| BackgroundUveitis is also called endophthalmitis, it refers to a wide group of inflammation occurs in uvea, retina, vitreous body and retina vascular. Uveitis more happens in young adults, with the characteristic of long duration and repeated attack. The etiology of uveitis is complex and the pathogenesis is not fully clear, its prevention becomes difficult and the treatment effect is not ideal. Therefore, looking for reasonable and effective drug treatment is an urgent problem in the field of ophthalmology. Both non-specific and specific immune responses could cause uveitis.EAU is a classic animal model of uveitis that represents human specific autoimmune posterior uveitis. It is well established that T cells mediated the pathology of uveitis, and CD4+T cells play a key role in the pathogenesis of uveitis. IRBP-specific Thl and (or) Th17 can drive ocular autoimmunity in EAU. Recent a body of evidence implicated that LXR as regulator of inflammation and autoimmunity. LXR agonist attenuates the inflammatory and immune response through inhibiting Thl- and Th17-cell differentiation in the animal models of EAE, CIA and DR. But the effect of LXR in the autoimmune uveitis is still not clear. MIF is a pleiotropy cytokine operating in innate and adaptive immunity. It exerts glucocorticoid antagonistic proinflammatory effect. We recently have demonstrated that the MIF gene has a strong association with BD and VKH syndrome. However, the role and the mechanism of MIF in the pathology of uveitis need our further study.Based on the above background, this subject includes the following two parts of experiments.(1) Investigate the expression of LXR and MIF in the retina of EAU, so as to determine whether LXR and MIF were involved in the development and progression of EAU. (2) Explore the effect and the mechanism of LXR and MIF in the intraocular inflammation of EAU. We aim to provide new ideas for the pathogenesis of uveitis through demonstrating the effect and the mechanism of LXR and MIF in EAU, and to provide new targets and new strategies for the prevention and treatment of uveitis.Part I The role of LXR in the pathogenesis of intraocular inflammation of EAUPurposeLXRs are ligand-activated transcription factors that involved in the cholesterol efflux and lipid metabolism. Increasing interest has been shown in LXRs as regulators of inflammatory and immune response. We aim to investigate whether a synthetic LXR agonist TO90 ameliorates ocular inflammation in EAU and to explore its underlying mechanism.MethodsEAU was induced with subcutaneous injection of IRBP in B10.RⅢ mice. TO90 or vehicle was administrated orally for successive 16 days or 8 days as prevention or effector phase, respectively. The severity of EAU was evaluated with clinical and histological scores. The levels of LXRs, p65, ABCA1 and proinflammatory cytokines in the retina were detected with real-time PCR and Western blotting. IRBP-specific lymphocyte proliferation was detected by MTT. Intracellular IFN-y and IL-17 in CD4+T cells were measured by flow cytometry.Results(1) Both LXRa and LXRβ were expressed in mouse retina. Compared to naive mice, LXRa expression was increased in vehicle and TO90 treated EAU mice, but the LXRβ expression was unchanged.(2) The protein level of ABCA1 was enhanced in TO90 treated naive and EAU mice but was unchanged in vehicle treated EAU mice, suggesting activation of LXRa by TO90 is ligand dependent.(3) TO90 mediated activation of LXRa improved the clinical and morphological scores in EAU mice.Meanwhile, activation of LXRa decreased the expressions of proinflammatory cytokines, including TNF-a, IL-1β, IL-6, MCP-1, IFN-γ and IL-17 in the retina.(4) TO90 treatment inhibited IRBP-specific immune responses. The proportions of Thl and Th17 that expressing IFN-y and IL-17 were reduced in TO90 treated EAU mice in both prevention and effector phases. Furthermore, TO90 significantly down-regulated the expressions of a NF-κB subunit p65 at protein and mRNA level.ConclusionsTO90 activates LXRa and potently attenuates ocular inflammation in EAU. Alleviation of ocular inflammation could partially result from inhibition of the NF-κB signaling pathway. TO90 reduces IFN-y and IL-17 expressing in both prevention and treatment scenarios. Our data suggests that LXR agonist may become a novel class of therapeutic agent for autoimmune uveitis.Part Ⅱ The role of MIF in the pathogenesis of intraocular inflammation of EAUPurposeMIF is a proinflammatory cytokine that operating in innate and adaptive immunity, it is associated with many immune-mediated diseases. We examined whether the intervention of MIF could modulate the intraocular inflammatory response in EAU and explored the underlying mechanism.MethodsMutant AAV8 (Y733F).CBA.MIF was delivered subretinally into B10.RIII mice, EAU was induced with a subcutaneous injection of IRBP three weeks after the vector delivery. Additionally, a MIF antagonist ISO-1 was injected intraperitoneally into EAU mice to inhibit the level of MIF. The mRNA level of proinflammatory cytokines was detected by real time-PCR. Retinal function was evaluated with electroretinography (ERG). The protein expression was detected by Western blotting.Intracellular IFN-y and IL-17 in CD4+T cells were measured by flow cytometry.Results(1) Compared to normal mice, the expression of MIF and its two receptors CD74 and CD44 was increased in the EAU mouse retina.(2) Compared to AAV8.CBA.eGFP injected and untreated EAU mice, the level of proinflammatory cytokines TNF-a, IL-1β and IL-6, the expression of Notch1, Notch4, D114, NICD and Hes-1 increased, but the ERG a- and b-wave amplitudes decreased in AAV8.CBA.MIF injected EAU mice. DAPT reduced the expression of NICD, Hes-1 and proinflammatory cytokines.(3) Further, ISO-1 attenuated intraocular inflammation, reduced intraocular macrophage recruitment and inhibited the differentiation of Thl and Th17 in EAU mice. ISO-1 inhibited the expression of MIF and the activity of the Notch signaling pathway.ConclusionsOver-expression of MIF exaggerated ocular inflammation, which was associated with the activation of the Notch signaling pathway. Our data suggest that the MIF-Notch axis may play an important role in the pathogenesis of EAU.Both MIF and Notch signaling pathways may be promising targets for developing novel therapeutic interventions for uveitis. |
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