Inhibition of experimental auto-immune uveitis by the A3 adenosine receptor agonist CF101

Abstract

Uveitis is an inflammation of the middle layer of the eye with a high risk of blindness. The Gi protein associated A3 adenosine receptor (A3AR) is highly expressed in inflammatory cells whereas low expression is found in normal cells. CF101 is a highly specific agonist at the A3AR known to induce a robust anti-inflammatory effect in different experimental animal models. The CF101 mechanism of action entails down-regulation of the NF-κB-TNF-α signaling pathway, resulting in inhibition of pro-inflammatory cytokine production and apoptosis of inflammatory cells. In this study the effect of CF101 on the development of retinal antigen interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveitis (EAU) was assessed. Oral treatment with CF101 (10 µg/kg, twice daily), initiated upon disease onset, improved uveitis clinical score measured by fundoscopy and ameliorated the pathological manifestations of the disease. Shortly after treatment with CF101 A3AR expression levels were down-regulated in the lymph node and spleen cells pointing towards receptor activation. Downstream events included a decrease in PI3K and STAT-1 and proliferation inhibition of IRPB auto-reactive T cells ex vivo. Inhibition of interleukin-2, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production and up-regulation of interleukin-10 was found in cultured splenocytes derived from CF101-treated animals. Overall, the present study data point towards a marked anti-inflammatory effect of CF101 in EAU and support further exploration of this small molecule drug for the treatment of uveitis.

Figure 1

Inhibition of EAU by CF101: fundoscopy. Mice were immunized with a uveitogenic regimen of IRBP and treated with CF101 from Day 7 after immunization. Eyes were examined for disease after pupil dilation under a binocular microscope. Shown are results on Day20 following immunization.

Figure 2

Inhibition of EAU by CF101: histopathology. Mice were immunized with a uveitogenic regimen of IRBP and treated with CF101 from Day 7 after immunization. On Day 20 eyes were collected, fixed and tissue sections were stained with hematoxylin and eosin. (A) Histopathology scores. (B) Pictures of representative slides.

Figure 3

A₃AR down-regulation upon CF101 treatment. Mononuclear cells from draining lymph nodes and splenocytes were collected from EAU mice upon treatment with vehicle or with CF101. Cell protein extracts were subjected to western blot analysis.

Figure 4

Modulation of signal transduction proteins upon CF101 treatment. Mononuclear cells from draining lymph nodes were collected from EAU mice upon treatment with vehicle or CF101. Protein cell extracts were subjected to western blot analysis. PI3K and STAT-1 were markedly down-regulated upon CF101 treatment.

Figure 5

CF101 treatment inhibits the ex vivo proliferation of antigen spe -cific lymphocytes. Draining lymph nodes were collected from vehicle and from the CF101-treated IRBP immunized mice, The cells were cultured for 48 h in the presence of different IRBP concentrations (0.2–20 μg/ml) and proliferation was evaluated by an ³[H]-thymidine incorporation assay.