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).

T 细胞介导的针对免疫特权视网膜抗原 (Ag) 的自身免疫的细胞和分子机制正在研究中。这些问题旨在阐明视网膜 Ag 自我耐受的自然发展和维持，并定义导致其病理崩溃的过程。目标是利用这些知识来设计新颖且合理的免疫治疗策略。实验方法利用实验性自身免疫性葡萄膜视网膜炎（EAU）模型，该模型类似于人类免疫介导的葡萄膜炎疾病，可导致失明。在小鼠和大鼠中，通过使用视网膜抗原（例如 IRBP、Arrestin (S-Ag) 或其组成肽表位）进行免疫，或通过输注识别这些抗原的培养淋巴细胞来诱导 EAU。使用细胞和分子方法在遗传、发育和免疫学水平上定义控制疾病易感性和发病机制的机制。根据这些发现设计了疾病调控的新方法。 为了加强我们认为的外周视网膜抗原耐受性缺陷，我们正在研究通过使用 10μg 编码 IRBP 片段的裸露 DNA 质粒进行流体动力 IV 疫苗接种来治疗成年小鼠的耐受性诱导。它在预防疾病方面非常有效，但在逆转疾病方面效果有限。我们目前正在研究导致保护的细胞机制。诱导外周耐受的另一种方法是输注经逆转录病毒转导的葡萄膜抗原的自体 B 细胞，从而获得针对 EAU 的长期保护。这可以在具有人类葡萄膜炎患者识别的 S-Ag 表位的 HLA Tg 小鼠中实现，这表明该方法具有临床意义 (4)。 人们认为对微生物的先天免疫反应在自身免疫的环境触发因素中发挥着作用。由于 EAU 诱导依赖于分枝杆菌佐剂，而分枝杆菌佐剂可提供强烈的先天刺激，因此我们研究了 EAU 诱导对 MyD88 以及 TLR2、TLR 4 和 TLR9 受体信号传导的依赖性。出乎意料的是，尽管MyD88 KO小鼠具有高度耐药性，但TLR2、4和9单KO以及双KO 2/4、4/9和2/9完全敏感，并且对IRBP有正常反应。相比之下，缺乏 IL-1R（但不缺乏 IL-18）（也通过 MyD88 发出信号的细胞因子）的小鼠具有耐药性。因此，IL-1 信号传导的缺失本身就可以解释 MyD88 KO 小鼠对 eau 的耐药性，并表明 IL-1R 作为葡萄膜炎的潜在治疗靶点 (2)。 我们之前已经表明，对 EAU 的易感性阈值部分由胸腺来源的“天然”CD4+CD25+ 调节性 T 细胞 (T-reg) 控制。我们研究了胸腺中同源 Ag (IRBP) 表达的要求以产生这些细胞。虽然需要 IRBP 的胸腺表达来产生 IRBP 特异性 T 调节细胞，但限制疾病的 EAU 相关 T 调节细胞不需要是 IRBP 特异性的。在 IRBP KO 小鼠中发现的多克隆 T 调节细胞可以被微生物（包括先天 TLR 介导的）刺激激活，以防止 EAU。这项研究还表明，在具有完整 T 细胞库的小鼠中将天然 T-reg 生成为天然自身抗原需要同源相互作用 (3)。 树突状细胞 (DC) 是诱导免疫和自身免疫反应的重要抗原呈递细胞 (APC)。我们开发了一种新的 EAU 模型，其中用体外成熟的 Ag 脉冲 DC（可以从流体动力学注射 Flt-3L 的小鼠中大量获得）诱导疾病。当成熟时（通过与 LPS 和抗 CD40 一起培养），用致葡萄膜炎的 IRBP 肽进行脉冲并注射到幼稚同系小鼠中，这些 DC 会引起眼部炎症。有趣的是，DC 诱导的 EAU 在临床表现、引起的效应反应类型以及眼部炎症浸润的性质（粒细胞与单核细胞）方面与免疫诱导的“传统”EAU 不同。这表明（i）不同形式的 EAU 会在不同环境下暴露于呈递给免疫系统的相同 Ag 后，（ii）提供替代的 EAU 模型，代表传统 EAU 模型无法很好代表的葡萄膜炎类型，以及（iii） ）有助于解释临床葡萄膜炎的异质性，因为人们主要针对相同的 Ag（视网膜抑制蛋白）（已提交手稿）。 我们与 NCI 的奥本海姆小组合作，继续研究自身抗原作为化学引诱剂的能力，这一发现可能与致病性有关。之前我们证明淋巴细胞和未成熟树突状细胞对视网膜 Ags S-Ag 和 IRBP 表现出趋化反应。趋化因子受体CXCR5和CXCR3介导IRBP的趋化作用，而S-Ag信号仅需要CXCR3。我们现在发现，自身抗原与 CXCR3 的结合区域与其同源配体 CXCL11 不同（已提交手稿）。 我们正在使用缺乏非共享受体（IL-12 的 p35 和 IL-23 的 p19）的小鼠，剖析 IL-12 和 IL-23 以及共享 p40 链的相关异二聚体细胞因子在 EAU 中的各自作用，以及各自的中和单克隆抗体。数据表明，IL-23（而非 IL-12）是诱导 EAU 的必需细胞因子。这可能部分是由于其促进 IL-17 反应的能力，最近已发现 IL-17 是自身免疫和炎症性疾病中重要的促炎细胞因子。与 IL-12 驱动的 IFN-α 不同，IL-23 驱动的 IL-17 是 EAU 中的效应细胞因子，并且在疾病传出阶段中和它具有治疗作用。然而，IL-17 效应 T 细胞可能是可有可无的，因为在输注仅产生表型稳定的 IFN-γ 的极化 Th1 IRBP 特异性 T 细胞后会出现严重的 EAU。相比之下，对 IL-23 的需求是非冗余的，因此可能超越 IL-23 的已知特性来促进 IL-17 反应（已提交手稿）。 眼部免疫豁免部分由眼内炎症过程的抑制组成，在葡萄膜炎中起着重要作用。我们之前已经证明，视网膜胶质细胞 Muller 细胞通过接触依赖性机制以 Ag 非特异性方式抑制 T 细胞的增殖。使用小鼠 Muller 细胞的原代培养物，我们检查了大量可能与这种现象有关的候选分子。初步数据表明，血小板反应蛋白-1（一种具有多种功能（包括激活 TGF-β）的分子）是 Muller 细胞抑制作用的部分原因。转化生长因子-？可能构成第二条途径，部分独立于 TSP-1。与 Muller 细胞相互作用的 T 细胞变得具有调节性，并且能够自身抑制新鲜 T 细胞的激活和增殖。 在我们对葡萄膜炎遗传易感性的研究范围内，我们使用以下组合方法定义了许多可能与易感性相关的基因：(i) 经典遗传学，在易感和抗性大鼠品系的 F2 杂交中辅以 SSLP 分析， (ii) 通过实时 PCR 和基因敲除小鼠品系验证这些品系表达的基因的微阵列分析，(iii) 使用SNP 数据库。此外，利用紧密间隔的 SSLP 标记，我们已经确定了几个与易感性相关的新遗传区域，这些区域与其他自身免疫和非自身免疫疾病报道的区域共享，其中包括实验性自身免疫性脑脊髓炎、关节炎和 2 型糖尿病。这表明葡萄膜炎与其他自身免疫性疾病和炎症性疾病使用相似的途径，这可能建议使用常见的治疗方法（已提交手稿）。