Genetic, Cellular and Molecular Mechanisms in Autoimmunity to Retina

视网膜自身免疫的遗传、细胞和分子机制

• 批准号: 7734587
• 负责人: Rachel R. Caspi
• 金额: $ 290.49万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734587
• 关键词: Alleles; Antigens; Arrestin; Arrestins; Arthritis; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Bioinformatics; Blood-Retinal Barrier; C57BL/6 Mouse; CD8-Positive T-Lymphocytes; Cells; Collaborations; Computer Simulation; Condition; DNA; DNA Sequencing Facility; Data; Dendritic Cells; Dependence; Development; Disease; Disease Resistance; Disease model; Disease susceptibility; Epitopes; Equilibrium; Experimental Autoimmune Encephalomyelitis; Experimental Models; Exposure to; Eye; GRB10 gene; Gene Transfer; Genes; Genetic; Genetic Predisposition to Disease; Glean; Goals; HLA-B27 Antigen; HLA-DQ8 antigen; HLA-DR Antigens; HLA-DR2 Antigen; Hand; Haplotypes; Heterogeneity; Human; IL10 gene; Immune; Immunization; Immunotherapy; In Vitro; Individual; Inflammatory; Infusion procedures; Interferon Type II; Interleukin 2 Receptor; Interleukin-10; Interleukin-12; Interleukin-17; Interleukin-4; Interleukin-6; Knowledge; Lead; Ligation; Light; Lymphocyte; Maintenance; Manuscripts; Maps; Mediating; Methods; Microarray Analysis; Modeling; Molecular; Monoclonal Antibodies; Muller' s cell; Mus; Mutant Strains Mice; Non-Insulin-Dependent Diabetes Mellitus; Numbers; Participant; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Peptides; Peripheral; Pharmacologic Substance; Physiologic pulse; Predisposition; Preparation; Process; Pulse taking; Rattus; Reporting; Research Design; Resistance; Resolution; Retina; Retinal; Rodent; Role; Self Tolerance; Specificity; T-Cell Proliferation; T-Cell Receptor; T-Lymphocyte; Therapeutic; Thrombospondin 1; Time; Transforming Growth Factor beta; Transgenic Organisms; Uveitis; Vaccination; alpha-galactosylceramide; cytokine; developmental genetics; in vivo; incomplete Freund' s adjuvant; insight; interest; interleukin-17C; interleukin-21; interleukin-23; macrophage; novel; novel strategies; peptide analog; prevent; protective effect; receptor; response; retinal S antigen peptide M; uveoretinitis

