and 5 mM DTT), 5.5mM MgCl2, 500 µM of each dNTPs, 0.4 U/µl of RNase inhibitor, 2.5 µM of random hexamers and 1.25 U/μl multiscribe Reverse transcriptase (Taqman® Reverse transcription Reagents, Applied Biosystems) and run under the following thermal cycler parameters: 25 ºC for 10 min, 48 ºC for 30 min, and 95 ºC for 5 min. Each sample was then collected, mixed thoroughly, aliquoted, and frozen at -80ºC. 3.3.7 Real time PCR In III (Fig. 5), real-time RT-PCR was performed using a LightCycler® 480 System (Roche) and FastStart SYBR Green Master mix (Roche). Each reaction was performed using 4 pmol of specific primers, 4 μl of cDNA template and 1x Mastermix in a volume of 10 μl and the PCR cycling conditions were: pre-incubation for 10 min at 95 °C, 38 cycles of 20 s at 95 °C, 15 s at 60 °C and 15 s at 72 °C. Fold inductions were calculated using the formula 2(ΔΔCt), where ΔΔCt is the ΔCt(stimulus)-ΔCt(solvent), ΔCt is Ct(target gene)-Ct(RPLP0), Ct is the cycle at which the threshold is crossed and RPLP0 is the housekeeping gene ribosomal Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 87 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD protein, large, P0. Quality of PCR products were monitored using post-PCR melt curve analysis. In I, II, IV and V, real-time RT-PCR of total cDNA using specific primers were performed under identical conditions using ABI PRISM 7000 Sequence Detection System (Applied Biosystems, USA) and TaqMan® chemistry with 2x TaqMan Universal PCR Master Mix (Applied Biosystems) from the same lot number. The primers used are commercially available from Applied Biosystems as assays-on-demand. Real-time RT-PCR runs were performed in 96-well optical plates, each containing 1x TaqMan Universal PCR Master Mix (Applied Biosystems), 0.4 pmol/µl of appropriate forward and reverse primer and 20 ng cDNA. The conditions for the amplification were as follows: 1 cycle of 2 min at 50 ºC and 1 cycle of 10 min at 95 ºC, followed by 40 cycles of 15 s at 95 ºC, 1 min at 60 ºC. Data were acquired at the end of each 60 ºC cycle. Fold inductions were calculated using the formula 2(ΔΔCt), where ΔΔCt is the ΔCt(stimulus)-ΔCt(control), ΔCt is Ct(target gene)-Ct(GAPDH), Ct is the cycle at which the threshold is crossed and GAPDH is the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. 3.4 In vivo experiments 3.4.1 Mice 8-10 week-old C57BL/6 mice (Charles River, Italy) were housed in plastic cages with water absorbent bedding. Litter was changed at least twice a week. The animal room was temperature controlled and had a 12 h light/dark cycle. Food and water were supplied ad libitum. Calcium and vitamin D3 content in the diet were respectively 8785 mg/kg and 1260 UI/kg. All procedures were reviewed and approved by local ethical committee. 3.4.2 Assessment of the MTD Eight-week-old female C57BL/6 mice (3 mice/group) were dosed orally (0.1 ml/mouse) with various concentrations of VDR agonists daily for four days. Analogues were formulated in miglyol for a final concentration of 0.01, 0.03, 0.1, 0.3, 1, 3, 10 30, 100 and 300 μg/kg, when given at 0.1 ml/mouse po. Blood for serum calcium assay was drawn by tail bleed on day five, the final day of the study. Serum calcium levels were determined using a colorimetric assay (Sigma Diagnostics, procedure no. 597). The highest dose of VDR agonist tolerated without inducing serum calcium levels above 10.7 mg/dl (threshold used in the clinic to define hypercalcemic patients) was taken as the MTD and expressed in μg/kg. 88 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Materials and Methods 3.4.3 Induction of experimental colitis Colitis was induced in 8-10-week-old male C57BL/6 mice (5 mice per group) by administration of 3% DSS (molecular mass 40 kDa; MP Biomedicals) in filter-purified (Millipore) drinking water for 5 days, after which the mice were resumed on water for the remainder of the experiment. Fresh DSS solutions were prepared every two days (Fig. 13). Water 3% DSS -1 0 5 9 Days after VDR agonist Fig. 13. DSS chemical structure and experimental set-up for the induction of colitis. Mice were pretreated with VDR agonists 24 h before addition of 3% DSS in drinking water. At day 0, 3% DSS was administered ad libitum in drinking water for 5 days and then removed until the end of the experiment. VDR agonists were administered daily intra-rectally in miglyol, until the end of the experiment. 3.4.4 Administration of VDR agonists For local treatment, 1 μg/kg of BXL-62 and 0.3µg/kg of 1α,25(OH)2D3, were dissolved in oil (miglyol 812,Sasol Germany GmbH) and 60µL were administered rectally to slightly anaesthetized mice through a 3.5 F catheter carefully inserted into the rectum. The catheter tip was inserted 4 cm proximal to the anal verge. To ensure distribution of the solution within the entire colon and caecum, mice were held in a vertical position for 1 min after the instillation. Treatments started one day prior DSS administration and continued every day thereafter for the duration of the experiment. Miglyol alone was administered as vehicle control. Prior observations confirmed that intra-rectal miglyol administration does not modify the bodyweight loss from mice undergoing DSS-induced colitis. 3.4.5 Assessment of inflammation All mice were observed daily for signs of gross toxicity, consistency of stools (formed, soft, mixed and diarrhea) and presence of gross blood (Haemoccult Sensa, Beckman Coulter, USA). The presence of blood was graded using a score of 0 for no color; 1 for a very light blue color taking over 30 seconds to appear; 2 for a blue color developing about 30 seconds; 3 for an immediate change in color occurring in less than 30 seconds and 4 for gross blood observable on the slide. Daily, mice were weighted and the body weight (BW) loss was Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 89 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD graded using a score of 0 for gain or no bodyweight loss; 1 for BW loss from 1 to 4.9%; 2 for BW loss from 5.0 to 9.9; 3 for BW loss from 10.0 to 15 and 4 for BW loss over 15. For each group, the disease activity index (DAI) was determined by combining scores of BW loss, haemoccult positivity and stool consistency (Table 6). On day 9, animals were euthanized by CO2 inhalation and necropsied. The colon was gently stretched and the distance from the colon-cecal junction to the end of the distal rectum was measured. Serum calcium levels were determined using a colorimetric assay (Sigma Diagnostics, procedure no. 597). Table 6. Scoring system for the disease activity index (DAI). The mean scores for each parameter and for each mouse represent the DAI score. Score 0 1 2 3 4 Weight loss (%) gain or 0 less than 5 less than 10 less than 15 more than 15 Stool consistency normal soft mixed (soft and liquid) liquid diarrhea Bloody stool (haemoccult test) no color light blue positivity around 30 s positivity lower than 30 s gross bleeding 3.4.6 Histology Entire colons were fixed in 4% paraformaldehyde for at least 48 h and were processed by the laboratory of Pathology Unit, "L. Sacco" Department Clinical Sciences, Milano. Colon tissues were embedded in paraffin and three µm sections were cut and stained with hematoxylin and eosin. Sections were scored blindly for the anus, the descendant and ascendant colon. For each section, inflammatory infiltrate, ulcerative lesion and regenerative hyperplasia were evaluated. In addition, the area with the histological lesion was measured. Total histological score was calculated as detailed in Table 7. Table 7. Scoring system for the histology score. Ulceration and regeneration were not graded, however the inflammation of the external wall (phlogosis) was quantified from absence to intensive infiltration. The total score represents the sum of all the four parameters. Score 0 1 2 3 4 5 90 Ulceration Regeneration Phlogosis absent absent no present present low medium intensive Lesion lenght (mm) absent less than 5 less than 10 less than 15 less than 20 20 and more Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Materials and Methods 3.5 Statistical analysis Data were analyzed with Graph Prism version 5.0 and curves were generated with the appropriated nonlinear fit regression. Statistical significance was determined by using the appropriate analysis of variance followed by a post-hoc test for multiple variable analyses or by the appropriate t-test for one to one comparison. Data had to follow a normal distribution before being tested for significance. Differences were considered significant at P < 0.05. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 91 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 4. Results 4.1 BPH cells can act as non-professional APCs to induce chronic prostate inflammation BPH, the most common age-related disease of the male, occurs clinically in about half of all men at 70 years of age (Webber 2006). A variety of growth factors and inflammatory chemokines like IL-8 have been implicated in the pathogenesis of BPH (Ropiquet, Giri et al. 1999; Giri and Ittmann 2001). However, the complex regulatory mechanisms of growth control in BPH are still incompletely understood. Inflammatory infiltrates in BPH have been found to consist primarily of T cells, mostly CD4+CD45RO+ cells, B cells and macrophages (Theyer, Kramer et al. 1992). Up-regulation of several pro-inflammatory cytokines has been described in BPH, in particular IL-2 and IFN-γ (Steiner, Stix et al. 2003), IL-15 (Handisurya, Steiner et al. 2001) and IL-17 (Steiner, Newman et al. 2003), leading to the hypothesis that BPH may represent an “immune inflammatory” disease (Kramer and Marberger 2006). This is an attractive hypothesis, because the association of BPH with chronic inflammation could offer a sound framework to understand the pathogenesis of the disease. However, immune mechanisms leading to chronic inflammation in BPH have not yet been clearly defined. In order to further characterize the immune mechanisms behind BPH pathogenesis, we have examined the capacity of prostate stromal cells obtained from BPH tissue to actively contribute to the organ specific inflammatory process by acting as APCs or as targets of TLR agonists, leading to the production of pro-inflammatory cytokines and chemokines able to mediate prostate chronic inflammation and hyperplasia. Analysis of TLR transcript expression by real-time RT-PCR on BPH cells shows that the 10 members of the family identified in human are constitutively expressed and are functional. With the exception of TLR9, BPH cells stimulated by any TLR agonist (Table 5) show a strong induction of pro-inflammatory cytokine (IL-8 and IL-6) and chemokine (CXCL10) production (I, Fig. 2B). The capacity of BPH cells to act as APCs was first examined by expression of MHC class II and co-stimulatory molecules, such as CD40, CD80, CD86 and CD134 on their surface. BPH cells constitutively express both MHC-I and -II molecules and co-stimulatory 92 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Results molecules, which are strongly up-regulated by a 48 h incubation with IFN-γ, as detected by cytofluorometry (I, Fig. 3A) and confirmed by confocal microscopy (I, Fig. 3B and Fig. 5A). Then, we examined ability of BPH cells to present alloantigen to alloreactive CD4+ T cells. Constitutive expression of MHC-II molecules by BPH cells, albeit relatively low, is already sufficient to induce proliferation of carboxyfluorescein succinimidyl ester (CFSE)labeled alloreactive CD4+ T cells, which is increased following IFN-γ treatment of BPH cells (I, Fig. 4A). Alloreactive CD4+ T cells co-cultured with IFN-γ-stimulated BPH cells produce not only IFN-γ but also IL-17 (I, Fig. 4D). Interestingly, IL-17 and IFN-γ produce by BPH-stimulated CD4+ T cells markedly upregulate production of IL-8, CXCL10 and IL-6 (I, Fig. 7). In addition, IL-8 is expressed in situ by epithelial and stromal prostate cells, and is functional, as shown by the recruitment of CXCR1- and CXCR2-positive leukocyte, as well as CD15+ neutrophils (I, Fig. 8). IL-8mediated BPH cell growth can be induced by a combination of IFN-γ and IL-17, thus establishing a possible relationship between the T-cell response induced by BPH cells and prostate cell growth (I, Fig. 9). In conclusion, our results show that human prostate cells can act as APCs, i.e. they are able to stimulate alloreactive CD4+ T cells to produce IFN-γ and IL-17. The induction of a BPH cell-driven autoimmune response, as well as triggering of TLRs expressed by BPH cells, up-regulate production of IL-8, IL-6 and CXCL10, which are key factors sustaining prostate inflammation, recruiting inflammatory leukocytes and promoting prostate cell hyperplasia. 