Idiopathic endogenous posterior uveitis encompasses a spectrum of chronic intraocular inflammatory disorders which are thought to be autoimmune in nature. The animal model experimental autoimmune uveoretinitis (EAU) is mediated by CD4+ T-lymphocytes, and has proved invaluable in the study of the underlying immunopathogenesis of uveitis and alternative immunosuppressive therapies, for example, oral tolerance induction. This thesis describes, in a model of retinal-extract induced EAU, the effects of intranasal administration of retinal antigens prior to induction of EAU with retinal extract. The thesis has demonstrated that immunisation with emulsified retinal extract and CFA (without pertussis) induces a dose-dependent intraocular inflammation, which at high doses leads ultimately to total loss of rod photoreceptor outer segments and retinal necrosis. A course of intranasal inoculations with retinal extract (tolerance induction), prior to immunisation with antigen suppresses the histological and clinical response of EAU. Animals which were tolerised with microgram quantities of antigen showed evidence of mild inflammation of the ciliary body and inner retinal vessels (vasculitis) but no evidence of direct photoreceptor damage when compared to controls. Intranasal inoculation with retinal extract suppressed S-Ag-induced EAU but not vice versa, despite the ability of S-Ag intranasal inoculations to suppress S-Ag induced disease. Tolerised animals demonstrated normal antibody responses to S-Ag, IRBP and retinal extract, and exhibited a significantly suppressed delayed hypersensitivity response to retinal extract but normal response to a non-specific antigen, PPD. Adoptive transfer of splenocytes from tolerised animals suppressed the induction of EAU in some naive recipients. These findings suggest that active suppressor mechanisms are involved in the induction of tolerance, which concurs with other findings of CD8+ T-lymphocyte mediated suppression in oral tolerance models. In order to study the future application of 'tolerance therapy', we attempted to suppress sensitised animals by intranasal tolerance which resulted in an incomplete suppression of EAU.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

葡萄膜炎是一組複雜的眼內炎性疾病，可導致嚴重的視力損害，約占世界範圍內工作年齡人群組致盲眼病的10%。孫然治療上已取得一定的進步，但找尋安全有效的治療方式仍是一個臨床難題。基於解剖學分類，葡萄膜炎分為前葡萄膜炎、中葡萄膜炎、后葡萄膜炎和全葡萄膜炎。其中，前葡萄膜炎 (AU) 為最常見的臨床形式。此外，基於病因學葡萄膜炎也可被歸類為感染性和非感染性兩大亞型。作為一種炎症性疾病，已有研究表明許多內源性的免疫機制及遺傳因素參與葡萄膜炎的形成。 / 基於對葡萄膜炎疾病進程的深入瞭解，兩條主要潛在的致病通路：T細胞反應途徑和補體系統顯示在分子水平與疾病密切相關。本研究涉及參與上述兩通路的諸多因子，旨在調查葡萄膜炎的遺傳易感性，揭示潛在致病機理，以及發現新的臨床診斷標記物。 / 本研究共納入501名參與者，包括98名AU患者，95名非感染性中後葡萄膜炎 (NIPU) 患者，病例收集自香港眼科醫院及威爾斯親王醫院。此外，308名年齡50歲以上，排除了主要眼科疾患及系統性免疫疾病的健康人被招募為對照組。全面詳細記錄病人資料及臨床信息。進一步剖析葡萄膜炎疾病特點及補體通路參與程度，深入研究補體基因的累加效應及相互作用，以期發現臨床標記物。最後，採用基因型表型相關性分析，探索其與疾病嚴重程度和進展的關係。 / 研究1：系統性綜述在葡萄膜炎基因學研究上的最新發現，研究表明多種基因與葡萄膜炎疾病相關，所涉基因包括白介素、趨化因子、腫瘤壞死因子，以及參與補體和氧化途徑的相關基因。我們廣泛調查了葡萄膜炎的遺傳易感性。基因多態性選擇基於疾病的免疫及炎性特徵。（1）Interleukin與CFH基因，分別參與T細胞反應及補體通路。（2）CFB，CFH的拮抗因子，共同參與了補體旁路的調控。（3）調查C1INH(SERPING1) 因子，闡明補體經典通路在葡萄膜炎形成中的作用。（4）C3和C5基因，分別作為補體系統的“中心“因子及下游調控因子，其與葡萄膜炎的相關性被深入調查。 / 研究 2：首先探索性地調查免疫相關基因的單核苷酸多態性，包括CFH，KIAA1109 與 IL27 基因，我們的結果顯示 CFH 基因多態性 (rs800292，rs1065489) 與前葡萄膜炎顯著相關。更重要的是，CFH-rs1065489 TT基因型被確認為臨床標記物，此基因型攜帶者表現更高的葡萄膜炎復發頻率。此外，易感基因與HLA-B27的交互作用以及性別敏感性差異亦被發現。本研究的第二部份，此三個候選基因在另一個葡萄膜炎亞組NIPU中被進一步調查，CFH基因多態性 (rs800292，rs1065489) 與NIPU相關。KIAA1109-rs4505848也被發現與白塞氏病密切相關。