DOI: 10.5061/dryad.qn42t
title: Data from: NK-CD11c+ cell crosstalk in diabetes enhances IL-6-mediated inflammation during mycobacterium tuberculosis infection

description: 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.
description: S1Figure 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.S2Figure 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 &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001.S3Figure 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 &lt; 0.05, **P &lt; 0.01, ***P &lt;
 0.001.S4Figure 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.S5Figure 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.S6Figure 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.