4.2 VDR agonist elocalcitol inhibits IL-8-dependent BPH cell proliferation and inflammatory response Little data assessing the clinical response of anti-inflammatory therapy in BPH is available, but treatment with elocalcitol has been found to arrest BPH development (Colli, Rigatti et al. 2006). VDR agonists, by promoting innate immunity and regulating adaptive immune responses, exert anti-inflammatory and immunoregulatory properties potentially useful in the treatment of diseases characterized by chronic inflammation and cell proliferation (Adorini and Penna 2008). The prostate is a target organ of VDR agonists and represents an extrarenal synthesis site of 1α,25(OH)2D3 (Flanagan, Young et al. 2006), but its capacity to respond to VDR agonists has been mostly probed clinically for the treatment of prostate cancer (Deeb, Trump et al. 2007). Based on the marked inhibitory activity of the Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 93 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD VDR agonist elocalcitol on basal and growth factor induced proliferation of human prostate cells (Crescioli, Ferruzzi et al. 2004), its anti-inflammatory properties in the treatment of EAP induced by injection of prostate homogenatecomplete Freund’s adjuvant in NOD male mice were tested (Penna, Amuchastegui et al. 2006). Administration of elocalcitol, at normocalcemic doses, for 2 weeks in already established EAP inhibits significantly the intraprostatic cell infiltrate, with reduced cell proliferation and increased apoptosis of resident and infiltrating cells (Penna, Amuchastegui et al. 2006). Th1 cell responses are decreased as well as production of IL-17 (Penna, Amuchastegui et al. 2006) emphasizing the potential of VDR agonists in the treatment of immuno-mediated diseases of the prostate (Adorini, Penna et al. 2007). To provide an understanding of the mechanism of action of elocalcitol potency in BPH, we analyzed in publication II its anti-proliferative and anti-inflammatory properties in IL-8 dependent mechanisms. In study II, we demonstrated that the increased expression of IL-8 and VDR transcripts promoted by T cell-derived inflammatory cytokines in BPH cells renders them more susceptible to the inhibitory action of elocalcitol (II, Fig. 1A). Moreover, elocalcitol inhibits dose dependently IL-8 production by BPH cells more effectively than calcitriol, whereas the 5-α reductase inhibitor finasteride has no effect on IL-8 production induced by proinflammatory cytokines in BPH cells (II, Fig. 1C). In addition, increasing concentrations (1017 to 10-6 M) of elocalcitol inhibit dose-dependently BPH cells proliferation induced by 10 nM IL-8 (II, Fig. 2B). Treatment with elocalcitol of cytokine-stimulated BPH cells does not significantly affect COX-1 expression but it significantly reduces, with an average decrease of about 50%, both COX-2 expression and PGE2 production (II, Fig. 3B and Fig. 4). To explain COX-2 inhibition, we further analyzed the NF-κB translocation after treatment with elocalcitol. Confocal microscopic analysis clearly showed translocation of NF-κB p65 to the nucleus in cytokine-stimulated BPH cells, whereas this is mostly retained into the cytoplasm in BPH cells treated with elocalcitol before cytokine addition (II, Fig. 5). Thus, elocalcitol inhibits NF-κB p65 nuclear translocation in BPH cells stimulated by inflammatory cytokines leading to inhibition of pro-inflammatory cytokine production. Activation of NF-κB signaling through the RhoA/ROCK pathway induces IL-8 production in a number of different cell types, including cervical stromal cells (Shimizu, Tahara et al. 2007), Kaposi sarcoma (Shepard, Yang et al. 2001; Zhao, Kuhnt-Moore et al. 94 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Results 2003), endothelial cells (Hippenstiel, Soeth et al. 2000) and colonic epithelial cells (Hippenstiel, Soeth et al. 2000). As RhoA activation results in its translocation to the plasma membrane (Somlyo and Somlyo 2003), we investigated by Western blot analysis its subcellular distribution (membrane vs. cytosol) in BPH cells, under basal conditions and after a 48 h stimulation with IL-8 (10 nM) in combination or not with elocalcitol (10-8 M). We could show that IL-8 stimulated RhoA membrane translocation was completely prevented by elocalcitol (II, Fig. 6A lower panel). The results were confirmed by confocal microscopy (II, Fig. 6B). These effects are consistent with the role of this calcium-sensitising signaling in the regulation of the inflammatory processes (Segain, Raingeard de la Bletiere et al. 2003) through the activation of NF-κB (Montaner, Perona et al. 1998) and the induction of IL-8 secretion (Shimizu, Tahara et al. 2007). Thus, these data provide a mechanistic explanation for the role of IL-8 in BPH pathogenesis, showing that a combination of Th1 and Th17 cell-derived inflammatory cytokines can markedly stimulate its secretion by BPH stromal cells. Moreover, our results suggest an IL-8-dependent link bridging inflammatory response and cell proliferation in BPH pathogenesis, which can be targeted by elocalcitol via multiple mechanisms of action. 4.3 Potent anti-inflammatory properties of 1α,25(OH)2-16-ene-20- cyclopropyl-vitamin D3 (BXL-62) in inflammatory bowel disease models Additions of 16-ene and/or a 20-epi moeity to vitamin D3 are known to represent an important chemical tools to increase the potency of VDR agonists with physiological calcium homeostasis (Uskokovic, Manchand et al. 2006). 20-epi-1α,25(OH)2D3 or 16-ene1α,25(OH)2D3 exhibit anti-proliferative activity from 200-5000 fold greater than the natural hormone coupled to more potent anti-inflammatory properties in a variety of cancer cell line and human keratinocytes (Uskokovic, Norman et al. 2001; Uskokovic, Manchand et al. 2006). Medicinal chemistry approaches lead to the proposition that a C20-cyclopropyl group could represent an interesting alternative to mimic the methyl group (Uskokovic, Manchand et al. 2006). These 20-cyclopropyl VDR agonists show a higher potency in the inhibition of IFN-γ production in MLR assays, with a 10-times lower capacity to induce hypercalcemia in mice (Uskokovic, Manchand et al. 2006). Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 95 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD A previous study combined these two modifications and showed that, among paired 16-ene-20-cyclopropyl VDR agonists, 1α,25(OH)2-16-ene-20-cyclopropyl-vitamin D3 (BXL62) is the most potent compound inhibiting IFN-γ in MLR assays (Uskokovic, Manchand et al. 2006). We undertook study III to evaluate the potency of 16-ene-20-cyclopropyl VDR agonists not only for IFN-γ inhibition in the MLR assay, but to extend our analysis to TNF-α inhibition in LPS-induced PBMC activation. We confirmed that BXL-62 is the most potent compound among the VDR agonists tested in the inhibition of both IFN-γ and TNF-α (III, Table1 and Fig. 6). BXL-62 is also more potent, compared to the natural hormone calcitriol, in the inhibition of two other key pro-inflammatory cytokines, IL-12/23p40 and IL-6 (III, Fig. 6). In addition, BXL-62 induces VDR primary response genes at a concentration 15 times lower than the natural hormone both in primary cells (PBMCs) (III, Fig. 4) and an immortal cell line (THP-1) without modification of its kinetic (III, Fig. 5). Moreover, despite the potent anti-inflammatory properties of BXL-62, this VDR agonist present a better safety as shown by its maximum tolerated dose 3 times higher than the natural hormone, confirming that this compound represents a potent VDR agonist (III, Fig. 8) with reduced calcemic liability compared to calcitriol. VDR expression is required to control inflammation of spontaneous and induced colitis mice model as demonstrated by exacerbation of the symptoms in Il-10/Vdr double deficient and in DSS-induced colitis mice (Froicu, Weaver et al. 2003; Froicu and Cantorna 2007). In addition, 1α,25(OH)2D3 has been shown to ameliorate spontaneous colitis, an effect mediated by direct and indirect inhibition of TNF-α (Zhu, Mahon et al. 2005) . Recently, a pilot clinical study was performed in IBD patients, showing short-term beneficial effects on bone metabolism and on disease activity after a one year administration of 1α,25(OH)2D3 (Miheller, Muzes et al. 2009). Recently, efficacy of VDR analogues were proposed in the IBD context, as the VDR agonist 19-nor-14,20-bisepi-23-yne-1α,25(OH)2-vitamin D3 (TX527) showed inhibition of TNF-α production and proliferation in PBMCs from CD patients (Stio, Martinesi et al. 2007). In mice models, the VDR agonist 22-ene-25-oxa-1α,25(OH)2D3 (ZK156979) was previously shown to improve symptoms in TNBS-induced colitis at normocalcemic doses by inhibiting TNF-α production and increasing level of anti-inflammatory cytokine (Daniel, Radeke et al. 96 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Results 2006). We undertook the project IV to support by pre-clinical data the potential used of our VDR agonist, BXL-62, for the treatment of IBD. To confirm the potential of this VDR agonist in the treatment of IBD, we have first studied its activity in the induction of VDR primary response genes in PBMCs purified from IBD patients. A dose-response titration of BXL-62 and 1α,25(OH)2D3 in unstimulated PBMCs from IBD patients shows a significantly higher increase of transcripts encoding CYP24A1 and CAMP induced by BXL-62 compared to 1α,25(OH)2D3 (IV, Fig. 1). These results confirm those obtained in PBMCs from healthy individuals (III), and demonstrate that PBMCs from IBD patients present an unaltered VDR-dependent signaling machinery. We also confirmed anti-inflammatory activites in LPS-activated PBMCs purified from IBD patients as observed in healthy donors (IV, Fig. 2B), but we also demonstrated that this protein inhibition is the consequence of an earlier mRNA inhibition (IV, Fig. 2A). We also extended this analysis and showed that in IBD patients, VDR agonists are able to inhibit proinflammatory cytokine production after any TLR agonist activation (Table 5) (IV, Fig. 3). These anti-inflammatory properties have been extended to lymphocytes purified from the inflamed target tissue, as shown by inhibition of TNF-α and IFN-γ production after activation of lymphocytes purified from LPMCs activated by antibodies targeting CD2 and CD28, potent activators of T cell responses (Targan, Deem et al. 1995) (IV, Fig. 4). To validate a potential use of BXL-62 as a therapeutic agent in IBD, we have analyzed its in vivo efficacy, compared to 1α,25(OH)2D3, in the DSS-induced colitis model, as previously described (Froicu and Cantorna 2007). Daily intra-rectal administration of BXL-62 prevents the body weight loss following DSS administration (IV, Fig. 5A) and induces a significant improvement in stool consistency (Fig. 5B) and in visible fecal blood (IV, Fig. 5C). In contrast, 1α,25(OH)2D3 treatment does not affect body weight loss (IV, Fig. 5A), improves stool consistency only early on in the disease course (IV, Fig. 5B) and ameliorates the bloody stool score (IV, Fig. 5C). The DAI summarizing the daily parameters observed, confirms that BXL-62 ameliorates significantly colitis symptoms, from day 4 until the end of the experiment, while 1α,25(OH)2D3 treatment leads to significant amelioration of disease only at day 4 (IV, Fig. 5D). At day 10 after initiation of DSS administration, BXL-62 shows a colon shortening significantly reduced compared to vehicle (IV, Fig. 6A) accompanied with a decrease of total colon lesions (IV, Fig. 6B). To control the treatment safety, we measured serum calcium levels and show that ten days of consecutive intra rectal administrations of VDR agonists do not induce hyper-calceamia in mice undergoing DSS-induced colitis (IV, Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 97 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Fig. 5A). In addition, BXL-62 activity is dose dependent and at equimolar dosage, BXL-62 conserved is higher potency than the natural hormone to ameliorate the stool consistency (IV, Fig. 6A) and the visible blood (IV, Fig. 6B). In conclusion, the strongest potential of BXL-62, compared to 1α,25(OH)2D3, to inhibit pro-inflammatory cytokines, in PBMCs and LPMCs from IBD patients, combined with its in vivo efficacy for amelioration of colitis symptoms in an experimental colitis model, suggest its potential use as a future therapy for IBD. 4.4 24-oxo BXL-62 metabolite exerts biological activities similar to its parent compound Modification of the catabolism of VDR agonists was proposed to explain, at least in part, their increase of potency compared to 1α,25(OH)2D3 (Lemire, Archer et al. 1994), as demonstrated by accumulation of a 24-oxo active metabolite from the 20-epi and 16-eneVDR agonists (Lemire, Archer et al. 1994; Siu-Caldera, Clark et al. 1996; Campbell, Reddy et al. 1997). However, little is known about 20-cyclopropyl-16-ene VDR agonists that exhibit stronger potency in proliferation inhibition and anti-inflammatory properties (Uskokovic, Manchand et al. 2006). Metabolic studies performed on a rat osteosarcoma cell line proposed that BXL-62 is protected, as the 16-ene-vitamin D3, from the C23 hydroxylation by CYP24A1, resulting to an accumulation of the 24-oxo metabolite (Uskokovic, Manchand et al. 2006). This observation could explain the higher potency of BXL-62, since 24-oxo-16-enevitamin D3 isolated from a culture supernatant presents similar anti-inflammatory activities than its parent compound (Lemire, Archer et al. 1994; Siu-Caldera, Rao et al. 2001). We undertook the study IV to achieve the characterization and synthesis of this 24-oxo metabolite and compared its activity to its parent VDR agonist. In order to confirm the previous observation (Uskokovic, Manchand et al. 2006), human cells derived from an ovarian carcinoma were treated 24 h with BXL-62 or 1α,25(OH)2D3 and their metabolites where identified by high pressure liquid chromatography. The pattern of metabolism of 1α,25(OH)2D3 into its various metabolites is similar to the previously reported (Reddy and Tserng 1989), but the pattern of the metabolism of BXL-62 is different. While 1α,25(OH)2D3 pattern presents 23-hydroxylate and 25-hydroxylate metabolites (III, Fig. 2 upper panel), they are absent from the BXL-62 profile (III, Fig. 2 lower panel). The BXL-62 pattern shows a higher amount of 24-oxo metabolite compared to 98 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Results 1α,25(OH)2D3 (III, Fig. 2 lower panel). Mass spectrometry analysis allowed us to characterize definitively this 24-oxo metabolite as 16-ene-20-cyclopropyl-24-oxo-1α,25(OH)2D3 (BXL143) (III, Fig. 3). Finally, in order to confirm that the potency of BXL-62 could be due to the accumulation of its 24-oxo metabolite, the chemical synthesis of BXL-143 was carried out (III, scheme 1). Characterization and synthesis of the 24-oxo metabolite from the active VDR agonist BXL-62 allowed us to compare their activity in order to confirm that BXL-62 activity could pass through accumulation of BXL-143. We first showed a similar ability of BXL-62 and its 24-oxo metabolite to induce primary VDR target genes, CYP24A1 and CAMP, in two different human cell culture systems, PBMCs (III, Fig. 4A) and THP-1 cells (III, Fig. 5B). To compare the anti-inflammatory properties of these VDR agonists, we performed two different assays, the MLR and the LPS-induced PBMC activation. In these two different assays, BXL62 and BXL-143 present similar inhibition of pro-inflammatory cytokine production, including IFN-γ (III, Fig. 6A), TNF-α (III, Fig. 6B), IL-12/23p40 (III, Fig. 7A) and IL-6 (III, Fig. 7B). Both compounds exhibit these activities at a lower dose than the natural hormone, indicating that BXL-62 activity could be mediated by accumulation of its 24-oxo metabolite. Interestingly, the normocalcemic activity of BXL-62 seems to be also a consequence of its specific metabolism as shown by the lower calcemic activity of its 24-oxo metabolite compared to its parent compound (III, Fig. 8). In conclusion, the potent VDR agonist BXL-62 is metabolized into a stable 24-oxo metabolite (BXL-143), which resists further metabolism. As a result, BXL-143 accumulates in tissues. By combining our observations of equipotency between BXL-62 and BXL-143 in transcript regulation and similar inhibition of cytokine production, we conclude that accumulation of BXL-143 can explain the strong potency of BXL-62, supporting the concept that specific differences in the target tissue metabolism of VDR agonists can play a critical role to increase their potency. 