此外，該基因多態性亦表現性別差異，較之對照組，顯性基因型頻率在男性NIPU組表現較高(GG/AG vs. AA)。 / 研究 3：CFB，作為CFH的拮抗因子，共同參與補體旁路的調控，其編碼基因被進一步調查。在AU研究中，位於C2/CFB區域的rs1048709被發現與其密切相關，該遺傳敏感性受HLA-B27影響。此外，我們還發現一個單體型 (AATA) 以及CFH與CFB的疊加效應均可導致AU風險度增高。同時，攜帶rs1048709(AG) 基因型患者傾向于更高程度的前房細胞數及KP比例。在NIPU研究中，類似的與CFB基因上的不同易感位點rs4151657相關性亦被檢測到。 / 研究 4, 5, 6: 在隨後的研究中，參與補體通路的其他三個候選基因 (SERPING1，C3和C5) 被進一步評估，儘管多種深入的分析方法被應用，但均未顯示出與疾病明確的相關性。 / 綜上，我們的結果首次揭示補體系統及其分子因素在葡萄膜炎進程中起著至關重要的作用。參與補體旁路調控的細胞因子 CFH 與 CFB，被確認為疾病風險因素。此外，分別參與補體經典通路或下游調控體系的其他三個候選基因 SERPING1(C1INH)，C3 和 C5， 以及參與T細胞反應通路的IL2_21區域和IL27基因在葡萄膜炎的發生發展中所起作用有限。因此，將來對於葡萄膜炎基因學及免疫學的研究應著眼于補體系統及其旁路途徑。 / Uveitis is a group of heterogeneous ocular inflammatory diseases with complex phenotypes, which causes substantial visual impairment and accounts for about 10% of blindness worldwide among the working age group. Despite considerable progress in treatment, safe and effective management is still a clinical challenge. Uveitis can be anatomically classified as anterior, intermediate, posterior, and panuveitis, anterior uveitis (AU) is the most common form. Based on etiology, uveitis can be also categorized as infectious and noninfectious subtypes. Uveitis is generally accepted as an inflammatory condition and regulated by various endogenous immunological mechanisms. Moreover, uveitis can occur in individuals with genetic predisposition coupled with environmental factors. / Based on our understanding of the critical checkpoints in the uveitis process, two major pathways, T-cell response and complement system, appear to be most related to uveitis in the molecular level. We therefore target on several molecular factors involved in the two major pathways to evaluate the genetic impact on susceptibility to uveitis, reveal potential disease mechanisms, and discover diagnostic markers. / We recruited a total of 501 unrelated Chinese individuals at the Hong Kong Eye Hospital and the Prince of Wales Hospital in Hong Kong, including 98 AU patients, 95 patients with noninfectious intermediate and posterior uveitis (NIPU), and 308 control subjects aged ≥ 50 years without major eye diseases or any systemic immune-related disorders. Clinical information and demographic conditions of the patients were documented. We moved on to depict the disease profile and estimate the contribution of each complement activation pathway in uveitis process. We also conducted interaction analysis among the complement factor genes to reveal the putative clinical markers for uveitis. Genotype-phenotype correlations were performed to explore their relationships with the disease severity and progression. / Study 1: In a systemic review to explore recent genetic findings in uveitis susceptibility, we found several genes persistently associated with uveitis and involved in various pathways. They are genes expressing interleukin, chemokine, tumor necrosis factor, and genes involved in complement and oxidation pathways. Genetic polymorphisms were selected based on the immunological and inflammatory properties of uveitis. (1) Interleukin and CFH genes, involving in the T-cell response and complement system respectively. (2) CFB, as a competitor of CFH, involved in the alternative pathway of complement cascade together. (3) Investigation of C1INH (SERPING1) in uveitis, with a view to elucidating the involvement of the classic pathway of complement cascade in uveitis development. (4) Evaluation