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: 10706090
• 负责人: Rachel R. Caspi
• 金额: $ 393.82万
• 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/10706090
• 关键词: Adopted; Affect; Agonist; Animal Model; Anti-Inflammatory Agents; Antibiotics; Arrestins; Aryl Hydrocarbon Receptor; Autoimmune Responses; Autoimmunity; Bioinformatics; Brain; Bruch' s basal membrane structure; COVID-19; COVID-19 patient; Cataract; Cells; Central Nervous System Diseases; Characteristics; Choroid; Clinic; Clinical; Collaborations; Corynebacterium; Crystalline Lens; Data; Development; Disease; Engineering; Equilibrium; Experimental Autoimmune Encephalomyelitis; Experimental Models; Extracellular Matrix; Eye; FOXP3 gene; Genes; Genetic; Germ-Free; Goals; Growth Factor; Homeostasis; Host Defense; Human; Immune; Immune response; Immunity; Immunization; Immunologics; Immunology; Immunotherapy; In Vitro; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Interleukin-1; Interleukin-17; Intervention; Intestines; Invaded; Knock-in Mouse; Laboratories; Lead; Leg; Legal; Leukocytes; Licensing; Literature; Lung; Lymphocyte; Lymphocyte Depletion; Measurable; Microglia; Molecular; Molecular Mimicry; Morbidity - disease rate; Mucosal Immune Responses; Mucous Membrane; Mus; Mutation; Nasopharynx; National Institute of Allergy and Infectious Disease; Neurons; Oral cavity; Organism; Participant; Pathogenesis; Pathogenicity; Patients; Peptides; Peripheral; Play; Predisposition; Preparation; Process; Publishing; Regulatory T-Lymphocyte; Retina; Role; Site; Spleen; Stimulus; Surface; T cell response; T-Cell Receptor; T-Lymphocyte; TLR2 gene; Therapeutic; Tissues; Transgenic Organisms; Tretinoin; United States National Institutes of Health; United States National Library of Medicine; Uveitis; Vagina; Virus; Vision; Vitamin A; Vitamins; Washington; autoimmune uveitis; autoinflammatory; clinical material; commensal microbes; cytokine; cytokine release syndrome; dietary; effector T cell; gain of function mutation; genome sequencing; gut microbes; gut microbiome; gut microbiota; human microbiota; humanized mouse; immune activation; interstitial retinol-binding protein; intraepithelial; lens capsule; microbial; microbiome; microbiota; mortality; mouse model; mucosal site; mutant; neuroinflammation; ocular surface; pathobiont; preservation; protective effect; receptor; reconstitution; resident commensals; response; sensor; transcriptome; whole genome

IMMUNITY AND AUTOIMMUNITY INVOLVING THE NEUORORETINA AND THE ROLE OF THE GUT MICROBIOME For most of these studies we utilize mouse models of autoimmune uveitis developed in our laboratory, (i) experimental autoimmune uveitis (EAU) induced in mice by immunization with the retinal antigen (Ag) IRBP, and (ii) spontaneous uveitis in R161H mice engineered to express a transgenic T cell receptor (TCR) specific for IRBP peptide 161-180. 1) Vitamin (VitA) derivatives are necessary for functional activation of immune cells (published literature). We previously demonstrated the importance of Vitamin A (VitA) and its metabolite, retinoic acid, in ocular immune privilege. Using mice made VitA deficient (VAD), we found that T cell effector function that was acquired before onset of VAD, is maintained in the VAD host. These findings may have clinical implications in regions where dietary VitA is limiting. (Horai, Zhou et al, in preparation). 2) Laquinimod is an aryl hydrocarbon receptor (AHR) agonist. We found that it has strong protective effects in the induced model of EAU by modulating multiple inflammatory and anti-inflammatory cytokines, and may affect composition of gut flora, which in turn affects development of disease. (Xu et al, in preparation) 3) "Licensing" of T cells for pathogenicity: It has been demonstrated that activated T cells specific to retina or brain infused into a new host first settle in peripheral tissues (spleen, lung) where they become "licensed" to invade the target tissue. We are currently examining whether the gut, which affects uveitis via its resident commensal flora, can serve as a licensing site that might be important in pathogenesis (Salvador et al., in preparation). 4) The role of IL-34 and microglia in EAU: IL-34 is a cytokine produced by neuronal tissue, which is a necessary growth factor for microglia, an immune-like call resident in the retina Preliminary data indicate that IL-34 depletion in the retina has a dampening effect on intraocular inflammation, suggesting that microglial cells may be active participants in the inflammatory process. (Peng, Mattapallil et al., in preparation) 5) Ocular immune privilege: We have previously shown that nave retina-specific T cells in the eye are primed locally, and after 7 days up to 50% convert into Foxp3+ T-regulatory cells (Tregs). We are currently examining the transcriptome of eye-induced Tregs and how it differs from Tregs primed at other tissue sites or in vitro, (Peng et al., in preparation). In collaboration with the Lin laboratory at Cleveland Clinic, we demonstrated that during uveitis, inflammatory cells are able to transmigrate between the choroid and retina through the RPE cell layer, which was not thought to occur, due to the presence of Bruchs membrane (ref.5). 