Fig 7. Interaction between natural killer and CD11c+ cells increases IL-6 production in Mtb-infected type 2 diabetic mice.

T2DM was induced as described in the Methods. Control and T2DM mice were infected with 50–100 CFU of aerosolized Mtb. A. After 6 months, lungs from Mtb-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. Lung mononuclear cells were isolated by gradient separation and cultured for 48 h with γ-Mtb (10 μg/ml). Some mononuclear cell populations were depleted of NK cells and cultured with γ-Mtb. 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. C. Expression of NKG2D and DNAM-1 by lung NK cells was determined by flow cytometry. D. Lung mononuclear cells were isolated as described in the methods section and cultured for 48 h with γ-Mtb 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 to D. 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. E. CD3-NK1.1+ and CD11C⁺ cells from pooled spleen, lymph node, and lung cells from Mtb-infected control and T2DM mice were isolated by magnetic selection and cultured (one NK cell and four CD11c+ cells) with or without γ-Mtb (10 μg/ml). Some of the -irradiated Mtb 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.