Lack of Th17 induction during WT M. tuberculosis infection is not due to duration or route of infection

(A-C) WT B6 mice were aerosol infected with WT M. tuberculosis Erdman strain and lungs were evaluated at 21 days post infection (dpi) for (A) bacterial burdens by CFU (B) IFN-γ+ CD4+ T cells and (C) IL-17A+ CD4+ T cells using flow cytometry. Cytokines were measured using ICS after restimulation with Ag85b or ESAT-6 peptide pools. (D-E) Mice were infected and analyzed at 170 dpi for (D) IFN-γ+ CD4+ T cells and (E) IL-17A+ CD4+ T cells in lungs. (F-H) WT B6 mice were either aerosol infected or intranasally infected with WT M. tuberculosis Erdman strain and evaluated 28 dpi for (F) bacterial burdens in lungs by CFU, (G) IFN-γ+ CD4+ T cells and (H) IL-17A+ CD4+ T cells in lungs by flow cytometry. Cytokines were measured using ICS after restimulation with ESAT-6 peptide pool. (I) WT B6 mice were aerosol infected with WT M. tuberculosis and treated with PBS control, recombinant murine IL-17A (IL-17A) or recombinant murine IL-17A fused to murine serum albumin (MSA-IL-17A) daily either using the intranasal (i.n.) or intraperitoneal (i.p.) routes between days 11 to 20 post infection. CFU in lungs was measured at 21 dpi. Results are representative of at least 2 biological replicates. For all graphs, each dot represents an individual mouse. *p<0.05, **p<0.01 (unpaired nonparametric Mann-Whitney U test).

Tbet−/− mice are protected from susceptibility to Mtb infection by the emergence of Th17 cells

WT B6, Ifng−/−, Tbet−/−, and Il17a−/−Tbet−/− mice were aerosol infected with WT M. tuberculosis Erdman strain. Tbet−/− and Il17a−/−Tbet−/− mice were littermate-controlled. Mice were infected with Mtb and evaluated 21 days post infection for (A) CFU in the lungs of indicated genotypes or (B-F) cellular immune responses using flow cytometry. CD4+ T cells were gated on single, live, MHC-II−, Ly6G−, CD3+, γδTCR−, CD4+, CD8a− cells for analysis of IFN-γ (B,C), IL-17 (B,D) and RORγT (E,F). Results in A-F are representative of 3 independent experiments. *, p < 0.05; **, p < 0.01 (unpaired nonparametric Mann-Whitney U test). confers significant protection against Mtb infection in Tbet−/− mice, at least during early stages of infection.

M. tuberculosis ESX-1 and PDIM virulence factors suppress Th17 response in mice

WT B6 mice were aerosol infected with indicated strains and evaluated at 21 days post infection. (A) Representative flow cytometry plots (cells gated on single, live, MHC-II−, Ly6G−, CD3+, γδTCR−, CD4+, CD8a−) of IFN-γ+ and IL-17A+ CD4+ T cell populations. Mice were sacrificed and evaluated with ESAT-6 peptide pool stimulated lung homogenates for (B) IFN-γ+ CD4+ T cells and (C) IL-17A+ CD4+ T cells by flow cytometry. Mice were sacrificed and lung homogenates were evaluated with BioLegend LEGENDplex for (D) IFN-γ and (E) IL-17A protein concentrations. (F-G) Littermate-controlled WT B6 and Il17a−/− mice were aerosol infected with indicated strains and sacrificed at 21 days post infection with lung homogenate plated for CFU. (H-I) Mice were infected with WT, mmpl4 mutant, or complemented mmpl4 mutant Mtb and evaluated for (H) day 21 CFU and (I) IL-17A+ CD4+ T cells by flow cytometry. Results are representative of 2 independent experiments. *, p < 0.05; **, p < 0.01 (Mann-Whitney U test).

Type I interferon signaling boosts Th1 induction and does not impact Th17 induction

WT B6, Ifnar−/−, Sp140−/−, Sp140−/−Ifnar−/−, Ifng−/−, and Tbet−/− mice were aerosol infected with WT M. tuberculosis Erdman strain. (B, C) Representative flow cytometry plots (cells gated on single, live, MHC-II−, Ly6G−, CD3+, γδTCR−, CD4+, CD8a−) of IFN-γ+ and IL-17A+ CD4+ T cells of these populations. 21 days post infection, lung homogenates were measured for (A) CFU and analyzed via flow cytometry for (D) IFN-γ+ CD4+ T cells and (E) IL-17A+ CD4+ T cells. Results in A, D, and E are representative of 2 independent experiments. *, p < 0.05; **, p < 0.01 (unpaired nonparametric Mann-Whitney U test).

