A cell penetrating peptide from SOCS-1 prevents ocular damage in experimental autoimmune uveitis

Abstract

We describe an immunosuppressive peptide corresponding to the kinase inhibitory region (KIR) of the intracellular checkpoint protein suppressor of cytokine signaling 1 (SOCS-1) that binds to the phospho-tyrosine containing regions of the tyrosine kinases JAK2 and TYK2 and the adaptor protein MAL, and thereby inhibits signaling downstream from these signaling mediators. The peptide, SOCS1-KIR, is thus capable of downregulating overactive JAK/STAT or NF-kB signaling in somatic cells, including those in many compartments of the eye. Attachment of poly-arginine to this peptide (R9-SOCS1-KIR) allows it to penetrate the plasma membrane in aqueous media. R9-SOCS1-KIR was tested in ARPE-19 cells and was found to attenuate mediators of inflammation by blocking the inflammatory effects of IFNγ, TNFα, or IL-17A. R9-SOCS1-KIR and also protected against TNFα or IL-17A mediated damage to the barrier properties of ARPE-19 cells, as evidenced by immunostaining with the tight junction protein, zona occludin 1 (ZO-1), and measurement of transepithelial electrical resistance (TEER). Experimental autoimmune uveitis (EAU) was generated in B10. RIII mice using a peptide of interphotoreceptor retinal binding protein (IRBP) as immunogen. Topical administration of R9-SOCS1-KIR, 2 days before (prophylactic), or 7 days after immunization (therapeutic) protected ocular structure and function as seen by fundoscopy, optical coherence tomography (OCT), and electroretinography (ERG). The ability R9-SOCS1-KIR to suppress ocular inflammation and preserve barrier properties of retinal pigment epithelium makes it a potential candidate for treatment of autoimmune uveitis.

A C-terminal peptide from type I interferon protects the retina in a mouse model of autoimmune uveitis

“…Cells were pretreated with 3 μM IFNα-C for 4 hr. Cells were then washed with PBS and total RNA was extracted using the RNeasy Mini Kit from QIAGEN, as described before [31]. One microgram of RNA was used to synthesize first strand cDNA using the iScript cDNA synthesis kit from Bio-Rad (Hercules, CA).…”

“…B10.RIII mice (female, 6 to 8 weeks old, n = 8) were immunized with 100 μg of human IRBP 160-181 peptide (GenScript, Piscataway, N.J.) dissolved 100 μl PBS emulsified in 100 μl of complete Freund's adjuvant. Mouse numbers were selected based on the effect size expected from our previous experiments with this model [31]. While sex is not reported to influence the susceptibility to disease, female mice have been typically used in this model [3].…”

“…These procedures were conducted exactly as described in our previous paper [31]. The health of mice in this study was monitored daily.…”

“…For these procedures mice were anesthetized with a mixture of ketamine (95 mg/kg) and xylazine (5-10 mg/kg), and at the conclusion of the study mice were euthanized by inhalation of CO 2 followed by cervical dislocation. We measured the number of infiltrating cells in the vitreous using Image J software from NIH (URL:https:// imagej.nih.gov/ij/), as described before [31]. Briefly, the area representing the vitreous was delineated in the OCT image.…”

“…Electroretinography measurements in dark adapted mice was conducted as described in Ahmed et al [31].…”