Small-Molecule Antagonist of VLA-4 (GW559090) Attenuated Neuro-Inflammation by Targeting Th17 Cell Trafficking across the Blood-Retinal Barrier in Experimental Autoimmune 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 by 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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Supplementary Fig.1 a Representative gating strategy to identify CD4+ T cell subsets in the retina for Fig.3. CD4+ T cells were first gated for single cells and live cells, and then for their expression of CD4. Within the CD4+ cell region, Th1 and Th17 were distinguished by intracellular expression of IFNγ and IL-17A respectively, within the FoxP3- region. b Representative gating strategy to identify myeloid cell subsets within the retina for Fig 4. Single live cells were gated for CD45 firstly, CD11b and CD11c, then CD64, Ly6G, and finally Ly6C.
Supplementary Fig. 2 Representative flow cytometry figure depicting VLA-4 expression in different CD4+ T cell subsets in healthy control (Ctrl) and EAU eye.
Posted 24 Sep, 2020
On 26 Oct, 2020
Received 21 Oct, 2020
Received 14 Oct, 2020
On 09 Oct, 2020
On 29 Sep, 2020
Invitations sent on 21 Sep, 2020
On 20 Sep, 2020
On 20 Sep, 2020
On 19 Sep, 2020
On 19 Sep, 2020
Small-Molecule Antagonist of VLA-4 (GW559090) Attenuated Neuro-Inflammation by Targeting Th17 Cell Trafficking across the Blood-Retinal Barrier in Experimental Autoimmune Uveitis
Posted 24 Sep, 2020
On 26 Oct, 2020
Received 21 Oct, 2020
Received 14 Oct, 2020
On 09 Oct, 2020
On 29 Sep, 2020
Invitations sent on 21 Sep, 2020
On 20 Sep, 2020
On 20 Sep, 2020
On 19 Sep, 2020
On 19 Sep, 2020
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 by 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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5