Lower expression of Th1-related cytokines and inducible nitric oxide synthase in mice with streptozotocin-induced diabetes mellitus infected with Mycobacterium tuberculosis

Abstract

Diabetes mellitus is an important predisposing factor for tuberculosis. The aim of this study was to investigate the mechanism underlying this association using a murine model. Mice with streptozotocin-induced diabetes mellitus were prone to Mycobacterium tuberculosis infection, as indicated by increased numbers of live bacteria in lung, liver and spleen. In diabetic mice, the levels of IL-12 and IFN-gamma in the lung, liver and spleen were lower than those in control animals on day 14 postinfection, while the opposite was true for IL-4 levels in the lung and liver. The expression pattern of inducible nitric oxide synthase (iNOS), in the two mice types was as for IL-12 and IFN-gamma. In addition, peritoneal exudate cells obtained from diabetic mice produced lower amounts of IL-12 and NO than those from control mice, when stimulated in vitro with M. bovis BCG. Spleen cells from diabetic mice infected with M. tuberculosis produced a significantly lower amount of IFN-gamma upon restimulation with purified protein derivatives (PPD) than those from infected nondiabetic mice. Interestingly, addition of high glucose levels (33 mM) to the cultures of PPD-restimulated spleen cells reduced the synthesis of IFN-gamma only in diabetic mice, and not in nondiabetic mice. Finally, control of blood glucose levels by insulin therapy resulted in improvement of the impaired host protection and Th1-related cytokine synthesis. Our results suggest that the reduced production of Th1-related cytokines and NO account for the hampered host defense against M. tuberculosis infection under diabetic conditions.

Fig. 1

Effect of diabetic condition on the expression of iNOS mRNA after M. tuberculosis infection. Diabetic and control mice were infected with M. tuberculosis. Total RNA was extracted from (a) lung, (b) liver and (c) spleen on day 7 postinfection, and real-time PCR was conducted to measure the expression of iNOS and GAPDH mRNA. The results are expressed as the relative values to GAPDH. Each symbol represents the result of each mouse. ○ control; • diabetic mice. NS, not significant; *P < 0·05, compared with control mice.

Fig. 2

Reduced production of (a) IL-12 and (b) NO by PEC in diabetic mice. PEC were prepared from diabetic and control mice on day 3 after intraperitoneal injection of M. bovis BCG. The cells were stimulated in vitro with various doses of M. bovis BCG for 48 h, and the concentrations of IL-12 and NO in the culture supernatants were measured. Each symbol represents the mean ± SD of triplicate cultures. ○ control; • diabetic mice. *P < 0·05, compared with control mice.

Fig. 3

Effect of diabetes on development of Th1 cells. Diabetic and control mice were infected with M. tuberculosis. Spleen cells were prepared on day 8 and re-stimulated with various doses of PPD for 48 h. The concentrations of IFN-γ in the culture supernatants were measured. Each symbol represents the mean ± SD of triplicate cultures. ○ control; • diabetic mice. *P < 0·05, compared with control mice.

Fig. 4

Reduced production of IFN-γ  by Th1 cells under high glucose conditions. (a) Control and (b) diabetic mice were infected with M. tuberculosis. Spleen cells were prepared on day 8 and re-stimulated with 1·0 and 3·3 µg/ml of PPD in high (33·3 m m) or normal (11·1 m m) glucose conditions for 48 h. The concentrations of IFN-γ in the culture supernatants were measured. Each bar represents the mean ± SD of triplicate cultures. □ normal; ▪ high glucose condition. CNT, control; DM, diabetic mice. *P < 0·05.