NEW MODELS OF OCULAR INFLAMMATION AND NEW WAYS TO STUDY THEM: (1) Birdshot chorioretinopathy (BC) is strongly associated with HLA-A29. The mechanistic basis for the disease may be clarified by developing and studying HLA-A29 transgenic mice, but repeated attempts to derive a strain with a phenotype using different types of constructs have not yet been fruitful. We now derived a new HLA-A29 construct. Founders derived with this construct are being observed for pathology. (2) We continue to collaborate with the group of Dr. Warren Strober (NIAID) on ocular inflammation in NLRP3 knock-in mice. This gene is associated with Muckle-Wells syndrome, which among other pathologies is also associated with inflammation at the ocular surface. Studies with these mice indicate existence of an abnormal response to what we believe to be the normal flora of the ocular surface. These studies may provide new insights into ocular involvement in Muckle-Wells syndrome. (3) A new model of conjunctivitis has been developed by challenging the mucosal ocular surface with innate stimuli. Findings reveal that the conjunctiva-associated lymphoid tissue (CALT) responds dynamically and contains a variety of leukocytes, among them innate-like lymphocytes (ILCs) and γδ T cells. This is an understudied area of research. We are defining the response and functionality of these cells in controlling infection at the ocular surface. We are also investigating whether there is a commensal ocular microbiome that functions to keep pathogenic organisms at bay. The insights may have important implications on treatment of conjunctivitis. FUNDAMENTAL MECHANISMS IN TOLERANCE, IMMUNITY AND AUTOIMMUNITY TO RETINAL ANTIGENS (1) Recent findings in IRBP T cell receptor transgenic R161H mice treated with antibiotics or made germ-free indicated that commensal flora contributes to development of spontaneous uveitis. Mechanistic studies revealed that signaling through the clonotypic TCR in the gut by a non-cognate antigen derived from gut microflora is a necessary (though possibly not sufficient) stimulus (Horai et al, Immunity 2015). Future work will attempt 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) 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 are studying the role of VitA in regulation of autoimmunity to retina. 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) IL-22 has been reported to have both pro-inflammatory and protective effects, depending on the tissue 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, produced by inflammatory cells and acting on neuronal and glial cells, has a local anti-inflammatory and tissue-protective role in the eye (Mattapallil et al., in preparation). (5) We examined the role of T regulatory (Treg) cells in limiting EAU. Our data suggest that Tregs found in uveitic eyes are (i) IRBP specific, (ii) functionally suppressive, and (iii) may play a role in natural resolution of disease and in the mainteenance of remission, which is at least in part due to local effects within the eye (Silver et al, J. Immunol. 2015). (6) In chronically inflamed eyes of R161H mice we identified structures resembling tertiary lymphoid tissue. These structures are functional germinal centers where immune cells may be activated for effector function and antibody production, affecting the course of disease. (J. Kielczewski, manuscript in revision). (7) Self reactivity has been proposed to have a role in neuroprotection, based on studies with adoptively transferred effector T cells (beneficial autoimmunity). R161H mice and their cells provide a way to examine this in a natural setting, where the autoreactive T cells are not initially primed and/or are endogenous to the host. Our data confirm existence of a neuroprotective effect, but it is minor and temporary. Based on our models, a significant neuroprotective role of beneficial autoimmunity in the retina is in question. 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 produce IL-17 independently of IL-6 and IL-21 (NKT17). We find that IL-17 production in NKT cells and in some other populations of innate lymphoid cells, may be produced via a unique signaling pathway that bypasses STAT3. In vivo experiments suggest that this pathway may have a function in protecting from infections at the ocular surface. (St.Leger, Hansen et al, in preparation). (2) The cytokine IFN-γ has both protective and proinflammatory in autoimmunity, which is an unsolved paradox. We uncovered a novel endogenous regulatory circuit based on crosstalk between NK cells and DCs, in which IFN-γ and IL-27 are produced as a self-amplifying regulatory feedback loop and are ultimately controlled by IL-10 produced by the interacting DC-NK cells themselves. The IL-27 from this interaction meanwhile dampens the adaptive response and limits autoimmunity by inducing regulatory T cells that control uveitogenic Th17 cells and disease by producing IL-10 (Chong et al, J. Exp. Med., in press). We speculate that NK-DC crosstalk may also limit adaptive immunity in responses to vaccines, and could explain the inhibitory effect of CD56-birght NK cells in patients treated for autoimmune disease with Daclizumab therapy. MUCOSAL IMMUNE RESPONSES AT THE OCULAR SURFACE: The composition and functionality of the mucosal conjunctiva-associated lymphoid tissue (CALT) has been understudied. A new research direction in the lab uncovered dynamic immune responsiveness of the CALT with the ability to recruit diverse adaptive and innate lymphoid elements. We have also obtained data that the ocular surface supports what appears to be a resident microbiome, whose elimination by antibiotics carries biological consequences. Local production of IL-17, driven by the microflora, recruits neutrophils to the conjunctiva and protects from infection by pathobionts such as candida albicans. Our data support the existence of an ocular surface microbiome that promotes immune homeostasis in the ocular mucosa.
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