Suppressor of Cytokine Signaling 3 Derived Peptide as a Therapeutic for Inflammatory, and Oxidative Stress Induced Damage to the Retina

Abstract

AbstractPurposeInflammation and oxidative stress are contributing factors to age-related macular degeneration (AMD) and other retinal diseases. We tested a cell penetrating peptide from the kinase inhibitory region of intracellular checkpoint inhibitor Suppressor of Cytokine Signaling 3 (R9-SOCS3-KIR) peptide for its ability to blunt the inflammatory or oxidative pathways leading to AMD.MethodsWe used Anaphylatoxin C5a to mimic the effect of activated complement, lipopolysaccharide (LPS) and TNFα to stimulate inflammation, and paraquat to induce mitochondrial oxidative stress. We used a human RPE cell line (ARPE-19) as proliferating cells and a mouse macrophage cell line (J774A.1) to follow cell propagation by microscopy or cell titer assays. We evaluated inflammatory pathways by monitoring nuclear translocation of NF-κB p65 and MAP kinase p38, and we used qRT-PCR and Western blots to evaluate induction of inflammatory markers. In differentiated ARPE-19 monolayers, we evaluated the integrity of tight junction proteins by microscopy and measurement of transepithelial electrical resistance. We used intraperitoneal injection of sodium iodate to test the ability of R9-SOC3-KIR to prevent RPE and retinal injury as assessed by fundoscopy, Optical Coherence Tomography (OCT) and histology.ResultsR9-SOCS3-KIR treatment suppressed C5a-induced nuclear translocation of the NF-kB activation domain p65 in undifferentiated ARPE-19 cells. TNF-mediated damage to tight junction proteins in RPE and the loss of transepithelial electrical resistance were prevented in the presence of R9-SOCS3-KIR. R9-SOCS3-KIR prevented the increased expression of genes related to inflammation in response to C5a treatment. R9-SOCS3-KIR also blocked lipopolysaccharide (LPS) induction of cyclooxygenase and inflammatory markers including IL-6, MCP1, COX-1 and IL-1β. R9-SOCS3-KIR prevented paraquat mediated cell death and enhanced the levels of antioxidant effectors. Daily eye drop instillation of R9-SOCS3-KIR protected against retinal injury caused by i.p. administration of sodium iodate.ConclusionR9-SOCS3-KIR blocks the induction of inflammatory signaling in cell culture and reduces retinal damage in a widely used model of RPE/retina oxidative injury. Since this peptide can be administered by corneal instillation, this treatment may offer a convenient way to slow the progression of ocular diseases arising from inflammation and chronic oxidative stress.