6) Commensal flora is necessary for development of spontaneous uveitis, in part due to molecular mimicry of IRBP161-180. However, innate mechanisms also play a role, as evidenced by the finding that antibiotics (ABX) starting a week before immunization for EAU (where the antigenic stimulus is provided), afforded measurable protection. That protection, however, was temporary and disappeared on prolonged ABX. Results suggest that prolonged ABX depletes intraepithelial lymphocytes (IEL), which are known to be microbiota-dependent, We hypothesize that IEL may have an inhibitory role in uveitis, an effect which is reversed after long-term ABX due to IEL depletion. (Salvador R et al., in preparation). 7) Human gut flora and uveitis: We reconstituted germ free R161H mice with gut microbes from 3 healthy human donors and established 3 humanized flora mouse lines Results showed that human gut flora supports development of spontaneous uveitis. Mice adopted only a subset of the human flora, but preserved the microbial footprint of the donor. We are currently identifying microbial taxa associated with low and high disease scores, with the aim of developing interventional strategies as an approach to regulating uveitis through directed manipulation of the microbiome (Horai, Zhou, Murphy et al, in preparation). 8) in collaboration with Drs. Sue Menko (Thomas Jefferson Univ) and Mary Ann Stepp (George Washington Univ) we uncovered that during uveitis immune cells invade the lens of the eye through the extracellular matrix of the lens capsule. The results may help explain inflammation-induced cataract formation (ref.2) 9) Examined the consequence of the Alpk1 mutation on susceptibility to EAU, since this mutation results in autoinflammatory disease, including uveitis, in humans. Demonstrated that the human mutant alpk1 knock-in mice, otherwise unperturbed, do not develop spontaneous ocular inflammation (ref.3) 10) In collaboration with the Egwuagu lab at the NEI, LI, demonstrated that IL-27-producing B-1a cells (Bregs) suppress EAU and EAE, indicating a role in neuroinflammation and CNS disease in a broader sense (ref.1). MUCOSAL IMMUNE RESPONSES TO COMMENSALS AT THE OCULAR SURFACE Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but presence of ocular surface microbiome was contentious. Previously, we isolated and purified a candidate ocular commensal, Corynebacterium mastitidis (C. mast). This organism elicits a commensal-specific IL-17 response from T cells in the ocular mucosa, tuning local host defense to afford protection from infectious pathogenic organisms. 1) We are examining the molecular sensors of C. mast in T cells. Data suggest that Vg4 T cells respond to C. mast mainly via their TCR, whereas Vg6 T cells respond through innate receptors such as TLR2, and are highly dependent on IL-1. TLR2 appears to be required not only in DC (where it contributes to IL-1 induction), but also in T cells. The mechanism of intrinsic T cell requirement for TLR2 is being investigated (Zhu & Xu et al. and Xu et al., in preparation). 2) We also examined commensal-elicited ocular surface responses in an immunologically abnormal host, mouse as well as human. In collaboration with the group of Dr. Warren Strober (NIAID) who developed knock-in mice expressing a gain-of-function mutation in the NLRP3 inflammasome gene, and Dr. Raphaela Goldbach-Mansky who treats patients with NLRP3 inflammasome related diseases, we obtained evidence to suggest that an ocular surface commensal can elicit ocular surface inflammation in an immunologically perturbed host, thus acting as a pathobiont. We are characterizing the local ocular immune response and the ocular surface microbiome in the mouse model and in patients with NLRP3 inflammasome mutations at the single-cell level (Siak, Mattapallil, St. Leger et al., in preparation). This will have implications for understanding and treatment of the characteristic ocular inflammation in patients with NLRP3-related diseases. 3) We performed whole genome sequencing, assembly and annotation of C. mast. https://dataview.ncbi.nlm.nih.gov/object/PRJNA758739?reviewer=dhmcn2fm0ngfj6bkh5jsq3u4d5 (Nagarajan et al., (ref4). COVID-19 related: EFFECTS OF THE MICROBIOME ON CYTOKINE STORM IN A "HUMANIZED" MOUSE MODEL. COVID-19 patients' morbidity and mortality is being attributed largely to elicitation of a cytokine storm due to an excessive immune response to the virus. The project uses immunodeficient NSG mice reconstituted with human leukocytes developed by Dr. Richard Flavell in collaboration with Regeneron, Inc. to study the effects of intestinal microbiome on cytokine storm. This project is currently stalled due to the inability of the NIH tech transfer to overcome legal barriers to obtaining these mice.

