Published ahead of print on June 21, 2007, doi:10.1165/rcmb.2006-0478OC
© 2007 American Thoracic Society DOI: 10.1165/rcmb.2006-0478OC Tuberculosis Susceptibility of Diabetic Mice1 Department of Medicine, and 2 Diabetes and Endocrinology Research Center, University of Massachusetts Medical School, Worcester, Massachusetts Correspondence and requests for reprints should be addressed to Dr. Hardy Kornfeld, UMASS Medical School, LRB-303, 55 Lake Avenue North, Worcester, MA 01655. E-mail: hardy.kornfeld{at}umassmed.edu
Increased susceptibility to infections, including tuberculosis (TB), is a major cause of morbidity and mortality in patients with diabetes. Despite the clinical importance of this problem, little is known about how diabetes impairs protective immunity. We modeled this phenomenon by infecting acute (  1 mo) or chronic ( 3 mo) diabetic mice with a low aerosol dose of Mycobacterium tuberculosis (Mtb) Erdman. Diabetes was induced by streptozotocin (STZ) treatment of C57BL/6 mice, while another mouse strain and diabetes model were used to confirm key observations. Lungs from acute diabetic and euglycemic mice had similar bacterial burdens, cytokine expression profiles, and histopathology. In contrast, chronic diabetic mice had > 1 log higher bacterial burden and more inflammation in the lung compared with euglycemic mice. The expression of adaptive immunity was delayed in chronic diabetic mice, shown by reduced early production of IFN- in the lung and by the presence of fewer Mtb antigen (ESAT-6)–responsive T cells compared with euglycemic mice within the first month of infection. However, after 2 months of TB disease proinflammatory cytokines levels were higher in chronic diabetic than euglycemic mice. Here we show that Mtb infection of STZ-treated mice provides a useful model to study the effects of hyperglycemia on immunity. Our data indicate that the initiation of adaptive immunity is impaired by chronic hyperglycemia, resulting in a higher steady-state burden of Mtb in the lung.
Key Words: diabetes mellitus • host defense • mouse • Mycobacterium tuberculosis
People with diabetes mellitus are prone to infection with a broad range of pathogens, including Mycobacterium tuberculosis (Mtb) (1, 2). The World Health Organization estimates that 170 million people worldwide currently have diabetes, a figure that will double by 2030. Tuberculosis (TB) remains among the leading causes of death from infection (3, 4). Many of the countries with the highest incidence of diabetes also have a high incidence of TB. Even a modest improvement in the management of diabetes-related infections has the potential to yield large benefits in terms of alleviating morbidity and reducing medical costs. Despite these considerations, surprisingly little is known about the basis for the increased susceptibility of patients with diabetes to infection.
Limited clinical studies of immunity in patients with diabetes described impaired T cell proliferation and a reduced capacity of T cells to respond to appropriate stimulation (5). A relatively small number of animal studies exploring the effects of hyperglycemia on host defense have been published. Reading and coworkers (6) reported increased Influenza A virus susceptibility of diabetic mice, possibly due to compromised collectin-mediated defense. Hyperglycemic mice are also more susceptible to trypanosomiasis and trichinosis, but less susceptible than euglycemic mice to blood-stage malaria infection (7–10). Diabetic mice infected with Mtb by high-dose (
Establishing a well-characterized diabetes infection model is a logical first step to investigate the mechanism of impaired host defense in diabetes. We used mice with acute (
Animals C57BL/6 and heterozygous Ins2Akita (Akita) mice were obtained from Jackson Laboratory (Bar Harbor, ME). Akita mice spontaneously become hypoinsulinemic and hyperglycemic by 3 to 4 weeks of age (13). ICR mice, an outbred mouse strain, were purchased from Taconic Farms (Hudson, NY). Mice were housed within the Animal Medicine facility at UMass Medical School, and the University of Massachusetts Medical School Institutional Animal Care and Use Committee approved these experiments. All mice were at least 8 weeks old when treated with STZ.
