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: 10930490
• 负责人: Rachel R. Caspi
• 金额: $ 422.64万
• 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/10930490
• 关键词: Actinobacteria class; Active Immunization; Adopted; Adoptive Transfer; Affect; Agonist; Animal Model; Anti-Inflammatory Agents; Antibiotics; Antigen Presentation; Antigens; Arrestins; Aryl Hydrocarbon Receptor; Autoimmune Responses; Autoimmunity; Automobile Driving; Bioinformatics; Blood - brain barrier anatomy; Blood Preservation; Brain; CD8-Positive T-Lymphocytes; Cell Survival; Cells; Characteristics; Clinic; Clinical; Coculture Techniques; Collaborations; Contact Lenses; Cornea; Corynebacterium; Data; Development; Disease; Endothelial Cells; Engineering; Equilibrium; Experimental Models; Eye; FOXP3 gene; Feces; Firmicutes; France; Fusobacteria; Genes; Genetic; Germ-Free; Goals; Homeostasis; Host Defense; Human; IL17 gene; Immune; Immune response; Immunity; Immunization; Immunologics; Immunology; Immunotherapy; In Vitro; Infiltration; Inflammasome; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Interferons; Interleukin-1; Interleukin-10; Intervention; Intestines; Knock-in Mouse; Laboratories; Lymphocyte; Lymphocyte Depletion; Maintenance; Measurable; Metabolic; Microbe; Molecular; Molecular Mimicry; Motor; Mucosal Immune Responses; Mucous Membrane; Mus; Mutation; Myeloid Cells; Nasopharynx; National Institute of Allergy and Infectious Disease; Onset of illness; Oral cavity; Organism; Pathogenicity; Pathway interactions; Patients; Peptides; Peripheral Blood Mononuclear Cell; Predisposition; Preparation; Process; Production; RNA; Regulatory T-Lymphocyte; Rest; Retina; Role; Signal Transduction; Site; Stereotyping; Stimulus; Structure of retinal pigment epithelium; Surface; System; T cell response; T-Cell Receptor; T-Lymphocyte; TLR2 gene; Therapeutic; Tissues; Transgenic Organisms; Uveitis; Vagina; Vision; Volatile Fatty Acids; autoimmune uveitis; autoinflammatory diseases; clinical material; commensal microbes; cytokine; exhaust; fatty acid oxidation; gain of function mutation; gene induction; gut microbes; gut microbiome; gut microbiota; human microbiota; in vivo; interstitial retinol-binding protein; intraepithelial; lens induction; microbial; microbiome; microbiota; mouse model; mucosal site; mutant; neuropathology; ocular surface; pathobiont; perforin; pharmacologic; preservation; protective effect; receptor; reconstitution; response; sensor; transcription factor; transcriptome; transcriptomics

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) 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. Laquinimod strongly inhibited EAU in the setting of active immunization and also had moderate effects in the setting of adoptive transfer. Data indicated that the major effect was exerted via myeloid cells, possibly by inhibiting antigen presentation and/or effector function during inflammation. Notably, it had inhibitory effect on immune response to recall antigens in human PBMC, suggesting potential clinical utility (B. Xu et al, in preparation) 2) Ocular immune privilege: We have previously shown that nave retina-specific T cells in the eye are primed locally, and after 7 days a proportion of them convert into Foxp3+ T-regulatory cells (Tregs). However, it was not clear what differentiation pathway was adopted by cells that were not converted to Tregs. Using single-cell transcriptomics of cells of fresh retina-specific lymphocytes injected into the healthy eye found that although all cells recognized their antigen, inflammation was not induced. About 30% of the cells adopted the profile of T-regulatory cells, actively secreting anti-inflammatory mediators TGF- and IL-10, whereas the rest were divided along a spectrum expressing stereotypic gene induction profiles consistent with non-pathogenic effector Th17 cells, exhausted, and anergic cells. IFN--expressing cells were notably absent. RNA velocity and trajectory indicated that these cell fates were largely independent and developed in parallel, rather than in a sequential fashion. This is the first molecular representation of the immune privilege phenomenon in the living eye and promises to shed new light how the eye controls inflammation. We are also 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). 3) 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 revision). 4) 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. Mice with high disease scores appeared to harbor more diverse gut flora than those with low scores. Verrucomicrobia, Actinobacteria, and Fusobacteria were enriched in mice with high disease scores, whereas Firmicutes appeared enriched in mice with low disease scores and were associated with higher content of short chain fatty acids in the feces. In-vivo association studies are underway to examine the ability of candidate microbes to modulate autoimmune uveitis. This may instruct interventional strategies as an approach to regulating uveitis through directed manipulation of the microbiome (Horai, Zhou, Murphy et al, in preparation). 5) in collaboration with the group of Feng Lin, Cleveland Clinic, demonstrated that CDCP1 regulates retinal pigmented epithelial barrier integrity for the development of experimental autoimmune uveitis (ref.2) 6) Collaborated with the group of Dan Kastner and colleagues on 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.4) 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 intrinsic TLR2 stimulation in V6 T cells is upregulates the transcription factor IAb, which in turn is required for metabolic support of IL-17A production in these cells through fatty acid oxidation. This study highlights the importance of intrinsic TLR2 signaling in driving the production of IL-17A in microbiome-specific gd T cells. (Z. Zhu & X. Xu et al., and X. 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. (Ref. 5). 3) A collaborative study with the lab of Roland Laulau, INSERM, France, in an in vitro co-culture system identified perforin as an essential molecule for killing of blood brain barrier endothelial cells (BBB-EC) by CD8 T cells. We then found that short-term pharmacologic inhibition of perforin commencing after disease onset restored motor function and inhibited the neuropathology. Perforin inhibition resulted in preserved BBB-EC viability, maintenance of the BBB, and reduced CD8 T-cell accumulation in the brain and retina. 4) In collaboration with S. Fleiszig and D Evans of UC Berkeley, we demonstrated that Contact lens-induced corneal parainflammation involves Ly6G+ cell infiltration and requires IL-17A and T cells (ref. 6).

