Yi Hsing Chen1,2,3, Malihe Eskandarpour1, Xiaozhe Zhang1, Grazyna Galatowicz1, John Greenwood1,4, Sue Lightman1,5, Virginia Calder6,7. 1. UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK. 2. Department of Ophthalmology, Chang Gung Memorial Hospital, Taoyuan, Taiwan. 3. College of Medicine, Chang Gung University, Taoyuan, Taiwan. 4. Moorfields Eye Hospital and UCL Biomedical Research Centre, London, UK. 5. Moorfields Eye Hospital, London, UK. 6. UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK. v.calder@ucl.ac.uk. 7. Moorfields Eye Hospital and UCL Biomedical Research Centre, London, UK. v.calder@ucl.ac.uk.
Abstract
BACKGROUND: The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. METHODS: Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. RESULTS: There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. CONCLUSIONS: This α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.
BACKGROUND: The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. METHODS:Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. RESULTS: There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. CONCLUSIONS: This α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.
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