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: 8556803
• 负责人: Rachel R. Caspi
• 金额: $ 248.38万
• 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/8556803
• 关键词: Affect; Age-Months; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Antibodies; Antigens; Ascaridil; Autoimmune Diseases; Autoimmune Process; Autoimmunity; Award; Binding; Birth; Bypass; C57BL/6N Mouse; CD8B1 gene; Cells; Characteristics; Communities; Complex; Data; Development; Disease; Disease remission; Disease susceptibility; Epitopes; Equilibrium; Eragrostis; Experimental Models; Eye; Familial amyloid nephropathy with urticaria and deafness; Feedback; GRB10 gene; Genes; Genetic; Glean; Goals; Host Defense; Human; Immune; Immune response; Immunity; Immunization; Immunotherapy; In Vitro; Inflammation; Inflammatory; Infusion procedures; Institutes; Interferon Type II; Interleukin-17; Interleukin-6; Investigation; Knock-in Mouse; Knowledge; Lamina Propria; Lead; Life; Ligation; Liquid substance; Lymphocyte; Maintenance; Manuscripts; Mediating; Memory; Modeling; Molecular; Mus; Mutate; Mutation; Names; National Institute of Allergy and Infectious Disease; Natural Killer Cells; Ocular Pathology; Paris, France; Pathogenesis; Pathology; Pathway interactions; Patients; Phenotype; Play; Population; Preparation; Process; Production; Publishing; Regulatory T-Lymphocyte; Reporter; Reporting; Resistance; Resolution; Retina; Retinal; Rodent; Role; STAT3 gene; Self Tolerance; Signal Pathway; Signal Transduction; Site; Source; T-Cell Receptor; T-Lymphocyte; Therapeutic; Time; Tissues; Transgenic Mice; Tretinoin; Uveitis; Vendor; Vision; Vision research; Work; ZNF145 gene; adaptive immunity; autoimmune uveitis; bench to bedside; cell population study; cytokine; design; developmental genetics; dimer; in vivo; insight; interleukin-22; interleukin-23; novel; overexpression; polarized cell; protective effect; receptor; response; transcription factor; uveoretinitis

NEW SPONTANEOUS AND HUMANIZED MODELS OF UVEITIS: (1) Birdshot chorioretinopathy (BC) is strongly associated with HLA-A29. To study the association between HLA-A29 and uveitis, we applied to and received a Bench to Bedside award for FYs 201112. As part of the project we developed HLA-A29 transgenic mice using a construct obtained from Jacques Cohen, INSERM, Paris, who cloned the HLA-A29 gene from a BC patient. The mice developed ocular pathology resembling BC within the first few months of life. Unexpectedly, this turned out to be due to the hitherto unknown presence of the rd8 mutation of the Crb1 gene expressed by the founder mouse from the C57BL/6N strain. Extensive investigation revealed that C57BL/6N mice from all major vendors (but not C57BL/6J kept by Jax) express the rd8 mutation and have the associated phenotype. This finding has grave implications for the vision research community, as some (many?) published ocular phenotypes may have been due to this mutation (Mattapallil et. al. IOVS 2012). We are currently rederiving this strain. (2) We continue to collaborate with Drs. Warren Strober (NIAID) and Guanxun Meng (Shanghai Pasteur Institute) on the study of ocular inflammation in mice with an inflammasome mutation. These mice are knock-in for a mutated NLRP3 gene associated with Muckle-Wells syndrome, which among its pathologies is also associated with ocular inflammation. Results indicate that there are effects on cytokine responses and on pathology associated with EAU and LPS induced uveitis, models in which inflammasome responses are heavily involved. (3) We have derived three lines of T cell receptor (TCR) Tg mice that express different copy numbers of a TCR specific to the major epitope of IRBP, named (from high expression to low) R161H, R161M and R161L. R161H and to a lesser extent R161M mice develop spontaneous uveitis by 2 months of age. R161L mice develop minimal or no spontaneous disease. These mice thus represent a new model that permits to study mechanisms of spontaneous uveitis and serve as a source of retinal antigen-specific T cells, permitting for the first time to study their development and function. Using these mice, insights are being gained into mechanisms of autoimmune uveitis. (Horai et al., in preparation) FUNDAMENTAL MECHANISMS IN TOLERANCE, IMMUNITY AND AUTOIMMUNITY TO RETINAL ANTIGENS (1) Interestingly, spontaneous uveitis was ameliorated in R161H mice treated with a broad-spectrum antibiotic mixture from before birth, resulting in a drastically altered gut bacterial flora. This suggests that endogenous bacterial flora contributes to development of spontaneous ocular autoimmune disease. IL-17-producing TCR Tg R161H T cells were enriched in the lamina propria of the gut of untreated (but not antibiotic-protected) mice, and we hypothesize that they are activated there by the bacterial flora and