In this study, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and identified factors that increase susceptibility of T2DM mice to infection by Mycobacterium tuberculosis (Mtb). All Mtb-infected T2DM mice and 40% of uninfected T2DM mice died within 10 months, whereas all control mice survived. In Mtb-infected mice, T2DM increased the bacterial burden and pro- and anti-inflammatory cytokine and chemokine production in the lungs relative to those in uninfected T2DM mice and infected control mice. Levels of IL-6 also increased. Anti-IL-6 monoclonal antibody treatment of Mtb-infected acute- and chronic-T2DM mice increased survival (to 100%) and reduced pro- and anti-inflammatory cytokine expression. CD11c+ cells were the major source of IL-6 in Mtb-infected T2DM mice. Pulmonary natural killer (NK) cells in Mtb-infected T2DM mice further increased IL-6 production by autologous CD11c+ cells through their activating receptors. Anti-NK1.1 antibody treatment of Mtb-infected acute-T2DM mice increased survival and reduced pro- and anti-inflammatory cytokine expression. Furthermore, IL-6 increased inflammatory cytokine production by T lymphocytes in pulmonary tuberculosis patients with T2DM. Overall, the results suggest that NK-CD11c+ cell interactions increase IL-6 production, which in turn drives the pathological immune response and mortality associated with Mtb infection in diabetic mice.
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Figure S1. Mtb infection enhances the dissemination of bacteria in T2DM. Control and T2DM mice were infected with 50-100 CFU of aerosolized Mtb H37Rv A & B. Bacterial burden in the spleen and liver six months post-infection. C & D. Random blood glucose levels and body weight were measured at monthly intervals for up to 6 months.
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Figure S2. Type 2 diabetes enhances pro- and anti-inflammatory responses during Mtb infection. Control and T2DM mice were infected with 50-100 CFU of aerosolized Mtb H37Rv. One and six months p.i., lung homogenates were collected from uninfected control and diabetic mice and from Mtb-infected control and diabetic mice and subjected to multiplex ELISA to determine the various cytokines and chemokines. Mean values, p-values and SEs are shown. *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure S3. Type 2 diabetes enhances pro- and anti-inflammatory responses during Mtb infection. Control and T2DM mice were infected with 50-100 CFU of aerosolized Mtb H37Rv. A to M. Six months p.i., lung cytokine mRNA expression was determined by real-time PCR. Mean values, p-values and SEs are shown. *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure S4. CD11c+ dendritic cells are the major source of IL-6 in Mtb-infected type 2 diabetic mice. Control and T2DM mice were infected with 50-100 CFU of aerosolized Mtb H37Rv. A. Six months p.i., pooled CD4+, CD8+, DX5+ and CD11c+ cells from the lung, spleen and lymph nodes were isolated by magnetic selection. IL-6 mRNA expression was determined by real-time PCR. The results of three independent experiments are shown. B & C. Splenic DX5+ and CD11C+ cells were isolated by magnetic selection and cultured (1 NK cell and 4 dendritic cells) with or without g-irradiated Mtb (10 µg/ml). Some of the g-irradiated Mtb H37Rv cultured cells were cultured in the presence of neutralizing antibodies (10 µg/ml) against DNAM-1 or rat IgG2a, κ (the isotype control antibody for the anti-DNAM-1 antibody) or against NKG2D or rat IgG1, κ (the isotype control antibody for the anti-NKG2D antibody). After 18 hours, cell-free culture supernatants were collected and IL-6 levels were measured by ELISA. Mean values, p-values and SEs are shown. *P ≤ 0.05, **P ≤0.01, ***P ≤ 0.001.
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Figure S5 Natural killer and dendritic cell interaction enhanced IL-6 production in Mtb-infected type 2 diabetic mice. Control and T2DM mice were infected with 50-100 CFU of aerosolized Mtb H37Rv. Six months p.i., lungs from Mtb-infected control and T2DM mice were isolated and formalin-fixed. Paraffin-embedded tissue sections were prepared and confocal microscopy analysis was performed to determine NK (pink), IL-6+ (green) and dendritic (red) cell co-localization. Scale bar: 20 µm (yellow bar) and 5 µm (white bar). Representative images of staining pattern of three independent experiments, each with three per group were shown.
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Figure S6. IL-6 is responsible for the increased pro-inflammatory cytokine production in type 2 diabetic patients with pulmonary tuberculosis. Blood from 20 pulmonary tuberculosis patients with T2DM and 20 pulmonary tuberculosis patients without diabetes was obtained. Whole blood was cultured with 10 µg/ml of purified protein derivative (PPD) as indicated in the methods section. A representative flow cytometric contour plot showing the frequency of cells expressing IFN-, TNF-, IL-17 and IL-2 is shown.