涉及神经视网膜的免疫和自身免疫以及肠道微生物组的作用 对于大多数这些研究，我们利用我们实验室开发的自身免疫性葡萄膜炎小鼠模型，(i) 通过视网膜抗原 (Ag) IRBP 免疫在小鼠中诱导实验性自身免疫性葡萄膜炎 (EAU)，以及 (ii) 工程化 R161H 小鼠中的自发性葡萄膜炎表达对 IRBP 肽 161-180 具有特异性的转基因 T 细胞受体 (TCR)。 1) 维生素（VitA）衍生物是免疫细胞功能激活所必需的（已发表文献）。我们之前证明了维生素 A (VitA) 及其代谢物视黄酸在眼部免疫豁免中的重要性。使用 VitA 缺乏 (VAD) 小鼠，我们发现 VAD 发生前获得的 T 细胞效应功能在 VAD 宿主中得以维持。这些发现可能对膳食维生素 A 限制的地区具有临床意义。 （Horai、Zhou 等人，正在准备中）。 2) 拉喹莫德是一种芳烃受体 (AHR) 激动剂。我们发现它通过调节多种炎症和抗炎细胞因子在EAU诱导模型中具有很强的保护作用，并可能影响肠道菌群的组成，进而影响疾病的发展。 （徐等人，准备中） 3）T细胞致病性的“许可”：已经证明，注入新宿主的视网膜或大脑特异性的活化T细胞首先定居在周围组织（脾脏、肺）中，在那里它们被“许可”侵入目标组织。我们目前正在研究通过其常驻共生菌群影响葡萄膜炎的肠道是否可以作为在发病机制中可能重要的许可位点（萨尔瓦多等人，正在准备中）。 4）IL-34和小胶质细胞在EAU中的作用：IL-34是神经元组织产生的细胞因子，是小胶质细胞必需的生长因子，小胶质细胞是视网膜中的免疫样细胞驻留物。初步数据表明，IL-34耗竭视网膜中的小胶质细胞对眼内炎症具有抑制作用，表明小胶质细胞可能是炎症过程的积极参与者。 （Peng、Mattapallil 等人，准备中） 5) 眼部免疫豁免：我们之前已经证明，眼睛中的视网膜特异性 T 细胞在局部启动，7 天后，高达 50% 的细胞转化为 Foxp3+ T 调节细胞 (Treg)。我们目前正在研究眼睛诱导的 Tregs 的转录组，以及它与在其他组织部位或体外引发的 Tregs 有何不同（Peng 等人，正在准备中）。我们与克利夫兰诊所的 Lin 实验室合作，证明在葡萄膜炎期间，炎症细胞能够通过 RPE 细胞层在脉络膜和视网膜之间迁移，由于 Bruchs 膜的存在，这种情况被认为不会发生（参考文献 1）。 5）。 6) 共生菌群对于自发性葡萄膜炎的发展是必需的，部分原因是IRBP161-180的分子模拟。然而，先天机制也发挥了作用，这一发现证明，在 EAU 免疫（提供抗原刺激）前一周开始使用抗生素 (ABX) 可以提供可测量的保护。然而，这种保护是暂时的，并且在长时间的 ABX 中消失。结果表明，长期 ABX 会消耗上皮内淋巴细胞 (IEL)，而上皮内淋巴细胞 (IEL) 已知是微生物群依赖性的。我们假设 IEL 可能对葡萄膜炎具有抑制作用，这种作用在长期 ABX 后由于 IEL 消耗而被逆转。 （Salvador R 等人，准备中）。 7) 人类肠道菌群和葡萄膜炎：我们用来自 3 名健康人类捐赠者的肠道微生物重建了无菌 R161H 小鼠，并建立了 3 个人源化菌群小鼠系。结果表明，人类肠道菌群支持自发性葡萄膜炎的发展。小鼠仅采用了人类菌群的一个子集，但保留了捐赠者的微生物足迹。