Topical administration of a suppressor of cytokine signaling-1 (SOCS1) mimetic peptide inhibits ocular inflammation and mitigates ocular pathology during mouse uveitis

Abstract

Uveitis is a diverse group of potentially sight-threatening intraocular inflammatory diseases and pathology derives from sustained production of pro-inflammatory cytokines in the optical axis. Although topical or systemic steroids are effective therapies, their adverse effects preclude prolonged usage and are impetus for seeking alternative immunosuppressive agents, particularly for patients with refractory uveitis. In this study, we synthesized a 16 amino acid membrane-penetrating lipophilic suppressor of cytokine signaling 1 peptide (SOCS1-KIR) that inhibits JAK/STAT signaling pathways and show that it suppresses and ameliorates experimental autoimmune uveitis (EAU), the mouse model of human uveitis. Fundus images, histological and optical coherence tomography analysis of eyes showed significant suppression of clinical disease, with average clinical score of 0.5 compared to 2.0 observed in control mice treated with scrambled peptide. We further show that SOCS1-KIR conferred protection from ocular pathology by inhibiting the expansion of pathogenic Th17 cells and inhibiting trafficking of inflammatory cells into the neuroretina during EAU. Dark-adapted scotopic and photopic electroretinograms further reveal that SOCS1-KIR prevented decrement of retinal function, underscoring potential neuroprotective effects of SOCS1-KIR in uveitis. Importantly, SOCS1-KIR is non-toxic, suggesting that topical administration of SOCS1-Mimetics can be exploited as a non-invasive treatment for uveitis and for limiting cytokine-mediated pathology in other ocular inflammatory diseases including scleritis.

Keywords: Biologics; Experimental autoimmune uveitis (EAU); Ocular inflammation; SOCS1; SOCS1 mimetic; Suppressor of cytokine signaling; Uveitis.

Figure 1. SOCS1-KIR suppresses the development of EAU

Fundus image of retina at day 14 (A) or day 21 (B) after EAU induction were taken using an otoendoscopic imaging system, and clinical score and assessment of disease severity were based on changes at the optic nerve disc or retinal vessels and retinal and choroidal infiltrates. Fundus images reveal inflammation with blurred optic disc margins and enlarged juxtapapillary area (black arrows), retinal vasculitis with moderate cuffing (red arrows), and yellow-whitish retinal and choroidal infiltrates (white arrows).

Figure 2. SOCS1-KIR inhibits uveitis and confers protection from ocular pathology

(A) Optical coherence tomography (OCT) analysis showing layered structure of the retina and ocular inflammation. Representative OCT images showing markedly increased inflammatory cells (white arrows) in the vitreous of mice treated with scrambled peptide. In addition, increased retinal vasculitis (yellow arrows), granulomatous lesions (red arrows), disturbance of the retinal layer structure, discontinuous and obscuring of the external limiting membrane (ELM) were observed in mice treated with scrambled peptide compared to SOCS1-KIR. (B) Histological analysis showing substantial numbers of inflammatory cells in the vitreous (black arrows), retinal folds (asterisk), photoreceptor cell lose (blue arrows) in retina of mice treated with scrambled peptide compared to SOCS1-KIR. The sections were stained with hematoxylin and eosin. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor layer; OpN, optic nerve.

Figure 3. SOCS1-KIR protects retinal function during uveitis

ERG analysis of the retina of mice treated with scrambled peptide or SOCS1-KIR on day 14 (A) or day 20 (B) after EAU induction. The averages of light- or dark-adapted ERG b-wave amplitudes are plotted as a function of flash luminance and values are means ± SEM from 4 animals in each group.

Figure 4. SOCS1-KIR suppresses trafficking of inflammatory cells into the retina

(A) Retinae of mice treated with scrambled peptide or SOCS1-KIR were isolated on day 21 post-immunization, digested with collagenase and analyzed for the expression of IL-23 (IL-12p40, IL-23p19) or IL-12 (IL-12p35, IL-12p40) by qRT-PCR. (B) Flow cytometric analysis of inflammatory cells in the retina on day 21 after EAU induction. Numbers in quadrants indicate percentage of CD45^+, CD3⁺ and/or CD11c⁺ cells in the retinae during EAU. Cells isolated from the spleen and LN of mice with EAU were re-stimulated in vitro with IRBP in medium containing SOCS1 KIR or scrambled peptides and analyzed for the expression integrins and chemokine receptors by qPCR (C) or FACS (D). Plots in (D) were gated on CD4⁺ T cells and numbers in quadrants indicate percent of CD4⁺ T cells expressing CD29, CD49d, CXCR3, CD11a, CCR6, or a4β7. Data are representative of three independent experiments.

Figure 5. SOCS1-KIR inhibits proliferation of uveitogenic T cells and up-regulates IL-10

Cells isolated from the spleen and LN of mice with EAU were re-stimulated in vitro with IRBP in medium containing SOCS1 KIR or scrambled peptide and analyzed by Thymidine-incorporation assay (A), intracellular cytokine staining assay (B), qPCR (C) or ELISA (D). Proliferative responses were analyzed in six replicate cultures and data presented as CPM and indicate the mean values of six replicate cultures (A) and numbers in quadrants indicate percentage of IL-17-expressing CD4⁺ T cells (B). Results represent at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test (two tailed)).

Figure 6. SOCS1-KIR has no effects on systemic immune response

(A) LN cells from unimmunized mice or mice immunized with IRBP and treated with SOCS1-KIR or scrambled peptide were analyzed by the intracellular cytokine staining assay. Numbers in quadrants indicate percent of CD4⁺ or CD8⁺ T cells expressing IL-17 or IFN-γ, Foxp3 or IL-10. (B–D) LN cells from unimmunized mice or mice immunized with IRBP and treated with SOCS1-KIR or scrambled peptide were analyzed by qPCR for the expression of chemokine or chemokine receptors (B), apoptotic proteins (C) and cytokines (D). (E) LN cells from mice described in (A) were re-stimulated with IRBP or ConA and proliferative responses were analyzed by the thymidine incorporation assay. Results represent at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test (two tailed).