Gut Microbiota Regulates Mincle Mediated Activation of Lung Dendritic Cells to Protect Against Mycobacterium tuberculosis

Abstract

Gut microbial components serve as ligand for various pattern recognition receptors (PRRs) present on immune cells and thereby regulates host immunity. Dendritic cells (DCs) are highly specialized innate cells involved in immune response to Mycobacterium tuberculosis (Mtb) infection. The gut-lung axis is a potential therapeutic target in tuberculosis; however, understanding of the innate immune mechanism underlying the interaction of gut microbiota and lung still remains obscure. We investigated if antibiotics (Abx) induced gut dysbiosis is able to affect the activation of innate receptor, macrophage inducible C-type lectin (mincle) in lungs during Mtb infection. We found that dysbiosis reduced the lung mincle expression with a concomitant increase in Mtb survival. Further, Abx diminished the effector and memory T cell population, while elevating frequency of regulatory T cells (Tregs) in the lungs. Here, we show that dysbiotic mice exhibited low mincle expression on lung DCs. These DCs with impaired phenotype and functions had reduced ability to activate naïve CD4 T cells, and thus unable to restrict Mtb survival. In vivo administration of trehalose-6,6-dibehenate (TDB: mincle ligand) efficiently rescued this immune defect by enhancing lung DCs function and subsequent T cell response. Further, gut microbial profiling revealed augmentation of Lactobacillus upon mincle stimulation in microbiota depleted animals. Accordingly, supplementation with Lactobacillus restored mincle expression on lung DCs along with anti-Mtb response. Our data demonstrate that gut microbiota is crucial to maintain DC-dependent lung immune response against Mtb, mediated by mincle. Abx interrupt this process to induce impaired T cell-response and increased susceptibility to Mtb.

Keywords: T cells; antibiotics; gut-lung axis; lung dendritic cells; mincle; tuberculosis.

Figures

Figure 1

Downregulation of mincle receptor in lungs of gut microbiota disrupted animals dampens anti-Mtb immunity. Mice were given ad libitum access to Abx supplemented drinking water for 4 wk, prior to aerosol challenge with Mtb (~100 CFU) and oral administration of TDB (50 μg). At 30 d post infection, lung cells were harvested and assessed for the (A) expression of mincle by qRT-PCR, depicted as fold change relative to control and normalized to β-actin and GAPDH reference gene; (B)Mtb burden by CFU assay. Further, (C–E) lung lymphocytes were stimulated with PPD (25 μg/ml) for 48 h. Thereafter, culture SNs was collected and quantified by ELISA for the secretion of (C) IFN-γ, (D) IL-17, and (E) IL-10. Data represented as mean ± SD are of three independent experiments (n = 5 mice/group). **p < 0.01, ***p < 0.001. CT: control mice without Abx treatment; Abx: mice treated with Abx; Mtb: Mtb challenged mice; Mtb-TDB: mice with Mtb infection and TDB administration; Abx-Mtb: mice treated with Abx prior to Mtb infection; Abx-Mtb-TDB: mice with disrupted gut microbiota prior to Mtb infection and TDB administration.

Figure 2

Mincle stimulation reduces Tregs population and frequency of PD-1+ CD4 T cells in gut microbiota disrupted animals. Mice were treated as described in the legend to Figure 1. Lymphocytes isolated from lungs were in vitro stimulated with PPD (25 μg/ml) for 72 h. Thereafter, expression of (A,B) intracellular FoxP3 and (C,D) PD-1 were assessed on CD4 T cells by flow cytometry. Contour plots and bar graphs represent the percentage of CD4 FoxP3+ and CD4 PD-1+ gated population. Data are shown as mean±SD of 2–3 independent experiments (n = 5 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Triggering through mincle restores the percentage of effector and memory CD4 T cells in mice with gut dysbiosis. Gut microbiota-disrupted mice were given Mtb challenge and TDB supplementation as mentioned in the legend to Figure 1. After 4 wk, lung lymphocytes were isolated and stained ex vivo for the T cells activation and memory markers, thereafter assessed by flow cytometry. CD4 T cells gated population was monitored for the (A,B) CD44hi expression representing activated CD4 T cells; (C–E) coexpression of CD44 and CD62L for effector (CD44hi CD62Llo) and central (CD44hi CD62Lhi) memory CD4 T cells; (F,G) CD127hi; (H,I) CD44hi CCR7hi cells. The results depicted as contour plots and bar diagram indicate percent population of the gated cells. The data represented as mean ± SD of three independent experiments (n = 5 mice/group). **p < 0.01, ***p < 0.001.

Figure 4

Stimulation through mincle reinstates the lung DCs function in gut microbiota depleted animals. Mtb infected mice with ablated gut microbiota were orally gavaged with TDB as indicated in legend to Figure 1. After 3 wk, lung cells from different groups of mice were sorted for CD11c+ population and (A–C) stained for the expression of (A) mincle receptor, bar graph depicts percentage of mincle positive population; (B) CD86; (C) MHC-II. Insets of flow cytometry represent percentage of cells gated on CD11c+ CD11b+ population. Further, (D,E) enriched lung DCs were pulsed with PPD (15 μg/ml) overnight. Later, cytokines (D) IL-12; (E) IL-6 were estimated in the cell culture SNs; (F)Mtb uptake by DCs was assessed through CFU assay. Data shown as means ± SD are of 2–3 independent experiments (n = 6 mice/group). *p < 0.05,**p < 0.01, ***p < 0.001.