4.5 Specific IL-10 production deficiency in inflammatory bowel disease patients compared to healthy controls Recent evidence supports the key role of innate immunity in the IBD onset, as shown by increased severity of chemically-induced colitis in TLR adaptor protein Myd88 KO vs. wild type mice (Araki, Kanai et al. 2005) and the description of the PRR member Nod2 as a Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 99 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD susceptible gene for CD (Cho and Abraham 2007). CD patients presenting the Nod2 mutation 3020insC were associated with a lower production of IL-10 in the periphery after costimulation with MDP and TLR1/2 agonist (Noguchi, Homma et al. 2009) confirming potential higher disease susceptibility in patients presenting Nod2 and IL-10 polymorphism (Marrakchi, Moussa et al. 2009). These observations highlight the potential link between innate immune system and IL-10 deficiency in IBD pathogenesis. Genetic studies using Il-10-deficient (Kuhn, Lohler et al. 1993) and IL-10 transgenic mice (Hagenbaugh, Sharma et al. 1997) established the unequivocal importance of IL-10 in controlling inflammation initiated and perpetuated by pro-inflammatory signals in acute and chronic diseases such as IBD (Moore, de Waal Malefyt et al. 2001). Despite the presence of polymorphisms in IBD patients (Aithal, Craggs et al. 2001), IL-10 regulatory properties for the inhibition of pro-inflammatory cytokine production seems to be maintained (Schmit, Carol et al. 2002). Interestingly, IL-10 production is shown to be lower in PHA-stimulatedLPMCs derived from inflamed colonic mucosal from IBD patients compared to its noninflamed conterpart (Gasche, Bakos et al. 2000). We undertook study V to further analyze the potential correlation between IL-10 production and IBD pathogenesis. We focussed on the IL10 production in the periphery and the inflamed tissue after TLR agonist stimulation. Previous observations have shown that TLR4 and TLR5 are the two stimuli, among the TLR agonist family tested, producing higher level of pro-inflammatory cytokine after 24 h activation (IV, Fig. 3). We demonstrated a significantly lower IL-10 production in both UC and CD compared to normal control, only in PBMCs stimulated by TLR4 agonist (V, Fig. 1A). This defect is not present for pro-inflammatory cytokines, such as TNF-α (V, Fig. 1), IL6 and IL-12/23p40 (V, Fig. 3). These data provide the first evidence that PBMCs purified from IBD patients present a reduction of IL-10 production specifically after TLR4 stimulation. As previously described (Schmit, Carol et al. 2002), IL-10 is able to regulate proinflammatory cytokine production since the blocking of its receptor by blocking antibody induced an up-regulation of TNF-α, ΙL-6 or IL-12/23p40 production after stimulation with TLR4 and TLR5 agonists (V, Fig. 2). While PBMCs stimulated with TLR5 in presence of the IL-10R blocking antibody respond with a significant increase of IL-10 production in both IBD subtypes and normal controls (V, Fig. 2A, right panel), PBMCs purified from IBD patients present a deficit in IL-10 production after TLR4 stimulation, while normal controls 100 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Results fail to present this defect (V, Fig. 2A, right panel). This observation confirms that IL-10 production is specifically defective in TLR4-stimulated PBMCs from IBD patients. As we have previously observed, TNF-α and IL-12/23p40 production by TLR4 or TLR5 PBMCs-activated are inhibited by 10 nM of 1α,25(OH)2D3 (IV, Fig. 3). In study V we show that this anti-inflammatory effect is independent of IL-10 activity, since 1α,25(OH)2D3 inhibits TNF-α and IL-12/23p40 production in presence of the blocking IL-10R antibody (V, Fig. 4). These observations are similar for IL-12/23p40 inhibition after TLR4 stimulation (V, Fig. 4). IL-10 regulatory properties for the inhibition of pro-inflammatory cytokine production is maintained, since IL-10 in LPMCs purified from IBD patients are able to inhibit IFN-γ and TNF-α production (Schmit, Carol et al. 2002). We next wanted to study the IL-10 production in the tissue site target of the inflammation. We purified LPMCs from four IBDs patients and performed a similar experiment than with PBMCs. Surprisingly, IL-10 production is increased after IL-10R blocking in LPMCs stimulated with LPS and Flagellin. These results present an intriguing disconcordance with the observations in PBMCs (V, Fig 5). These results highlight a specific TLR-induced defect in the periphery of IBD patients, inducing a lower production of IL-10, an important anti-inflammatory cytokine. Despite the lack of lower IL-10 production in LPMCs from the inflamed tissue, these findings contribute to confirm that IL-10 represents a key cytokine in the pathogenesis of IBD. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 101 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 5. Discussion 5.1 Prostatic cells as inducers and targets of chronic inflammation The pathophysiological mechanisms underlying BPH-related end-stage bladder decompensation are still unclear, but it is likely that both increased urethral narrowing and bladder smooth muscle overactivity are involved (Andersson and Arner 2004). Hence, in the development of BPH and its related symptoms, at least three distinct components can be defined: a static component, related to the overgrowth of the prostate gland, a dynamic component, associated with smooth muscle hypercontractility, and an inflammatory component. The static component is mostly responsible for obstructive symptoms, because the enlarged prostate is a mechanic obstacle to the physiological urinary outflow resulting in complaints of weak stream, intermittent urinary flow and/or straining to void. The dynamic component is responsible for the occurrence of storage (irritative) symptoms as urinary frequency, urgency and nocturia. Recently, chronic inflammation has emerged as the third component of BPH pathogenesis, taking part with the androgen receptor signaling in the induction of the tissue remodelling typical of the advanced stages of the disease and the prostatic inflammatory infiltrates observed in a large percentage of BPH surgical specimens (Nickel 2008). Previous work in our laboratory has shown significantly increased levels of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6 and IL-12p70 and the chemokines CCL1, CCL4, CCL22 and IL-8 in the seminal plasma from BPH patients (Penna, Mondaini et al. 2007). The concomitant increase of several inflammatory cytokines and chemokines in BPH patients is consistent with an important chronic inflammatory component in disease pathogenesis and expression profiling data demonstrate a strong correlation between inflammation and symptomatic BPH (Prakash, Pirozzi et al. 2002). Results in study I demonstrate for the first time that IL-8-mediated BPH cell growth can be induced by a combination of IFN-γ and IL-17, thus establishing a possible relationship between the T-cell response induced by BPH cells and prostate cell growth. Therefore, these data provide a mechanistic explanation for the role of IL-8 in BPH pathogenesis, showing that a combination of Th1 and Th17 cell-derived inflammatory cytokines can markedly stimulate its secretion by BPH stromal cells. Thus, BPH can be seen as a form of chronic prostatitis, whose pathogenesis may be triggered by infection. The release of prostatic self-antigens following tissue damage may sensitise the immune system and start autoimmune responses 102 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Discussion and among pro-inflammatory cytokines and chemokines produced by the prostatic microenvironment, stromal-derived IL-8 may be considered a key link between chronic inflammation and stromal cell proliferation. 5.2 VDR agonists inhibit intraprostatic inflammatory responses Pharmacological management of BPH is a novel application of vitamin D analogs, prompted by the detection of VDR expression in cultured prostatic and bladder stromal cells derived from BPH patients. Reducing prostate overgrowth by decreasing intra-prostatic androgen signaling, without directly interfering with systemic androgen action, would obviate the adverse systemic side effects of anti-androgens, such as 5α-reductase inhibitors. In addition, VDR agonists can modulate the dynamic component of LUTS pathogenesis, and exert anti-inflammatory activities. Thus, this class of agents could represent an interesting therapeutic option for the pharmacological treatment of BPH. We have already discussed in study I that among the pro-inflammatory cytokines and chemokines produced by the prostatic microenvironment, stromal-derived IL-8 may be considered a key link between chronic inflammation and stromal cell proliferation. Consistent with an IL-8-dependent link bridging inflammatory response and cell growth in BPH cells, the VDR agonist elocalcitol, a well-defined anti-inflammatory agent inhibiting prostate growth in experimental models (Crescioli, Ferruzzi et al. 2004) and arresting prostate growth in BPH patients (Colli, Rigatti et al. 2006) has been shown to inhibit IL-8-mediated prostate growth and inflammation through multiple mechanisms of action (Penna, Amuchastegui et al. 2006; Morelli, Vignozzi et al. 2007). VDR expression is promoted by T cell-derived inflammatory cytokines in BPH cells, rendering them more susceptible to the inhibitory action of elocalcitol. In addition, elocalcitol inhibits IL-8 production induced by pro-inflammatory cytokines secreted by prostateinfiltrating CD4+ T cells (IFN-γ, IL-17 and TNF-α) in human prostatic stromal cells, accompanied by reduced COX-2 expression and PGE2 production and by arrest of the nuclear translocation of NF-κB, a transcriptional factor that directly regulates IL-8 production. Elocalcitol dose-dependently counteracts IL-8-dependent BPH cell proliferation via inhibition of the RhoA/ROCK pathway. These effects are consistent with the role of this calcium-sensitising signaling in the regulation of the inflammatory processes (Segain, Raingeard de la Bletiere et al. 2003), through the activation of NF-κB (Montaner, Perona et Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 103 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD al. 1998) and the induction of IL-8 secretion (Zhao, Kuhnt-Moore et al. 2003). In particular, the effect on this calcium-sensitizing pathway may represent a common denominator for the therapeutic efficacy of this drug on all the three components of BPH: static, dynamic and inflammatory. Consistent with the role of RhoA/ROCK signaling in regulating human and rat bladder contraction and tone, in particular in generating involuntary contractions (Peters, Schmidt et al. 2006), elocalcitol affects bladder contractility via inhibition of the calcium sensitizing RhoA/ROCK pathway by interfering with RhoA activation (Morelli, Vignozzi et al. 2007). The reduction of RhoA/ROCK-mediated inappropriate bladder contraction induced by elocalcitol does not interfere with the overall detrusor motility, preventing urinary retention due to voiding impairment. In conclusion, combined results on elocalcitol activity indicate possible beneficial effects of elocalcitol on bladder overactivity by two mechanisms: counteracting the enhanced expression and signaling of growth factors involved in bladder smooth muscle hypertrophy and hyperplasia (Crescioli, Morelli et al. 2005) and increasing the contractile efficiency of bladder muscle cells through the modulation of smooth muscle gene expression and the down-regulation of smooth muscle myofilament sensitization to calcium (Morelli, Vignozzi et al. 2007). Based on these preclinical premises, a phase IIa, double blind, placebo-controlled study has shown clear efficacy signals on the primary endpoint (mean volume voided per micturition) and on symptoms, including frequency, nocturia and incontinence episodes (Colli, Digesu et al. 2007). A multi-center phase IIb trial shows that elocalcitol arrests prostate growth and positive effects on the secondary endpoints of LUTS (ie, urgency and frequency of urination, and nocturia) and International Prostate Symptom Score (IPSS) were also observed (Fibbi, Penna et al. 2009). Unfortunately, the additional potential benefits of elocalcitol in men with BPH and associated LUTS and/or bladder outlet obstruction were further obviated because of negative results from a phase IIb study in OAB. Despite these results, elocalcitol will still have the status of 'first-in-target-class' of vitamin D3 analogs, given its encouraging data on several endpoints (Tiwari 2009). 