Involvement of Th17 cells and the effect of anti-IL-6 therapy in autoimmune uveitis

Abstract

Objectives: Human endogenous uveitis is one of the sight-threatening diseases associated with variety of systemic disorders, such as Behcet's disease and sarcoidosis. Recently, biosynthesized antibodies against inflammatory cytokines have been recognized to be useful to control the regional inflammation. In this study, we focused on the possibility of IL-6-based biological therapies for endogenous uveitis. We initially confirmed the significant increase of several inflammatory soluble factors including IL-6 in the vitreous fluids from refractory/chronic engogenous uveitis patients.

Methods: To investigate the role of IL-6 in the formation of refractory ocular inflammation, we used the mouse experimental autoimmune uveitis (EAU) model. Both IL-6 and IL-23 are required for the development of IL-17-producing helper T subset (Th17) from naïve CD4(+) T cells. Results. In the EAU model, neither IL-6-deficient mice nor IL-23-deficient mice could induce Th17 cells and the EAU score was decreased in these mice in the entire time course. We also confirmed that systemic administration of anti-il-6 receptor antibody ameliorates EAU By suppressing both systemic and regional TH17 responses.

Conclusions: IL-6 is responsible for causing ocular inflammation, and it is, at least partially, due to IL-6-dependent Th17 differentiation. IL-6 may be a target for therapy of refractory endogenous uveitis in humans.

Fig. 1.

Concentrations of IL-6, IL-8 and MCP-1 in the vitreous fluid. Vitreous samples were collected from patients with non-proliferative control diseases (epiretinal membrane and macular hole: n = 83) and chronic uveitis (Behcet's disease, sarcoidosis, Vogt–Koyanagi–Harada disease and unknown uveitis: n = 35). Concentrations of indicated cellular mediators were analysed. *P < 0.01.

Fig. 2.

EAU in IL-6-deficient and IL-23p19-deficient mice. (A) Clinical scores of EAU in IL-6-deficient mice. WT B6 mice (open circles; n = 24) and IL-6-deficient mice (closed circles; n = 24) were immunized with IRBP as described in Materials and methods section, and from Day 10, disease severity was examined. The values represent means ± s.e.m. *P < 0.05. The data represent combined results of three independent experiments. (B) Clinical scores of EAU in IL-23p19-deficient mice. WT B6 mice (open triangles; n = 14) and IL-23p19-deficient mice (closed triangles; n = 14) were immunized as above, and from Day 10, disease severity was examined. The values represent means ± s.e.m. *P < 0.05. The data represent combined results of three independent experiments. (C and D) Typical pictures of histological sections in WT B6 mice (left) and IL-6-deficient mice (C, right) and IL-23p19-deficient mice (D, right). The arrows indicate inflammatory cell-infiltration sites.

Fig. 3.

IRBP-specific cytokine production by LN cells of WT B6, IL-6-deficient and IL-23p19-deficient mice. On Days 11 and 21, LN from WT B6 mice, IL-6-deficient mice and IL-23p19-deficient mice were collected. Purified CD4⁺ T cells were cultured with 10 μg/ml of IRBP and irradiated (20 Gy) WT APCs. After 48 h, supernatants were analysed for IFN-γ or IL-17 production by ELISA. The values represent means ± s.d. *P < 0.05.

Fig. 4.

Effect of administrating IL-6RAb (MR16-1) in EAU. (A) Comparison of the funduscopic severity between control Ig-treated (open circles; n = 10) and MR16-1-administered (closed circles; n = 10) mice. Each antibody was administered intravitreously on Day 7 after IRBP immunization. At Day 14, funduscopic examinations of the mice were performed. (B) Intraperitoneal administration protocols of MR16-1 and control Abs. Control IgGs and MR16-1 at the dose of 10 μg/g body weight were administered intrapenitoneally four times on days 0, 3, 9 and 15 after IRBP immunization. (C) EAU symptoms in the eyes were evaluated 18 days after IRBP immunization. Data shown are control IgGs-treated (open triangles; n = 13) and MR16-1-administered (closed triangles; n = 12). *P < 0.05 (D) Photomicrographs of EAU eye. Typical pictures of ocular fundus of the eye are shown. The EAU scores are indicated in the fundus photographs. (E) Photomicrographs of histological sections of the eye. Typical pictures of histological sections in control Ig-administered mice (left) and MR16-1-administered mice (right). The arrows indicate inflammatory cell infiltration sites. Re: retina.

Fig. 5.

IRBP-specific cytokine production and Foxp3 expression by LN cells of MR16-1-administered WT B6 mice. On Day 19, LN cells from control IgG-administered mice or MR16-1-administered mice were collected. (A) Purified CD4⁺ T cells were cultured with 10 μg/ml of IRBP and irradiated (20 Gy) WT APCs. After 48 h, supernatants were analysed for IFN-γ or IL-17 production by ELISA. (B) Foxp3-positive CD4⁺ T was analysed by FACS (n = 7). The values represent means ± s.d. *P < 0.05.

Fig. 6.

Cytokines and chemokines in ocular fluid of EAU mice. (A) On Day 18, ocular fluid protein from WT B6 mice (n = 8), IL-6-deficient mice (n = 7) and IL-23p19-deficient mice (n = 8) was collected and analysed. (B) On Day 19, ocular fluid protein from eyes of control IgG-administered mice (n = 7) and MR16-1-administered mice (n = 7) was prepared, then the concentrations of indicated cytokine/chemokine production were analysed. The values represent mean ± s.d. *P < 0.05.