migrate to the eye to elicit uveitis. Data (Horai et al, manuscript in preparation). We are currently attempting to dissect which component(s) of the flora is(are) responsible. (2) crossing R161H mice to IFN-g deficient or IL-17 deficient mice unexpectedly revealed a major role for IFN-g in the spontaneous disease. IFN-g-/- R161H mice, but not IL-17-/- R161H mice, had severely reduced spontaneous uveitis scores. These findings raise the possibility that IL-17 produced by R161H cells in the gut, as described above, is a marker of the pathogenic cells but not necessarily the pathogenic cytokine itself. (3) Using IRBP specific T cells from R161H mice crossed to FoxP3-GFP reporter mice we re-examined the concept of local immune privilege in vitro and in vivo. Our data show that uncommitted T cells rapidly convert in ocular fluids as well as within the living eye to functional FoxP3+ Tregs in a process involving retinal antigen recognition, de novo FoxP3 induction and proliferation. The process is in part dependent on retinoic acid within the eye, which thus fulfills a dual role: in vision and in ocular immune privilege. Importantly, only nave T cells could be converted, but effector-memory retina-specific T were resistant to conversion, and instead caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. (Zhou et al, J Immunol 2011 and Zhou et al., J. Immunol 2012). (4) Our previous work showed that autoimmunity to retina could be either Th17 or Th1 driven. Because immune responses have inherent plasticity, targeting only Th1 or only Th17 could drive the response down the other pathway while continuing to fuel pathology. We therefore sought an approach that would concurrently inhibit both. IL27p28 binds to gp130 and can block both IL-6 and IL-27 signaling, which are involved in Th17 and Th1 responses, respectively. Overexpression of IL27p28 in mice leads to protection from EAU induced by either Th1 or by Th17 cells. Furthermore, similar effects of IL-27p28 in vitro are seen on human Th1 and Th17 polarized cells. This could point to IL-27 and/or its subunits as a therapeutic mechanism in immunologically complex diseases such as uveitis. (Chong et al, submitted). (5) IL-22 is a cytokine produced by Th17 cells and is present in inflammatory sites; however, its effects on the tissue are controversial. It has been reported to have both pro-inflammatory and protective effects, depending on the site and the model. We used IL-22 and IL-22-receptor deficient mice and anti-IL-22 antibodies to examine effects of IL-22 modulation on EAU. Our data suggest that IL-22 has a local anti-inflammatory and tissue-protective role in the eye (Mattapallil et al., in preparation). (6) We examined whether T regulatory (Treg) cells found in uveitic eyes are (i) IRBP specific, (ii) functionally suppressive, and (iii) may play a role in natural resolution of disease. Using IRBP-MHC dimers as well as FoxP3 reporter and FoxP3 deleter mice, we found that the T cell infiltrate in uveitic eyes of mice that have a polyclonal T cell repertoire is highly enriched in IRBP-specific Treg and Teff cells. Unlike what has been reported for Treg in other inflammatory sites, Treg in uveitic eyes are unimpaired functionally. Finally, FoxP3+ Treg appear to play a role in the natural resolution of EAU and in the maintenance of remission. (Silver et al, in preparation) EFFECTS OF INNATE IMMUNE RESPONSES ON AUTOIMMUNITY: The innate immune response directly affects immunopathogenic processes and also impacts on adaptive immunity. (1) We previously identified a population of NKT cells that express the IL-23R constitutively and produce IL-17 independently of IL-6 and IL-21 (NKT17). Recent data indicate that IL-17 production in these cells may involve a unique signaling pathway that bypasses STAT3 under some conditions. Furthermore, have identified a novel population of non-NKT innate T cells that rapidly produce high amounts of IL-17 upon T cell receptor and IL-23 receptor ligation, similarly to NKT17. These cells lack both CD4 and CD8 expression (double negative = DNT) and express the unique PLZF transcription factor characteristic of innate T cells. We are currently studying this cell population with the goal of defining its role in host defense and tissue pathology (A. Hansen, in preparation). (2) The cytokine IFN-g can be either protective or proinflammatory in autoimmunity, which is an unsolved paradox. Our recent data suggest that there is a positive feedback loop between NK cell derived IFN-g acting on DC to produce IL-27, which in turn augments the IFN-g; response. This is in line with earlier data published by our lab (Grajewski, Hansen et al., J Immunol 2008) that innate IFN-g; production dampens subsequent adaptive IFN-g and IL-17 responses and protects from EAU (Chong et al, in preparation).