5.3 TLR specific deficiency for IL-10 production IL-10 represents clearly a key cytokine in the pathogenesis of IBD as demonstrated by Il-10 deficient mice that develop spontaneous colitis (Kuhn, Lohler et al. 1993). In addition, an inappropriate regulation of IL-10 production in the periphery could represent an important event for the pathogenesis since transgenic CD4+CD45RBhigh T cell, purified from 104 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Discussion splenocytes producing high level of IL-10, transfered in a SCID mice recipient do not exhibit wasting colitis symptoms as observed in transfer with wild type T cells (Hagenbaugh, Sharma et al. 1997). Consistent with this observation, T cell-induced severe colitis was totally abrogated in mice treated with recombinant IL-10 (Powrie, Leach et al. 1994). Recently, the concept of lower IL-10 levels circulating in sera of IBD patients was correlated with the presence of neutralizing antibodies (Ebert, Schott et al. 2009), but despite some report showing that IL-10 production is lower in 3020insC Nod2 mutated patients after MDP and TLR2 co-activation (Noguchi, Homma et al. 2009), no clear evidence of an impaired of IL-10 secretion in the periphery was reported. In study V, we demonstrated that IL-10 production in PBMCs purified from IBD patients is defective after TLR4 stimulation but not after TLR5 stimulation. Surprisingly, LPMCs do not seem to exhibit this defect. These findings highlight a possible difference in response to TLR4 in the periphery and in tissue site of inflammation in IBD patients, confirming that IL-10 production in the periphery from IBD patients could play a key role in the disease onset. Compared to PBMCs, lamina propria macrophages present a low expression of TLR2 and TLR4 (Hausmann, Kiessling et al. 2002) and isolated lamina propria macrophages do not express CD14 and are unresponsive to LPS (Smith, Smythies et al. 2001). In addition, colonic epithelial cells express low levels of TLR4 and MD2 and are poorly responsive to LPS (Abreu, Vora et al. 2001; Naik, Kelly et al. 2001; Suzuki, Hisamatsu et al. 2003). These findings support the concept that in humans the TLR4 co-receptor CD14 promoter polymorphisms could contribute to disease development (de Buhr, Hedrich et al. 2009). In the DSS-induced colitis model, evidence suggests that TLR4 may play a partial role in the development of severe disease, at least in certain mouse strains (Lange, Delbro et al. 1996). TLR4 mutation in C3H/HeJ mice renders them more susceptible to the spontaneous development of chronic colitis (Elson, Cong et al. 2000) suggesting, as observed in humans, that genetic factors can influence the relative importance of TLR4 in the development of colitis (Sepulveda, Beltran et al. 2008). A role for TLR4 in the development of chronic colitis has been demonstrated clearly in mice that have a myeloid-specific deletion of Stat3 and present enhanced Th1 responses and develop chronic colitis, probably due to the inability of myeloid cells to respond to IL-10 (Takeda, Clausen et al. 1999). In these mice, colitis development seems to depend on the presence of TLR4, IL-12/23p40 and T cells, because conditional Stat3 KO mice that are also Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 105 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD deficient in any of these molecules or cells do not develop colitis (Takeda, Clausen et al. 1999). These observations were confirmed by the blocking of TLR4 complex signaling that result in decreased intestinal inflammation in the Mdr1a-deficient mice, another model of colitis that is characterized by the presence of Th1-type T cells (Panwala, Jones et al. 1998) These data demonstrate that TLR4 signaling in response to the presence of bacteria in periphery could have an important role in the onset of IBD by reducing production of IL-10, a potent anti-inflammatory cytokine. 5.4 BXL-62 ameliorates symptoms in experimental model of colitis Because supraphysiologic concentrations of 1α,25(OH)2D3 and its analogues are usually required to exert immunosuppressive and anti-proliferative effects, more than 3000 vitamin D3 analogues with several different structural modifications have been synthesized during the past two decades (Deluca and Cantorna 2001; Uskokovic, Norman et al. 2001; Uskokovic, Manchand et al. 2006). We have already discussed about elocalcitol that presents an enhanced biological activity with less calcemic liability compared to 1α,25(OH)2D3 (Colli, Rigatti et al. 2006). Previous studies showed that the potency of 20-cyclopropyl-vitamin D3 analogues can be increased both in terms of their calcemic and immunomodulatory activities by the addition of a 16-ene moiety (Uskokovic, Manchand et al. 2006). In study III we have further characterized the potency of 16-ene-20-cyclopropyl family members with respect to their anti-inflammatory properties by studying the inhibition of IFN-γ production in MLR assays and TNF-α in LPS-activated PBMCs. We have identified BXL-62 as the most potent anti-inflammatory compound among all the members of the 16ene-20-cyclopropyl family tested. The MLR assay is used as an in vitro model of adaptive immunity and is widely applied to monitor diseases, such as AIDS (Clerici, Stocks et al. 1989) to predict transplant rejection, especially in renal transplantation (Kerman, Susskind et al. 1997) and to screen for novel immunosuppressive drugs (Matsumoto, Marui et al. 1993). One of the mechanisms regulating innate immunity involves the TLRs (Athman and Philpott 2004), so in our experiments we focused on TLR4, because its ligand, the bacterial endotoxin LPS, is a potent inducer of immune responses (Medzhitov 2007). We have analyzed the proinflammatory cytokine TNF-α, which is produced after the activation of TLR4. TNF-α plays a key role in many disorders, such as rheumatoid arthritis, acute lung injury and IBD (Bradley 106 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Discussion 2008). Anti-TNF-α biologicals are clinically used for the treatment of several autoimmune diseases including IBD (Baumgart and Sandborn 2007). Our observations, documenting the capacity our selected VDR agonists to markedly inhibit TNF-α, suggest this class of agents as a potential treatment of IBD. In order to validate its potentcy in IBD context, we wanted to confirm in vitro potency of our selected VDR agonist in PBMCs purified from IBD patients. Inhibition of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-12/23p40, by VDR agonists after any TLR activation in PBMCs purified from IBD patients could represent an important mechanism for the regulation of the abnormal immune response to the bacteria, as commonly though as starting point of the disease in genetic predisposed individual (Danese and Fiocchi 2006). Additionaly, our VDR agonist present an important regulation of adaptive immunity, since these three pro-inflammatory cytokines, especially IL-12/23p40, are potent regulators of Th1 and Th17-mediated T cell response (Steinman 2007). Pro-inflammatory cytokine production is controlled by the inducible NF-κB, which binds to regulatory regions inflammatory genes and up-regulates the transcription of proinflammatory cytokines (Oeth, Parry et al. 1994). NF-κB nuclear translocation is the main event downstream the TLR, inducing transcription of pro-inflammatory genes. Coupled to the direct transcript inhibition mediated by the VDR, the indirect effect on NF-κB confirmed the potency of this agonist as alternative of the biological agent in the regulation of the proinflammatory response mediated by TLR members (Lips 1996). Finally, since IL-10 represents an important cytokine for IBD onset, we demonstrated that despite a deficiency observed in PBMCs from IBD patients, the VDR agonist BXL-62 maintains its antiinflammatory poteny for TNF-α and IL-12/23p40. No study has been previously carried out on anti-inflammatory properties exterted by VDR agonists on human LPMCs purified from inflamed tissue. To extend our previous results, it was interesting to study the anti-inflammatory potency of BXL-62 on cells purified from tissues directly involved in disease pathogenesis. A similar inhibition of proinflammatory cytokines was observed in PBMCs and LPMCs, indicating that VDR expression in LMPCs is functional. These results are promising to further determine the potency of BXL-62 in the DSSinduced colitis model. DSS-induced colitis is a model of wasting disease based on epithelial damage mimicking the increase of the permeability leading to the abnormal presentation of the Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 107 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD bacteria flora (Xavier and Podolsky 2007). Recent work has shown the important role of the VDR in the onset of spontaneous and induced colitis in mice and the amelioration of DSSinduced colitis symptoms after intra-rectal injection of calcitriol (2.5 µg/kg) (Cantorna, Munsick et al. 2000; Froicu, Weaver et al. 2003; Zhu, Mahon et al. 2005; Froicu and Cantorna 2007). It was interesting to see that in this model, the amelioration was directly mediated by TNF-α inhibition (Zhu, Mahon et al. 2005). In study IV, we demonstrated that at the maximum tolerated dose established by four consecutive oral administrations of compound, intra-rectal administration of BXL-62 (1 µg/kg, 20 ng/mouse) prevents symptoms of colitis while calcitriol (0.3 µg/kg, 6.7 ng/mice) failed to ameliorate disease. The strong BXL-62 efficacy on the fecal blood could be explained by the anti-inflammatory activity of the compound but also by the critical role of VDR in the maintenance of the integrity if the intestinal mucosal barrier, as demonstrated by a greater loss of intestinal transepithelial electric resistance in Vdr KO mice compared to wild type (Kong, Zhang et al. 2008). The regulation of inflammation, confirmed by an increase of the colon length after BXL-62 treatment, could result, as previously discussed, in a stronger effect of this compound in the regulation of TLR-mediated inflammation. Taken together, these pre-clinical in vitro and in vivo data confirm the potential therapeutic used of VDR agonists for the treatment of IBD. 5.5 24-oxo metabolite accumulation, a key event for BXL-62 potency It is now well established that 1α,25(OH)2D3 is metabolized by CYP24A1, a multi-catalytic enzyme, in various target tissues (Deeb, Trump et al. 2007). Over a decade ago, differences were identified in the metabolism between 1α,25(OH)2-16-ene-vitamin D3 and 1α,25(OH)2D3 and it was recognized that minor alterations in the structure of 1α,25(OH)2D3 can produce major changes in its target tissue metabolism, which allows efficient C24-hydroxylation and C24-oxidation but not C23-hydroxylation (Lemire, Archer et al. 1994; Siu-Caldera, Clark et al. 1996). As a result, the 24-oxo metabolite of 1α,25(OH)2-16-ene-vitamin D3 accumulates in increasing amounts in target tissues when compared to the corresponding 24-oxo metabolite of 1α,25(OH)2D3. The biological activity of the stable 24-oxo metabolite of 1α,25(OH)2-16ene-vitamin D3 seems to be similar to some of the actions of 1α,25(OH)2-16-ene-vitamin D3, such as induction of cell growth inhibition and promotion of RWLeu-4 human myeloid leukemic cell differentiation (Siu-Caldera, Clark et al. 1996). These data thus support the 108 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Discussion concept that one of the important mechanisms responsible for the increase in the potency of some of the vitamin D analogues can be due to their metabolism through alternative pathways leading to the production of stable and bioactive metabolites. On the basis of these earlier observations, the metabolism of 1α,25(OH)2-20-cyclopropyl-vitamin D3 with that of BXL-62 was compared, showing that 1α,25(OH)2-20-cyclopropyl-vitamin D3 is rapidly metabolized through three different pathways (24-oxidation, C3-epimerization and C1-esterification pathways) (Uskokovic, Manchand et al. 2006), with a pattern of metabolism similar to that of 1α,25(OH)2-20-epi-vitamin D3 (Siu-Caldera, Sekimoto et al. 1999). Conversely, BXL-62 is mainly metabolized through only two pathways (C24-oxidation and C3-epimerization pathways). In study III, we have investigated the metabolism of 1α,25(OH)2-20-cyclopropyl-16ene-vitamin D3 in human ovarian carcinoma derived cell line and confirmed that the 24-oxo metabolite BXL-143 is indeed the final, stable metabolite of BXL-62. The chemical synthesis of BXL-143 allowed us to assess its various biological properties. We demonstrated that VDR primary response gene induction, as well as inhibition of pro-inflammatory cytokine production, are similar between BXL-62 and BXL-143. Expression of CAMP, a primary VDR target gene, is also modulated by BXL-62 and BXL-143 in the same way as that of CYP24A1 (Wang, Nestel et al. 2004). As CAMP plays an important role in innate immunity (Wang, Nestel et al. 2004; Liu, Stenger et al. 2006; Schauber, Dorschner et al. 2007), these data indicate comparable properties for BXL-62 and BXL-143 in the regulation of innate immune responses. Collectively, these findings indicate that the strong potency of BXL-62 can be explained by the accumulation of its stable 24-oxo metabolite that displays immunoregulatory and anti-inflammatory properties superimposable to those exerted by BXL-62 itself. However, we found that the MTD of BXL-143 is three times higher than its parent compound BXL-62, indicating that the potency of the metabolite compared to the parent compound to induce hypercalcemia was reduced. Based on these findings, we have proposed that BXL-143 represents a superior anti-inflammatory agent to its parent BXL-62, due to its wider therapeutic window. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 109 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 6. Summary and conclusions In conclusion, the five publications, on which this thesis is based, contribute to the comprehension of the pathogenesis of two chronic inflammatory disorders, BPH and IBD. The developments of alternative therapeutic agents are critical for ameliorating the patient’s quality of life and represent an important challenge. Work in this thesis on patient samples and animal experimental models proposes VDR agonists as promising therapeutic agents that should be further investigated and developed. Chronic inflammation induced by stromal BPH cells In study I, we show that stromal cell purified from biopsies from patients suffering of BPH express functional receptors involving in innate immunity, the TLRs, as well as the complex machinery required for induction of adaptive immune responses. We demonstrate that TLR activation of stromal BPH cells induces pro-inflammatory cytokine production, such as IL-8, which promotes BPH cells growth, and CXCL10 that recruits imflammatory leukocytes. Additionally, BPH cells can activate CD4+ T cells inducing production of pro-inflammatory cytokines typical of Th1 and Th17-mediated immune responses, such as IFN-γ and IL-17. Thus, in study I we provide, for the first time, direct demonstration of the immunological mechanisms leading to an inflammatory component in BPH pathogenesis, and highlight possible new therapeutic agents for this disease. Elocalcitol inhibit inflammatory reponse in BPH cells Following the discovery of an inflammatory component in BPH, we studied in publication II the inhibitory potency of elocalcitol, a VDR agonist proposed for BPH treatment. We demonstrated that IL-8-dependent BPH cell growth is inhibited by elocalcitol, as well as proinflammatory factors, such as COX-2 and PGE2. These inhibitiory activities are directly mediated by a reduction of the nuclear translocation of NF-κB and a down-regulation of Rho/Rho kinase pathway. Thus, elocalcitol could ameliorate static, dynamic, and inflammatory components of BPH pathogenesis. IBD patients present in the periphery a IL-10 production defect In study V, we investigated the production of anti- and pro-inflammatory cytokines after TLR4 and TLR5 stimulation in the periphery and the tissue site of inflammation in IBD patients. We demonstrated that despite a similar pro-inflammatory cytokine production profile in the periphery between IBD patients and healthy controls, the production of the anti- 110 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) Summary and conclusions inflammatory cytokine IL-10 is defective in PBMCs from IBD patients stimulated by a TLR4 agonist. We could not confirm this IL-10 defect in the inflamed tissue, highlighting the potential relevance of peripheral IL-10 production in IBD pathogenesis. BXL-62 as a potent anti-inflammatory VDR agonist in IBD Following the definition of BXL-62 as the most potent anti-inflammatory VDR agonist among its family members tested, we decided to study its potency to ameliorate colitis symptoms both in mononuclear cells purified from patients and in a chemically-induced colitis model in the mouse. We confirmed the strong anti-inflammatory potency of this VDR agonist compared to the natural hormone in IBD patients, both in the periphery and in inflamed intestinal tissue. We demonstrated also that VDR agonists are able to inhibit proinflammatory cytokines after stimulation by any TLR agonist, confirming VDR agonists as potent regulators of the abnormal bacteria recognition. In addition, DSS-induced colitis symptoms in mice were significantly more ameliorated by daily treatment with BXL-62 compared to the natural ligand, confirming a potential interest for its development as a therapeutics agent in IBD. BXL-62 potency is mediated by its 24-oxo metabolite accumulation In study III, we confirm previous observations and show that BXL-62 is protected from CYP24A1 hydroxylation, leading to an accumulation of a 24-oxo-BXL-62 (BXL-143). This VDR agonist shows similar VDR primary response gene induction and anti-inflammatory potency to its parent compound, suggesting that BXL-62 activity is mediated by this 24-oxo metabolite. Additionaly, the latter compound exhibits a maximum tolerated dose 3 times lower than its parent compound, suggesting BXL-143 as an even more interesting VDR agonist. General conclusion In conclusion, these studies confirm elocalcitol as a potent VDR agonist and a potential drug to ameliorate BPH. Elocalcitol targets BPH cells, inhibiting proliferation and inflammation induced by their capacity to act as APCs and to recognize bacterial components via TLRs. In addition, we characterize a novel potent VDR agonist, BXL-62, that exhibits strong antiinflammatory and immunoregulatory properties both in innate and adaptive immunity, and propose this VDR agonist as a potential therapeutic agent for IBD, a chronic inflammatory disease of the gastrointestinal tract. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 111 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD 7. Future aspects IBD is a complex disease where the cross talk between environmental and genetic factors contributes to disease pathogenesis. The precise ethiology of IBD remains unknown, despite all the progress in genome-wide association studies. The current understanding of IBD pathogenesis has nevertheless permitted the development of effective therapies, although none of them leads to complete remission. Drugs with the capacity to regulate abnormal inflammatory responses could lead to the selection of novel candidates. In this context, in addition to promising pre-clinical activity in diverse inflammatory models, VDR agonists represent potential candidates for the treatment of IBD, among other chronic inflammatory disorders. The VDR agonist BXL-62 identified in this thesis could represent an interesting candidate for future development. Further characterization of its potency in spontaneous and induced colitis models and the confirmation of its mechanisms of action mediated by its 24oxo metabolite will lead to the possible identification of novel treatment opportunities, since undesirable effects induced by VDR agonists appear to be predictable and manageable. 112 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References 8. References Abe, E., C. Miyaura, et al. (1981). "Differentiation of mouse myeloid leukemia cells induced by 1 alpha,25-dihydroxyvitamin D3." Proc Natl Acad Sci U S A 78(8): 4990-4. Abreu, M. T., P. Vora, et al. (2001). "Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide." J Immunol 167(3): 1609-16. Adorini, L. (2002). "Immunomodulatory effects of vitamin D receptor ligands in autoimmune diseases." Int Immunopharmacol 2(7): 1017-28. Adorini, L., N. Giarratana, et al. (2004). "Pharmacological induction of tolerogenic dendritic cells and regulatory T cells." Semin Immunol 16(2): 127-34. Adorini, L. and G. Penna (2008). "Control of autoimmune diseases by the vitamin D endocrine system." Nat Clin Pract Rheumatol 4(8): 404-12. Adorini, L., G. Penna, et al. (2007). "Inhibition of prostate growth and inflammation by the vitamin D receptor agonist BXL-628 (elocalcitol)." J Steroid Biochem Mol Biol 103(3-5): 689-93. Adorini, L., G. Penna, et al. (2004). "Dendritic cells as key targets for immunomodulation by Vitamin D receptor ligands." J Steroid Biochem Mol Biol 89-90(1-5): 437-41. Adorini, L., G. Penna, et al. (2003). "Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting allograft rejection and autoimmune diseases." J Cell Biochem 88(2): 227-33. Aithal, G. P., A. Craggs, et al. (2001). "Role of polymorphisms in the interleukin-10 gene in determining disease susceptibility and phenotype in inflamatory bowel disease." Dig Dis Sci 46(7): 1520-5. Akira, S. and K. Takeda (2004). "Toll-like receptor signalling." Nat Rev Immunol 4(7): 499511. Akira, S., K. Takeda, et al. (2001). "Toll-like receptors: critical proteins linking innate and acquired immunity." Nat Immunol 2(8): 675-80. Ali, M. M. and V. Vaidya (2007). "Vitamin D and cancer." J Cancer Res Ther 3(4): 225-30. Anderson, G., N. C. Moore, et al. (1996). "Cellular interactions in thymocyte development." Annu Rev Immunol 14: 73-99. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 113 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Andersson, K. E. and A. Arner (2004). "Urinary bladder contraction and relaxation: physiology and pathophysiology." Physiol Rev 84(3): 935-86. Andoh, A., H. Takaya, et al. (2001). "Cooperation of interleukin-17 and interferon-gamma on chemokine secretion in human fetal intestinal epithelial cells." Clin Exp Immunol 125(1): 56-63. Araki, A., T. Kanai, et al. (2005). "MyD88-deficient mice develop severe intestinal inflammation in dextran sodium sulfate colitis." J Gastroenterol 40(1): 16-23. Ardizzone, S., A. Cassinotti, et al. (2009). "Immunomodulatory effects of 1,25dihydroxyvitamin D3 on TH1/TH2 cytokines in inflammatory bowel disease: an in vitro study." Int J Immunopathol Pharmacol 22(1): 63-71. Arriagada, G., R. Paredes, et al. (2007). "Phosphorylation at serine 208 of the 1alpha,25dihydroxy Vitamin D3 receptor modulates the interaction with transcriptional coactivators." J Steroid Biochem Mol Biol 103(3-5): 425-9. Artis, D. (2008). "Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut." Nat Rev Immunol 8(6): 411-20. Athman, R. and D. Philpott (2004). "Innate immunity via Toll-like receptors and Nod proteins." Curr Opin Microbiol 7(1): 25-32. Baecher-Allan, C. and D. A. Hafler (2006). "Human regulatory T cells and their role in autoimmune disease." Immunol Rev 212: 203-16. Baker, A. R., D. P. McDonnell, et al. (1988). "Cloning and expression of full-length cDNA encoding human vitamin D receptor." Proc Natl Acad Sci U S A 85(10): 3294-8. Banchereau, J., F. Briere, et al. (2000). "Immunobiology of dendritic cells." Annu Rev Immunol 18: 767-811. Banchereau, J. and R. M. Steinman (1998). "Dendritic cells and the control of immunity." Nature 392(6673): 245-52. Bandholtz, L., G. J. Ekman, et al. (2006). "Antimicrobial peptide LL-37 internalized by immature human dendritic cells alters their phenotype." Scand J Immunol 63(6): 4109. Barrat, F. J., D. J. Cua, et al. (2002). "In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines." J Exp Med 195(5): 603-16. 114 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Batista, F. D. and N. E. Harwood (2009). "The who, how and where of antigen presentation to B cells." Nat Rev Immunol 9(1): 15-27. Baumgart, D. C. and S. R. Carding (2007). "Inflammatory bowel disease: cause and immunobiology." Lancet 369(9573): 1627-40. Baumgart, D. C. and W. J. Sandborn (2007). "Inflammatory bowel disease: clinical aspects and established and evolving therapies." Lancet 369(9573): 1641-57. Bell, N. H. (1998). "Renal and nonrenal 25-hydroxyvitamin D-1alpha-hydroxylases and their clinical significance." J Bone Miner Res 13(3): 350-3. Bemiss, C. J., B. D. Mahon, et al. (2002). "Interleukin-2 is one of the targets of 1,25dihydroxyvitamin D3 in the immune system." Arch Biochem Biophys 402(2): 249-54. Benn, B. S., D. Ajibade, et al. (2008). "Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k." Endocrinology 149(6): 3196-205. Bierhoff, E., U. Walljasper, et al. (1997). "Morphological analogies of fetal prostate stroma and stromal nodules in BPH." Prostate 31(4): 234-40. Binderup, L., S. Latini, et al. (1991). "20-epi-vitamin D3 analogues: a novel class of potent regulators of cell growth and immune responses." Biochem Pharmacol 42(8): 1569-75. Bookout, A. L., Y. Jeong, et al. (2006). "Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network." Cell 126(4): 789-99. Boonstra, A., F. J. Barrat, et al. (2001). "1alpha,25-Dihydroxyvitamin d3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells." J Immunol 167(9): 4974-80. Bouillon, R., G. Eelen, et al. (2006). "Vitamin D and cancer." J Steroid Biochem Mol Biol 102(1-5): 156-62. Bouillon, R., S. Van Cromphaut, et al. (2003). "Intestinal calcium absorption: Molecular vitamin D mediated mechanisms." J Cell Biochem 88(2): 332-9. Boyce, B. F. and L. Xing (2008). "Functions of RANKL/RANK/OPG in bone modeling and remodeling." Arch Biochem Biophys 473(2): 139-46. Bradley, J. R. (2008). "TNF-mediated inflammatory disease." J Pathol 214(2): 149-60. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 115 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Brenza, H. L. and H. F. DeLuca (2000). "Regulation of 25-hydroxyvitamin D3 1alphahydroxylase gene expression by parathyroid hormone and 1,25-dihydroxyvitamin D3." Arch Biochem Biophys 381(1): 143-52. Brown, A. J., M. Zhong, et al. (1995). "The roles of calcium and 1,25-dihydroxyvitamin D3 in the regulation of vitamin D receptor expression by rat parathyroid glands." Endocrinology 136(4): 1419-25. Budarf, M. L., C. Labbe, et al. (2009). "GWA studies: rewriting the story of IBD." Trends Genet 25(3): 137-46. Burrows, P. D. and M. D. Cooper (1997). "B cell development and differentiation." Curr Opin Immunol 9(2): 239-44. Campbell, M. J., G. S. Reddy, et al. (1997). "Vitamin D3 analogs and their 24-oxo metabolites equally inhibit clonal proliferation of a variety of cancer cells but have differing molecular effects." J Cell Biochem 66(3): 413-25. Cantorna, M. T. and B. D. Mahon (2004). "Mounting evidence for vitamin D as an environmental factor affecting autoimmune disease prevalence." Exp Biol Med (Maywood) 229(11): 1136-42. Cantorna, M. T., C. Munsick, et al. (2000). "1,25-Dihydroxycholecalciferol prevents and ameliorates symptoms of experimental murine inflammatory bowel disease." J Nutr 130(11): 2648-52. Carlberg, C. (2003). "Current understanding of the function of the nuclear vitamin D receptor in response to its natural and synthetic ligands." Recent Results Cancer Res 164: 2942. Carlberg, C., I. Bendik, et al. (1993). "Two nuclear signalling pathways for vitamin D." Nature 361(6413): 657-60. Carlberg, C. and S. Seuter (2009). "A genomic perspective on vitamin D signaling." Anticancer Res 29(9): 3485-93. Chaplin, D. D. (2003). "1. Overview of the immune response." J Allergy Clin Immunol 111(2 Suppl): S442-59. Chaplin, D. D. (2006). "1. Overview of the human immune response." J Allergy Clin Immunol 117(2 Suppl Mini-Primer): S430-5. Chawla, A., J. J. Repa, et al. (2001). "Nuclear receptors and lipid physiology: opening the Xfiles." Science 294(5548): 1866-70. 