Genetic, Cellular And Molecular Mechanisms In Autoimmune

自身免疫的遗传、细胞和分子机制

• 批准号: 7321836
• 负责人: Rachel R. Caspi
• 金额: --
• 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/7321836
• 关键词: Genetic; Cellular; Molecular; Mechanisms; Autoimmune

Cellular and molecular mechanisms involved in T cell-mediated autoimmunity against immunologically privileged retinal antigens (Ags) are being studied. The questions are aimed at elucidating the natural development and maintenance of self-tolerance to retinal Ags, and defining the processes that lead to their pathological breakdown. The goal is to use this knowledge for designing novel and rational strategies for immunotherapy. The experimental approaches utilize the model of experimental autoimmune uveoretinitis (EAU), which resembles immune-mediated uveitic diseases in humans that can lead to blindness. EAU is induced in mice and rats by immunization with retinal Ags such as IRBP, Arrestin (S-Ag), or their component peptide epitopes, or by infusion of cultured lymphocytes that recognize these Ags. The mechanisms controlling disease susceptibility and pathogenesis are being defined at the genetic, developmental, and immunological levels using cellular and molecular approaches. Novel approaches to disease regulation are devised based on these findings. As an attempt to strengthen what we believe to be deficient peripheral tolerance to retinal Ags, we are studying therapeutic induction of tolerance in adult mice by hydrodynamic IV vaccination with a 10 ?g of naked DNA plasmid encoding an IRBP fragment. It is highly effective in preventing disease, but only moderately effective in reversing it. We are currently studying the cellular mechanisms leading to the protection. Another approach to induce peripheral tolerance is infusion of autologous B cells retrovirally transduced with a uveitogenic antigen, which leads to long term protection from EAU. This can be achieved in HLA Tg mice with an epitope of S-Ag recognized by human uveitis patients, suggesting clinical relevance of this approach (4). Innate immune responses to microbes are thought to play a role as environmental triggers of autoimmunity. Because EAU induction is dependent on mycobacterial adjuvant, which provides strong innate stimuli, we studied the dependence of EAU induction on signaling through the MyD88 as well as TLR2, TLR 4 and TLR9 receptors. Unexpectedly, although MyD88 KO mice were highly resistant, TLR2, 4 and 9 single KOs and double KOs 2/4, 4/9 and 2/9 were fully susceptible and had normal responses to IRBP. In contrast, mice deficient in IL-1R (but not in IL-18), cytokines which also signal through MyD88, were resistant. Thus, absence of IL-1 signaling can by itself account for resistance of MyD88 KO mice to eau, and points to IL-1R as a potential therapeutic target for uveitis (2). We have previously shown that the threshold of susceptibility to EAU is in controlled in part by thymic derived "natural" CD4+CD25+ regulatory T cells (T-regs). We investigated the requirement for expression of the cognate Ag (IRBP) in the thymus to generate these cells. While thymic expression of IRBP was needed to generate IRBP-specific T-regs, EAU-relevant T-regs that limit disease did not need to be IRBP specific. Polyclonal T-regs found in IRBP KO mice could be activated by microbial (including innate TLR-mediated) stimuli to protect from EAU. This study also demonstrated that generation of natural T-regs to a native auto-Ag in a mouse with a full T cell repertoire requires a cognate interaction (3). Dendritic cells (DC) are important Ag presenting cells (APC) in induction of immune and autoimmune responses. We developed a new model EAU model where disease is induced