of C3 and C5 genes in uveitis, due to their respective role of center component and downstream factor in the complement cascade. / Study 2: Genetic variations in the CFH, KIAA1109 and IL27 genes were examined. Our results showed an association between AU and CFH polymorphisms (rs800292 and rs1065489). The frequency of the CFH-rs800292 184G allele and GG homozygosity was higher in female patients than in controls. CFH-rs1065489 TT genotype was identified as a clinical marker associated with higher uveitis recurrence frequency. Interactions with HLA-B27 status in AU patients and different gender susceptibility were observed. In the second part of this study, these three candidate genes were examined in the other uveitic entity, NIPU, in our study cohort. CFH gene polymorphisms (rs1065489 and rs800292) were associated with NIPU patients. Specific association between KIAA1109-rs4505848 polymorphism and Behçet’s disease was identified. There was also gender specific genetic difference. The dominant genotype of KIAA1109-rs4505848 in male NIPU patients was significantly more frequent than in male controls (GG/AG vs. AA). / Study 3: CFB, a competitor of CFH and participated in the same complement alternative pathway, was investigated. SNP rs1048709 in the C2/CFB region was associated with AU, and this genetic influence was affected by HLA-B27 status. Furthermore, one haplotype block across CFB (AATA) significantly predisposed AU with an increased risk of 1.97 (95% CI: 1.41-2.76; Pcorr=0.0005). Joint effects of CFB-rs1048709 with (CFH-rs800292 and CFH-rs1065489) were identified to be at a risk of 7.48 and 7.0 respectively. In addition, patients carrying rs1048709 (AG) were predicted to develop a higher degree of anterior chamber cells and higher proportion of keratic precipitate (KP) during AU course. For NIPU, association with CFB was detected for a different SNP, rs4151657, in female patients only. / Studies 4, 5, and 6: Three candidate genes (SERPING1, C3 and C5) across the complement cascade were orderly evaluated in the whole study cohort of AU, NIPU and controls. They did not show any significant associations with both two uveitis entities, although multiple in-depth analyses have been performed. / Collectively, our results provide evidence for the involvement of the complement system in the disease pathogenesis of uveitis. CFH and CFB, involved in the complement alternative pathway, are identified as genetic risk factors for uveitis. Other complement pathway genes, SERPING1 (C1INH), C3 and C5, as well as IL2_21 region and IL27 involving in the T-cell response, confer either no or limited risk for the development of uveitis. Future genetic and immunologic investigations in uveitis should therefore be focused on the complement system and the alternative pathway. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yang, Mingming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 148-163). / Abstracts also in Chinese. / Title page --- p.i / Abstract --- p.iii / 摘要 --- p.vii / Acknowledgement --- p.x / Table of Contents --- p.xi / List of Tables --- p.xvi / List of Figures --- p.xx / Abbreviations --- p.xxi / Publications and Conference Presentation --- p.xxiv / Awards Received --- p.xxvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Uveitis-one of the most challenging dilemmas in ophthalmology --- p.1 / Chapter 