AUTOIMMUNE AND INFLAMMATORY RESPONSES AFFECTING THE RETINA Most of these studies emphasize mouse models of autoimmune uveitis developed in our laboratory, (i) experimental autoimmune uveitis (EAU) induced in mice by immunization with the retinal Ag IRBP, and (ii) spontaneous uveitis, which develops in R161H mice that express a transgenic T cell receptor (TCR) specific for the IRBP epitope encoded by residues 161-180, on the B10.RIII background. To better study basic mechanisms, we have backcrossed numerous gene-manipulated strains onto the B10.RIII/R161H background. (1) Recent findings in R161H mice indicated that gut commensal flora is necessary for development of spontaneous uveitis. Interestingly, while long term depletion of gut flora by antibiotic treatment, starting before birth, inhibits disease development in the spontaneous model, it had no effect on the (immunization induced) EAU model. However, a short-term antibiotic treatment did afford measurable protection. We found that long term antibiotic treatment depletes intraepithelial lymphocytes (IEL), which are known to be microbiota-dependent. These cells are able to inhibit activation of conventional lymphocytes, including CD4+ TCR uveitogenic T lymphocytes. We hypothesize that this may be connected to their regulatory function in uveitis, which is abrogated by long-term antibiotic treatment due to depletion of IEL. (Salvador et al., in preparation). (2) The role of various cytokines in pathogenesis and regulation of uveitis is being studied using both the classical and the spontaneous uveitis models, with emphasis on the balance between Th1 and Th17 responses, and their control by T-regulatory (Treg) cells. (a) Although previous studies indicated a critical role for the Th17 lineage cytokines in pathogenesis of immunization-induced EAU, crossing R161H mice to IFN-g deficient or IL-17 deficient mice revealed a major role for IFN- in the spontaneous disease. (b) Using mice deficient in both IL-17 and IFN-, we demonstrated that disease is still induced, but is now mediated by distinct effector cytokine profile. GM-CSF appears to be a critical pathogenic cytokine, which may drive disease in the absence of both IFN- and IL-17 (S Bing et al, manuscript in preparation). (c) On the other hand, IL-22 appears to have a protective role, which may at least in part be due to be due to a neuroprotective effect of IL-22. Data indicate that this is likely an indirect effect that requires other retinal cells (Mattapallil et al, J. Autoimmun 2019). (d) We reveal a novel regulatory pathway elicited by IL-17, a major proinflammatory cytokine. By binding to the IL-17R on activated T cells, IL17 elicits production of IL-24, which suppresses production of other Th17-related cytokines, including, importantly, GM-CSF. We speculate that the loss of this regulatory pathway in uveitis patients treated with anti-IL-17 neutralizing antibody (Secukinumab) may underlie the unexpectedly disappointing effects of this therapy (WP Chong et al., submitted). (e) In collaboration with Dr. Benjamin Sredni of Bar Ilan University, Israel, we showed that the immunomodulatory organotellurium compound, AS-101, ameliorates experimental autoimmune uveitis by regulating Th1 and Th17 responses and inducing Treg cells. (S Bing et al, J Autoimmun 2019). (3) 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 geographical regions where dietary VitA is limiting. (Horai, Zhou et al, in preparation). (4) In collaboration with Dr. Daniela Verthelyi and her group at FDA/CBER we are studying ocular effects of neonatal infection with Zika virus or Ebola virus constructs. The data suggest that immune privilege plays a role in promoting persistence of infectious agents within the CNS by protecting it from elimination by systemic immune mechanisms. These studies have the potential to help elucidate the mechanisms underlying the damage that these viral agents cause to the eyes and brain, and aid in the development of effective therapeutics (McWilliams et al., Cell Rep. 2019). EFFECTS OF INNATE IMMUNE RESPONSES ON OCULAR IMMUNITY AND AUTOIMMUNITY: Cellular and molecular elements of the innate immune system can affect immunopathogenic processes directly as well as indirectly, by affecting adaptive immunity. Innate immunity receptors have a major role in controlling susceptibility to autoimmune uveitis. A collaborative study with Dr. Holly Rosenzweig lab at OHSU, Portland, revealed that, unexpectedly, NOD2, usually thought of as an inflammation-promoting receptor, limits autoimmunity to the neuroretina. This appears to occur through a T cell intrinsic mechanism, by downregulating IL-17 production from T cells, but 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.. (Manuscript in revision). 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 in a C1d-dependent fashion, and as a source of IFN-. TLR2 appears to be required not only in T cells, but also in DC, for a maximal t cell IL-17 response. 2) A second direction of study examines commensal-elicited responses in an immunologically abnormal host, mouse and patient. 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 Gabriela Goldbach-Mansky who treats patients with NLRP3 inflammasome mutations, we have preliminary evidence to suggest that, in a host with an overactive inflammasome, an ocular surface commensal may elicit ocular surface inflammation, thus acting as a pathobiont. We are currently characterizing the local ocular immune response a in the mouse model and in patients with NLRP3 inflammasome mutation at the single-cell level.
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