Blockade of interleukin-6 signaling suppresses experimental autoimmune uveoretinitis by the inhibition of inflammatory Th17 responses,☆☆

https://doi.org/10.1016/j.exer.2010.04.009Get rights and content

Abstract

The aim of this study was to investigate the effect of anti-mouse IL-6 receptor monoclonal antibody (MR16-1) treatment on CD4 T cell differentiation and compared it to the effect of anti-TNF mAb treatment with using a murine model of experimental autoimmune uveoretinitis (EAU). C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) to induce ocular inflammation treatment with control IgG or MR16-1 or anti-TNF mAb. Helper T cells differentiation was analyzed during the development of EAU. Immunization with IRBP increased the frequency of Th17 cells rather than Th1 cells in the early stage of EAU. Treatment with MR16-1 on the same day as immunization (day 0) or one day after (day 1) suppressed ocular inflammation in EAU mice. Treatment with MR16-1 on day 0 inhibited the induction of Th17 cells in vivo, and inhibited not only IRBP-responsive Th17 cells but also their Th1 counterparts and induced IRBP-responsive regulatory T (Treg) cells in vitro. The administration of anti-TNF mAb had no significant protective effect in EAU mice. The protective effect of anti-IL-6R mAb treatment, but not anti-TNF mAb treatment on EAU correlated with the inhibition of Th17 differentiation. This finding suggests that IL-6 blockade may have a therapeutic effect on human ocular inflammation which is mediated via mechanisms distinct from those of TNF blockade. IL-6 blockade may thus represent an alternative therapy for patients with ocular inflammation who are refractory to anti-TNF mAb therapy.

Introduction

Experimental autoimmune uveoretinitis (EAU) serves as a model for several ocular inflammatory diseases in humans with a putative autoimmune etiology (e.g. Behçet’s disease, Vogt–Koyanagi–Harada disease and sarcoidosis (Wacker et al., 1977, Yamaki et al., 2000)). EAU is induced in genetically susceptible murine strains by immunization with retinal proteins or by the adoptive transfer of T cells specific for these antigens into naive syngenetic recipients (Caspi et al., 1986, Gregerson et al., 1986, Mochizuki et al., 1985, Rizzo et al., 1996, Sanui et al., 1986). Initially the development of EAU was considered to be mediated by activation of Th1 cells (Tarrant et al., 1998, Xu et al., 1997). However, recent studies (Amadi-Obi et al., 2007, Bettelli et al., 2007, Luger et al., 2008, McInnes and Schett, 2007, Park et al., 2005, Yoshimura et al., 2008) have suggested that highly proinflammatory interleukin-17 producing Th17 cells, rather than Th1 cells, are central to the pathology of experimental autoimmune diseases. Several reports (Amadi-Obi et al., 2007, Chi et al., 2007, Yoshimura et al., 2009) also have suggested a pathogenic role for Th17 cells in human uveitis and scleritis.

Interleukin-6 (IL-6) is a potent pleiotropic cytokine that regulates cell growth and differentiation and plays an important role in the immune response (Akira et al., 1993). Recent studies have indicated that IL-6 promotes the differentiation of Th17 cells. In vitro experiments using cultured murine CD4 T cells (Bettelli et al., 2006, Mangan et al., 2006, Veldhoen et al., 2006) have revealed that stimulation of CD4 T cells with IL-6 plus TGF-β potently induces Th17 differentiation, whereas stimulation with TGF-β alone triggers the development of immunosuppressive regulatory T (Treg) cells. Although the precise role of IL-6 in the development of Th17 cells and other helper T cells in vivo is yet to be fully clarified, mice deficient in STAT3 are resistant to both EAU and experimental autoimmune encephalomyelitis and feature a paucity of Th17 cells (Liu et al., 2008), while deficiency of IL-6 in mice suppresses IL-17 production in EAU (Yoshimura et al., 2009). These reported findings thus indicate an important role for IL-6 in the differentiation of Th17 cells in vivo.

Anti-IL-6 receptor monoclonal antibody (anti-IL-6R mAb) is available for clinical use and has demonstrated its therapeutic efficacy in the treatment of severe rheumatoid arthritis. While Yoshimura et al. recently reported that anti-IL-6R mAb treatment could suppress the induction of ocular inflammation in EAU murine models (Yoshimura et al., 2009), it has not yet been demonstrated whether this treatment is also an effective therapy for ocular inflammatory diseases in human. In this study, we used anti-IL-6R mAb to investigate the effect of IL-6 blockade on the development of ocular inflammation and the differentiation of helper T cells in EAU.

Section snippets

Mice

WT C57BL/6 mice were purchased from Japan SLC Inc. (Shizuoka, Japan), and were maintained in specific pathogen-free conditions at Osaka University, Japan. Female mice (8–10 weeks old) were used in all experiments. All animals were treated humanely, and all experiments conformed to the provisions of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

EAU induction and clinical evaluation

C57BL/6 female mice were immunized with 100 μg of human interphotoreceptor

The protective effect of IL-6 receptor blockade in early-phase EAU

We first evaluated the effect of MR16-1 treatment on EAU development in terms of EAU clinical scores. In our model, the onset of ocular inflammation was observed on day 11 ± 1, and ocular inflammation gradually increased thereafter in control mice. Inflammation peaked on day 20 ± 1 after immunization, and then started decrease. MR16-1 treatment suppressed the development of uveitis when administered on day 0 or day 1 after immunization (Fig. 1A and B). Peak EAU clinical scores were

Discussion

This study demonstrated three significant findings regarding the development of EAU and the differentiation of helper T cells in EAU. First, treatment with an anti-IL-6R mAb (MR16-1) effectively suppressed the development and severity of EAU when MR16-1 was administered at the same day or one day after IRBP (1–20) immunization. However, MR16-1 treatment on day 3 or day 5 after immunization failed to suppress EAU. Second, MR16-1 inhibited the induction of Th17 cells in vivo, while it inhibited

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    The authors have no proprietary interest in any aspect of this report.

    ☆☆

    Supported in part by research grants from the Ministry of Education, Science and Culture, Tokyo, Japan.

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