Diabetes mellitus increases human TB susceptibility, but the immunological basis for this diabetic complication remains poorly understood. We combined STZ treatment and low-dose aerosol Mtb challenge of mice to investigate the impact of hyperglycemia on protective immunity. We found that control of Mtb infection was significantly impaired by chronic diabetes, while acute STZ-induced diabetes had no discernible effect on TB susceptibility. Mtb challenge of chronic diabetic mice resulted in a > 1 log higher plateau lung bacillary burden compared with euglycemic mice (). This increased TB susceptibility could hypothetically result from defects in leukocyte recruitment to the lung, reduced expression of cytokines essential for TB defense, or a reduced capacity of macrophages to respond to activating cytokines. We found no noticeable differences in the proportion of lung T cells, macrophages/monocytes or granulocytes between diabetic and euglycemic mice with TB (), no evidence for deficient IFN-γ levels in established TB disease (16 wk after infection), nor any evidence of decreased macrophage responsiveness to IFN-γ in diabetic mice as reflected by iNOS expression in vivo (, and ). The increased lung leukocytes and histopathology seen in chronic diabetic mice at 16 weeks after infection presumably reflects their higher bacterial load rather than any direct effect of hyperglycemia on inflammation, since there was no difference in leukocyte recruitment to the lungs of acute diabetic versus euglycemic mice with TB that had comparable lung bacillary burden. While chronic diabetic mice ultimately expressed a robust immune response to Mtb in the chronic phase of TB disease, a key finding in our study was a relative delay in IFN-γ responses detected between 1 and 4 weeks after infection ().
Host control of TB requires an effective Th1 adaptive immune response in the lungs. After aerosol challenge, Mtb
grows logarithmically in the lung for about 3 weeks until cell-mediated immunity causes bacillary load to plateau, largely due to IFN-γ activation of macrophages to restrict bacillary replication. Any delay in the expression of Th1 immunity translates into a higher plateau lung bacillary burden. In our study, chronic diabetic mice reached a higher plateau cfu level than euglycemic mice, but they retained the capacity to ultimately restrict logarithmic Mtb
growth (). As compared with euglycemic controls, chronic diabetic mice had a reduced level of IFN-γ in lung homogenates 2 weeks after infection, and reduced IFN-γ production by purified lung T cells re-stimulated ex vivo
with anti-CD3 mAb, Con A, or ESAT-6 peptide at 1 and 4 weeks after infection ( and ). These results point to an adverse effect of chronic diabetes on the acquisition of immunity to Mtb
, and that could be due to impaired dendritic cell function. The TB susceptibility phenotype of chronic diabetic mice in our study was quite similar to that reported by Tian and colleagues (18
) in experiments in which dendritic cells were transiently depleted in nondiabetic mice before Mtb
We used the STZ diabetes model as it uniformly produces hyperglycemia and because it allowed us to control the duration of diabetes. Further, the STZ diabetes model let us focus on the immunosuppressive effects of hyperglycemia without any of the confounding immunologic factors associated with the NOD mice, including defects in antigen-presenting cell function, T cell repertoire regulation, and natural killer cell function (19
). It was recently reported that STZ treatment produces a transient (~ 9 d) period of lymphopenia and reversible immune suppression in mice (22
). Our finding of impaired TB defense in mice with chronic but not acute diabetes is the opposite of what would be expected if STZ was somehow directly responsible for the TB susceptibility phenotype, since the interval between STZ treatment and Mtb
challenge was the shortest in the acute diabetes group. Moreover, Akita mice with chronic diabetes exhibited increased TB susceptibility in the absence of STZ treatment.
Within the limits of our experimental system, acute diabetes had no adverse impact on TB defense. Lung bacterial burden, histopathology, and iNOS expression were similar between acute diabetic and euglycemic C57BL/6 or ICR mice ( and data not shown). These data indicate that hyperglycemia per se
does not directly promote Mtb
growth or degrade protective immunity. Irreversible formation of advanced glycation end products (AGE) has been linked to many of the complications associated with diabetes, including atherosclerosis, glomerulopathy, impaired wound healing, and depressed neutrophil function (23
). Metabolites from the polyol and hexosamine pathways as well as dysfunctional protein kinase C activation may also contribute to these diabetic complications (28
). AGE accumulate over time in humans with hyperglycemia and in mice. Our finding that TB susceptibility increases with the duration of diabetes suggests that AGE might contribute to the observed impairment of protective immunity. Prior studies indicate that 3 months of hyperglycemia, the period we used to model chronic diabetes in our studies, is sufficient for significant AGE accumulation and AGE-related pathology to develop in mice (29
Recently, Yamashiro and coworkers (11
) reported increased TB susceptibility of ICR mice with acute STZ-induced hyperglycemia, as evidenced by increased lung Mtb
burden as early as 14 days after infection. The conditions of that study differed significantly from ours. They used a 1,000-fold higher dose of Mtb
and delivered the bacteria by intravenous injection. The conditions of diabetes were also different. Most STZ-treated ICR mice reported by Yamashiro and colleagues had a fasting blood glucose over 600 mg/dl, while STZ-treated ICR mice in our study had average fasting blood glucose of 403 mg/dl. We excluded the possibility of ketoacidosis influencing TB susceptibility the STZ-treated mice for our study, while this parameter was not mentioned in the report by Yamashiro and coworkers. Diabetic ketoacidosis might cause immune suppression by mechanisms different than hyperglycemia, as exemplified by its distinct association with rhinocerebral mucormycosis (2
Diabetes and TB are globally important diseases with far-reaching health and economic consequences. We have established a reliable mouse model to study protective immunity against Mtb in the context of diabetes. This study extends previously reported findings of TB susceptibility in diabetic mice by contrasting acute and chronic diabetes, by evaluating lung histopathology and lung leukocyte recruitment, by excluding diabetic ketoacidosis as a contributing factor to immunosuppression, by surveying a broad array of cytokines relevant to TB defense, and by using a pathophysiologically relevant dose and route of Mtb infection. To our knowledge, this is the first report of increased TB susceptibility caused by chronic hyperglycemia in mice. Our data argue against a direct adverse effect of acute hyperglycemia and suggest instead that impaired host defense is a consequence of persistent hyperglycemia, as is the case for the vascular and renal complications of diabetes. Specifically, the data point to an adverse impact of chronic hyperglycemia on the initiation of adaptive immunity rather than on the magnitude or efficacy of its eventual expression. Based on these findings, our future studies will focus on dendritic cell trafficking and antigen presentation after aerosol Mtb challenge of diabetic mice. Although STZ treatment produces insulin-deficient diabetes, the resulting hyperglycemia is a common feature of type 1 and 2 diabetes and one that has been linked to a shared spectrum of diabetic complications. It is therefore reasonable to hypothesize that the adverse effect of chronic hyperglycemia on host defense could occur in the context of type1 or type 2 diabetes.