Genetic, Cellular and Molecular Mechanisms in Autoimmunity to Retina

National Eye Institute

Investigators

Linked publications, trials & patents

Abstract

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.