Interleukin 27 induces the expression of complement factor H (CFH) in the retina

Abstract

Complement factor H (CFH) is a central regulator of the complement system and has been implicated in the etiology of age-related macular degeneration (AMD), a leading cause of blindness in the elderly. In view of previous studies showing that reduced expression of CFH in the retina is a risk factor for developing AMD, there is significant interest in understanding how CFH expression is regulated in the retina. In this study, we have shown that the anti-inflammatory cytokine, IL-27, induced CFH expression in mouse retinal cells and human retinal pigmented epithelial cells (RPE) through STAT1-mediated up-regulation of Interferon Regulatory Factor-1 (IRF-1) and IRF-8. We further show that cells in the ganglion and inner-nuclear layers of the retina constitutively express IRF-1 and IRF-8 and enhanced CFH expression in the retina during ocular inflammation correlated with significant increase in the expression of IRF-1, IRF-8 and IL-27 (IL-27p28 and Ebi3). Our data thus reveal a novel role of IL-27 in regulating complement activation through up-regulation of CFH and suggest that defects in IL-27 signaling or expression may contribute to the reduction of CFH expression in the retina of patients with AMD.

Figure 1. CFH is constitutively expressed in the retina and up-regulated in RPE cells by IL-27 through STAT1-dependent mechanism.

(A) Immunohistochemical localization of CFH expression in the mouse retina. Retina pigmented epithelium (RPE); Photoreceptors (pho); outer nuclear layer (ONL); outer plexiform layer (OPL); inner plexiform layer (IPL). (B) Whole cell extracts prepared from the retina or liver of C57BL/6 mice were analyzed for expression of CFH by Western blot assay. (C) ARPE-19 cells were stimulated for 24 hours in medium containing human IL-27 (10 ng/ml). ARPE-19 cells were cultured overnight in complete medium, washed and starved in serum free medium (SFM) for 2 hours and then stimulated in SFM containing human recombinant IL-27 (10 ng/ml) for 24 hours (C, D, E) or 30 minutes (F). (C) RNA was isolated and analyzed by qRT-PCR. (D) The ARPE-19 cells were stimulated with several concentrations of human IL-27 and then subjected to qRT-PCR analysis. (E, F) Whole cell extracts were analyzed by Western blot assay.

Figure 2. IL-27 induces CFH expression in the retina through STAT1-dependent mechanism.

(A) Characterization of retina from mice with specific deletion of STAT3 in the retina (Ret-STAT3^−/−). (B) Freshly isolated retinas from wild type (WT), STAT1 knockout (STAT1^−/−), (Ret-STAT3^−/−), or from mice with targeted over-expression of SOCS1 in the retina (SOCS1Tg) were analyzed by RT-PCR. (C, D) Freshly isolated retinal cells from WT or STAT1^−/− mice were cultured overnight, washed, starved for 2 hours in serum-free medium and then cultured for an additional 22 hours in medium containing IL-27 (10 ng/ml). We analyzed RNA and total cell extracts from the cells by RT-PCR (C) or Western blotting (D).

Figure 3. IL-27 induces expression of CFH through STAT1-mediated up-regulation of IRF-1 and IRF-8.

(A–C) ARPE-19 cells were starved for 2 hours in serum free medium and cultures were then stimulated with IL-27 (10 ng/ml) for 30, 90 or 720 min. (B–D) In other cultures, cells were treated with CHX (35 ug/ml) for 30 min prior treatment with IL-27 for 30 min (B) or 24 h (C, D). Whole cell extracts were analyzed by Western blot assay and the antibodies used are indicated. (D) CFH mRNA transcripts were detected and quantified by qRT-PCR.

Figure 4. siRNA-mediated silencing of IRF-1 or IRF-8 abrogated the induction of CFH by IL-27.

ARPE-19 cells were stimulated with human IL-27 in the presence or absence of control siRNA, IRF-1 siRNA or IRF-8 siRNA. (A) Detection of CFH expression by Western blot analysis. (B) Blots were analyzed and relative levels of CFH protein were quantified by densitometry; samples were normalized to the GAPDH expression level.

Figure 5. Expression of IRF-1, IRF-8 and IL-27 (IL27p28/EBi3) by retinal cells was up-regulated during experimental autoimmune uveitis (EAU).

(A) Fundus images of the retina of un-immunized mouse and mouse with EAU. (B, C) Retinas were isolated from control or EAU mice and analyzed for IRF-1, IRF-8, CFH, IL27p28 or EBi3 expression by qRT-PCR.

Figure 6. Detection and localization of IRF-1 and IRF-8 proteins in the retina.

Retinas were isolated from control or EAU mice and whole cell extracts were analyzed for IRF-1 (A) or IRF-8 expression by Western blot analysis. (C) Immunohistochemical localization of IRF-8 expression was detected in the normal mouse retina or retina of mice with EAU using an IRF-8-specific antibody as described in Methods section. Retina pigmented epithelium (RPE); Photoreceptors (pho); outer nuclear layer (ONL); outer plexiform layer (OPL); inner plexiform layer (IPL).