Downregulation of cathepsin G reduces the activation of CD4+ T cells in murine autoimmune diabetes

Abstract

Type 1 diabetes mellitus (T1DM) is an autoimmune disease due to progressive injury of islet cells mediated by T lymphocytes (T cells). Our previous studies have shown that only cathepsin G (CatG), not other proteases, is involved in the antigen presentation of proinsulin, and if the presentation is inhibited, the activation of CD4+ T cells induced by proinsulin is alleviated in T1DM patients, and CatG-specific inhibitor reduces the activation of CD4+ cells induced by proinsulin in T1DM patients. Therefore, we hypothesize that CatG may play an important role in the activation of CD4+ T cells in T1DM. To this end, mouse studies were conducted to demonstrate that CatG impacts the activation of CD4+ T cells in non-obese diabetic (NOD) mice. CatG gene expression and the activation of CD4+ T cells were examined in NOD mice. The effect of CatG inhibitor was investigated in NOD mice on the activation of CD4+ T cells, islet β cell function, islet inflammation and β-cell apoptosis. Furthermore, NOD mice were injected with CatG siRNA in early stage to observe the effect of CatG knockdown on the activation status of CD4+ T cells and the progression of diabetes. During the pathogenesis of diabetes, the expression level of CatG in NOD mice gradually increased and the CD4+ T cells were gradually activated, resulting in more TH1 cells and less TH2 and Treg cells. Treatment with CatG-specific inhibitor reduced the blood glucose level, improved the function of islet β cells and reduced the activation of CD4+ T cells. Early application of CatG siRNA improved the function of islet β cells, reduced islet inflammation and β cell apoptosis, and lowered the activation level of CD4+ T cells, thus slowing down the progression of diabetes.

Keywords: CD4+ T cells; NOD mice; cathepsin G; type 1 diabetes mellitus.

Figure 1

Gene expression of CatG and the activation of CD4+ T cells in NOD mice. A. mRNA level of CatG; B. Upper panel: cytometry assay of T cells, lower panel: percentage of CD4+ T subsets. ** and *** denote P < 0.01 and 0.001 vs normal.

Figure 2

Effect of cathepsin G inhibitor on blood glucose, insulin levle and islet β cell apoptosis in NOD mice. A. Blood glucose level over time; B. Serum insulin level over time, C. Islet H&E staining; D. Flow cytometry assay of apoptotic cells and apoptotic rate. ** Denotes P < 0.01 vs control.

Figure 3

Effect of cathepsin G inhibitor on the activation of CD4+ T cells in NOD mice. A. Cytometry assay of T cells and percentage of CD4+ T subsets in spleen and peripheral blood; B. mRNA level of CatG. **P < 0.01 vs control.

Figure 4

Effect of CatG siRNA on the blood glucose, insulin levels and β cell apoptosis in NOD mice. A. Relative GatG mRNA level; B and C. Blood glucose and insulin levels over time; D. Islet H&E staining; E. Flow cytometry assay and percentage of apoptosis. ** Denotes P < 0.01 vs control.

Figure 5

Effect of CatG siRNA treatment on the activation of CD4+ T cells in pre-DM NOD mice. Upper panel: cytometry assay of T cells, lower panel: percentage of CD4+ T subsets in spleen and peripheral blood. ** Denotes P < 0.01 vs control.