1.1.1 --- Classification of uveitis --- p.4 / Chapter 1.1.2 --- Clinical characteristics of uveitis --- p.5 / Chapter 1.1.3 --- Epidemiology of uveitis --- p.6 / Chapter 1.1.4 --- Etiology of uveitis --- p.9 / Chapter 1.1.5 --- Current management of uveitis and future perspectives --- p.11 / Chapter 1.2 --- The Descriptive Complexity of Uveitis --- p.13 / Chapter 1.3 --- Uveitis Genetics Research Strategies --- p.14 / Chapter 1.3.1 --- Candidate gene association study --- p.14 / Chapter 1.3.2 --- The identification of new genes --- p.15 / Chapter 1.4 --- Statistical Genetics for Uveitis --- p.16 / Chapter 1.4.1 --- Hardy-Weinberg equilibrium test --- p.16 / Chapter 1.4.2 --- Univariate analysis --- p.16 / Chapter 1.4.3 --- Linkage disequilibrium --- p.17 / Chapter 1.4.4 --- Haplotype analysis --- p.18 / Chapter 1.4.5 --- Multivariable analysis --- p.19 / Chapter Chapter 2 --- Objectives --- p.21 / Chapter Chapter 3 --- General Materials and Methods --- p.23 / Chapter 3.1 --- Overall Study Design --- p.23 / Chapter 3.2 --- Research Ethics --- p.23 / Chapter 3.3 --- Study Subjects Recruitment --- p.23 / Chapter 3.4 --- Demographic and Clinical Characteristics of Patients --- p.24 / Chapter 3.4.1 --- Anterior uveitis (AU) --- p.24 / Chapter 3.4.2 --- Non-infectious intermediate and posterior uveitis (NIPU) --- p.25 / Chapter 3.5 --- General Methods --- p.26 / Chapter 3.5.1 --- Total genomic DNA extraction in study subjects --- p.26 / Chapter 3.5.2 --- Taqman SNP genotyping --- p.27 / Chapter 3.5.3 --- Nested polymerase chain reaction (nPCR) --- p.27 / Chapter 3.6 --- Statistical Analysis --- p.28 / Chapter 3.6.1 --- Hardy-Weinberg equilibrium test --- p.28 / Chapter 3.6.2 --- Individual SNP association analysis --- p.28 / Chapter 3.6.3 --- Pairwise linkage disequilibrium and haplotype analysis --- p.30 / Chapter 3.6.4 --- Genotype-phenotype correlation analysis --- p.31 / Chapter 3.6.5 --- Gene-gene interaction analysis --- p.31 / Chapter Chapter 4 --- Investigation into Genetic Determinants of Uveitis --- p.32 / Chapter 4.1 --- A Critical Review on The Roles of Genetic Factors in Uveitis --- p.32 / Chapter 4.1.1 --- Human leukocyte antigens (HLA) --- p.33 / Chapter 4.1.2 --- Interleukin (IL) genes --- p.36 / Chapter 4.1.3 --- Chemokine and chemokine receptor genes --- p.37 / Chapter 4.1.4 --- Tumor necrosis factor (TNF) genes --- p.39 / Chapter 4.1.5 --- Other genes implicated in susceptibility to uveitis --- p.40 / Chapter 4.1.6 --- Complement system --- p.42 / Chapter 4.1.7 --- Conclusions and directions --- p.43 / Chapter 4.2 --- Interleukin and CFH Polymorphisms in Uveitis --- p.49 / Chapter 4.2.1 --- Introduction --- p.49 / Chapter 4.2.2 --- Study subjects --- p.50 / Chapter 4.2.3 --- SNP selection and genotyping --- p.50 / Chapter 4.2.4 --- Statistical analysis --- p.51 / Chapter 4.2.5 --- Association of interleukin and CFH polymorphisms with AU --- p.51 / Chapter 4.2.5.1 --- Association between SNPs and AU --- p.51 / Chapter 4.2.5.2 --- Association between SNPs and AU stratified by gender --- p.52 / Chapter 4.2.5.3 --- Association between SNPs and AU stratified by HLA-B27 status --- p.52 / Chapter 4.2.5.4 --- Genotype-phenotype correlation analysis --- p.53 / Chapter 4.2.6 --- Association of interleukin and CFH polymorphisms with NIPU --- p.53 / Chapter 4.2.6.1 --- Association between SNPs and NIPU --- p.53 / Chapter 4.2.