Tuberculosis susceptibility of diabetic mice

Abstract

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 (< or = 1 mo) or chronic (> or = 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-gamma 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.

Figure 1.

Chronic but not acute diabetes increases tuberculosis (TB) susceptibility in mice. We grouped mice as acute or chronic based on the duration of streptozotocin (STZ)-induced diabetes, ⩽ 1 mo or ⩾ 3 mo, respectively, before aerosol infection with Mycobacterium tuberculosis (Mtb) Erdman (∼ 50 colony-forming units [cfu]). Lung Mtb load was determined by plating serial dilutions of lung homogenates at the indicated times and counting cfu after 3 wk incubation. (A) Acute diabetic (STZ) versus euglycemic control (CTL) mice 4 weeks after infection. (B) Chronic STZ diabetic versus euglycemic mice 8 and 16 weeks after infection. Data are presented as mean log₁₀ cfu ± SD. Open squares: STZ-treated mice with chronic diabetes; closed diamonds: euglycemic control mice. Data are presented as mean cfu ± SD. *P < 0.05, n = 5.

Figure 2.

Chronic diabetic mice with TB have a similar pattern but increased extent of lung inflammation compared with euglycemic controls. (A) Lung area involved with inflammation of euglycemic and chronic diabetic C57BL/6 mice at different time points after aerosol challenge with Mtb Erdman. Total cross-sectional lung area from all tissues sections was examined and the combined areas of inflammation within these sections were delineated using a Nikon Eclipse E400 microscope (×20 magnification) with Spot Insight v 3.5 software. Percent total area involved with inflammation for two mice per group was calculated as (total area of inflammation/total lung area surveyed) ×100. Open squares: STZ-treated mice with chronic diabetes; closed diamonds: euglycemic control mice. (B, C) Representative hematoxylin and eosin (H&E)-stained lung sections from euglycemic C57BL/6 mouse 12 weeks after infection taken at ×20 and ×200 magnification, respectively. (D, E) Representative H&E-stained lung section from chronic diabetic C57BL/6 mouse 12 weeks after infection at ×20 and ×200 magnification, respectively.

Figure 3.

Chronic diabetic mice have greater leukocyte recruitment to the lungs than euglycemic mice after 16 weeks of TB disease. Isolated lung leukocytes were stained with anti-CD3, -CD4, -CD8, −Gr-1, and -F4/80 mAb for analysis by flow cytometry. (A) The proportion of different leukocyte populations is presented as the mean % surface marker positive cells ± SD (n = 3). Open columns: euglycemic C57BL/6 mice; closed columns: STZ-treated mice with chronic diabetes. (B) The total number of leukocytes of each subset in the lung was determined by multiplying the % surface marker positive cells by the total number of leukocytes isolated from each mouse. Open columns: euglycemic mice; closed columns: STZ-treated mice with chronic diabetes. Data are presented as the mean cell number (×10⁶) ± SD. *P < 0.05, n = 3.

Figure 4.

Lung IFN-γ and inflammatory cytokine expression are increased in acute and chronic diabetic mice late in Mtb infection, while chronic diabetic mice have reduced IFN-γ expression shortly after infection. Lung homogenates were prepared as described in Materials and Methods. (A) Cytokines present in pooled lung lysates prepared from acute diabetic or euglycemic C57BL/6 mice 8 weeks after infection. Closed bars: STZ-treated mice with acute diabetes; open bars: euglycemic control mice; n = 5. (B) Cytokines present in pooled lung lysates prepared from chronic diabetic or euglycemic mice 16 weeks after infection. Closed bars: STZ-treated mice with chronic diabetes; open bars: euglycemic control mice; n = 5. (C) IFN-γ content in lung lysates from individual chronic diabetic C57BL/6 mice 7, 14, 21, and 28 days after infection. Open diamonds: euglycemic control mice; closed diamonds: STZ-treated mice with chronic diabetes; *P < 0.05, n = 5. (D) Cytokines present in pooled lung lysates prepared from chronic diabetic Akita mice or euglycemic C57BL/6 mice 16 weeks after infection. Closed bars: Akita mice with chronic diabetes; open bars: euglycemic control mice; n = 5.

Figure 5.

Lung iNOS expression in established TB lesions is similar between euglycemic and chronic diabetic mice. A euglycemic control C57BL/6 mouse (A), an STZ-treated chronic diabetic mouse (B), a euglycemic wild-type C57BL/6J control for the Akita mice (C), and a chronic diabetic Akita mouse (D) were infected with Mtb Erdman by aerosol. Lung sections prepared 16 weeks after infection were stained with anti-iNOS/NOS II. Cells expressing iNOS are identified by brown staining. Images are ×40 magnification.

Figure 6.

Antigen-specific and nonspecific IFN-γ responses are reduced in chronic diabetic mice at early time points in TB disease. Chronic diabetic C57BL/6 mice (STZ) and euglycemic controls (CTL) were infected with Mtb Erdman by aerosol. Lung leukocytes harvested 7 and 28 days after infection were cultured on IFN-γ ELIspot plates in the presence of control media, Con A, anti-CD3 mAb, Mtb Erdman CFP, or an ESAT-6 MHC class II peptide. Results are expressed as the mean number of spot-forming cells (SFC) per million cells. *P < 0.05, n = 4.