MicroRNA-146a Alleviates Experimental Autoimmune Anterior Uveitis in the Eyes of Lewis Rats

Purpose. This study aimed to determine the effect and roles of microRNA (miRNA, miR) treatment in experimental autoimmune anterior uveitis (EAAU). Materials and Methods. Uveitis was induced in Lewis rats by simultaneous injections of bovine melanin-associated antigen into the hind footpad and the intraperitoneal cavity. The animals were injected intravitreally with low-dose (0.5 μg) or high-dose (1.5 μg) miR-146a. The clinical scores, leukocyte count in the aqueous humor, and histology were assessed. Cytokine changes were evaluated by relative mRNA expression and enzyme-linked immunosorbent assay (ELISA). The expression of nuclear factor kappa B (NF-κB) was assessed by immunofluorescence and Western blotting. Evaluation of the DNA-binding activity of NF-κB was performed by electrophoretic mobility shift assay (EMSA). Results. Treatment with miR-146a significantly attenuated clinical scores and leukocyte infiltration in a dose-dependent manner, a result that was compatible with histological findings. Following miR-146a injections, downregulation of interleukin- (IL-) 1β, IL-6, and IL-12 and interferon- (IFN-) γ and upregulation of IL-10 and IL-17 were noted. The decreased NF-κB expression on immunofluorescence and Western blotting and reduced DNA-binding activity on EMSA were demonstrated following miR-146a treatment. Conclusions. miR-146a effectively reduced intraocular inflammation in EAAU through the inhibition of NF-κB. miR-146a might be a new treatment choice for uveitis.

Expression of MicroRNAs in the Eyes of Lewis Rats with Experimental Autoimmune Anterior Uveitis

Purpose.This study aimed to determine the dynamic changes of NF-κB-related microRNAs (miRNAs) and cytokines over the course of experimental autoimmune anterior uveitis (EAAU) and elucidate the possible immunopathogenesis.Materials and Methods.Uveitis was induced in Lewis rats using bovine melanin-associated antigen. The inflammatory activity of the anterior chamber was clinically scored, and leukocytes in the aqueous humor were quantified. RNA was extracted from the iris/ciliary bodies and popliteal lymph nodes to reveal the dynamic changes of eight target miRNAs (miR-155-5p, miR-146a-5p, miR-182-5p, miR-183-5p, miR-147b, miR-21-5p, miR-9-3p, and miR-223-3p) and six cytokine mRNAs (IFN-γ, IL-17, IL-12A, IL-1β, IL-6, and IL-10).In situhybridization of miRNA and enzyme-linked immunosorbent assay quantification of cytokines were performed to confirm the results.Results. Disease activity and leukocyte quantification were maximum at day 15 after immunization. The profiling of miRNA revealed downregulation of miR-146a-5p, miR-155-5p, miR-223-3p, and miR-147b and upregulation of miR-182-5p, miR-183-5p, and miR-9-3p. Cytokine analysis revealed IFN-γ, IL-17, IL-12A, IL-1β, and IL-6 overexpression, with IL-10 downregulation.Conclusions.Dynamic changes of miRNAs were observed over the course of EAAU. By initiating NF-κB signaling, the expressions of downstream cytokines and effector cells from the Th17 and Th1 lineages were sequentially activated, contributing to the disease.

Chitosan Oligosaccharides Attenuate Ocular Inflammation in Rats with Experimental Autoimmune Anterior Uveitis

We investigated the protective effects and mechanisms of chitosan oligosaccharides (COS) on experimental autoimmune anterior uveitis (EAAU) in rats. EAAU was induced in Lewis rats by footpad and intraperitoneal injections of melanin-associated antigen. The rats received intraperitoneal injections of low-dose (5 mg/kg) or high-dose (10 mg/kg) COS or PBS daily after the immunization. The effects of COS were evaluated by determining the clinical scores and the morphology of the iris/ciliary body (ICB). The expression of inflammatory mediators was evaluated using western blot, immunofluorescence, and ELISA. Treatment with COS significantly attenuated the clinical scores and the leukocyte infiltration in the ICB in a dose-dependent manner. COS effectively reduced the expression of inflammatory mediators (TNF-α, iNOS, MCP-1, RANTES, fractalkine, and ICAM-1). Moreover, COS decreased the IκB degradation and p65 presence in the ICB, which resulted in the inhibition of NF-κB/DNA binding activity. In an in vitro study, sensitized spleen-derived lymphocytes of the COS-treated group showed less chemotaxis toward their aqueous humor and decreased secretion of the above inflammatory mediators in the culture media. COS treated EAAU by inhibiting the activation of NF-κB and reducing the expression of inflammatory mediators. COS might be a potential treatment for acute anterior uveitis.