We have examined the respective roles in EAU of the Th1 and the Th17 effector pathways in uveitis by using monoclonal antibodies and mutant mice deficient in various components of these pathways. We found that IL-23 and not IL-12 were necessary for development of EAU. Depending on the model, autoimmunity to retina could be either Th17 or Th1 driven, to the point of mediating disease as a standalone effector. That the conditions that determine which effector type will predominate appear to include the innate context in which Ag exposure occurs. The conditions that lead to a Th17 driven disease are strong TLR stimulation (CFA) and a diversity of APC, but when disease is induced in the context of a more focused stimulation, such as in vitro matured Ag pulsed dendritic cells, Th1 is dominant. These data shed light on the heterogeneity of human uveitis and may impact on therapy by suggesting that either the Th17 or the Th1 pathways may be appropriate targets in different types of human uveitis (1). We found that mouse as well as human NKT cells make a very rapid IL-17 response upon ligation of the T cell receptor (TCR) and/or the IL-23 Receptor. The two pathways are independent and additive. NKT have constitutive expression of IL-23R and RORγt, and their IL-17 response is independent of IL-6 and appears independent of IL-21. All known NKT subtypes (type I, II and III, defined by response to α−GalCer and dependence on CD1d) make IL-17. However, unlike IFN-gamma and IL-4, IL-17 is made only by the NK1.1-negative subset. The relevance, if any, of this innate IL-17 response to autoimmunity and to EAU, is unknown at this time (2). The role of NKT-produced IFN-g appears can, on the other hand, be protective. Triggering NKT in vivo by treatment with alpha-GalCer at the time of uveitogenic immunization inhibits development of EAU. This was causally related to inhibition down the line of the adaptive Th1 and Th17 responses. This contrasts with reports in some other autoimmune disease models, which connected the protective effect of NKT to induction of IL-4 and Th2 polarization. These findings are doubly interesting in view of the reported role of NKT as participants in the process of eye-induced tolerance, contributing to ocular immune privilege, and suggest that NKT cells can protect from EAU at more than one level. (3). Sequestration of retinal Ags behind the blood-retinal barrier hinders peripheral tolerance and renders the individual susceptible to EAU. Exposure to IRBP in the periphery under tolerogenic conditions, by "vaccination for tolerance" can prevent and to a lesser extent can reverse EAU. In the previous review year we used naked DNA vaccination. In this review year we studied the tolerogenic effect of inhibitory peptide analogs administered in incomplete Freund's adjuvant. Characterization of the mechanisms revealed involvement of T regulatory cells (Tregs) and Th2 skewing. Tolerogenic vaccination, or transfer of in vitro expanded Treg cells, may offer a new approach to Ag-specific therapy of uveitis. (4) L-10 is a known regulatory cytokine that our previous studies connected to resolution of EAU as well as to genetic resistance to the disease. We now show that transgenic IL-10 expression in macrophages, and to a lesser extent in T cells, protects from EAU at several levels. It inhibits priming of new uveitogenic effector T cells from naïve precursors, and also inhibits the function of already mature effectors. These data suggest that increasing endogenous expression of IL-10 by gene transfer could be developed as a therapeutic approach. (5). We were able to identify several new pathogenic peptides for the C57BL/6 (H-2b) and B10.RIII (H-2r) strains. Each peptide elicited responses of both CD4 and CD8 cells. Some of these represent specificities recognized only by IRBP KO but not WT mice (i.e., become reduced or eliminated through central tolerance in WT mice of the respective strains). C57BL/6 mice recognized more KO-only epitopes, suggesting more extensive repertoire culling in this strain. These findings will be useful for immunological studies that require knowledge of multiple epitopes and will help to understand the pathogenic potential of IRBP as an auto-antigen (6). Within the scope of our studies on genetic susceptibility to uveitis in the rat EAU model using an F2 cross of EAU-susceptible and resistant strains, we have defined a number of genes that may be involved in susceptibility. The methods use combined approaches of classical genetics, microarray analysis of expressed genes and in silico analysis (the latter in collaboration with Roche Pharmaceuticals). Some susceptibility regions are unique, and could be mapped to specific genes using microarray and in silico approaches, and some are shared with other autoimmune and non-autoimmune diseases, among them EAE, arthritis and type 2 diabetes. This indicates that uveitis shares common pathways with other autoimmune and inflammatory diseases, and may suggest use of common therapeutic approaches (7, 8). Immunological responses to S-Ag have been implicated in human uveitis, however, direct study of uveitogenic epitopes in humans is not possible. We have established a "humanized" EAU model in HLA Tg mice. Using bioinformatic methods for epitope prediction we are studying recognition and pathogenicity of S-Ag epitopes, and in some cases identifying their core sequences We have identified permissive HLA-DR3(0301), HLA-DR4(0402), HLA-DQ8 and non-permissive HLA-DR4(0401), HLA-DR2 (*1501, *1502, and *1503) alleles of the HLA-DR and DQ genes, and characterized some of the allele-specific uveitogenic epitopes of human S-Ag. Importantly, the sequences of these epitopes overlap with peptide M and peptide N of S-Ag, and the S-Ag crossreactive B27PD peptide derived from HLA-B27 molecule, that were reported to be recognized by lymphocytes of uveitis patients. We are currently developing MHC tetramers to study lymphocytes with T cell receptors specific to these epitopes in the HLA Tg mice and in uveitis patients. These data will provide new insights into the pathogenesis if uveitis and may help to develop therapeutic strategies tailored to the specific HLA haplotype of the patient. (Mattapallil et al., manuscript in preparation). We continue to examine the effect of retinal glial Müller cells on uveitogenic T lymphocytes. We have previously shown that retinal glial Muller cells inhibit proliferation of T cells in an Ag-independent fashion by a contact dependent mechanism. Using primary cultures of murine Muller cells and genetically manipulated mice, we have identified thrombospondin-1 and TGF-beta as partly responsible for the suppressive effect of Muller cells. T cells that have interacted with Muller cells downregulate any preexisting expression of IL-2 receptor and FoxP3. We are currently studying other functional consequences on the T cells that came in contact with Müller cells. (Cortes et al., manuscript in preparation).