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.54 / Chapter 4.2.6.3 --- Association between SNPs and NIPU stratified by gender --- p.54 / Chapter 4.2.7 --- Discussion --- p.55 / Chapter 4.2.7.1 --- Association of interleukin and CFH polymorphisms with AU --- p.55 / Chapter 4.2.7.2 --- Association of interleukin and CFH polymorphisms with NIPU --- p.58 / Chapter 4.3 --- C2/CFB Polymorphisms in Uveitis --- p.70 / Chapter 4.3.1 --- Introduction --- p.70 / Chapter 4.3.2 --- Study subjects --- p.70 / Chapter 4.3.3 --- SNP selection and genotyping --- p.70 / Chapter 4.3.4 --- Statistical analysis --- p.71 / Chapter 4.3.5 --- Association of C2/CFB polymorphisms with AU --- p.71 / Chapter 4.3.5.1 --- Association between SNPs and AU --- p.71 / Chapter 4.3.5.2 --- Association between SNPs and AU stratified by HLA-B27 status --- p.72 / Chapter 4.3.5.3 --- Linkage disequilibrium and haplotype association analysis --- p.73 / Chapter 4.3.5.4 --- Genotype-phenotype correlation analysis --- p.73 / Chapter 4.3.5.5 --- Joint-effect analysis between CFH and CFB in AU --- p.73 / Chapter 4.3.6 --- Association of C2/CFB polymorphisms with NIPU --- p.74 / Chapter 4.3.6.1 --- Association between SNPs and NIPU --- p.74 / Chapter 4.3.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.74 / Chapter 4.3.6.3 --- Association between SNPs and NIPU stratified by gender --- p.75 / Chapter 4.3.6.4 --- Linkage disequilibrium and haplotype association analysis --- p.75 / Chapter 4.3.7 --- Discussion --- p.75 / Chapter 4.3.7.1 --- Association of C2/CFB polymorphisms with AU --- p.75 / Chapter 4.3.7.2 --- Association of C2/CFB polymorphisms with NIPU --- p.77 / Chapter 4.4 --- SERPING1 Gene Polymorphisms in Uveitis --- p.94 / Chapter 4.4.1 --- Introduction --- p.94 / Chapter 4.4.2 --- Study subjects --- p.94 / Chapter 4.4.3 --- SNP selection and genotyping --- p.95 / Chapter 4.4.4 --- Statistical analysis --- p.95 / Chapter 4.4.5 --- Association of SERPING1 polymorphisms with AU --- p.95 / Chapter 4.4.5.1 --- Association between SNPs and AU --- p.95 / Chapter 4.4.5.2 --- Association between SNPs and AU stratified by gender --- p.96 / Chapter 4.4.5.3 --- Association between SNPs and AU stratified by HLA-B27 status --- p.96 / Chapter 4.4.5.4 --- Association between SNPs and AU stratified by clinical features --- p.96 / Chapter 4.4.6 --- Association of SERPING1 polymorphisms with NIPU --- p.96 / Chapter 4.4.6.1 --- Association between SNPs and NIPU --- p.97 / Chapter 4.4.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.97 / Chapter 4.4.6.3 --- Association between SNPs and NIPU stratified by gender --- p.97 / Chapter 4.4.7 --- Discussion --- p.98 / Chapter 4.5 --- C3 Gene Polymorphisms in Uveitis --- p.109 / Chapter 4.5.1 --- Introduction --- p.109 / Chapter 4.5.2 --- Study subjects --- p.109 / Chapter 4.5.3 --- SNP selection and genotyping --- p.110 / Chapter 4.5.4 --- Statistical analysis --- p.110 / Chapter 4.5.5 --- Association of C3 polymorphisms with AU --- p.110 / Chapter 4.5.5.1 --- Association between SNPs and AU --- p.110 / Chapter 4.5.5.2 --- Association between SNPs and AU stratified by gender --- p.111 / Chapter 4.5.5.3 --- Association between SNPs and AU stratified by HLA-B27 status --- p.111 / Chapter 4.5.5.4 --- Association between SNPs and AU stratified by clinical features --- p.111 / Chapter 4.5.6 --- Association of C3 polymorphisms