Reagents
Induction of Diabetes
Infection with Mtb
Bacterial Load
Lung Histology
Flow Cytometry of Lung Leukocytes One to two million lung leukocytes were treated with Fc blocking mAb (clone 2.4G2; BD Bioscience Pharmingen, San Diego, CA) then stained with anti-CD3-APC-Cy7 (clone 145–2C11), CD4-PerCP (clone RM4–5), CD8-PE (clone 53.6–7), and Gr-1-PE-Cy7 (clone RB6–8C5; all from BD Bioscience Pharmingen, San Diego, CA) and F4/80-APC (clone BM8; eBioscience, San Diego, CA). Stained cells were analyzed on a LSRII flow cytometer (BD Bioscience Pharmingen). Fifty thousand leukocyte-gated events were collected, and data analysis was done with FlowJo PC (TreeStar, Inc., Ashland, OR). Isotype control antibodies were purchased from BD Bioscience Pharmingen and eBioscience.
Lung Cytokine Expression
Ex Vivo Lung T Cell Restimulation
Statistical Analysis
Induction of Diabetes in Mice Nonfasting blood glucose was measured after STZ treatment (except for heterozygous Akita mice, which become spontaneously diabetic), before Mtb Erdman infection, and at the conclusion of an experiment. Nonfasting blood glucose was measured because we felt this was a better indicator than fasting values of typical blood glucose levels during infection. Nonfasting blood glucose of euglycemic C57BL/6 was less than 200 mg/dl. Some of the STZ-treated C57BL/6 mice had nonfasting blood glucose above the upper limit of glucometer detection (> 600 mg/dl), while the remaining mice had an average blood glucose of 463 mg/dl. Interestingly, half of the ICR mice not treated with STZ had nonfasting blood glucose over 200 mg/dl (range, 175–329 mg/dl), but their blood glucose was less than 200 mg/dl after a 16-hour fast, consistent with previously reported fasting blood glucose values for this strain (11). With the exception of one STZ-treated ICR mouse that became euglycemic, all of the STZ-treated ICR mice had a blood glucose greater than 200 mg/dl after a 16-hour fast. None of the diabetic mice developed ketoacidosis as determined by urine ketone dipstick tests. Blood glucose of acute and chronic diabetic mice was not significantly influenced by Mtb Erdman infection (data not shown).
TB Susceptibility of Diabetic Mice
We saw no noticeable difference in the pattern or extent of TB pathology in the lungs of acute diabetic and euglycemic mice (data not shown). Similarly, there was no consistent difference in the pattern of lung TB pathology between euglycemic and chronic diabetic C57BL/6 mice, but the extent of inflammation was increased in the diabetic group. We compared the extent of inflammation by analyzing serial sections through the entire lung of two mice per group, measuring the total cross-sectional area of lung tissue sections for each mouse and the total area within those sections involved with inflammation. A considerably greater area of the lung was occupied with inflammation in chronic diabetic mice with than in euglycemic controls (Figures 2A–2E). The histopathology results demonstrate that mice with diabetes are capable of responding to Mtb infection and recruiting a normal appearing but exaggerated leukocyte response to the lung, consistent with the 10-fold greater bacillary burden.
Lung Leukocyte Populations To further characterize the inflammatory response of chronic diabetic and eugylcemic mice with pulmonary TB, we isolated lung leukocytes from the left lung and right caudal lung lobe by enzymatic digestion and measured T cell (CD3+, CD4+, CD8+), macrophage/monocyte (F4/80+), and granulocyte (GR-1+, CD3–, F4/80–) populations by flow cytometry 16 weeks after infection. Chronic diabetic and euglycemic mice had the same relative proportion of T cell subsets, macrophages/monocytes, and granulocytes. However, chronic diabetic had more total lung leukocytes and therefore more total cells of each population than euglycemic controls (Figures 3A and 3B). This finding is consistent with the histopathology results. We saw no evidence of increased early inflammation in chronic diabetic mice, as they had a number of lung leukocytes and splenocytes comparable with those of euglycemic mice 7, 14, and 21 days after infection (data not shown). A comparison of acute diabetic and euglycemic C57BL/6 mice with TB revealed no difference in total lung leukocytes or proportion of different leukocyte subsets at any time point (data not shown).
Lung Cytokine Expression Protective immunity to TB depends on a T helper (Th)1 cell–mediated immune response. It is well established that IFN- is critically important for TB defense in mice and in the human host (14–16). We assayed pooled lung lysates from acute diabetic, chronic diabetic, and euglycemic mice with TB for Th1, Th2, and pro-inflammatory cytokines. Expression of IFN- and IL-1 was higher in lungs from acute diabetic C57BL/6 mice compared with euglycemic mice 8 weeks after infection (Figure 4A). Acute diabetic ICR mice had lower IL-12p40 expression in their lungs than euglycemic mice but a comparable amount of IFN- 8 weeks after infection, which agrees with previously reported observations (11, and data not shown).
We measured IFN- in lung lysates from individual C57BL/6 mice with chronic STZ-induced diabetes on Weeks 1, 2, 3, and 4 after Mtb infection. IFN- levels were comparable between groups at 1 week after infection and increased in both groups by 2 weeks after infection, but the increase in the eugylcemic mice was significantly greater than in the diabetic mice (Figure 4C). While IFN- increased more slowly in the diabetic than in euglycemic mice during the first 3 weeks after Mtb infection, the levels measured in pooled lung lysates from chronic diabetic C57BL/6 and Akita mice were higher than eugylcemic controls 16 weeks after infection. The levels of IL-1 and TNF were also higher in chronic diabetic mice relative to euglycemic mice 16 weeks after infection (Figures 4B and 4D). These data indicate that mice with chronic diabetes are fully capable of mounting a strong IFN- response to chronic Mtb infection in the lung, albeit with delayed kinetics.
One possible explanation for impaired control of Mtb growth in the lungs despite the high level of IFN-
Ex Vivo Antigen Restimulation Compared with euglycemic mice, IFN- expression was reduced in the lungs of chronic diabetic mice 14 days after infection but increased 16 weeks after infection, suggesting that the increased TB susceptibility of these mice stems from delayed expression of adaptive immunity rather than an intrinsically faulty effector response. Consistent with that concept, Mtb growth is held at a plateau level in the lungs of chronic diabetic mice but at a higher bacillary burden than in euglycemic controls. To evaluate the kinetics of adaptive immunity, we isolated lung leukocytes from chronic diabetic and euglycemic C57BL/6 mice 7 and 28 days after infection. Lung leukocytes were cultured 24 hours on IFN- ELIspot plates and stimulated with Con A, anti-CD3 mAb, Mtb Erdman CFP, an MHC class II restricted Mtb ESAT-6 epitope peptide, or control media. As shown in Figure 6, by 7 days after infection there was a higher frequency of IFN-–producing lung T cells responsive to Con A or anti-CD3 in euglycemic C57BL/6 mice compared with chronic diabetic mice. By 28 days after infection lung leukocytes from euglycemic mice had a significantly higher frequency of IFN-–producing lung T cells responsive to the Mtb-specific antigen ESAT-6 than mice with chronic diabetes. These results demonstrate a temporal delay in the expression of protective immunity to Mtb in chronic diabetic mice.
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 (Figure 1). 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 (Figure 3A), no evidence for deficient IFN- levels in established TB disease (16 wk after infection), nor any evidence of decreased macrophage responsiveness to IFN- in diabetic mice as reflected by iNOS expression in vivo (Figures 4A, 4B, 4D, and 5). 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- responses detected between 1 and 4 weeks after infection (Figure 6).
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-
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 (19–21). It was recently reported that STZ treatment produces a transient ( 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 (Figure 1 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 (23–27). 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, 30). 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.
The authors thank Birgit Stein, Madhumathi Thiruvengadam, Jonathan Eskander, and Linda Paquin for technical assistance with data collection and animal care.
* These authors contributed equally to this work.  This work was supported by NIH grants DK 32520 and DK 53006 (to Aldo Rossini and D.G.), and HL 081149 (to H.K.). Originally Published in Press as DOI: 10.1165/rcmb.2006-0478OC on June 21, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form December 29, 2006 Accepted in final form May 25, 2007
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Abstract
CLINICAL RELEVANCE
50 cfu) aerosol infection model with Mtb Erdman. The bulk of our work was done with C57BL/6 mice, but other mouse strains and a different diabetes model were used to provide corroborative data. Acute diabetes did not measurably impair TB defense. In contrast, chronic diabetic mice had a higher bacterial lung burden, increased lung inflammation, and a delayed adaptive immune response to Mtb compared with euglycemic mice. 
, IL-1
