Suppressor of cytokine signaling 3-derived peptide as a therapeutic for inflammatory and oxidative stress-induced damage to the retina

Abstract

Purpose: Inflammation and oxidative stress contribute to age-related macular degeneration (AMD) and other retinal diseases. We tested a cell-penetrating peptide from the kinase inhibitory region of an 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.

Methods: We used anaphylatoxin C5a to mimic the effect of activated complement, lipopolysaccharide (LPS), and tumor necrosis factor alpha (TNFα) to stimulate inflammation and paraquat to induce mitochondrial oxidative stress. We used a human retinal pigment epithelium (RPE) cell line (ARPE-19) as proliferating cells and a mouse macrophage cell line (J774A.1) to follow cell propagation using microscopy or cell titer assays. We evaluated inflammatory pathways by monitoring the nuclear translocation of NF-κB p65 and mitogen-activated protein kinase p38. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to evaluate the induction of inflammatory markers. In differentiated ARPE-19 monolayers, we evaluated the integrity of tight junction proteins through microscopy and the measurement of transepithelial electrical resistance (TEER). We used intraperitoneal injection of sodium iodate in mice to test the ability of R9-SOC3-KIR to prevent RPE and retinal injury, as assessed by fundoscopy, optical coherence tomography, and histology.

Results: R9-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 TEER was prevented in the presence of R9-SOCS3-KIR. Treatment with the R9-SOCS3-KIR peptide blocked the C5a-induced expression of inflammatory genes. The R9-SOCS3-KIR treatment also blocked the LPS-induced expression of interleukin-6, MCP1, cyclooxygenase 2, and interleukin-1 beta. R9-SOCS3-KIR prevented paraquat-mediated cell death and enhanced the levels of antioxidant effectors. Daily eye drop treatment with R9-SOCS3-KIR protected against retinal injury caused by i.p. administration of sodium iodate.

Conclusions: R9-SOCS3-KIR blocks the induction of inflammatory signaling in cell culture and reduces retinal damage in a widely used RPE/retinal oxidative injury model. As this peptide can be administered through corneal instillation, this treatment may offer a convenient way to slow down the progression of ocular diseases arising from inflammation and chronic oxidative stress.