新的自发性和人性化葡萄膜炎模型： (1) 鸟弹状脉络膜视网膜病变 (BC) 与 HLA-A29 密切相关。为了研究 HLA-A29 与葡萄膜炎之间的关联，我们申请并获得了 201112 财年的 Bench to Bedside 奖。作为该项目的一部分，我们使用从巴黎 INSERM 的 Jacques Cohen 获得的构建体开发了 HLA-A29 转基因小鼠。从 BC 患者身上克隆了 HLA-A29 基因。这些小鼠在出生后的最初几个月内出现了类似 BC 的眼部病变。出乎意料的是，这是由于 C57BL/6N 品系的创始小鼠所表达的 Crb1 基因存在迄今为止未知的 rd8 突变所致。广泛的调查显示，所有主要供应商的 C57BL/6N 小鼠（但 Jax 饲养的 C57BL/6J 除外）均表达 rd8 突变并具有相关表型。这一发现对视觉研究界具有重大影响，因为一些（许多？）已发表的眼部表型可能是由于这种突变造成的（Mattapallil 等人 IOVS 2012）。我们目前正在重新衍生该菌株。 (2) 我们继续与博士合作。 Warren Strober (NIAID) 和孟冠勋（上海巴斯德研究所）对炎症小体突变小鼠的眼部炎症进行了研究。这些小鼠被敲入了与 Muckle-Wells 综合征相关的 NLRP3 突变基因，该综合征的病理学也与眼部炎症有关。结果表明，对细胞因子反应以及与 EAU 和 LPS 诱导的葡萄膜炎相关的病理学有影响，其中炎症体反应密切相关。 (3)我们衍生了三系T细胞受体(TCR) Tg小鼠，它们表达不同拷贝数的特异于IRBP主要表位的TCR，分别命名为(从高表达到低表达)R161H、R161M和R161L。 R161H 和较小程度的 R161M 小鼠在 2 个月大时出现自发性葡萄膜炎。 R161L 小鼠很少或没有自发性疾病。因此，这些小鼠代表了一种新模型，可以研究自发性葡萄膜炎的机制，并作为视网膜抗原特异性 T 细胞的来源，从而首次可以研究其发育和功能。使用这些小鼠，我们可以深入了解自身免疫性葡萄膜炎的机制。 （Horai 等人，准备中） 视网膜抗原耐受、免疫和自身免疫的基本机制 (1) 有趣的是，R161H 小鼠在出生前接受广谱抗生素混合物治疗，自发性葡萄膜炎得到改善，导致肠道细菌菌群发生显着改变。这表明内源性细菌菌群有助于自发性眼部自身免疫性疾病的发展。产生 IL-17 的 TCR Tg R161H T 细胞在未经治疗（但不受抗生素保护）的小鼠肠道固有层中富集，我们假设它们被细菌菌群激活并迁移到眼睛引发葡萄膜炎。数据（Horai 等人，手稿正在准备中）。我们目前正在尝试剖析植物区系的哪些成分负责。 (2) 将 R161H 小鼠与 IFN-g 缺陷或 IL-17 缺陷小鼠杂交，意外地揭示了 IFN-g 在自发性疾病中的主要作用。 IFN-g-/- R161H 小鼠，而非 IL-17-/- R161H 小鼠，自发性葡萄膜炎评分严重降低。这些发现提出了一种可能性，即如上所述，肠道中 R161H 细胞产生的 IL-17 是致病细胞的标记物，但不一定是致病细胞因子本身。 (3)使用来自R161H小鼠的IRBP特异性T细胞与FoxP3-GFP报告小鼠杂交，我们重新检验了体外和体内局部免疫特权的概念。我们的数据表明，未定型的 T 细胞在眼液以及活体眼睛内迅速转化为功能性 FoxP3+ Tregs，这一过程涉及视网膜抗原识别、从头 FoxP3 诱导和增殖。该过程部分依赖于眼内的视黄酸，从而发挥双重作用：视觉和眼部免疫特权。