with in vitro-matured, Ag pulsed DC (which can be obtained in large numbers from mice injected hydrodynamically with Flt-3L). When matured (by culture with LPS and anti-CD40), pulsed with a uveitogenic IRBP peptide and injected into naive syngeneic mice, these DC elicit ocular inflammation. Interestingly, DC-induced EAU differs from "traditional" EAU induced by immunization in terms of clinical manifestations, the type of effector response elicited and the nature of the inflammatory infiltrate in the eye (granulocytic vs. monocytic). This shows (i) that distinct forms of EAU follow exposure to the same Ag presented to the immune system in a different context, (ii) provide an alternative EAU model representing types of uveitis not well represented by the traditional EAU model, and (iii) help explain the heterogeneity of clinical uveitis in the face of recognition directed predominantly at the same Ag (retinal arrestin) (manuscript submitted). In collaboration with the Oppenheim group at NCI we continue to study the ability of auto-Ags to act as chemoattractants, a finding which may be connected to pathogenicity. Previously we demonstrated that lymphocytes and immature dendritic cells exhibit chemotactic responses to the retinal Ags S-Ag and IRBP. The chemokine receptors CXCR5 and CXCR3 mediated the chemotactic effect of IRBP, while only CXCR3 was required for the S-Ag signal. We now found that auto-Ag bind to a different region of CXCR3 than does its cognate ligand CXCL11 (manuscript submitted). We are dissecting the respective roles in EAU of IL-12 and IL-23, related heterodimeric cytokines which share the p40 chain, using mice deficient in the non-shared receptors (p35 for IL-12 and p19 for IL-23), and the respective neutralizing monoclonal antibodies. Data indicate that IL-23, not IL-12, is an obligatory cytokine for induction of EAU. This may in part be due to its ability to promote the IL-17 response, which has recently been identified as an important proinflammatory cytokine in autoimmune and inflammatory disease. Unlike the IL-12-driven IFN-?, the IL-23 driven IL-17 is an effector cytokine in EAU and its neutralization during the efferent phase of disease is therapeutic. However, the IL-17 effector T cell can be dispensable, as severe EAU follows infusion of polarized Th1 IRBP specific T cells that produce only IFN-?, whose phenotype is stable. In contrast the requirement for IL-23 is a nonredunant and may therefore transcend the known property of IL-23 to promote the IL-17 response (manuscript submitted). Ocular immune privilege, composed in part of inhibition of inflammatory processes in the eye, plays an important role in uveitis. We have previously shown that retinal glial Muller cells inhibit proliferation of T cells in an Ag-nonspecific fashion by a contact dependent mechanism. Using primary cultures of murine Muller cells, we have examined a large number of candidate molecules that might be involved in this phenomenon. Preliminary data identify thrombospondin-1, a molecule with many functions including activation of TGF-?, as partly responsible part of the suppressive effect of Muller cells. TGF-? may constitute a 2nd pathway, in part independent of TSP-1. T cells that have interacted with Muller cells become regulatory and are able to themselves inhibit activation and proliferation of fresh T cells. Within the scope of our studies on genetic susceptibility to uveitis, we have defined a number of genes that may be involved in susceptibility, using combined approaches of (i) classical genetics aided by SSLP analysis in an F2 cross of susceptible and resistant rat strains, (ii) microarray analysis of genes expressed by these strains validated by real-time