Hepatic immunophenotyping for streptozotocin-induced hyperglycemia in mice

Abstract

Emerging evidence revealed that diabetes induces abnormal immune responses that result in serious complications in organs. However, the effect of hyperglycemia on hepatic immunity remains obscure. We evaluated the population and function of hepatic immune cells in streptozotocin (STZ)-induced hyperglycemic mice. CC chemokine receptor 2 (CCR2)-knockout mice and mice with a depletion of regulatory T cells (DEREG) were used to investigate the migration and role of regulatory T cells (Tregs) in hyperglycemic mice. The inflammatory cytokines and hepatic transaminase levels were significantly increased in the hyperglycemic mice. The population and number of infiltrating monocytes, granulocytes, and Tregs were enhanced in the livers of the hyperglycemic mice. Hepatic monocytes other than macrophages showed the increased expression of inflammatory cytokines and chemokines in the hyperglycemic mice. The CCR2 knockout and DEREG chimeric mice exhibited increased populations of activated T cells and neutrophils compared to the WT chimeric mice, which promoted hepatic inflammation in the hyperglycemic mice. The migration of CCR2 knockout Tregs into the liver was significantly reduced compared to the WT Tregs. We demonstrated that hyperglycemia contributes to increase in infiltrating monocytes and Tregs, which are associated with hepatic immune dysfunction in mice. CCR2-mediated migration of Tregs regulates hyperglycemia-induced hepatic inflammation.

Figure 1. STZ-induced hyperglycemia increases monocytes and neutrophils in the mouse liver.

Hyperglycemia was induced in C57BL/6 mice with low dose STZ (50 mg/kg) administration for five consecutive days. (a) IFN-γ, and TNF-α levels were measured in sera collected from mice treated with the vehicle or STZ. (b) AST, ALT, triglyceride, and TC were measured in sera collected from the mice. (c) T cell population and activation in mouse peripheral blood mononuclear cells. (d) Liver mononuclear cells were subjected to flow cytometry to analyze lymphocytes, monocytes, and neutrophils. (e) Percentages of hepatic immune cells in mice treated with the vehicle or STZ. All data are representative of 3 independent experiments (n = 6 per group). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 compared with the corresponding controls.

Figure 2. STZ-induced hyperglycemia promotes CCR2-mediated migration of Tregs in the liver.

(a) The population of hepatic monocytes and macrophages in mice treated with the vehicle or STZ. (b) The population of hepatic monocytes in mice treated with the vehicle or STZ. (c,d) The population and absolute number of Tregs in the mesenteric lymph nodes of mice treated with the vehicle or STZ. (e,f) The population and absolute number of Tregs in the livers of mice treated with the vehicle or STZ. (g) CCR2 expression in hepatic Tregs from mice treated with the vehicle or STZ. (h) Glucose, AST, and ALT were measured in sera collected from the mice treated with saline or insulin. (i,j) The population of hepatic monocytes, macrophages, and Tregs in the STZ-induced hyperglycemic mice treated with the saline or insulin. All data are representative of 3 independent experiments (n = 6 per group). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 compared with the corresponding controls.

Figure 3. STZ-induced hyperglycemia enhances the expression of proinflammatory cytokines and chemokines in monocytes but not liver resident macrophages.

(a) Morphological analysis of hepatic monocytes and macrophages using FACS. (b) Sorted hepatic monocytes and macrophages from mice treated with vehicle or STZ were subjected to real-time PCR analysis. (c) Sorted hepatic neutrophils from mice treated with vehicle or STZ were subjected to real-time PCR analysis. (d) HSCs and hepatocytes isolated from mice treated with vehicle or STZ were subjected to real-time PCR. (e) HSCs were treated with STZ in vitro. All data are representative of 3 independent experiments (n = 6 per group). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 compared with the corresponding controls.

Figure 4. CCR2-mediated migration of Tregs regulates hyperglycemia-induced hepatic inflammation.

(a) At week 8 after the transplantation of WT, CCR2 KO, and DEREG bone marrow into WT mice, the chimeric mice were injected with diphtheria toxin and STZ. (b) AST, ALT, triglyceride, and TC levels were measured in sera collected from the mice. (c) IFN-γ and TNF-α were measured in sera collected from the three groups of mice. (d,e) Hepatic T cell population and absolute number in the three groups of mice. (f) IFN-γ⁺ T cell population in the livers of in WT, CCR2 knockout, and DEREG mice. All data are representative of 3 independent experiments (n = 6–8 per group). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 compared with the corresponding controls.

Figure 5. CCR2 is critical for the migration of Tregs into the livers of hyperglycemic mice.

(a,b) The population and absolute number of hepatic monocytes and neutrophils. (c) CCR2 expression in hepatic monocytes from WT and DEREG mice. (d) FACS analysis of infiltrating hepatic monocytes in WT, CCR2 knockout, and DEREG mice. (e) Tregs in the livers of WT and CCR2 chimeric mice. (f) Tregs in the mesenteric lymph nodes of WT and CCR2 chimeric mice. (g) Schematic model for the ex vivo closed circulation method. (h) Migration assay of WT or CCR2 KO Tregs using the ex vivo closed circulation methods with 4 independent experiments. All data excluding closed circulation experiements are representative of 3 independent experiments (n = 6–8 per group). Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01 compared with the corresponding controls.

Figure 6. A summary figure depicting the infiltration of inflammatory cells and the protective role of Tregs in hyperglycemia-induced hepatic inflammation.

The infiltration of monocytes and neutrophil facilitates inflammatory reactions in the liver in response to STZ-induced hyperglycemia, followed by the migration of CCR2⁺ Tregs into the inflamed liver, thereby improving hepatic inflammation.