Figure 5

Lung DCs from gut microbiota ablated mice fail to stimulate Mtb specific naïve CD4 T cells. Mice treated with Abx were infected with Mtb and orally administered TDB as illustrated in the legend to Figure 1. After 3 wk, sorted CD11c+ lung cells enriched for DCs were pulsed with PPD (15 μg/ml) overnight prior to coculture with Mtb specific naïve CD4 T cells which were prior labeled with CFSE (2 μM) at a ratio of 1:10. After 72 h, proliferation of CD4 T cells was examined by flow cytometry. (A) Contour plots and (B) Bar graph represent the percent population of CFSE+ CD4 T cells. Later, (C) IFN-γ, (D) IL-17, and (E) IL-10 level was measured in the culture SNs by ELISA. Data are shown as mean ± SD of 2–3 independent experiments (n = 6 mice/group). **p < 0.01, ***p < 0.001.

Figure 6

Adoptive transfer of mincle stimulated DCs restricts Mtb survival in Abx treated mice. Lung DCs (5 × 106) from Mtb infected mice were PPD pulsed and stimulated with TDB (20 μg/ml) overnight. Cells were adoptively transferred to Abx treated mice, 24 h prior to Mtb infection. After 3 wk, Mtb burden was monitored in the lungs by CFU assay. Data represented as mean ± SD are of 2 independent experiments (n = 5 mice/group). **p < 0.01, ***p < 0.001.

Figure 7

Blocking of mincle receptor in lung DCs limits their ability to restrict the growth of Mtb. Lung DCs from CT and Abx treated mice were infected with Mtb (at multiplicity of infection 5) in vitro and incubated with or without anti-mincle blocking antibody (α mincle; 10 μg/ml) for 1 h prior to TDB stimulation (20 μg/ml). After 48 h, (A)Mtb killing ability of DCs was examined by CFU assay; and (B) IL-12 secretion in the culture SNs quantified by ELISA. Data represented as mean ± SD are of 2 independent experiments (n = 5 mice/group). **p < 0.01, ***p < 0.001., UI: uninfected DCs; UT: Mtb infected DCs; UT- α mincle: DCs infected with Mtb and treated with anti-mincle blocking antibody; TDB: Mtb infected DCs stimulated with TDB; TDB-α mincle: Mtb infected DCs treated with anti-mincle blocking antibody prior to TDB stimulation.

Figure 8

Triggering through mincle induces distinct shifts in the gut microbiota composition. Mice were treated with broad-spectrum Abx for 4 wk, followed by Mtb infection and TDB supplementation. After 30 d post infection, fecal DNA was isolated and subjected to (A–D) qPCR analysis, Bar graphs depict (A) total gut bacterial load; (B)Lactobacillus; (C)Bacteroides; (D)Enterococcus. Relative abundance depicted as fold change normalized with a universal bacterial primer, *p < 0.05, **p < 0.01, ***p < 0.001. Further, (E,F) 16S rRNA gene sequencing of fecal DNA was performed on Illumina MiSeq platform. (E) Relative abundance at phylum level was depicted in pie chart; (F) Bar diagram represents relative abundance of bacterial genera. Data are from two independent experiments (n = 5–6 mice/group).

Figure 9

Lactobacillus plantarum restores the expression of mincle in lung along with compromised immunity against Mtb in mice with gut dysbiosis. Mice were subjected to Abx in drinking water for 4 wk followed by L. plantarum (108 CFU per mice) administration every other day for 2 wk prior to Mtb infection until sacrifice. After 4 wk, lung tissue was harvested and examined for the (A)Mtb load by CFU assay. Further, (B,C) lung lymphocytes were cultured with PPD (25 μg/ml) for 72 h. Later, cells were evaluated for the expression of FoxP3 on CD4 T cells by flow cytometry. (D–G) Lung cells were monitored ex vivo for the expression of CD44 and CD62L to evaluate the (D,E) activation (CD44hi); and (F,G) effector memory response (CD62LloCD44hi) by flow cytometry. Contour plots and bar graphs represent the percentage population of cells gated on CD4 T cells. Data represented as mean±SD are of 2 independent experiments (n = 5 mice/group). CT: control mice with no Abx; Mtb: Mtb challenged mice; Abx-Mtb: mice treated with Abx prior to Mtb infection; Abx-Mtb-LP: Abx treated mice infected with Mtb and supplemented with Lactobacillus plantarum. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 10

Conceptual model of the study. During homeostasis, a healthy gut through microbial products such as glycolipids (synthetic analog, TDB that binds mincle) regulates lung immune response during Mtb infection. Gut commensals bacteria derived glycolipids reach lung via blood stream. In Mtb infected lungs, they bind to the mincle receptor expressed on DCs leading to their activated phenotype and functions such as production of immunoregulatory cytokines (IL-6, IL-12), which in turn elicits the CD4 T cells differentiation to Th1 and Th17 cells via the release of IFN-γ and IL-17 cytokines, respectively. Further, there is generation of memory response and protective immunity against Mtb in lungs. This immunoregulation through gut microbiota is disturbed upon the Abx induced dysbiosis. Abx depletes the beneficial commensals population, which is responsible for the impairment of DCs function and hence the dysregulated lung anti-Mtb immunity.