PCR and gene knockout mouse strains, (iii) and in silico analysis of inbred mouse strains of varying EAU susceptibility using a SNP database. In addition, using closely spaced SSLP markers we have identified several new genetic regions associated with susceptibility, that are shared with regions reported for other autoimmune and non-autoimmune diseases, among them experimental autoimmune encephalomyelitis, arthritis and type 2 diabetes. This indicates that uveitis uses similar pathways as other autoimmune and inflammatory diseases, which may suggest use of common therapeutic approaches (manuscript submitted).

正在研究参与T细胞介导的自身免疫的细胞和分子机制针对免疫特权视网膜抗原（AGS）。这些问题旨在阐明自然发展和维持视网膜AG的自我耐受性，并定义导致其病理崩溃的过程。目的是利用这些知识来设计免疫疗法的新颖和理性策略。实验方法利用了实验性自身免疫性葡萄膜炎（EAU）的模型，该模型类似于人类中免疫介导的葡萄膜疾病，可能导致失明。通过用视网膜AG（例如IRBP，抑制蛋白（S-AG）或其成分肽表位）或通过输注识别这些AGS的培养的淋巴细胞来诱导EAU。控制疾病易感性和发病机理的机制正在使用细胞和分子方法在遗传，发育和免疫学水平上定义。根据这些发现，设计了新的疾病调节方法。 为了增强我们认为对视网膜AG的外围耐受性不足的尝试，我们正在研究成年小鼠的耐受性通过流体动力IV疫苗接种，并用10？g编码IRBP破坏的裸型DNA质粒疫苗接种。它在预防疾病方面非常有效，但仅在逆转疾病方面有效。我们目前正在研究导致保护的细胞机制。诱导外围耐受性的另一种方法是输注用葡萄膜抗原逆转转导的自体B细胞，从而导致EAU的长期保护。这可以在HLA TG小鼠中以人类葡萄膜炎患者认可的S-AG表位来实现，这表明这种方法的临床相关性（4）。 对微生物的先天免疫反应被认为是自身免疫的环境触发器的作用。由于EAU诱导取决于提供强大先天刺激的分枝杆菌佐剂，因此我们研究了EAU诱导对通过MyD88以及TLR2，TLR 4和TLR9受体的依赖性。出乎意料的是，尽管MyD88 KO小鼠具有高度抗性，但TLR2、4和9单个KOS和双KOS 2/4、4/9和2/9完全易感，并且对IRBP的反应正常。相反，缺乏IL-1R的小鼠（但在IL-18中不存在IL-18），细胞因子也通过MyD88发出信号，具有抗性。因此，没有IL-1信号传导本身可以解释MyD88 KO小鼠对EAU的耐药性，并将IL-1R视为葡萄膜炎的潜在治疗靶标（2）。 我们先前已经表明，对EAU的敏感性的阈值部分由胸腺衍生的“天然” CD4+ CD25+调节T细胞（T-REGS）控制。我们研究了对胸腺中同源Ag（IRBP）表达以产生这些细胞的要求。虽然需要IRBP的胸腺表达来产生IRBP特异性T-REG，但限制疾病的EAU与EAU相关的T-REG不需要特定于IRBP。在IRBP KO小鼠中发现的多克隆T-REG可以通过微生物（包括先天TLR介导的）刺激激活以保护EAU。这项研究还表明，在具有全T细胞库的小鼠中，天然T-Reg产生的天然自动-AG需要相关相互作用（3）。 树突状细胞（DC）是重要的AG呈现细胞（APC），以诱导免疫和自身免疫反应。我们开发了一种新的模型EAU模型，其中疾病是通过体外成熟的Ag脉冲DC诱导的（可以从大量的小鼠中以FLT-3L注射流体动力学）。当成熟（通过LPS培养和抗CD40培养）时，用葡萄剂IRBP肽脉冲并注入天真的合成小鼠时，这些DC引起了眼部炎症。有趣的是，DC诱导的EAU不同于免疫在临床表现方面引起的“传统” EA，引起的效应子反应的类型以及眼睛中炎症性浸润的性质（粒细胞与单核细胞）。 This shows (i) that distinct forms of EAU follow exposure to the same Ag presented to the immune system in a different context, (ii) provide an alternative EAU model representing types of uveitis not well represented by the traditional EAU model, and (iii) help explain the heterogeneity of clinical uveitis in the face of recognition directed predominantly at the same Ag (retinal arrestin) (manuscript submitted). 在与NCI的Oppenheim组合作的情况下，我们继续研究自动吸引力作为化学吸引剂的能力，这一发现可能与致病性有关。以前，我们证明淋巴细胞和未成熟的树突状细胞表现出对视网膜AGS S-AG和IRBP的趋化反应。趋化因子受体CXCR5和CXCR3介导了IRBP的趋化作用，而S-AG信号仅需要CXCR3。现在，我们发现自动-AG与CXCR3的其他区域结合，其同源配体CXCL11（手稿提交）。 我们正在剖析IL-12和IL-23的EAU中各自的作用，相关的异二聚体细胞因子，它们共享p40链，使用缺乏非共享受体的小鼠（IL-125的p35和p19的IL-23）和相应的中和单核抗体。数据表明，IL-23而不是IL-12是诱导EAU的强制性细胞因子。这可能部分是由于它促进IL-17反应的能力，该反应最近被确定为自身免疫性和炎症性疾病中重要的促炎细胞因子。与IL-12驱动的IFN-？不同，IL-23驱动的IL-17是EAU的效应细胞因子，其在疾病传出期间的中和具有治疗性。但是，IL-17效应T细胞可以是可分配的，因为严重的EA遵循仅产生IFN-？的极化Th1 IRBP特异性T细胞，谁的表型是稳定的。相比之下，IL-23的要求是一种非还原药，因此可以超越IL-23的已知特性以促进IL-17响应（手稿提交）。 眼部免疫特权部分是由眼睛中炎症过程抑制的一部分组成的，在葡萄膜炎中起着重要作用。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AG非特异性方式抑制T细胞的增殖。使用鼠类穆勒细胞的一级培养物，我们检查了可能参与这种现象的大量候选分子。初步数据鉴定了具有许多功能的分子，包括激活TGF-？，这是Muller细胞抑制作用的部分原因。 tgf-？可以构成第二途径，部分独立于TSP-1。与Muller细胞相互作用的T细胞成为调节性，并能够抑制新鲜T细胞的激活和增殖。 Within the scope of our studies on genetic susceptibility to uveitis, we have defined a number of genes that may be involved in susceptibility, using combined approaches of (i) classical genetics aided by SSLP analysis in an F2 cross of susceptible and resistant rat strains, (ii) microarray analysis of genes expressed by these strains validated by real-time PCR and gene knockout mouse strains, (iii) and in silico analysis of使用SNP数据库的不同EAU敏感性的近交小鼠菌株。此外，使用紧密间隔的SSLP标记，我们已经确定了与易感性相关的几个新遗传区域，这些区域与其他自身免疫性和非自动免疫性疾病报告的区域共享，其中包括实验性自身免疫性脑脊髓炎，关节炎，关节炎和2型糖尿病。这表明葡萄膜炎使用与其他自身免疫性和炎症性疾病相似的途径，这可能表明使用常见的治疗方法（手稿提交）。