B7‐H4 Inhibits the Development of Primary Sjögren’s Syndrome by Regulating Treg Differentiation in NOD/Ltj Mice

Abstract

Background . This study is aimed at exploring the role of B7‐H4 in the pathogenesis of primary Sjögren’s syndrome (pSS) in NOD/Ltj mouse. Methods . B7‐H4 expression in salivary glands was examined by IHC, and lymphocyte infiltration was showed by H&E. Next, anti‐B7‐H4 mAb or immunoglobulin isotype was injected into NOD/Ltj mice. Cytokine levels were measured by quantitative RT‐PCR, and immunoglobulins were measured by ELISA. T cell subsets were analyzed by flow cytometry. Last, we treated NOD/Ltj mice with B7‐H4Ig and control Ig. CD4+Foxp3+ T cells were assessed by immunohistochemistry. Two‐tailed Student’s t ‐tests were used to detect the statistical difference in various measures between the two groups. Results . B7‐H4 expression was remarkably reduced in salivary glands of NOD/Ltj mice at 15 weeks compared with the NOD/Ltj mice at 8 weeks. Anti‐B7‐H4 mAb treatment increased lymphocyte infiltration in salivary glands. Inflammatory cytokines including IL‐12, IL‐18, IL‐1 α , TNF‐ α , IFN‐ α , and BAFF were upregulated markedly in anti‐B7‐H4 mAb‐treated mice compared to IgG isotype‐treated mice. Flow cytometry analysis showed that anti‐B7‐H4 mAb‐treated mice had lower levels of CD4+Foxp3+/CD4+ T cells in spleen. Moreover, Foxp3 mRNA levels of salivary glands were diminished in anti‐B7‐H4 mAb‐treated mice. Flow cytometry analysis showed that anti‐B7‐H4 mAb inhibited CD4+Foxp3+/CD4+ T cell production, while B7‐H4Ig would promote naïve CD4+ T into Treg differentiation. Administration with B7‐H4Ig displayed significantly decreased lymphocyte infiltration in salivary glands and low levels of total IgM and IgG in serum. Analysis of inflammatory cytokines in salivary glands after B7‐H4Ig treatment revealed that the mRNA levels of IL‐12, IL‐6, IL‐18, IL‐1 α , TNF‐ α , and IFN‐ α were significantly downregulated in B7‐H4Ig‐treated mice compared to control Ig treatment. B7‐H4Ig‐treated mice had significantly higher levels of CD4+Foxp3+/CD4+ T cells in spleen. IHC in salivary gland revealed that CD4+Foxp3+ T cells of B7‐H4Ig treatment mouse were more than control Ig treatment. Conclusions . Our findings implicate that B7‐H4 has a protective role for salivary gland epithelial cells (SGECs) and therapeutic potential in the treatment of pSS.