A20 Inhibits Intraocular Inflammation in Mice by Regulating the Function of CD4+T Cells and RPE Cells

Abstract

A20 is a negative regulator of inflammation and immunity and plays a role in several autoimmune and inflammatory diseases. Here, we demonstrate that A20 overexpression significantly ameliorates severity of EAU by inhibiting the infiltration of Th1 and Th17 cells, and by protecting integrity of the blood retinal barrier. In vitro studies showed that A20 silencing could promote CD4+T cells toward a Th1 and Th17 phenotype. A decreased expression of A20 in CD4+T cells was noticed in active BD patients but not in VKH patients. Furthermore, silencing of A20 in hRPE cells induced the production of IL-6, IL-8, and MCP-1 and downregulated ZO-1 and occludin expression which is mediated by inhibition of MAPK and NF-κB pathways. This study reveals a mechanism by which A20 prevents autoimmune uveitis.

Keywords: A20; Behcet’s disease; CD4+T cells; EAU; blood-retinal-barrier.

Figure 1

A20 overexpression in the intraocular tissue by AAV-TNFAIP3 injection. (A) Relative mRNA expression of A20 in the retina and choroid-complex from EAU mice in different periods by RT-qPCR (n≥4 per group). (B) The protein level of A20 in the retina and choroid-complex from EAU mice in different periods was tested by Western-blot. (C) The expression of A20 in the retina after injecting AAV-TNFAIP3 was tested by RT-qPCR (n=4 per group). (D) The expression of A20 in the other organs after injecting AAV-TNFAIP3 was tested by RT-qPCR (n=4 per group). (E) The protein level of A20 in the retina after injecting AAV-TNFAIP3 was detected by immunoblot. Data are shown as mean ± SD. One-way ANOVA was used for statistical analyis, ns p<0.05, *p<0.05, **p<0.01, ***p<0.001.

Figure 2

A20 overexpression in early onset suppressed the severity of EAU. (A) Both eyes were injected with AAV-TNFAIP3 or AAV-GFP, EAU was induced by IRBP161-180 immunization fourteen days later. (B) Representative slit-lamp images of EAU mice at the 14^th day after immunization from the frontal and lateral view. (C) Representative histological image is shown of eyes harvested at the 14th day after immunization. (D) The clinical scores were measured every two days from day 6 after IRBP immunization. The clinical score at the peak of EAU peak (day 14) is shown in the right panel (n≥6 per group, p value was compared between AAV-TNFAIP3 group and AAV-GFP group). (E) Histological scores were assessed by hematoxylin and eosin (H&E) staining of paraffin-embedded sections. (F) Relative mRNA expression of Th1 and Th17 cytokines IFN-γ and IL-17 by RT-qPCR (n≥4 per group). (G) Relative mRNA expression of transcription factors T-bet, RORγT, GATA3, and FOXP3 in CD4+T cells by RT-qPCR (n≥4 per group). Scale bar = 50 μm. Data were shown as mean ± SD. One-way ANOVA was used, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3

A20 expression was decreased in CD4+T cell from BD patients and A20 knockdown shifted CD4+T cells toward Th1 and Th17 phenotype. (A) Immunoblot showing A20 protein inhibition in CD4+T cells treated with A20 siRNA. (B) Representative dot plots gated on CD4+ T cells showing IFN-γ and IL-17 expression. (C) FACS analysis of the IL-17A expression in activated CD4+ T after A20 was inhibited by siRNA (n=8). (D) The production of IL-17 and IFN-γ by A20 knockdown CD4+T cells was tested by ELISA (n=8). (E) The mRNA expression of A20 in CD4+T cells from BD patients and VKH patients was tested by RT-qPCR. (F) Spearman’s rank correlation coefficient of mRNA expression of A20 vs IL17 and A20 vs IFN-γ (n=24). Data are shown as mean ± SD. One-way ANOVA, Spearman test, Mann Whitney test and paired -T test was used, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4

A20 overexpression 14 days prior to IRBP immunization inhibited intraocular inflammation and maintained blood-retinal-barrier (BRB) integrity and tight junction protein expression. BRB integrity of EAU mice was tested by Evans-blue. (A) Representative Evans-blue image of the retina from EAU mice in whom A20 overexpression was induced 21 days before IRBP immunization. (B) Representative immunostaining of Occludin in retinal sections of EAU mice in whom A20 overexpression was induced 21 days before IRBP immunization. (C) Representative Immunostaining of ZO-1 in retinal sections of EAU mice in whom A20 overexpression was induced 21 days before IRBP immunization. (D) Western blot tested the expression of ZO-1 and Occludin in the retinal of EAU mice in whom A20 overexpression was induced 21 days before IRBP immunization. (E) The expression of ZO-1 and Occludin was tested by Western blot. (F) Relative mRNA expression of the cytokines IL-6, MCP-1, IL-1β, and TNF-α by RT-qPCR (n≥4 per group). (G) The mRNA expression of A20 in TNF-α-induced stimulation of hRPE cells was tested by RT-qPCR (n=4 per group). (H) A20 protein inhibition in hRPE cells by A20 siRNA as tested by immunoblot. (I) The expression of IL-6, IL-8, and MCP-1 in culture supernatants was measured by ELISA (n=6 per group). Scale bar = 50 μm. Data are shown as mean ± SD. One-way ANOVA and student’s t test was used, ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5

A20 regulates inflammation via MAPK and NF-κb pathways in vitro and in vivo. (A) Immunoblot tested the activation of the NF-κb pathway in TNF-α stimulated hRPE cells following A20 siRNA treatment in vitro. (B) Immunoblot tested the activation of the MAPK pathway in TNF-α stimulated hRPE cells following A20 siRNA treatment. (C) Immunoblot analysis testing the activation of the NF-κb pathway in the retina of EAU mice following A20 overexpression in vivo. (D) Immunoblot analysis testing the activation of the MAPK pathway in the retina of EAU mice following A20 overexpression in vivo.