我们通过使用单克隆抗体和突变小鼠在这些途径的各种成分中使用的单克隆抗体和突变小鼠，研究了TH1和Th17效应子途径中各自的作用。我们发现IL-23而不是IL-12对于开发EAU是必需的。根据模型，视网膜自身免疫性可以是Th17或Th1驱动的，以介导疾病作为独立效应子。确定哪种效应类型将占主导地位的条件似乎包括发生AG暴露的先天环境。导致Th17驱动疾病的条件是强烈的TLR刺激（CFA）和APC的多样性，但是当疾病在更为集中的刺激的背景下诱导疾病时，例如体外成熟的Ag脉冲树突状细胞时，TH1是主导的。这些数据阐明了人类葡萄膜炎的异质性，并可能通过暗示TH17或TH1途径可能是不同类型的人类葡萄膜炎的适当靶标（1），可能会影响治疗。 我们发现，在T细胞受体（TCR）和/或IL-23受体连接后，小鼠和人NKT细胞产生了非常快速的IL-17反应。这两个途径是独立的和加法的。 NKT具有IL-23R和RORγT的组成型表达，其IL-17响应与IL-6无关，并且与IL-21无关。所有已知的NKT亚型（I型，II和III，由对α-级别的响应和对CD1D的依赖性的响应定义）使IL-17成为IL-17。但是，与IFN-gamma和IL-4不同，IL-17仅由NK1.1阴性子集制成。目前尚不清楚这种先天IL-17对自身免疫性和EAU的响应的相关性（如果有的话）（2）。 另一方面，出现NKT生产的IFN-G的作用可能是保护性的。在葡萄球菌免疫时用α-galcer治疗触发NKT在体内触发NKT会抑制EAU的发展。这与抑制自适应Th1和Th17反应的抑制作用有关。这与其他一些自身免疫性疾病模型中的报道形成鲜明对比，后者将NKT的保护作用与IL-4和Th2极化的诱导相关联。鉴于NKT在眼睛引起的耐受性过程中报道的作用，有助于眼部免疫特权，并暗示NKT细胞可以在多个级别的多个级别上保护EAU，因此这些发现非常有趣。 （3）。 血液视网膜屏障后面的视网膜AG的隔离阻碍外围耐受性，并使个人容易受到EAU的影响。通过“耐受性疫苗接种”可以预防，并且在较小程度上可以逆转EAU。在上一个审查年中，我们使用了裸体DNA疫苗接种。在本审查年中，我们研究了在不完整的Freund辅助剂中施用的抑制性肽类似物的耐受作用。机制的表征揭示了T调节细胞（TREG）和TH2偏斜的参与。 耐受性疫苗接种或体外扩张的Treg细胞转移，可能为葡萄膜炎的Ag特异性治疗提供了一种新方法。 （4） L-10是一种已知的调节细胞因子，我们以前的研究与EAU的分辨率以及对疾病的遗传性有关。现在，我们表明巨噬细胞中的转基因IL-10表达在T细胞中较小程度上可以在多个级别保护EAU。它抑制从幼稚前体启动新的葡萄构效应T细胞，并抑制已经成熟效应子的功能。这些数据表明，通过基因转移增加IL-10的内源性表达可以作为治疗方法开发。 （5）。 我们能够鉴定C57BL/6（H-2B）和B10.RIII（H-2R）菌株的几种新的致病肽。 每个肽都会引起CD4和CD8细胞的反应。其中一些代表了仅由IRBP KO识别的特异性，而不是WT小鼠（即，通过在各自菌株的WT小鼠中通过中央耐受性降低或消除）。 C57BL/6小鼠识别出更多仅KO的表位，这表明在这种菌株中更广泛的曲目cull。 这些发现对于需要了解多个表位的免疫学研究很有用，并将有助于理解IRBP作为自动抗原的致病潜力（6）。 在我们使用EAU易感和抗性菌株的F2交叉中，我们对大鼠EAU模型中葡萄膜炎的遗传敏感性的研究范围内，我们定义了许多可能涉及易感性的基因。该方法使用经典遗传学的联合方法，表达基因的微阵列分析和计算机分析（后者与Roche Pharmaceuticals合作）。一些易感区域是独特的，可以使用微阵列和计算机方法映射到特定基因，其中一些与其他自身免疫性和非自动免疫性疾病共享，其中包括EAE，关节炎，关节炎和2型糖尿病。这表明葡萄膜炎与其他自身免疫性和炎症性疾病共同途径，并可能建议使用常见的治疗方法（7，8）。 对S-AG的免疫反应已与人类葡萄膜炎有关，但是，对人类的葡萄膜源性表位的直接研究是不可能的。我们已经在HLA TG小鼠中建立了一个“人性化” EAU模型。使用生物信息学预测的生物信息学方法，我们正在研究S-AG表位的识别和致病性，在某些情况下，我们已经确定了我们已经确定的核心序列的HLA-DR3（0301），HLA-DR4，HLA-DR4（0402），HLA-DQ8，HLA-DQ8，HLA-DQ8和非渗透性HLA-HLA-HLA-DRAD4（HLA-DRA2） *1502和 *1503）HLA-DR和DQ基因的等位基因，并表征了人类S-AG的某些等位基因特异性的葡萄象作用表位。重要的是，这些表位的序列与S-AG的肽M和肽N的序列重叠，以及源自HLA-B27分子的S-AG交叉反应性B27PD肽，这些肽据报道被葡萄炎患者的淋巴细胞识别。我们目前正在开发MHC四聚体以研究HLA TG小鼠和葡萄膜炎患者中特有这些表位的T细胞受体的淋巴细胞。如果葡萄膜炎，这些数据将为发病机理提供新的见解，并可能有助于开发针对患者特定HLA单倍型量身定制的治疗策略。 （Mattapallil等人，准备中的手稿）。 我们继续研究视网膜神经胶质细胞对葡萄膜造成T淋巴细胞的影响。我们先前已经表明，视网膜神经胶质细胞通过接触依赖机制以非依赖性的方式抑制T细胞的增殖。我们使用鼠毛细胞的原代培养物和遗传操纵小鼠，我们已经确定了血小板蛋白-1和TGF-β是部分原因，部分原因是穆勒细胞的抑制作用。与Muller细胞相互作用的T细胞下调了IL-2受体和FOXP3的任何先前表达。我们目前正在研究与Müller细胞接触的T细胞的其他功能后果。 （Cortes等人，准备中的手稿）。