with NIPU --- p.111 / Chapter 4.5.6.1 --- Association between SNPs and NIPU --- p.111 / Chapter 4.5.6.2 --- Association between SNPs and NIPU stratified by subtypes and gender --- p.112 / Chapter 4.5.7 --- Discussion --- p.112 / Chapter 4.6 --- C5 Gene Polymorphisms in Uveitis --- p.126 / Chapter 4.6.1 --- Introduction --- p.126 / Chapter 4.6.2 --- Study subjects --- p.126 / Chapter 4.6.3 --- SNP selection and genotyping --- p.127 / Chapter 4.6.4 --- Statistical analysis --- p.127 / Chapter 4.6.5 --- Association of C5 polymorphisms with AU --- p.127 / Chapter 4.6.5.1 --- Association between SNPs and AU --- p.127 / Chapter 4.6.5.2 --- Association between SNPs and AU stratified by gender --- p.128 / Chapter 4.6.5.3 --- Association between SNPs and AU stratified by HLA-B27 status and clinical features --- p.128 / Chapter 4.6.6 --- Association of C5 polymorphisms with NIPU --- p.128 / Chapter 4.6.6.1 --- Association between SNPs and NIPU --- p.128 / Chapter 4.6.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.129 / Chapter 4.6.6.3 --- Association between SNPs and NIPU stratified by gender --- p.129 / Chapter 4.6.7 --- Discussion --- p.129 / Chapter Chapter 5 --- Conclusions and Future Perspectives --- p.143 / Chapter 5.1 --- General Conclusion --- p.143 / Chapter 5.2 --- Future Research in Uveitis Molecular Genetics --- p.144 / REFERENCES --- p.148

The primary aim of this research is to test the therapeutic potential of certain new generation corticosteroid drugs in order to develop safe and effective treatment for eye diseases that result in oedema, or swelling. The rising incidence of diabetes and the ageing population of developed countries mean that the prevalence of uveitis, diabetic retinopathy and age related macular degeneration will rise. Often, oedema is one of the reasons for vision loss. Corticosteroids are often used to reduce inflammation. Inflammation is one of several sources of oedema. Glucocorticoids, a class of corticosteroids that have anti-inflammatory properties, are thus used to treat ocular oedema. There is an unmet need to support clinical experience of the efficacy of steroids for ocular inflammation and oedema with more substantial scientific evidence. None of the drugs under investigation, with the exceptions of dexamethasone and triamcinolone, have been used for any ocular therapeutic purpose before. This thesis investigates “repurposing” fludrocortisone to the ophthalmic area. 11-Desoxycorticosterone (11D) and Deoxycorticosterone (DCS), other potentially valuable mineralocorticoids, remain completely untested. Lastly, Kenacort ®, or triamcinolone acetonide (TCA), is only used off-label by ophthalmologists. Methods: In the first study, corticosteroids, and especially mineralocorticoids, were investigated for their treatment efficacy in experimental uveitis, or intraocular inflammation (using a model known as endotoxin induced uveitis). In the second study, endothelial cells from choroidal blood vessels in the back of the eye were used in vitro to study whether corticosteroids reduce paracellular (between cells) permeability. Lastly, since endophthalmitis due to frequent injections is a side effect of corticosteroid use, the pharmacokinetics of different size formulations of corticosteroids were studied in an effort to find a formula that would have a prolonged dwell time within the eye.

