sirolimus and Retinitis

The determination of the molecular mechanism underlying retinal pathogenesis and visual dysfunction during innate inflammation, and the treatment effect of rapamycin thereon.. The endotoxin-induced uveitis and retinitis mouse model was established by injecting lipopolysaccharide. The mice were subsequently treated with rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. The rhodopsin mRNA and protein expression level in the retina and the photoreceptor outer segment (OS) length in immunohistochemical stainings were measured, and visual function was recorded by electroretinography. Inflammatory cytokines, their related molecules, mTOR, and LC3 levels were measured by real-time PCR and/or immunoblotting. Leukocyte adhesion during inflammation was analyzed using concanavalin A lectin.. The post-transcriptional reduction in the visual pigment of rod photoreceptor cells, rhodopsin, OS shortening, and rod photoreceptor cell dysfunction during inflammation were suppressed by rapamycin. Activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and induction of inflammatory cytokines, such as interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and the activation of the downstream signaling protein, signal transducer and activator of transcription 3 (STAT3), which reduces rhodopsin in the retina during inflammation, were attenuated by rapamycin. Increased leukocyte adhesion was also attenuated by rapamycin. Interestingly, although mTOR activation was observed after NF-κB activation, mTOR inhibition suppressed NF-κB activation at the early phase, indicating that the basal level of activated mTOR was sufficient to activate NF-κB in the retina. In addition, the inhibition of NF-κB suppressed mTOR activation, suggesting a positive feedback loop of mTOR and NF-κB during inflammation. The ratio of LC3II to LC3I, which reflects autophagy induction, was not changed by inflammation but was increased by rapamycin.. Our results propose the potential use of rapamycin as a neuroprotective therapy to suppress local activated mTOR levels, related inflammatory molecules, and the subsequent visual dysfunction during retinal inflammation.

Topics: Animals; Inflammation; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; NF-kappa B; Retina; Retinitis; Signal Transduction; Sirolimus; Uveitis

The efficacy and action mechanism of everolimus in the treatment of experimental autoimmune uveoretinitis (EAU) was analyzed. Disease was induced in B10.RIII mice by immunization with human interphotoreceptor-retinoid-binding protein peptide 161-180 (hIRBPp161-180). Everolimus was administered by oral gavage (5 mg/kg/d) beginning either two days before or 14 days after immunization. Everolimus significantly reduced the histopathological uveitis score compared to sham-treated mice. To examine the effect on the antigen-specific immune response, proliferation ([(3)H]-thymidine test) and delayed-type hypersensitivity (DTH) response were measured. Furthermore, content of T-helper-1, -2, and -17 cytokines were analyzed intraocularly (Bead Array) and in cell culture supernatants from splenocytes (sandwich ELISA). To study the effect on the humoral immune response the presence of antigen-specific serum antibodies was tested (indirect ELISA). The DTH, the humoral immune response, the proliferation of splenocytes and the intraocular Th1, Th2, Th17 cytokine content and in vitro production of Th1 and Th17 cytokines were impaired after everolimus treatment. The study of CD4+CD25+FoxP3+ regulatory T cells (T(reg)) in peripheral blood, draining lymph nodes, and spleen by flow cytometry showed an increased number of splenic T(reg) in mice of the everolimus therapy group. Furthermore the T(reg) of these mice had a higher suppressive capacity than cells from sham-treated mice. These results indicate that the immunosuppressive effect of everolimus on EAU was associated with the suppression of pathogenic effector responses and induction of regulatory T cells.

Topics: Animals; Antibodies; Autoimmune Diseases; Cell Proliferation; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Everolimus; Eye Proteins; Flow Cytometry; Forkhead Transcription Factors; Hypersensitivity, Delayed; Immunosuppressive Agents; Mice; Retinitis; Retinol-Binding Proteins; Sirolimus; Spleen; T-Lymphocytes, Regulatory; Uveitis, Posterior

Immunosuppressive drugs currently available for the treatment of autoimmune diseases display a narrow therapeutic window between efficacy and toxic side effects. The use of combinations of drugs that have a synergistic effect may expand this window and reduce the risk of toxicity. We evaluated the combination effect of rapamycin (Rapa) and cyclosporin A (CsA) in an autoimmune disease model of the eye. The dose-effect relationship of Rapa with CsA was measured in vitro on the inhibition of proliferation of retinal S-Ag-primed lymphocytes. A median effect analysis was performed and a combination index (CI) calculated for 50% inhibition of proliferation. Rapa and CsA were markedly synergistic over a wide dose range (lowest CI = 0.31). Calculated dose reduction factors indicated that Rapa could be reduced nine-fold and CsA reduced five-fold when these drugs were used in combination. These reduced doses were tested in vivo for the treatment of experimental autoimmune uveoretinitis (EAU). Twelve of 15 rats treated with CsA, 2 mg/kg/day, developed EAU with a median severity of 2.5. Fourteen of 15 rats treated with Rapa, 0.01 mg/kg/day, developed EAU with a median severity of 3.25. Complete inhibition of EAU was achieved in all 15 animals treated with the combination of Rapa and CsA (combined vs CsA alone, p < 0.0002; combined vs Rapa alone, p < 0.00001). The demonstrated synergistic relationship between Rapa and CsA will allow the use of reduced doses of each drug to achieve a therapeutic effect. The use of lower doses may reduce the toxicity of these drugs for the treatment of autoimmune uveitis.

Topics: Animals; Antigens; Arrestin; Autoimmune Diseases; Cyclosporine; Drug Synergism; Eye Proteins; Immunosuppressive Agents; Lymph Nodes; Lymphocyte Activation; Male; Polyenes; Rats; Rats, Inbred Lew; Retinitis; Sirolimus; Uveitis

Rapamycin (RAPA) is a macrolide antibiotic with unique immunosuppressive properties. RAPA inhibits T-cell function by interfering with IL-2 and IL-4 signal transduction. It does not prevent IL-2 production or IL-2R expression. The efficacy of RAPA in the treatment of autoimmune diseases was evaluated using the experimental autoimmune uveoretinitis (EAU) model. EAU was actively induced in Lewis rats by immunization with S-antigen in Hunter's adjuvant. RAPA and control vehicle were administered by continuous intravenous infusion over a 14 day period by miniosmotic pump. RAPA treatment initiated on the day of immunization or 7 days later was found to efficiently inhibit EAU induction. The minimal effective dose was 0.1 mg/kg/d. EAU inhibition was correlated with reduced number of cells in the immunization site draining lymph nodes, as well as with a shift and lowering of the peak of the lymphocyte proliferative response curve. The anti-S-antigen antibody response was delayed by 3 days under RAPA treatment and the serum levels lowered in a dose dependent manner. An initial body weight loss was observed during the first week of drug administration, but there was a normal weight gain afterward.

Topics: Animals; Antigens; Arrestin; Disease Models, Animal; Eye Proteins; Immunoglobulin G; Immunosuppressive Agents; Lymphocyte Activation; Male; Polyenes; Rats; Rats, Inbred Lew; Retinitis; Signal Transduction; Sirolimus; T-Lymphocytes; Uveitis