ESX-1 and PDIM alter conventional dendritic cell cytokine signaling in the mediastinal lymph node

WT B6 bone-marrow-derived macrophages were infected with designated Mtb Erdman strains at MOI = 10 or uninfected for 24 hours with (A) IL-23 ELISA taken from cultured supernatants. WT B6 mice were aerosol infected with indicated strains and analyzed at 21 days post infection (C,E) Flow cytometry schematic for gating of (B) cDC1 (gated on single, live, CD3−, CD19−, CD11c+, MHC-II+, XCR1+, CD11b−) and (D) cDC2 (gated on single, live, CD3−, CD19−, CD11c+, MHC-II+, XCR1−, CD11b+) populations of mediastinal lymph nodes with representative flow cytometry plots of IL-12 p35+ and IL-23 p19+ gating of cDC1 and cDC2 populations respectively. 21 days post infection, mediastinal lymph nodes from WT B6 mice were aerosol infected with either WT, ΔeccC1 (ΔESX-1), complemented ΔeccC1::eccC1, transposon-inserted fadD28::tn (PDIM-lacking),or complemented transposon-inserted fadD28::tn + pfadD28 M. tuberculosis Erdman strain were extracted and analyzed for IL-12 p35+ cDC1s (C) and IL-23 p19+ cDC2s (E) by flow cytometry. Results in A, C, and E are representative of 2 independent experiments. *, p < 0.05; **, p < 0.01 (unpaired nonparametric Mann-Whitney U test).

Mice were infected with either the Erdman or Beijing strain of Mtb via the aerosol route and evaluated at 21dpi for (A) CFU in the lungs or (B-G) IL-17A+ CD4+ T cells in the lungs. (B,C) unstimulated, (D,E) restimulated with Antigen 85B peptide, (F,G) restimulated with ESAT-6 peptide. Representative experiment of 2.

Mtb Erdman PDIM mutants secrete ESAT-6 and ESX-1 mutants produce PDIM

(A) WT, ΔeccC1, complemented ΔeccC1::eccC1 (ΔeccC1 comp), fadD28::tn, or fadD28::tn + pfadD28 (fadD28::tn comp) M. tuberculosis Erdman strain were cultured in Sauton’s complete media without Tween-80 for 5 days. Cell lysate and culture supernatant were processed through SDS-PAGE and Western blot analysis. Anti-ESAT-6 antibody was used for detecting ESAT-6. Anti-Mtb GroEL2 antibody (BEI Resources NR-13657) was used as a loading control for the cell lysate fraction. Anti-Mtb Mpt32 antibody (BEI Resources NR-13807) was used as a loading control for the culture filtrate. Results shown are from 1 experiment representative of 2 independent experiments. (B) WT, ΔeccC1 (ESX), complemented ΔeccC1::eccC1 (ESX(C), fadD28::tn (PDIM), or fadD28::tn + pfadD28 (PDIM(C)) M. tuberculosis Erdman strain were cultured in 7H9 media. Outer leaflet of mycomembrane was removed via hexanes wash, concentrated and separated on TLC plate with 98:2 petroleum ether:acetone mobile phase. TLC plate was stained with 0.2% Coomassie blue in 20% methanol. Results shown are from 1 experiment representative of 2 independent experiments.

Attenuation of ESX-1 and PDIM mutants at 21 days post infection.

WT B6 mice were aerosol infected with either WT, ΔeccC1, ΔeccC1::eccC1, fadD28::tn, or fadD28::tn + pfadD28 M. tuberculosis Erdman strain. 21 days post infection, mice were sacrificed, and CFU were enumerated from lung lysates.

Antigen 85b stim resembles ESAT-6 stimulation for IFN-γ and IL-17A staining of CD4+ T cells.

As in Figure 3, WT B6 mice were aerosol infected with either WT, ΔeccC1, ΔeccC1::eccC1, fadD28::tn, or fadD28::tn + pfadD28 M. tuberculosis Erdman strain. 21 days post infection, mice were sacrificed, lung single cell homogenates were stimulated with Antigen 85b peptide pool and measured for lung (A) IFN-γ+ CD4+ T cells and (B) IL-17A+ CD4+ T cells by flow cytometry. Results in A and B are representative of 2 independent experiments. *, p < 0.05; **, p < 0.01 (unpaired nonparametric Mann-Whitney U test).

ΔmmpL4 Mtb induces similar IFN-γ+ CD4+ T cells as WT or complemented Mtb.

Mice were aerosol infected with WT, ΔmmpL4 mutant, or complemented ΔmmpL4 Mtb Erdman strain and evaluated for IFN-γ+ CD4+ T cells after restimulation with ESAT-6 peptide by flow cytometry. Results are representative of 2 independent experiments. *, p < 0.05; **, p < 0.01 (unpaired nonparametric Mann-Whitney U test).

There is little to no IL-23 p19 expression in cDC1s and IL-12 p35 expression in cDC2s during Mtb infection.

As in Figure 5, WT B6 mice were aerosol infected with WT, ΔeccC1, ΔeccC1::eccC1, fadD28::tn, or fadD28::tn + pfadD28 M. tuberculosis Erdman strain. 21 days post infection, mice were sacrificed, mediastinal lymph nodes were extracted and processed for ICS. Analysis of (A) IL-23 p19+ cDC1s and (B) IL-12 p35+ cDC2s by flow cytometry. Results in A and B are representative of 2 independent experiments. (unpaired nonparametric Mann-Whitney U test).