116 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Cho, J. H. (2008). "The genetics and immunopathogenesis of inflammatory bowel disease." Nat Rev Immunol 8(6): 458-66. Cho, J. H. and C. Abraham (2007). "Inflammatory bowel disease genetics: Nod2." Annu Rev Med 58: 401-16. Cippitelli, M. and A. Santoni (1998). "Vitamin D3: a transcriptional modulator of the interferon-gamma gene." Eur J Immunol 28(10): 3017-30. Claus, S., R. Berges, et al. (1997). "Cell kinetic in epithelium and stroma of benign prostatic hyperplasia." J Urol 158(1): 217-21. Clerici, M., N. I. Stocks, et al. (1989). "Detection of three distinct patterns of T helper cell dysfunction in asymptomatic, human immunodeficiency virus-seropositive patients. Independence of CD4+ cell numbers and clinical staging." J Clin Invest 84(6): 1892-9. Cohen, J. J., R. C. Duke, et al. (1992). "Apoptosis and programmed cell death in immunity." Annu Rev Immunol 10: 267-93. Colli, E., G. A. Digesu, et al. (2007). "Overactive bladder treatments in early phase clinical trials." Expert Opin Investig Drugs 16(7): 999-1007. Colli, E., P. Rigatti, et al. (2006). "BXL628, a novel vitamin D3 analog arrests prostate growth in patients with benign prostatic hyperplasia: a randomized clinical trial." Eur Urol 49(1): 82-6. Colonna, M., G. Trinchieri, et al. (2004). "Plasmacytoid dendritic cells in immunity." Nat Immunol 5(12): 1219-26. Colston, K., M. J. Colston, et al. (1982). "1,25-dihydroxyvitamin D3 receptors in human epithelial cancer cell lines." Cancer Res 42(3): 856-9. Crescioli, C., P. Ferruzzi, et al. (2004). "Inhibition of prostate cell growth by BXL-628, a calcitriol analogue selected for a phase II clinical trial in patients with benign prostate hyperplasia." Eur J Endocrinol 150(4): 591-603. Crescioli, C., A. Morelli, et al. (2005). "Human bladder as a novel target for vitamin D receptor ligands." J Clin Endocrinol Metab 90(2): 962-72. D'Ambrosio, D., F. Sinigaglia, et al. (2003). "Special attractions for suppressor T cells." Trends Immunol 24(3): 122-6. Daeron, M. (1997). "Fc receptor biology." Annu Rev Immunol 15: 203-34. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 117 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Danese, S. and C. Fiocchi (2006). "Etiopathogenesis of inflammatory bowel diseases." World J Gastroenterol 12(30): 4807-12. Danese, S., M. Sans, et al. (2004). "Inflammatory bowel disease: the role of environmental factors." Autoimmun Rev 3(5): 394-400. Danese, S., S. Semeraro, et al. (2005). "Extraintestinal manifestations in inflammatory bowel disease." World J Gastroenterol 11(46): 7227-36. Daniel, C., H. H. Radeke, et al. (2006). "The new low calcemic vitamin D analog 22-ene-25oxa-vitamin D prominently ameliorates T helper cell type 1-mediated colitis in mice." J Pharmacol Exp Ther 319(2): 622-31. Daniel, C., N. A. Sartory, et al. (2008). "Immune modulatory treatment of trinitrobenzene sulfonic acid colitis with calcitriol is associated with a change of a T helper (Th) 1/Th17 to a Th2 and regulatory T cell profile." J Pharmacol Exp Ther 324(1): 23-33. de Buhr, M. F., H. J. Hedrich, et al. (2009). "Analysis of Cd14 as a genetic modifier of experimental inflammatory bowel disease (IBD) in mice." Inflamm Bowel Dis. Deeb, K. K., D. L. Trump, et al. (2007). "Vitamin D signalling pathways in cancer: potential for anticancer therapeutics." Nat Rev Cancer 7(9): 684-700. DeLuca, H. F. (2004). "Overview of general physiologic features and functions of vitamin D." Am J Clin Nutr 80(6 Suppl): 1689S-96S. Deluca, H. F. and M. T. Cantorna (2001). "Vitamin D: its role and uses in immunology." FASEB J 15(14): 2579-85. Delves, P. J. and I. M. Roitt (2000). "The immune system. First of two parts." N Engl J Med 343(1): 37-49. Dhodapkar, M. V., R. M. Steinman, et al. (2001). "Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells." J Exp Med 193(2): 233-8. Di Nardo, A., M. H. Braff, et al. (2007). "Cathelicidin antimicrobial peptides block dendritic cell TLR4 activation and allergic contact sensitization." J Immunol 178(3): 1829-34. Donjacour, A. A. and G. R. Cunha (1991). "Stromal regulation of epithelial function." Cancer Treat Res 53: 335-64. Ebert, A. K., G. Schott, et al. (2009). "Long-term follow-up of male patients after reconstruction of the bladder-exstrophy-epispadias complex: Psychosocial status, continence, renal and genital function." J Pediatr Urol. 118 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Ellmeier, W., S. Sawada, et al. (1999). "The regulation of CD4 and CD8 coreceptor gene expression during T cell development." Annu Rev Immunol 17: 523-54. Elson, C. O., Y. Cong, et al. (2005). "Experimental models of inflammatory bowel disease reveal innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota." Immunol Rev 206: 260-76. Elson, C. O., Y. Cong, et al. (2000). "The C3H/HeJBir mouse model: a high susceptibility phenotype for colitis." Int Rev Immunol 19(1): 63-75. Fantini, M. C., A. Rizzo, et al. (2007). "IL-21 regulates experimental colitis by modulating the balance between Treg and Th17 cells." Eur J Immunol 37(11): 3155-63. Faubion, W. A., Jr., E. V. Loftus, Jr., et al. (2001). "The natural history of corticosteroid therapy for inflammatory bowel disease: a population-based study." Gastroenterology 121(2): 255-60. Fernandez-Garcia, N. I., H. G. Palmer, et al. (2005). "1alpha,25-Dihydroxyvitamin D3 regulates the expression of Id1 and Id2 genes and the angiogenic phenotype of human colon carcinoma cells." Oncogene 24(43): 6533-44. Ferreira, G. B., E. van Etten, et al. (2009). "Proteome analysis demonstrates profound alterations in human dendritic cell nature by TX527, an analogue of vitamin D." Proteomics 9(14): 3752-64. Fibbi, B., G. Penna, et al. (2009). "Chronic inflammation in the pathogenesis of benign prostatic hyperplasia." Int J Androl. Fina, D., M. Sarra, et al. (2008). "Regulation of gut inflammation and th17 cell response by interleukin-21." Gastroenterology 134(4): 1038-48. Flanagan, J. N., M. V. Young, et al. (2006). "Vitamin D metabolism in human prostate cells: implications for prostate cancer chemoprevention by vitamin D." Anticancer Res 26(4A): 2567-72. Fort, M. M., A. Mozaffarian, et al. (2005). "A synthetic TLR4 antagonist has antiinflammatory effects in two murine models of inflammatory bowel disease." J Immunol 174(10): 6416-23. Froicu, M. and M. T. Cantorna (2007). "Vitamin D and the vitamin D receptor are critical for control of the innate immune response to colonic injury." BMC Immunol 8: 5. Froicu, M., V. Weaver, et al. (2003). "A crucial role for the vitamin D receptor in experimental inflammatory bowel diseases." Mol Endocrinol 17(12): 2386-92. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 119 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Fukata, M., K. S. Michelsen, et al. (2005). "Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis." Am J Physiol Gastrointest Liver Physiol 288(5): G1055-65. Gasche, C., S. Bakos, et al. (2000). "IL-10 secretion and sensitivity in normal human intestine and inflammatory bowel disease." J Clin Immunol 20(5): 362-70. Gaya, D. R., R. K. Russell, et al. (2006). "New genes in inflammatory bowel disease: lessons for complex diseases?" Lancet 367(9518): 1271-84. Geier, M. S., R. N. Butler, et al. (2007). "Inflammatory bowel disease: current insights into pathogenesis and new therapeutic options; probiotics, prebiotics and synbiotics." Int J Food Microbiol 115(1): 1-11. Gilad, L. A., T. Bresler, et al. (2005). "Regulation of vitamin D receptor expression via estrogen-induced activation of the ERK 1/2 signaling pathway in colon and breast cancer cells." J Endocrinol 185(3): 577-92. Giri, D. and M. Ittmann (2001). "Interleukin-8 is a paracrine inducer of fibroblast growth factor 2, a stromal and epithelial growth factor in benign prostatic hyperplasia." Am J Pathol 159(1): 139-47. Gombart, A. F., N. Borregaard, et al. (2005). "Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3." FASEB J 19(9): 1067-77. Grant, W. B. (2006). "Epidemiology of disease risks in relation to vitamin D insufficiency." Prog Biophys Mol Biol 92(1): 65-79. Gregori, S., M. Casorati, et al. (2001). "Regulatory T cells induced by 1 alpha,25dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance." J Immunol 167(4): 1945-53. Gregori, S., N. Giarratana, et al. (2002). "A 1alpha,25-dihydroxyvitamin D(3) analog enhances regulatory T-cells and arrests autoimmune diabetes in NOD mice." Diabetes 51(5): 1367-74. Griffin, M. D., X. Dong, et al. (2007). "Vitamin D receptor-mediated suppression of RelB in antigen presenting cells: a paradigm for ligand-augmented negative transcriptional regulation." Arch Biochem Biophys 460(2): 218-26. Griffin, M. D., W. Lutz, et al. (2001). "Dendritic cell modulation by 1alpha,25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo." Proc Natl Acad Sci U S A 98(12): 6800-5. 120 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Hackstein, H. and A. W. Thomson (2004). "Dendritic cells: emerging pharmacological targets of immunosuppressive drugs." Nat Rev Immunol 4(1): 24-34. Hagenbaugh, A., S. Sharma, et al. (1997). "Altered immune responses in interleukin 10 transgenic mice." J Exp Med 185(12): 2101-10. Hanauer, S. B., B. G. Feagan, et al. (2002). "Maintenance infliximab for Crohn's disease: the ACCENT I randomised trial." Lancet 359(9317): 1541-9. Handisurya, A., G. E. Steiner, et al. (2001). "Differential expression of interleukin-15, a proinflammatory cytokine and T-cell growth factor, and its receptor in human prostate." Prostate 49(4): 251-62. Harzstark, A. L. and C. J. Ryan (2008). "Therapies in development for castrate-resistant prostate cancer." Expert Rev Anticancer Ther 8(2): 259-68. Hashimoto, C., K. L. Hudson, et al. (1988). "The Toll gene of Drosophila, required for dorsalventral embryonic polarity, appears to encode a transmembrane protein." Cell 52(2): 269-79. Hausmann, M., S. Kiessling, et al. (2002). "Toll-like receptors 2 and 4 are up-regulated during intestinal inflammation." Gastroenterology 122(7): 1987-2000. Haussler, M. R., G. K. Whitfield, et al. (1998). "The nuclear vitamin D receptor: biological and molecular regulatory properties revealed." J Bone Miner Res 13(3): 325-49. Hippenstiel, S., S. Soeth, et al. (2000). "Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells." Blood 95(10): 3044-51. Holick, M. F. (2007). "Vitamin D deficiency." N Engl J Med 357(3): 266-81. Hollis, B. W. (2005). "Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: implications for establishing a new effective dietary intake recommendation for vitamin D." J Nutr 135(2): 317-22. Hsieh, C. S., S. E. Macatonia, et al. (1993). "Development of TH1 CD4+ T cells through IL12 produced by Listeria-induced macrophages." Science 260(5107): 547-9. Huntly, B. J. and D. G. Gilliland (2005). "Leukaemia stem cells and the evolution of cancerstem-cell research." Nat Rev Cancer 5(4): 311-21. Inohara, Chamaillard, et al. (2005). "NOD-LRR proteins: role in host-microbial interactions and inflammatory disease." Annu Rev Biochem 74: 355-83. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 121 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Ishigooka, M., S. Hayami, et al. (1996). "Relative and total volume of histological components in benign prostatic hyperplasia: relationships between histological components and clinical findings." Prostate 29(2): 77-82. Itano, A. A. and M. K. Jenkins (2003). "Antigen presentation to naive CD4 T cells in the lymph node." Nat Immunol 4(8): 733-9. Izcue, A., S. Hue, et al. (2008). "Interleukin-23 restrains regulatory T cell activity to drive T cell-dependent colitis." Immunity 28(4): 559-70. Janeway, C. A., Jr. and R. Medzhitov (2002). "Innate immune recognition." Annu Rev Immunol 20: 197-216. Jensen, P. E. (2007). "Recent advances in antigen processing and presentation." Nat Immunol 8(10): 1041-8. Jurutka, P. W., L. Bartik, et al. (2007). "Vitamin D receptor: key roles in bone mineral pathophysiology, molecular mechanism of action, and novel nutritional ligands." J Bone Miner Res 22 Suppl 2: V2-10. Kallay, E., P. Pietschmann, et al. (2001). "Characterization of a vitamin D receptor knockout mouse as a model of colorectal hyperproliferation and DNA damage." Carcinogenesis 22(9): 1429-35. Kamao, M., S. Tatematsu, et al. (2004). "C-3 epimerization of vitamin D3 metabolites and further metabolism of C-3 epimers: 25-hydroxyvitamin D3 is metabolized to 3-epi-25hydroxyvitamin D3 and subsequently metabolized through C-1alpha or C-24 hydroxylation." J Biol Chem 279(16): 15897-907. Kandler, K., R. Shaykhiev, et al. (2006). "The anti-microbial peptide LL-37 inhibits the activation of dendritic cells by TLR ligands." Int Immunol 18(12): 1729-36. Kang, P. B., A. K. Azad, et al. (2005). "The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis." J Exp Med 202(7): 987-99. Kanneganti, T. D., M. Lamkanfi, et al. (2007). "Intracellular NOD-like receptors in host defense and disease." Immunity 27(4): 549-59. Kaplan, M. H., Y. L. Sun, et al. (1996). "Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice." Nature 382(6587): 174-7. Kerman, R. H., B. Susskind, et al. (1997). "Postrenal transplant MLR hypo-responders have fewer rejections and better graft survival than MLR hyper-responders." Transplant Proc 29(1-2): 1410-1. 