我们目前正在识别与低和高疾病评分相关的微生物类群，目的是制定干预策略，作为通过直接操纵微生物组来调节葡萄膜炎的方法（Horai、Zhou、Murphy 等人，正在准备中）。 8）与博士合作。 Sue Menko（托马斯杰斐逊大学）和 Mary Ann Stepp（乔治华盛顿大学）发现，在葡萄膜炎期间，免疫细胞通过晶状体囊的细胞外基质侵入眼睛的晶状体。结果可能有助于解释炎症诱发的白内障形成（参考文献2） 9) 检查了 Alpk1 突变对 EAU 易感性的影响，因为这种突变会导致人类自身炎症性疾病，包括葡萄膜炎。证明人类突变体 alpk1 敲入小鼠在其他方面不受干扰的情况下不会出现自发性眼部炎症（参考文献 3） 10) 与 NEI 的 Egwuagu 实验室合作，LI 证明了产生 IL-27 的 B-1a 细胞 (Bregs) 抑制 EAU 和 EAE，表明在更广泛的意义上在神经炎症和 CNS 疾病中的作用（参考文献 1） 。 对眼表面共生体的粘膜免疫反应 肠道、口腔、鼻咽和阴道等粘膜部位都有相关的共生菌群。眼睛表面也是粘膜部位，但眼表面微生物组的存在存在争议。此前，我们分离并纯化了一种候选眼部共生菌——乳腺棒状杆菌（C. mast）。这种生物体会引起眼粘膜 T 细胞的共生特异性 IL-17 反应，调整局部宿主防御以提供免受传染性病原生物体侵害的保护。 1) 我们正在检查 T 细胞中 C. mast 的分子传感器。数据表明，Vg4 T 细胞主要通过 TCR 响应 C. mast，而 Vg6 T 细胞通过 TLR2 等先天受体响应，并且高度依赖 IL-1。 TLR2 似乎不仅在 DC 中（它有助于 IL-1 诱导）需要，而且在 T 细胞中也需要。 T 细胞内在 TLR2 需求的机制正在研究中（Zhu & Xu 等人以及 Xu 等人，正在准备中）。 2）我们还检查了免疫异常宿主（小鼠和人类）中共生体引起的眼表反应。我们与 Warren Strober 博士（NIAID）团队和 Raphaela Goldbach-Mansky 博士合作，他们开发了表达 NLRP3 炎性体基因功能获得性突变的敲入小鼠，而 Raphaela Goldbach-Mansky 博士则治疗 NLRP3 炎性体相关疾病患者。获得的证据表明，眼表共生体可以在免疫紊乱的宿主中引发眼表炎症，从而充当病原体。我们正在单细胞水平上表征小鼠模型和 NLRP3 炎性体突变患者的局部眼部免疫反应和眼表微生物组（Siak、Mattapallil、St. Leger 等人，正在准备中）。这对于理解和治疗 NLRP3 相关疾病患者的特征性眼部炎症具有重要意义。 3）我们对C. mast进行了全基因组测序、组装和注释。 https://dataview.ncbi.nlm.nih.gov/object/PRJNA758739?reviewer=dhmcn2fm0ngfj6bkh5jsq3u4d5（Nagarajan 等人，（参考 4）。 COVID-19 相关：微生物组对“人性化”小鼠模型中细胞因子风暴的影响。 COVID-19 患者的发病率和死亡率很大程度上归因于对病毒的过度免疫反应引发的细胞因子风暴。该项目使用 Richard Flavell 博士与 Regeneron, Inc. 合作开发的用人类白细胞重建的免疫缺陷 NSG 小鼠来研究肠道微生物组对细胞因子风暴的影响。由于美国国立卫生研究院的技术转让无法克服获得这些小鼠的法律障碍，该项目目前陷入停滞。