重要的是，只有幼稚 T 细胞可以被转化，但效应记忆视网膜特异性 T 细胞对转化具有抵抗力，反而引起严重的葡萄膜炎。因此，未承诺的 T 细胞可以被解除武装，但免疫特权无法保护其免受在进入眼睛之前已获得效应功能的 T 细胞的侵害。 （Zhou 等人，J.Immunol 2011；Zhou 等人，J.Immunol 2012）。 (4) 我们之前的工作表明，视网膜自身免疫可能是 Th17 或 Th1 驱动的。由于免疫反应具有固有的可塑性，因此仅针对 Th1 或仅针对 Th17 可能会沿着其他途径驱动反应，同时继续加剧病理学。因此，我们寻求一种能够同时抑制两者的方法。 IL27p28 与 gp130 结合，可以阻断分别参与 Th17 和 Th1 反应的 IL-6 和 IL-27 信号传导。小鼠中 IL27p28 的过度表达可防止 Th1 或 Th17 细胞诱导的 EAU。此外，在体外，IL-27p28 对人 Th1 和 Th17 极化细胞也有类似的作用。这可能表明 IL-27 和/或其亚基可以作为葡萄膜炎等免疫复杂疾病的治疗机制。 （Chong 等人提交）。 (5)IL-22是Th17细胞产生的细胞因子，存在于炎症部位；然而，它对组织的影响存在争议。据报道，它具有促炎和保护作用，具体取决于部位和模型。我们使用 IL-22 和 IL-22 受体缺陷小鼠和抗 IL-22 抗体来检查 IL-22 调节对 EAU 的影响。我们的数据表明，IL-22 在眼睛中具有局部抗炎和组织保护作用（Mattapallil 等人，正在准备中）。 (6) 我们检查了在葡萄膜炎眼中发现的 T 调节 (Treg) 细胞是否 (i) IRBP 特异性，(ii) 功能抑制，以及 (iii) 可能在疾病的自然消退中发挥作用。使用 IRBP-MHC 二聚体以及 FoxP3 报告小鼠和 FoxP3 删除小鼠，我们发现具有多克隆 T 细胞库的小鼠葡萄膜眼中浸润的 T 细胞高度富集 IRBP 特异性 Treg 和 Teff 细胞。与其他炎症部位 Treg 的报道不同，葡萄膜炎眼中的 Treg 功能未受损。最后，FoxP3+ Treg 似乎在 EAU 的自然消退和维持缓解中发挥作用。 （Silver 等人，正在准备中） 先天免疫反应对自身免疫的影响： 先天免疫反应直接影响免疫致病过程，也影响适应性免疫。 (1) 我们之前鉴定了一个组成型表达 IL-23R 并独立于 IL-6 和 IL-21 (NKT17) 产生 IL-17 的 NKT 细胞群。最近的数据表明，这些细胞中 IL-17 的产生可能涉及在某些条件下绕过 STAT3 的独特信号通路。此外，还发现了一种新的非 NKT 先天 T 细胞群，与 NKT17 类似，它们在 T 细胞受体和 IL-23 受体连接后快速产生大量 IL-17。这些细胞缺乏 CD4 和 CD8 表达（双阴性 = DNT），并表达先天 T 细胞特有的独特 PLZF 转录因子。我们目前正在研究该细胞群，目的是确定其在宿主防御和组织病理学中的作用（A. Hansen，正在准备中）。 (2) 细胞因子IFN-g在自身免疫中既可以起到保护作用，也可以起到促炎作用，这是一个尚未解决的悖论。我们最近的数据表明，NK 细胞衍生的 IFN-g 作用于 DC 产生 IL-27，进而增强 IFN-g；回复。这与我们实验室早期发表的数据（Grajewski、Hansen 等人，JImmunol 2008）一致，即先天性 IFN-g；产生抑制随后的适应性 IFN-g 和 IL-17 反应并防止 EAU（Chong 等人，正在准备中）。