122 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Kong, J., Z. Zhang, et al. (2008). "Novel role of the vitamin D receptor in maintaining the integrity of the intestinal mucosal barrier." Am J Physiol Gastrointest Liver Physiol 294(1): G208-16. Korn, T., E. Bettelli, et al. (2009). "IL-17 and Th17 Cells." Annu Rev Immunol 27: 485-517. Korzenik, J. R. and D. K. Podolsky (2006). "Evolving knowledge and therapy of inflammatory bowel disease." Nat Rev Drug Discov 5(3): 197-209. Kramer, G. and M. Marberger (2006). "Could inflammation be a key component in the progression of benign prostatic hyperplasia?" Curr Opin Urol 16(1): 25-9. Krishnan, A. V., J. Moreno, et al. (2007). "Novel pathways that contribute to the antiproliferative and chemopreventive activities of calcitriol in prostate cancer." J Steroid Biochem Mol Biol 103(3-5): 694-702. Kuhn, R., J. Lohler, et al. (1993). "Interleukin-10-deficient mice develop chronic enterocolitis." Cell 75(2): 263-74. Kurosaki, T. (1997). "Molecular mechanisms in B cell antigen receptor signaling." Curr Opin Immunol 9(3): 309-18. Kutuzova, G. D., F. Sundersingh, et al. (2008). "TRPV6 is not required for 1alpha,25dihydroxyvitamin D3-induced intestinal calcium absorption in vivo." Proc Natl Acad Sci U S A 105(50): 19655-9. Lala, S., Y. Ogura, et al. (2003). "Crohn's disease and the NOD2 gene: a role for paneth cells." Gastroenterology 125(1): 47-57. Lande, R., J. Gregorio, et al. (2007). "Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide." Nature 449(7162): 564-9. Lange, S., D. S. Delbro, et al. (1996). "The role of the Lps gene in experimental ulcerative colitis in mice." APMIS 104(11): 823-33. Larriba, M. J., E. Martin-Villar, et al. (2009). "Snail2 cooperates with Snail1 in the repression of vitamin D receptor in colon cancer." Carcinogenesis 30(8): 1459-68. LeBien, T. W. and T. F. Tedder (2008). "B lymphocytes: how they develop and function." Blood 112(5): 1570-80. Lee, K. L. and D. M. Peehl (2004). "Molecular and cellular pathogenesis of benign prostatic hyperplasia." J Urol 172(5 Pt 1): 1784-91. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 123 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Lemire, J. M., D. C. Archer, et al. (1994). "1,25-Dihydroxy-24-OXO-16ene-vitamin D3, a renal metabolite of the vitamin D analog 1,25-dihydroxy-16ene-vitamin D3, exerts immunosuppressive activity equal to its parent without causing hypercalcemia in vivo." Endocrinology 135(6): 2818-21. Lin, V. K., D. Wang, et al. (2000). "Myosin heavy chain gene expression in normal and hyperplastic human prostate tissue." Prostate 44(3): 193-203. Lips, P. (1996). "Vitamin D deficiency and osteoporosis: the role of vitamin D deficiency and treatment with vitamin D and analogues in the prevention of osteoporosis-related fractures." Eur J Clin Invest 26(6): 436-42. Liu, P. T., S. Stenger, et al. (2006). "Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response." Science 311(5768): 1770-3. Liu, Y. J. (2005). "IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors." Annu Rev Immunol 23: 275-306. Lutz, M. B. and G. Schuler (2002). "Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?" Trends Immunol 23(9): 445-9. Maggi, M., C. Crescioli, et al. (2006). "Pre-clinical evidence and clinical translation of benign prostatic hyperplasia treatment by the vitamin D receptor agonist BXL-628 (Elocalcitol)." J Endocrinol Invest 29(7): 665-74. Mahon, B. D., A. Wittke, et al. (2003). "The targets of vitamin D depend on the differentiation and activation status of CD4 positive T cells." J Cell Biochem 89(5): 922-32. Mangan, P. R., L. E. Harrington, et al. (2006). "Transforming growth factor-beta induces development of the T(H)17 lineage." Nature 441(7090): 231-4. Manicassamy, S. and B. Pulendran (2009). "Modulation of adaptive immunity with Toll-like receptors." Semin Immunol. Marrack, P. and J. Kappler (2004). "Control of T cell viability." Annu Rev Immunol 22: 76587. Marrakchi, R., A. Moussa, et al. (2009). "Interleukin 10 promoter region polymorphisms in inflammatory bowel disease in Tunisian population." Inflamm Res 58(3): 155-60. Martinesi, M., C. Treves, et al. (2008). "Vitamin D derivatives induce apoptosis and downregulate ICAM-1 levels in peripheral blood mononuclear cells of inflammatory bowel disease patients." Inflamm Bowel Dis 14(5): 597-604. 124 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Mathieu, C., M. Waer, et al. (1995). "Prevention of type I diabetes in NOD mice by nonhypercalcemic doses of a new structural analog of 1,25-dihydroxyvitamin D3, KH1060." Endocrinology 136(3): 866-72. Matsumoto, S., S. Marui, et al. (1993). "A novel immunosuppressant, IR-1116, which has a different biological mechanism from that of cyclosporine A." Eur J Immunol 23(9): 2121-8. Mattner, F., S. Smiroldo, et al. (2000). "Inhibition of Th1 development and treatment of chronic-relapsing experimental allergic encephalomyelitis by a non-hypercalcemic analogue of 1,25-dihydroxyvitamin D(3)." Eur J Immunol 30(2): 498-508. McCollum, E. V., N. Simmonds, et al. (1995). "Studies on experimental rickets. XVII. The effects of diets deficient in calcium and in fat-soluble A in modifying the histological structure of the bones. 1921." Am J Epidemiol 141(4): 280-96; discussion 279. McNeal, J. (1990). "Pathology of benign prostatic hyperplasia. Insight into etiology." Urol Clin North Am 17(3): 477-86. McVary, K. T. (2006). "BPH: epidemiology and comorbidities." Am J Manag Care 12(5 Suppl): S122-8. Medzhitov, R. (2001). "Toll-like receptors and innate immunity." Nat Rev Immunol 1(2): 135-45. Medzhitov, R. (2007). "Recognition of microorganisms and activation of the immune response." Nature 449(7164): 819-26. Medzhitov, R., P. Preston-Hurlburt, et al. (1997). "A human homologue of the Drosophila Toll protein signals activation of adaptive immunity." Nature 388(6640): 394-7. Mellanby, E. (1976). "Nutrition Classics. The Lancet 1:407-12, 1919. An experimental investigation of rickets. Edward Mellanby." Nutr Rev 34(11): 338-40. Miheller, P., G. Muzes, et al. (2009). "Comparison of the effects of 1,25 dihydroxyvitamin D and 25 hydroxyvitamin D on bone pathology and disease activity in Crohn's disease patients." Inflamm Bowel Dis. Montaner, S., R. Perona, et al. (1998). "Multiple signalling pathways lead to the activation of the nuclear factor kappaB by the Rho family of GTPases." J Biol Chem 273(21): 12779-85. Moore, K. W., R. de Waal Malefyt, et al. (2001). "Interleukin-10 and the interleukin-10 receptor." Annu Rev Immunol 19: 683-765. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 125 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Morelli, A., L. Vignozzi, et al. (2007). "BXL-628, a vitamin D receptor agonist effective in benign prostatic hyperplasia treatment, prevents RhoA activation and inhibits RhoA/Rho kinase signaling in rat and human bladder." Prostate 67(3): 234-47. Mullen, A. C., F. A. High, et al. (2001). "Role of T-bet in commitment of TH1 cells before IL-12-dependent selection." Science 292(5523): 1907-10. Naderi, N., A. Farnood, et al. (2008). "Association of vitamin D receptor gene polymorphisms in Iranian patients with inflammatory bowel disease." J Gastroenterol Hepatol 23(12): 1816-22. Nagpal, S., S. Na, et al. (2005). "Noncalcemic actions of vitamin D receptor ligands." Endocr Rev 26(5): 662-87. Nagy, L. and J. W. Schwabe (2004). "Mechanism of the nuclear receptor molecular switch." Trends Biochem Sci 29(6): 317-24. Naik, S., E. J. Kelly, et al. (2001). "Absence of Toll-like receptor 4 explains endotoxin hyporesponsiveness in human intestinal epithelium." J Pediatr Gastroenterol Nutr 32(4): 449-53. Neurath, M. F., I. Fuss, et al. (1995). "Antibodies to interleukin 12 abrogate established experimental colitis in mice." J Exp Med 182(5): 1281-90. Nickel, J. C. (2008). "Inflammation and benign prostatic hyperplasia." Urol Clin North Am 35(1): 109-15; vii. Niess, J. H., F. Leithauser, et al. (2008). "Commensal gut flora drives the expansion of proinflammatory CD4 T cells in the colonic lamina propria under normal and inflammatory conditions." J Immunol 180(1): 559-68. Noguchi, E., Y. Homma, et al. (2009). "A Crohn's disease-associated NOD2 mutation suppresses transcription of human IL10 by inhibiting activity of the nuclear ribonucleoprotein hnRNP-A1." Nat Immunol 10(5): 471-9. Norman, A. W., J. Y. Zhou, et al. (1990). "Structure-function studies on analogues of 1 alpha,25-dihydroxyvitamin D3: differential effects on leukemic cell growth, differentiation, and intestinal calcium absorption." Cancer Res 50(21): 6857-64. Novac, N. and T. Heinzel (2004). "Nuclear receptors: overview and classification." Curr Drug Targets Inflamm Allergy 3(4): 335-46. Nurieva, R., X. O. Yang, et al. (2007). "Essential autocrine regulation by IL-21 in the generation of inflammatory T cells." Nature 448(7152): 480-3. 126 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References O'Garra, A. and N. Arai (2000). "The molecular basis of T helper 1 and T helper 2 cell differentiation." Trends Cell Biol 10(12): 542-50. Oeth, P. A., G. C. Parry, et al. (1994). "Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site." Mol Cell Biol 14(6): 3772-81. Ogawa, A., A. Andoh, et al. (2004). "Neutralization of interleukin-17 aggravates dextran sulfate sodium-induced colitis in mice." Clin Immunol 110(1): 55-62. Ohyama, Y., K. Ozono, et al. (1994). "Identification of a vitamin D-responsive element in the 5'-flanking region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene." J Biol Chem 269(14): 10545-50. Orholm, M., P. Munkholm, et al. (1991). "Familial occurrence of inflammatory bowel disease." N Engl J Med 324(2): 84-8. Palmer, H. G., J. M. Gonzalez-Sancho, et al. (2001). "Vitamin D(3) promotes the differentiation of colon carcinoma cells by the induction of E-cadherin and the inhibition of beta-catenin signaling." J Cell Biol 154(2): 369-87. Palmer, H. G., M. J. Larriba, et al. (2004). "The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer." Nat Med 10(9): 917-9. Panwala, C. M., J. C. Jones, et al. (1998). "A novel model of inflammatory bowel disease: mice deficient for the multiple drug resistance gene, mdr1a, spontaneously develop colitis." J Immunol 161(10): 5733-44. Pappa, H. M., R. J. Grand, et al. (2006). "Report on the vitamin D status of adult and pediatric patients with inflammatory bowel disease and its significance for bone health and disease." Inflamm Bowel Dis 12(12): 1162-74. Pedersen, A. E., E. G. Schmidt, et al. (2009). "Dexamethasone/1alpha-25-dihydroxyvitamin D3-treated dendritic cells suppress colitis in the SCID T-cell transfer model." Immunology 127(3): 354-64. Peehl, D. M. and R. G. Sellers (1997). "Induction of smooth muscle cell phenotype in cultured human prostatic stromal cells." Exp Cell Res 232(2): 208-15. Penna, G. and L. Adorini (2000). "1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation." J Immunol 164(5): 2405-11. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 127 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Penna, G., S. Amuchastegui, et al. (2006). "Treatment of experimental autoimmune prostatitis in nonobese diabetic mice by the vitamin D receptor agonist elocalcitol." J Immunol 177(12): 8504-11. Penna, G., S. Amuchastegui, et al. (2007). "Vitamin D receptor agonists in the treatment of autoimmune diseases: selective targeting of myeloid but not plasmacytoid dendritic cells." J Bone Miner Res 22 Suppl 2: V69-73. Penna, G., N. Mondaini, et al. (2007). "Seminal plasma cytokines and chemokines in prostate inflammation: interleukin-8 as a predictive biomarker in Chronic Prostatitis/Chronic Pelvic Pain Syndrome and Benign Prostatic Hyperplasia." Eur Urol 51: 524-533. Penna, G., A. Roncari, et al. (2005). "Expression of the inhibitory receptor ILT3 on dendritic cells is dispensable for induction of CD4+Foxp3+ regulatory T cells by 1,25dihydroxyvitamin D3." Blood 106(10): 3490-7. Peters, S. L., M. Schmidt, et al. (2006). "Rho kinase: a target for treating urinary bladder dysfunction?" Trends Pharmacol Sci 27(9): 492-7. Pike, J. W., L. A. Zella, et al. (2007). "Molecular actions of 1,25-dihydroxyvitamin D3 on genes involved in calcium homeostasis." J Bone Miner Res 22 Suppl 2: V16-9. Podolsky, D. K. (2002). "Inflammatory bowel disease." N Engl J Med 347(6): 417-29. Powrie, F., M. W. Leach, et al. (1994). "Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells." Immunity 1(7): 553-62. Prakash, K., G. Pirozzi, et al. (2002). "Symptomatic and asymptomatic benign prostatic hyperplasia: molecular differentiation by using microarrays." Proc Natl Acad Sci U S A 99(11): 7598-603. Provvedini, D. M., C. D. Tsoukas, et al. (1983). "1,25-dihydroxyvitamin D3 receptors in human leukocytes." Science 221(4616): 1181-3. Quack, M. and C. Carlberg (2000). "Ligand-triggered stabilization of vitamin D receptor/retinoid X receptor heterodimer conformations on DR4-type response elements." J Mol Biol 296(3): 743-56. Raff, M. C. (1973). "T and B lymphocytes and immune responses." Nature 242(5392): 19-23. Raghavan, M. and P. J. Bjorkman (1996). "Fc receptors and their interactions with immunoglobulins." Annu Rev Cell Dev Biol 12: 181-220. Ravetch, J. V. and S. Bolland (2001). "IgG Fc receptors." Annu Rev Immunol 19: 275-90. 