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) 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。拉喹莫德在主动免疫的情况下强烈抑制 EAU，并且在过继转移的情况下也有中等效果。数据表明，主要作用是通过骨髓细胞发挥的，可能是通过抑制炎症期间的抗原呈递和/或效应器功能。值得注意的是，它对人 PBMC 中回忆抗原的免疫反应具有抑制作用，表明其具有潜在的临床实用性（B. Xu 等人，正在准备中） 2) 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，其中一部分转化为 Foxp3+ T 调节细胞 (Treg)。然而，尚不清楚未转化为Treg的细胞采用何种分化途径。使用注入健康眼睛的新鲜视网膜特异性淋巴细胞的单细胞转录组学发现，尽管所有细胞都识别其抗原，但并未诱导炎症。大约 30% 的细胞采用 T 调节细胞的特征，积极分泌抗炎介质 TGF-和 IL-10，而其余细胞则沿着表达与非致病性效应 Th17 细胞一致的刻板基因诱导特征的谱进行划分，精疲力尽，细胞无能。明显不存在表达IFN的细胞。 RNA速度和轨迹表明这些细胞的命运在很大程度上是独立的并且是平行发展的，而不是以连续的方式。这是活体眼睛中免疫特权现象的第一个分子表征，有望为眼睛如何控制炎症提供新的线索。我们还在检查眼睛诱导的 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）。 3) 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，修订中）。 4) 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。疾病评分高的小鼠似乎比评分低的小鼠拥有更多样化的肠道菌群。疣微菌门、放线菌门和梭杆菌在疾病评分高的小鼠中富集，而厚壁菌门在疾病评分低的小鼠中富集，并且与粪便中短链脂肪酸含量较高有关。体内关联研究正在进行中，以检查候选微生物调节自身免疫性葡萄膜炎的能力。这可能会指导介入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）。 5）与克利夫兰诊所的 Feng Lin 团队合作，证明了 CDCP1 调节视网膜色素上皮屏障完整性以促进实验性自身免疫性葡萄膜炎的发生（参考文献 2） 6) 与 Dan Kastner 及其同事小组合作研究 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 4） 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。 V6 T 细胞中内在的 TLR2 刺激会上调转录因子 IAb，而 IAb 又是这些细胞中通过脂肪酸氧化产生 IL-17A 的代谢支持所必需的。这项研究强调了内在 TLR2 信号传导在驱动微生物组特异性 gd T 细胞产生 IL-17A 方面的重要性。 （Z. Zhu 和 X. Xu 等人，以及 X. 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。 （参考文献 5）。 3) 与法国 INSERM 的 Roland Laulau 实验室在体外共培养系统中进行的一项合作研究，确定穿孔素是 CD8 T 细胞杀死血脑屏障内皮细胞 (BBB-EC) 的重要分子。然后我们发现，在疾病发作后开始对穿孔素进行短期药理抑制可恢复运动功能并抑制神经病理学。穿孔素抑制可保留 BBB-EC 活力，维持 BBB，并减少大脑和视网膜中 CD8 T 细胞的积累。 4) 与加州大学伯克利分校的 S. Fleiszig 和 D Evans 合作，我们证明隐形眼镜引起的角膜副炎症涉及 Ly6G+ 细胞浸润，并且需要 IL-17A 和 T 细胞（参考文献 6）。

项目成果

Glutamate joins the ranks of immunomodulators.

• DOI: 10.1038/nm0810-856
• 发表时间: 2010-08
• 期刊: Nature medicine
• 影响因子: 82.9

TMP778, a selective inhibitor of RORγt, suppresses experimental autoimmune uveitis development, but affects both Th17 and Th1 cell populations.

• DOI: 10.1002/eji.201747029
• 发表时间: 2018-11
• 期刊: European journal of immunology
• 影响因子: 5.4
• 作者: Lyu C;Bing SJ;Wandu WS;Xu B;Shi G;Hinshaw SJ;Lobera M;Caspi RR;Lu L;Yang J;Gery I
• 通讯作者: Gery I

Ocular immune privilege.

• DOI: 10.3410/b2-3
• 发表时间: 2010-01-18
• 期刊: F1000 biology reports
• 作者: Zhou, Ru;Caspi, Rachel R
• 通讯作者: Caspi, Rachel R

ZIKA virus infection causes persistent chorioretinal lesions.

• DOI: 10.1038/s41426-018-0096-z
• 发表时间: 2018-05-25
• 期刊: Emerging microbes & infections
• 影响因子: 13.2
• 作者: Manangeeswaran M;Kielczewski JL;Sen HN;Xu BC;Ireland DDC;McWilliams IL;Chan CC;Caspi RR;Verthelyi D
• 通讯作者: Verthelyi D

IL-12p35 induces expansion of IL-10 and IL-35-expressing regulatory B cells and ameliorates autoimmune disease.

• DOI: 10.1038/s41467-017-00838-4
• 发表时间: 2017-09-28
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Dambuza IM;He C;Choi JK;Yu CR;Wang R;Mattapallil MJ;Wingfield PT;Caspi RR;Egwuagu CE
• 通讯作者: Egwuagu CE