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

mTOR Signaling pathway regulates the IL-12/IL-10 axis in Leishmania donovani infection

Leishmania-induced interleukin-12 (IL-12) expression is negatively regulated by the phosphatidylinositol 3-kinase (PI3K) and extracellular signal regulated kinase (ERK) 1/2 pathways in human monocyte derived macrophages (MDMs). To extend these studies, we examined the pathways downstream from PI3K in L. donovani-induced reciprocal regulation of IL-12/IL-10 axis in THP-1-derived macrophages. We show for the first time that in THP-1-derived macrophages and human monocytes, mTOR inhibition by rapamycin reversed L. donovani-induced IL-12 and IL-10 modulation. L. donovani-induced phosphorylation of P70S6K, a correlate of mTOR activity, in TLR-stimulated THP-1 derived macrophages. This increase in P70S6K phosphorylation was completely blocked by rapamycin (mTOR inhibitor) and partially by wortmannin (PI3K inhibitor). These observations suggest that a PI3K independent pathway is operative in the modulation of IL-12 and IL-10. Blocking of TLR2 significantly attenuated IL-10 induced by the parasite, but did not affect IL-12 production. Thus, our data suggests that intracellular network of PI3K and mTOR pathway control IL-12/IL-10 modulation by L. donovani. mTOR inhibitors may be attractive molecules to reverse this modulation and may result in control of disease

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

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

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

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

CD11c+CD11b- cells are the major source of IL-6 in <i>Mtb</i>-infected type 2 diabetic mice.

<p>Control and T2DM mice were infected with 50–100 CFU of aerosolized <i>Mtb</i>. <b>A.</b> At 6 months p.i., lungs from uninfected control and diabetic mice and from <i>Mtb</i>-infected control and diabetic mice were isolated and immunohistochemical analysis of IL-6 expression was performed. Representative images of staining patterns in multiple fields (at 100× and 400×) are shown. <b>B.</b> Histological scores for IL-6 expression in lung sections. <b>C.</b> The frequencies of IL-6-producing lung cell populations were determined by intracellular flow-cytometry staining at 1 and 6 months p.i. <b>A to C.</b> Data are representative of two independent experiments (n = 5 mice per group per experiment). Data are expressed as the mean ± SE. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.</p

List of primers used in the study.

<p>List of primers used in the study.</p

Anti-NK1.1 antibody reduces pro- and anti-inflammatory responses and increases survival of <i>Mtb</i>-infected type 2 diabetic mice.

<p>One month after STZ/NA treatment, T2DM mice were infected with 50–100 CFU of aerosolized <i>Mtb</i> H37Rv. Mice were then treated with an anti-NK1.1 mAb or an isotype-matched control mAb (0.3 mg per mouse, starting at 6 months p.i., every 4 days for 1 month). <b>A.</b> Schematic representation of <i>Mtb</i> infection and anti-NK1.1 mAb treatment of T2DM mice. <b>B.</b> Survival of <i>Mtb</i>-infected diabetic mice treated with an anti-NK1.1 mAb or an isotype-matched control mAb. Data were pooled from two independent experiments (n = 3 mice per group per experiment). Survival curves were compared using the log rank test (P < 0.001). <b>C.</b> Bacterial burden in the lungs. <b>D.</b> Cytokine mRNA expression in the above lung samples, expressed as -fold differences compared with that in untreated diabetic mice. <b>C and D.</b> The bacterial burden in the lungs of four isotype-matched control antibody-treated mice was examined, and RNA was isolated for subsequent mRNA analysis immediately after death. The remaining two isotype-matched control antibody-treated mice and anti-NK1.1 antibody-treated mice were sacrificed after 240 days p.i., to determine the bacterial burden in the lung and cytokine mRNA levels. T2DM mice and <i>Mtb</i>-infected T2DM mice treated with PBS were subjected to the same procedures. <b>E.</b> At 6 months p.i., <i>Mtb</i>-infected T2DM mice were treated with either an anti-NK1.1 mAb or an isotype-matched control mAb or PBS. Lungs were isolated and formalin-fixed. Paraffin-embedded tissue sections were prepared, and hematoxylin and eosin staining was performed. Data were pooled from two independent experiments (n = 3 mice per group per experiment). Data are expressed as the mean ± SE. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.</p

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

<div><p>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 <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>). All <i>Mtb</i>-infected T2DM mice and 40% of uninfected T2DM mice died within 10 months, whereas all control mice survived. In <i>Mtb</i>-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 <i>Mtb</i>-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 <i>Mtb</i>-infected T2DM mice. Pulmonary natural killer (NK) cells in <i>Mtb</i>-infected T2DM mice further increased IL-6 production by autologous CD11c+ cells through their activating receptors. Anti-NK1.1 antibody treatment of <i>Mtb</i>-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 <i>Mtb</i> infection in diabetic mice.</p></div