128 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Reddy, G. S. and K. Y. Tserng (1989). "Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway." Biochemistry 28(4): 1763-9. Robey, E. and B. J. Fowlkes (1994). "Selective events in T cell development." Annu Rev Immunol 12: 675-705. Robinson-Rechavi, M., H. Escriva Garcia, et al. (2003). "The nuclear receptor superfamily." J Cell Sci 116(Pt 4): 585-6. Ropiquet, F., D. Giri, et al. (1999). "FGF7 and FGF2 are increased in benign prostatic hyperplasia and are associated with increased proliferation." J Urol 162(2): 595-9. Rumpold, H., G. Untergasser, et al. (2002). "The development of benign prostatic hyperplasia by trans-differentiation of prostatic stromal cells." Exp Gerontol 37(8-9): 1001-4. Sakaguchi, S., K. Wing, et al. (2007). "Regulatory T cells - a brief history and perspective." Eur J Immunol 37 Suppl 1: S116-23. Schatz, D. G., M. A. Oettinger, et al. (1992). "V(D)J recombination: molecular biology and regulation." Annu Rev Immunol 10: 359-83. Schauber, J., R. A. Dorschner, et al. (2007). "Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism." J Clin Invest 117(3): 803-11. Schmit, A., M. Carol, et al. (2002). "Dose-effect of interleukin-10 and its immunoregulatory role in Crohn's disease." Eur Cytokine Netw 13(3): 298-305. Schrader, M., K. M. Muller, et al. (1994). "Vitamin D3-thyroid hormone receptor heterodimer polarity directs ligand sensitivity of transactivation." Nature 370(6488): 382-6. Schrader, M., S. Nayeri, et al. (1995). "Natural vitamin D3 response elements formed by inverted palindromes: polarity-directed ligand sensitivity of vitamin D3 receptorretinoid X receptor heterodimer-mediated transactivation." Mol Cell Biol 15(3): 115461. Schwartz, G. G. (2005). "Vitamin D and the epidemiology of prostate cancer." Semin Dial 18(4): 276-89. Schwartz, R. H. (1997). "T cell clonal anergy." Curr Opin Immunol 9(3): 351-7. Schwartz, R. H., D. L. Mueller, et al. (1989). "T-cell clonal anergy." Cold Spring Harb Symp Quant Biol 54 Pt 2: 605-10. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 129 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Schwarz, B. A. and A. Bhandoola (2006). "Trafficking from the bone marrow to the thymus: a prerequisite for thymopoiesis." Immunol Rev 209: 47-57. Schwinn, M. K. and H. F. DeLuca (2007). "Differential recruitment of coactivators to the vitamin D receptor transcriptional complex by 1alpha,25-dihydroxyvitamin D3 analogs." Arch Biochem Biophys 465(2): 443-51. Segain, J. P., D. Raingeard de la Bletiere, et al. (2003). "Rho kinase blockade prevents inflammation via nuclear factor kappa B inhibition: evidence in Crohn's disease and experimental colitis." Gastroenterology 124(5): 1180-7. Sepulveda, S. E., C. J. Beltran, et al. (2008). "[Inflammatory bowel diseases: an immunological approach]." Rev Med Chil 136(3): 367-75. Shepard, L. W., M. Yang, et al. (2001). "Constitutive activation of NF-kappa B and secretion of interleukin-8 induced by the G protein-coupled receptor of Kaposi's sarcomaassociated herpesvirus involve G alpha(13) and RhoA." J Biol Chem 276(49): 4597987. Shevach, E. M., R. A. DiPaolo, et al. (2006). "The lifestyle of naturally occurring CD4+ CD25+ Foxp3+ regulatory T cells." Immunol Rev 212: 60-73. Shevach, E. M. and G. L. Stephens (2006). "The GITR-GITRL interaction: co-stimulation or contrasuppression of regulatory activity?" Nat Rev Immunol 6(8): 613-8. Shimizu, S., M. Tahara, et al. (2007). "Involvement of nuclear factor-kB activation through RhoA/Rho-kinase pathway in LPS-induced IL-8 production in human cervical stromal cells." Mol Hum Reprod 13(3): 181-7. Sicinska, W. and P. Rotkiewicz (2009). "Structural changes of vitamin D receptor induced by 20-epi-1alpha,25-(OH)2D3: an insight from a computational analysis." J Steroid Biochem Mol Biol 113(3-5): 253-8. Sigmundsdottir, H., J. Pan, et al. (2007). "DCs metabolize sunlight-induced vitamin D3 to 'program' T cell attraction to the epidermal chemokine CCL27." Nat Immunol 8(3): 285-93. Simmons, J. D., C. Mullighan, et al. (2000). "Vitamin D receptor gene polymorphism: association with Crohn's disease susceptibility." Gut 47(2): 211-4. Siu-Caldera, M. L., J. W. Clark, et al. (1996). "1alpha,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1alpha,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells." J Steroid Biochem Mol Biol 59(5-6): 405-12. 130 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Siu-Caldera, M. L., D. S. Rao, et al. (2001). "Tissue specific metabolism of 1alpha,25dihydroxy-20-epi-vitamin D3 into new metabolites with significant biological activity: studies in rat osteosarcoma cells (UMR 106 and ROS 17/2.8)." J Cell Biochem 82(4): 599-609. Siu-Caldera, M. L., H. Sekimoto, et al. (1999). "Production of 1alpha,25-dihydroxy-3-epivitamin D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed." Bone 24(5): 457-63. Smith-Garvin, J. E., G. A. Koretzky, et al. (2009). "T cell activation." Annu Rev Immunol 27: 591-619. Smith, P. D., L. E. Smythies, et al. (2001). "Intestinal macrophages lack CD14 and CD89 and consequently are down-regulated for LPS- and IgA-mediated activities." J Immunol 167(5): 2651-6. Soderholm, J. D., G. Olaison, et al. (2002). "Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's disease." Gut 50(3): 307-13. Somlyo, A. P. and A. V. Somlyo (2003). "Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase." Physiol Rev 83(4): 1325-58. Starr, T. K., S. C. Jameson, et al. (2003). "Positive and negative selection of T cells." Annu Rev Immunol 21: 139-76. Steiner, G. E., M. E. Newman, et al. (2003). "Expression and function of pro-inflammatory interleukin IL-17 and IL-17 receptor in normal, benign hyperplastic, and malignant prostate." Prostate 56(3): 171-82. Steiner, G. E., U. Stix, et al. (2003). "Cytokine expression pattern in benign prostatic hyperplasia infiltrating T cells and impact of lymphocytic infiltration on cytokine mRNA profile in prostatic tissue." Lab Invest 83(8): 1131-46. Steinman, L. (2007). "A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of T cell-mediated tissue damage." Nat Med 13(2): 139-45. Steinman, R. M. and J. Banchereau (2007). "Taking dendritic cells into medicine." Nature 449(7161): 419-26. Steinman, R. M., D. Hawiger, et al. (2003). "Tolerogenic dendritic cells." Annu Rev Immunol 21: 685-711. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 131 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Stio, M., A. G. Bonanomi, et al. (2001). "Suppressive effect of 1,25-dihydroxyvitamin D3 and its analogues EB 1089 and KH 1060 on T lymphocyte proliferation in active ulcerative colitis." Biochem Pharmacol 61(3): 365-71. Stio, M., M. Martinesi, et al. (2007). "The Vitamin D analogue TX 527 blocks NF-kappaB activation in peripheral blood mononuclear cells of patients with Crohn's disease." J Steroid Biochem Mol Biol 103(1): 51-60. Stio, M., C. Treves, et al. (2002). "Synergistic inhibitory effect of cyclosporin A and vitamin D derivatives on T-lymphocyte proliferation in active ulcerative colitis." Am J Gastroenterol 97(3): 679-89. Sugimoto, K., A. Ogawa, et al. (2008). "IL-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis." J Clin Invest 118(2): 534-44. Suzuki, M., T. Hisamatsu, et al. (2003). "Gamma interferon augments the intracellular pathway for lipopolysaccharide (LPS) recognition in human intestinal epithelial cells through coordinated up-regulation of LPS uptake and expression of the intracellular Toll-like receptor 4-MD-2 complex." Infect Immun 71(6): 3503-11. Tai, E. K., W. K. Wu, et al. (2007). "A new role for cathelicidin in ulcerative colitis in mice." Exp Biol Med (Maywood) 232(6): 799-808. Takeda, K., B. E. Clausen, et al. (1999). "Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils." Immunity 10(1): 39-49. Tang, J., R. Zhou, et al. (2009). "Calcitriol suppresses antiretinal autoimmunity through inhibitory effects on the Th17 effector response." J Immunol 182(8): 4624-32. Targan, S. R., R. L. Deem, et al. (1995). "Definition of a lamina propria T cell responsive state. Enhanced cytokine responsiveness of T cells stimulated through the CD2 pathway." J Immunol 154(2): 664-75. Theilgaard-Monch, K., B. T. Porse, et al. (2006). "Systems biology of neutrophil differentiation and immune response." Curr Opin Immunol 18(1): 54-60. Theyer, G., G. Kramer, et al. (1992). "Phenotypic characterization of infiltrating leukocytes in benign prostatic hyperplasia." Lab Invest 66(1): 96-107. Tinker, A. C. and A. V. Wallace (2006). "Selective inhibitors of inducible nitric oxide synthase: potential agents for the treatment of inflammatory diseases?" Curr Top Med Chem 6(2): 77-92. Tiwari, A. (2009). "Elocalcitol, a vitamin D3 analog for the potential treatment of benign prostatic hyperplasia, overactive bladder and male infertility." IDrugs 12(6): 381-93. 132 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Tocchini-Valentini, G., N. Rochel, et al. (2001). "Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands." Proc Natl Acad Sci U S A 98(10): 5491-6. Towers, T. L. and L. P. Freedman (1998). "Granulocyte-macrophage colony-stimulating factor gene transcription is directly repressed by the vitamin D3 receptor. Implications for allosteric influences on nuclear receptor structure and function by a DNA element." J Biol Chem 273(17): 10338-48. Towers, T. L., T. P. Staeva, et al. (1999). "A two-hit mechanism for vitamin D3-mediated transcriptional repression of the granulocyte-macrophage colony-stimulating factor gene: vitamin D receptor competes for DNA binding with NFAT1 and stabilizes cJun." Mol Cell Biol 19(6): 4191-9. Tysk, C., E. Lindberg, et al. (1988). "Ulcerative colitis and Crohn's disease in an unselected population of monozygotic and dizygotic twins. A study of heritability and the influence of smoking." Gut 29(7): 990-6. Underhill, D. M. and A. Ozinsky (2002). "Phagocytosis of microbes: complexity in action." Annu Rev Immunol 20: 825-52. Untergasser, G., S. Madersbacher, et al. (2005). "Benign prostatic hyperplasia: age-related tissue-remodeling." Exp Gerontol 40(3): 121-8. Uskokovic, M. R., P. Manchand, et al. (2006). "C-20 cyclopropyl vitamin D3 analogs." Curr Top Med Chem 6(12): 1289-96. Uskokovic, M. R., A. W. Norman, et al. (2001). "Highly active analogs of 1alpha,25dihydroxyvitamin D(3) that resist metabolism through C-24 oxidation and C-3 epimerization pathways." Steroids 66(3-5): 463-71. van Abel, M., J. G. Hoenderop, et al. (2003). "Regulation of the epithelial Ca2+ channels in small intestine as studied by quantitative mRNA detection." Am J Physiol Gastrointest Liver Physiol 285(1): G78-85. Van Cromphaut, S. J., M. Dewerchin, et al. (2001). "Duodenal calcium absorption in vitamin D receptor-knockout mice: functional and molecular aspects." Proc Natl Acad Sci U S A 98(23): 13324-9. van Etten, E. and C. Mathieu (2005). "Immunoregulation by 1,25-dihydroxyvitamin D3: basic concepts." J Steroid Biochem Mol Biol 97(1-2): 93-101. Veldman, C. M., M. T. Cantorna, et al. (2000). "Expression of 1,25-dihydroxyvitamin D(3) receptor in the immune system." Arch Biochem Biophys 374(2): 334-8. Volkman, A. and J. L. Gowans (1965). "The Origin of Macrophages from Bone Marrow in the Rat." Br J Exp Pathol 46: 62-70. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 133 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Walport, M. J. (2001). "Complement. First of two parts." N Engl J Med 344(14): 1058-66. Walport, M. J. (2001). "Complement. Second of two parts." N Engl J Med 344(15): 1140-4. Wang, T. T., F. P. Nestel, et al. (2004). "Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression." J Immunol 173(5): 2909-12. Watts, C. (2004). "The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules." Nat Immunol 5(7): 685-92. Webber, R. (2006). "Benign prostatic hyperplasia." Clin Evid(15): 1213-26. Weinberger, C., S. M. Hollenberg, et al. (1985). "Identification of human glucocorticoid receptor complementary DNA clones by epitope selection." Science 228(4700): 740-2. White, J. H. (2004). "Profiling 1,25-dihydroxyvitamin D3-regulated gene expression by microarray analysis." J Steroid Biochem Mol Biol 89-90(1-5): 239-44. Windaus A, S. F., von Werder F. (1936). "Uber das antirachitisch wirksame Bestrahlungsprodukt aus 7-Dehydrocholesterin. (Concerning the antirachitic activity of the irradiation product of 7-dehydrocholesterol.)." Hoppe-Seyler’s Z Physiol Chem 241: 3. Xavier, R. J. and D. K. Podolsky (2007). "Unravelling the pathogenesis of inflammatory bowel disease." Nature 448(7152): 427-34. Yamazaki, S., T. Iyoda, et al. (2003). "Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen-processing dendritic cells." J Exp Med 198(2): 235-47. Yang, Z. Z., A. J. Novak, et al. (2006). "Intratumoral CD4+CD25+ regulatory T-cellmediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma." Blood 107(9): 3639-46. Yasmin, R., R. M. Williams, et al. (2005). "Nuclear import of the retinoid X receptor, the vitamin D receptor, and their mutual heterodimer." J Biol Chem 280(48): 40152-60. Ylikomi, T., I. Laaksi, et al. (2002). "Antiproliferative action of vitamin D." Vitam Horm 64: 357-406. Yu, J., N. Mookherjee, et al. (2007). "Host defense peptide LL-37, in synergy with inflammatory mediator IL-1beta, augments immune responses by multiple pathways." J Immunol 179(11): 7684-91. Zenewicz, L. A., A. Antov, et al. (2009). "CD4 T-cell differentiation and inflammatory bowel disease." Trends Mol Med 15(5): 199-207. 134 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) References Zhang, Z., M. Zheng, et al. (2006). "Critical role of IL-17 receptor signaling in acute TNBSinduced colitis." Inflamm Bowel Dis 12(5): 382-8. Zhao, D., S. Kuhnt-Moore, et al. (2003). "Neurotensin stimulates IL-8 expression in human colonic epithelial cells through Rho GTPase-mediated NF-kappa B pathways." Am J Physiol Cell Physiol 284(6): C1397-404. Zheng, Y., P. A. Valdez, et al. (2008). "Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens." Nat Med 14(3): 282-9. Zhou, L., J. E. Lopes, et al. (2008). "TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function." Nature 453(7192): 236-40. Zhu, Y., B. D. Mahon, et al. (2005). "Calcium and 1 alpha,25-dihydroxyvitamin D3 target the TNF-alpha pathway to suppress experimental inflammatory bowel disease." Eur J Immunol 35(1): 217-24. Zidovetzki, R., B. Rost, et al. (1998). "Role of transmembrane domains in the functions of Band T-cell receptors." Immunol Lett 64(2-3): 97-107. Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009) 135 Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Appendix: Original publications Publication I Human Benign Prostatic Hyperplasia Stromal Cells as Inducers and Targets of Chronic Immuno-mediated Inflammation Penna G, Fibbi B, Amuchastegui S, Cossetti C, Aquilano F, Laverny G, Gacci M, Crescioli C, Maggi M, Adorini 2009 Journal of Immunology 182(7):4056-64
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