项目成果

Fas/Fas ligand-associated apoptosis in experimental autoimmune uveoretinitis in rodents: role of proinflammatory corticotropin-releasing hormone.

啮齿动物实验性自身免疫性葡萄膜视网膜炎中 Fas/Fas 配体相关细胞凋亡：促炎性促肾上腺皮质激素释放激素的作用。

• DOI: 10.1006/exer.2001.0992
• 发表时间: 2001
• 期刊: Experimental eye research
• 影响因子: 3.4
• 作者: Poulaki,V;Mitsiades,N;Mastorakos,G;Caspi,RR;Chrousos,GP;Bouzas,E
• 通讯作者: Bouzas,E

Rodent models of experimental autoimmune uveitis.

• DOI: 10.1007/978-1-60761-720-4_22
• 发表时间: 2012
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Agarwal, Rajeev K;Silver, Phyllis B;Caspi, Rachel R
• 通讯作者: Caspi, Rachel R

Genetic control of susceptibility in clinical and experimental uveitis.

• DOI: 10.1080/08830180212059
• 发表时间: 2002-03-01
• 期刊: International reviews of immunology
• 影响因子: 5
• 作者: Pennesi, Giuseppina;Caspi, Rachel R
• 通讯作者: Caspi, Rachel R

Ocular autoimmunity: the price of privilege?

• DOI: 10.1111/j.1600-065x.2006.00439.x
• 发表时间: 2006-10-01
• 期刊: IMMUNOLOGICAL REVIEWS
• 影响因子: 8.7
• 作者: Caspi, Rachel R.

Genetic factors involved in central nervous system/immune interactions.

遗传因素涉及中枢神经系统/免疫相互作用。

• DOI: 10.1007/0-306-47611-8_7
• 发表时间: 2001
• 期刊: Advances in experimental medicine and biology
• 作者: Wilder,RL;Griffiths,MM;Cannon,GW;Caspi,R;Gulko,PS;Remmers,EF
• 通讯作者: Remmers,EF