Everolimus improves experimental autoimmune uveoretinitis

Abstract

The efficacy and action mechanism of everolimus in the treatment of experimental autoimmune uveoretinitis (EAU) was analyzed. Disease was induced in B10.RIII mice by immunization with human interphotoreceptor-retinoid-binding protein peptide 161-180 (hIRBPp161-180). Everolimus was administered by oral gavage (5 mg/kg/d) beginning either two days before or 14 days after immunization. Everolimus significantly reduced the histopathological uveitis score compared to sham-treated mice. To examine the effect on the antigen-specific immune response, proliferation ([³H]-thymidine test) and delayed-type hypersensitivity (DTH) response were measured. Furthermore, content of T-helper-1, -2, and -17 cytokines were analyzed intraocularly (Bead Array) and in cell culture supernatants from splenocytes (sandwich ELISA). To study the effect on the humoral immune response the presence of antigen-specific serum antibodies was tested (indirect ELISA). The DTH, the humoral immune response, the proliferation of splenocytes and the intraocular Th1, Th2, Th17 cytokine content and in vitro production of Th1 and Th17 cytokines were impaired after everolimus treatment. The study of CD4+CD25+FoxP3+ regulatory T cells (T_reg) in peripheral blood, draining lymph nodes, and spleen by flow cytometry showed an increased number of splenic T_reg in mice of the everolimus therapy group. Furthermore the T_reg of these mice had a higher suppressive capacity than cells from sham-treated mice. These results indicate that the immunosuppressive effect of everolimus on EAU was associated with the suppression of pathogenic effector responses and induction of regulatory T cells.

Introduction

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease (Atalla et al., 1990) which serves as a model for noninfectious posterior uveitis in humans (Forrester et al., 1990). EAU can be induced in rats and mice by injecting a retinal antigen such as retinal S-antigen or interphotoreceptor retinoid-binding protein (IRBP) (Caspi et al., 1990, 1988; Chan et al., 1990; Mochizuki et al., 1985; Xu et al., 2000). Here, the ocular inflammation peaks 14 days after immunization (Abbas et al., 2007; Caspi et al., 1986; Jiang et al., 1999) and is characterized by a massive leukocyte infiltration (Caspi et al., 1988). The infiltrate consists mainly of CD4⁺ T cells, which mediate disease initiation. With disease exacerbation, mainly activated macrophages and granulocytes invade the ocular tissue (Caspi et al., 1993). Further histological features of EAU are vasculitis, retinal granuloma formation, folding and detachment of the retina, and destruction of the photoreceptors (Caspi et al., 1988).

The antigens responsible for the development of human uveitis have not yet been identified. Nevertheless, as patients with posterior uveitis often show a strong cellular immune response to the retinal antigens that are used for EAU induction in experimental animal models, the observations from rodent EAU models can possibly be applied to human disease (Chan et al., 1990).

Patients with endogenous, noninfectious uveitis are currently first treated with topical or systemic corticosteroids. If the disease is refractory to this treatment, additional immunosuppressive agents are used (e.g. antimetabolites, T-cell inhibitors, and alkylating agents) (Jabs et al., 2000). The mTor inhibitor rapamycin has demonstrated efficacy in preventing intraocular inflammation from developing in a rat EAU model (Roberge et al., 1993). Inhibition of mTor blocks the growth factor-driven signal transduction in T- and B-cells, resulting in cell cycle arrest in G1 phase (Schuler et al., 1997; Schuurman et al., 1997). In addition to suppressing effector T cells, rapamycin induces the de novo generation of T_reg (Battaglia et al., 2005; Valmori et al., 2006). This could be an advantage for the treatment of autoimmune diseases with known malfunction or dysregulation of T_reg (Haxhinasto et al., 2008; Kang et al., 2008; Sauer et al., 2008; Zeiser et al., 2008).

Everolimus (mTor inhibitor), a derivate of rapamycin, has a higher bioavailability, reaches the steady-state level faster without a loading dose, and has a shorter half-life than rapamycin (Schuler et al., 1997). Its immunosuppressive efficacy has also been demonstrated in transplant studies, showing prolonged allograft survival in primates after lung transplantation (Hausen et al., 2000a, 2000b; Schuurman et al., 2000; Schuurman et al., 1998) and in clinical studies for heart transplantation (Ross et al. 2010). Therefore, we hypothesized that everolimus may also be suitable for treating T-cell-mediated uveitis.

In the present study we analyzed the efficacy of everolimus for the prevention and treatment of EAU in mice. Furthermore, the inhibitory capacity of everolimus therapy on uveitogenic effector cells and the induction of CD4+CD25+FoxP3+ T cells were tested.