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Un organe est dit immunologiquement privilégié lorsqu’il permet la survie d’allogreffes. Le privilège immun de l’œil a d’abord été décrit pour la chambre antérieure de l’œil puis étendu à l’espace sous-rétinien qui est délimité par les photorécepteurs et les cellules de l’épithélium pigmentaire (EPR). Vers le milieu des années nonante, Janet Liverdsidge a montré que les cellules de l’EPR étaient capables d’inhiber l’activation lymphocytaire (effet immunosuppressif des cellules de l’EPR). Parallèlement, plusieurs équipes ont montré que les uvéites postérieures non infectieuses (UPNI) étaient associées à une activation pathologique des mêmes cellules de l’EPR. Le but de notre travail a été dans un premier temps de préciser les mécanismes responsables de l’effet immunosuppressif des cellules de l’EPR et dans un deuxième temps de mieux caractériser les processus impliqués dans leur activation, afin d’imaginer de nouvelles stratégies pour traiter les UPNI. Nous avons d’abord observé que les cellules de l’EPR étaient incapables d’activer des lymphocytes T naïfs mais au contraire inhibaient la prolifération de lymphocytes T activés et induisaient leur apoptose. Nous avons corrélé cette inhibition à un phénotype de cellules présentatrices d’antigène déviantes puisque si, après stimulation par interféron γ (IFNγ), l’EPR exprimaient des molécules du CMH de classe II et du CD40, elles étaient incapables d’exprimer le B7.1 ou le B7.2, ni de sécréter de l’IL-12, même après stimulation par le CD40 ligand. Ensuite, nous avons pu mettre en évidence que les cellules de l’EPR phagocytaient les lymphocytes T et que cette phagocytose était en partie responsable de l’inhibition de la prolifération lymphocytaire. De plus, cette phagocytose était associée à une diminution de la transcription du gène de l’Il-1β induite par le TNFα. Enfin, nous avons étudié l’effet de la 15-deoxy-delta 12,14-prostaglandin J2 (15 PGJ2), un nouvel immunomodulateur potentiel, sur l’activation des cellules de l’EPR par l’IFNγ. Nous avons ainsi pu démontrer que, indépendamment du PPARγ, la 15 PGJ2 était capable d’inhiber l’induction de l’expression du CMH de class II par l’IFNγ. Parallèlement, nous avons mis en évidence que la stimulation des cellules de l’EPR par l’IFNγ induisait une activation de STAT1, ainsi que la transcription du class II transactivator (CIITA) et de SOCS1. Néanmoins, nos résultats ont aussi montré que l’effet inhibiteur de la 15PGJ2 n’était pas dépendant d’une modulation de l’activité de STAT1 ou de la transcription de SOCS1 ou du CIITA et n’impliquait pas la voie d’ERK1/2 ou de la PKB. Enfin, étant donné le rôle important du TNFα dans l’activation pathologique des cellules de l’EPR et le développement d’UPNI, nous avons analysé l’effet d’une approche thérapeutique anti-TNFα chez des patients atteints d’UPNI. Nos données ont montré que l’inhibition du TNFα par une protéine de fusion du fragment p55 du récepteur au TNFα améliorait l’état clinique de patients souffrant d’UPNI. De plus, nous avons corrélé cet effet bénéfique à une augmentation de la sécrétion intra-cytoplasmique d’IL-10 par les lymphocytes T CD4 + périphériques ainsi qu’à une augmentation du ratio entre les lymphocytes secrétant de l’IL-10 et ceux secrétant de l’IFNγ. En conclusion, nous avons pu observer que les cellules de l’EPR étaient capables d’inhiber la prolifération de lymphocytes T activés par des mécanismes de présentation déviante d’antigène, induction d’apoptose et phagocytose. Néanmoins, nos résultats ont démontré qu’à coté de ces propriétés immunosuppressives, les cellules de l’EPR pouvaient également être activées par l’IFNγ et le TNFα. Différentes approches biologiques ont été investigués pour bloquer cette activation pathologique. / Doctorat en sciences médicales / info:eu-repo/semantics/nonPublished

