NK-CD11c+ Cell Crosstalk in Diabetes Enhances IL-6-Mediated Inflammation during Mycobacterium tuberculosis Infection

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

Data from: NK-CD11c+ cell crosstalk in diabetes enhances IL-6-mediated inflammation during mycobacterium tuberculosis infection

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 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 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 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 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 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

Type 2 diabetes increases the bacterial burden and reduces survival of <i>Mtb</i>-infected mice.

<p><b>A.</b> Schematic representation of T2DM induction and <i>Mtb</i> infection. <b>B.</b> Bacterial burden in lungs at 1, 3, and 6 months p.i. Data are representative of two independent experiments (n = 5 mice per group). <b>C.</b> Alveolar macrophages from control and T2DM mice (at 1, 3, and 6 months after the induction of diabetes) were infected with <i>Mtb</i> at a MOI of 1:2.5. After 2 h, macrophages were washed to remove extracellular bacteria and cultured. After 5 days, intracellular <i>Mtb</i> levels were measured. Data points represent the mean value of three independent experiments. Pooled lung alveolar macrophages from two mice per group per time point were used for each independent experiment. <b>D.</b> Survival curves for control (black square), T2DM (red triangle), <i>Mtb</i>-infected control (blue circle), and <i>Mtb</i>-infected T2DM mice (green diamond). Data were pooled from two independent experiments (n = 7–8 mice per group per experiment). Survival curves were compared using the log rank test (P < 0.001). Data are expressed as the mean ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001.</p

IL-6 drives increased pro-inflammatory cytokine production in type 2 diabetic patients with pulmonary tuberculosis.

<p>Blood was obtained from 20 diabetic pulmonary tuberculosis patients (DM) and from 20 non-diabetic pulmonary tuberculosis patients (NDM). Whole blood was stimulated or not (UNS) with 10 μg/ml PPD. <b>A.</b> The frequency of IFN-γ-, TNF-α-, IL-17-, and IL-2-producing CD4⁺ T cells was determined by flow cytometry. <b>B and C.</b> In some wells, cells from pulmonary tuberculosis patients with T2DM or cells from healthy volunteers were cultured in the presence of anti-IL-6 receptor neutralizing antibodies or isotype-matched control antibodies (2.5 μg/ml). After 18 h, the frequency of IFN-γ-, IL-2-, TNF-α-, and IL-17-producing cells was determined by flow cytometry. <b>B.</b> Pulmonary tuberculosis patients with T2DM. <b>C.</b> Healthy volunteers. Data are expressed as the mean ± SE. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.</p

Interaction between natural killer and CD11c+ cells increases IL-6 production in <i>Mtb</i>-infected type 2 diabetic mice.

<p>T2DM was induced as described in the Methods. Control and T2DM mice were infected with 50–100 CFU of aerosolized <i>Mtb</i>. <b>A.</b> After 6 months, lungs from <i>Mtb</i>-infected T2DM mice were isolated and formalin-fixed. Paraffin-embedded tissue sections were analyzed by confocal microscopy to determine colocalization of NK cells (pink), IL-6+ cells (green), and CD11c+ cells (red). Scale bars, 20 μm (yellow) and 5 μm (white). White arrows point to IL-6-expressing CD11c+ cells, while yellow arrows point to NK1.1+ natural killer cells. A representative staining pattern from three independent experiments is shown (n = 3 mice per group per experiment). <b>B.</b> Lung mononuclear cells were isolated by gradient separation and cultured for 48 h with γ-<i>Mtb</i> (10 μg/ml). Some mononuclear cell populations were depleted of NK cells and cultured with γ-<i>Mtb</i>. The frequency of IL-6-expressing CD11c+ cells was determined by intracellular flow cytometry, and IL-6 levels in culture supernatants were measured by ELISA. <b>C.</b> Expression of NKG2D and DNAM-1 by lung NK cells was determined by flow cytometry. <b>D</b>. Lung mononuclear cells were isolated as described in the methods section and cultured for 48 h with γ-<i>Mtb</i> in the presence or absence of blocking NKG2D or anti-DNAM-1 neutralizing antibodies or an isotype-matched control antibody. The frequency of IL-6+ CD11c+ cells was determined by intracellular staining, and IL-6 levels in culture supernatants were measured by ELISA. <b>B to D.</b> Data points represent the mean values from three independent experiments. Pooled lung mononuclear cell populations from two mice per group were used for each independent experiment. <b>E.</b> CD3-NK1.1+ and CD11C⁺ cells from pooled spleen, lymph node, and lung cells from <i>Mtb</i>-infected control and T2DM mice were isolated by magnetic selection and cultured (one NK cell and four CD11c+ cells) with or without γ-<i>Mtb</i> (10 μg/ml). Some of the -irradiated <i>Mtb</i> H37Rv-cultured cells were cultured in the presence of blocking antibodies (10 μg/ml) against DNAM-1 or a rat IgG2a κ (the isotype control antibody for the anti-DNAM-1 antibody) or with blocking antibodies against NKG2D or a rat IgG1 κ (the isotype control antibody for the anti-NKG2D antibody). After 18 h, cell-free culture supernatants were collected and IL-6 levels were measured by ELISA. *P ≤0.05, **P ≤ 0.01, and ***P ≤ 0.001.</p