Genetic, Cellular and Molecular Mechanisms in Autoimmunity to Retina

National Eye Institute

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Abstract

AUTOIMMUNE AND INFLAMMATORY RESPONSES AFFECTING THE RETINA Most of these studies utilized 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 that express a transgenic T cell receptor (TCR) specific for the IRBP epitope encoded by residues 161-180. 1) Commensal flora is necessary for development of spontaneous uveitis, but not of immunization-induced EAU. Wild type (WT) mice treated with antibiotics (ABX) before birth, developed full blown EAU. However, ABX starting a week before EAU challenge, afforded measurable protection. We found that long term ABX depletes intraepithelial lymphocytes (IEL), which are known to be microbiota-dependent. These cells appear to inhibit activation of uveitis-inducing R161H lymphocytes. We hypothesize that they may have a regulatory role in uveitis, which is reversed by long-term antibiotic treatment due to depletion of IEL. (Salvador, R. et al., ref 5, and Salvador R et al., in preparation). 2) 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). 3) Laquinimod is an aryl hydrocarbon receptor (AHR) agonist. We found that it has strong protective effects in the induced model of EAU, and marked protective effects in the spontaneous uveitis model in R161H mice. Its action is 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, and Tang et al, in preparation) 4) Tristetraprolin (TTP) is an inducible endogenous anti-inflammatory protein that controls expression of cytokines. In collaboration with P. Blackshear, NIEHS, we studied induced mutant mice that overexpress TTP. We found that these mice are resistant to EAU, have reduced proinflammatory responses and enhanced regulatory responses. The primary effect seems to be on T cell priming, by affecting antigen-presenting cells. (Xu et al., Ref 6). 6) 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). 5) Tofacitinib as a novel therapeutic of uveitis: Tofacitinib is a pan-JAK inhibitor that suppresses cytokine signaling. Treatment with tofacitinib suppressed the development of EAU and reduced the levels of secreted IFN-but not of IL17. Because EAU is both Th1 and Th17 dependent, these data indicate that each arm is nonredundant and contributes independently to pathology. (Bing SJ et al., ref 1). 6) B-regulatory cells and their product IL-35 as modulators and potential therapy for uveitis: In collaboration with the group of Egwuagu et al, we are studying the regulatory properties of IL-35-producing regulatory B cells. Furthermore, the exosome-contained IL-35 is being investigated as a potential biotherapeutic for uveitis using the EAU model. (Kang et al, ref. 2, and Oladipupo et al, ref. 4) EFFECTS OF INNATE IMMUNE RESPONSES ON OCULAR IMMUNITY AND AUTOIMMUNITY: The innate immune system can be involved in tissue pathology directly, or indirectly by affecting adaptive immunity. In collaboration with Dr. Holly Rosenzweig, OHSU, Portland, OR, we found that, unexpectedly, NOD2, which is usually thought of as an inflammation-promoting receptor, limits autoimmunity to the neuroretina. This involves downregulation of IL-17 production from T cells via a novel, T cell intrinsic pathway, and not through conventional APC priming. This study uncovers a hitherto unrecognized role for Nod2 as an inherent genetic modifier of T cell function in uveitis.. (Napier et al, 2020 ref 3). THE OCULAR SURFACE MICROBIOME AND MUCOSAL IMMUNE RESPONSES 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 V4 T cells respond to C. mast mainly via their TCR, whereas V6 T cells respond through innate receptors such as TLR2, and are highly dependent on IL-1. DC are involved in this response in a dual capacity as APC that present C. mast components and as a source of IL-1. To elicit an efficient Th17 response, 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 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 have obtained evidence to suggest that an ocular surface commensal can elicit ocular surface inflammation, thus acting as a pathobiont. We are currently 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., in preparation). 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 and treated with broad spectrum antibiotics to eliminate endogenous microbiota, to examine whether its presence affects the cytokine storm elicited by GVH (chronic) or by OKT3 antibody (acute). Preliminary data indicate that there are measurable effects on immune and physiological parameters relating to both responses. This may suggest that the status of the commensal microbiome is a factor in the cytokine storm response of COVID-19 patients. We are currently attempting to obtain improved humanized mice, which have enhanced engraftment of the myeloid compartment, for a better representation of the human immune response. Unfortunately, since January 2021, NIH tech transfer has been unable to overcome legal barriers to obtaining these mice. (Mattapallil et al., in progress).