Immunology plays an important role in many ocular disorders. With Evolution, some major organs are able to hide from the immune system. Ocular immune privilege (OIP) can be defined as the ability to raise immune tolerance against an antigen (Ag) when this Ag is placed in specific areas of the eye. Despite the presence of OIP, RPE cells transplanted to the subretinal space (SRS) encounter immune rejection. Specifically, posterior segment autoimmune uveitis (AIU) is a sight-threatening disorder affecting the working-age population. It could be defined as the alteration of OIP that allows retinal auto-antigen recognition by the immune system. Blood-retinal barrier (BRB) breakdown plays a central role in AIU, leading to invasion of leukocytes to the eye. Animal models of experimental autoimmune uveitis (EAU) play a major place in the comprehension of AIU, with correlations to human clinic. Using anti-inflammatory gene transfer to the eye with secreted proteins, different groups significantly reduced EAU development. SOCS1, being a natural intracellular down-regulator of IFNγ pathway and interacting on other cascades, appeared to be an interesting candidate.<p><p>We herein propose to study different therapeutical paradigms for intraocular inflammation using anti-inflammatory gene transfer to the retina.<p>Transfer of immuno-modulatory genes in RPE cells prior to their transplantation into the subretinal space could be useful to reduce immune rejection. We thus compared in vitro adeno-associated viral (AAV) gene transfer to a human immortalised RPE cell-line (ARPE-19) and primary cells (hRPE), to modify their genetic properties. We investigated 3 different serotypes and promoters in vitro, before evaluating a SOCS1 gene transfer to decrease immunogenicity of ARPE-19 cells in a xenograft rat model. We showed that AAV2 efficiently transduced at least 60% of ARPE-19 and hRPE cells, by comparison with the AAV1 and 5. In dividing ARPE-19 cells, mean-fluorescent intensity of CMV-driven gene expression was higher as compared to chicken beta-actin (CAG) and tetracycline inducible (TetON) promoters, but quickly decreased with time whereas CAG was more stable. AAV2-CAG-SOCS1 infection of ARPE-19 cells significantly decreased IFNγ-induced MHC II expression. In a last experiment, we infected in vitro ARPE-19 cells, using AAV2-CAG-SOCS1, prior to their delivery into the SRS of Lewis rats, and compared it with AAV2-CAG-eGFP-infected cells or non-infected cells. Since our preliminary results were not conclusive due to technical limitations, more extended investigations are necessary.<p>In another part, we developed a clinical grading system (CGS) to efficiently score EAU development in mice fundus. Particularly, we introduced the concept of active and inactive inflammation. However, some differences between CGS and histological (HGS) grading systems were pointed out to better characterise weaknesses of each method. We thus enhanced our CGS to reduce discrepancies with HGS but will need further investigations to obtain comparable grading systems.<p>Finally, we examined in vivo effects of a SOCS1 overexpression on EAU development, following AAV2-CAG-SOCS1 intravitreal (IVit) delivery in right eyes. We first tried two different intraocular routes of injections in this inflammatory model and showed IVit delivery to be the less traumatic. Due to important animal variabilities in EAU, SOCS1 overexpression did not lead to a significant reduction of inflammation when compared to GFP as a whole. However, our design study, allowing to compare injected versus non injected eyes, furthermore revealed IVit injection side effects with pro-inflammatory reaction due to the injection of AAV2-CAG-eGFP itself. In order to reduce the impact of inter-animal variability, we standardized the data by comparing the mean of ratios of injected over non-injected eyes (I/NI) for each animal rather than absolute values. We showed a significant reduction of the clinical and histological scores of the SOCS1 group as compared to the GFP group that was even stronger in the AAV2-targeted parts of the eyes. However, we missed a saline control to corroborate using our GFP group as a control and will need to introduce in a close future some bilateral injections to validate the use of the mean of grading ratios of I/NI in our experiments. Particularly, we showed a different pattern of MHC II positive invading cells in the ciliary body between SOCS1 treated and non-treated eyes. Further investigations are necessary to confirm and characterise SOCS1 protective mechanism in EAU. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished