Diabetic mice display a delayed adaptive immune response to Mycobacterium tuberculosis

Abstract

Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB) but the defect in protective immunity responsible for this has not been defined. We previously reported that streptozotocin-induced DM impaired TB defense in mice, resulting in higher pulmonary bacterial burden, more extensive inflammation, and higher expression of several proinflammatory cytokines known to play a protective role in TB. In the current study, we tested the hypothesis that DM leads to delayed priming of adaptive immunity in the lung-draining lymph nodes (LNs) following low dose aerosol challenge with virulent Mycobacterium tuberculosis. We show that M. tuberculosis-specific IFN-gamma-producing T cells arise later in the LNs of diabetic mice than controls, with a proportionate delay in recruitment of these cells to the lung and stimulation of IFN-gamma-dependent responses. Dissemination of M. tuberculosis from lung to LNs was also delayed in diabetic mice, although they showed no defect in dendritic cell trafficking from lung to LNs after LPS stimulation. Lung leukocyte aggregates at the initial sites of M. tuberculosis infection developed later in diabetic than in nondiabetic mice, possibly related to reduced levels of leukocyte chemoattractant factors including CCL2 and CCL5 at early time points postinfection. We conclude that TB increased susceptibility in DM results from a delayed innate immune response to the presence of M. tuberculosis-infected alveolar macrophages. This in turn causes late delivery of Ag-bearing APC to the lung draining LNs and delayed priming of the adaptive immune response that is necessary to restrict M. tuberculosis replication.

FIGURE 1

Detection of Mtb-specific IFN-γ-producing T cells in STZc and control mice. Ex vivo reactivity of pulmonary LN (A) and lung leukocytes (B) to Mtb Ag85B was measured by ELISPOT. Bars illustrate the number of IFN-γ spots per 10⁵ CD4⁺ cells at the indicated times after aerosol Mtb infection of non-diabetic control mice (filled bars) and diabetic STZc mice (empty bars). Bars represent mean ± SD, n = 3–8. Lines within each bar correspond to the value for unstimulated cells for each group and time point. * p < 0.05, ** p < 0.01.

FIGURE 2

Levels of IFN-γ and host defense factors induced by IFN-γ in the lungs of control and diabetic mice. A, Lungs were harvested from non-diabetic control mice (filled circles) and STZc mice (empty circles) 15 d after low dose aerosol Mtb infection. Lung lysates were prepared for measurement of IFN-γ by ELISA (left panel) and nitrate/nitrate as a reflection of NO (right panel). * p < 0.05. B, Levels of the IFN-γ-inducible chemokine CXCL9 were measured by ELISA in lung homogenates of diabetic and non-diabetic mice 15 d p.i.

FIGURE 3

Dissemination of Mtb from lung to LN. Non-diabetic control mice (filled circles) and STZc mice (open circles) were infected with ~100 CFU of Mtb Erdman by aerosol. Lung-draining LN were isolated on the indicated days, homogenized, and plated for determination of CFU. * p < 0.05.

FIGURE 4

DC migration from lung to LN in response to LPS. Pulmonary DC were simultaneously labeled with Far Red and activated with LPS by i.t. instillation. After 20 h, the lung-draining LN were removed and leukocytes were analyzed by flow cytometry. A, Gating strategy to identify newly emigrated DC as Far Red⁺ CD11c⁺ cells. B, Proportion of Far Red⁺ CD11c⁺ cells in LN of control and STZc mice 20 h after LPS stimulation. C, Expression of CD40, CD80, CD86, and MHC class II on a gated population of Far Red⁺ CD11c⁺ cells.

FIGURE 5

Pulmonary lesions at early time points after Mtb infection. Control and STZc mice were challenged with ~100 CFU Mtb Erdman by aerosol and the lungs were subsequently harvested, inflated and fixed with formalin and then processed for H&E staining. A, Leukocyte aggregates (arrows) were easily identified in non-diabetic control mice (Ctrl) but not in mice with chronic DM (STZc) by microscopy at X 20 magnification. B, Total cross-sectional lung area from all tissues sections examined (3 to 6 mice per group at different time points) and the combined areas of inflammation within these sections were measured by video microscopy. Percent total area involved with inflammation (top panel)was calculated as [(total area of inflammation / total lung area surveyed) × 100]. The mean values for non-diabetic control mice (filled circles) and for STZc mice (open circles) diabetes are indicated by horizontal lines. The number lesions of any size visible at X 20 magnification on H&E stained lung sections was counted (bottom panel). Horizontal lines indicate mean values for control and STZc groups. * P < 0.05.

FIGURE 6

Anti-PPD immunohistochemistry of lung sections from diabetic (STZc) and control (Ctrl) mice with TB. Mice were infected with ~100 CFU Mtb Erdman by aerosol and then lungs were isolated 15 d later for immunohistochemistry with H&E counter stain. Mtb-infected macrophages are distinguished by brown staining (X 400 magnification).

FIGURE 7

Pulmonary chemokines in diabetic and control mice with TB. Mice were infected with Mtb Edrman and lung lysates were prepared 15 d p.i. for analysis by ELISArray of samples pooled from 4 mice per group (A), or by ELISA of samples from individual mice (B). * p < 0.05.