PMNs harbours substantial amount of Mtb and provide an immune-privileged niche to Mtb.

(A) FACS gating strategy used to sort PMNs, Macrophages and MSCs from the lungs of mice. (B) Mtb burden in individual sorted cells (PMNs, Macrophages and MSCs), plotted as CFU/104 sorted cells from the lungs of Mtb infected mice at 4-,8-& 12-weeks post-infection time point. 2-way ANOVA statistical test was applied to compare all the groups at different time points. Sample size (n)= 5 mice at each time point. (C) IFA images of the lungs of Mtb-infected mice showing the co-localization of PMNs (Ly6G+ cells shown in red colour) and Mtb antigen Ag85B (shown in green colour). (D) Study design for BCG vaccination experiment. C57BL/6 mice were vaccinated intradermally with 106 BCG bacilli, followed by aerosol infection with H37Rv (100-200 CFU) 8 weeks post BCG vaccination. At 4,8,12 weeks post-infection, mice were euthanized for various studies. (E) Determination of Mtb burden in individual sorted cells, plotted as CFU/104 sorted cells at 4,8,12 weeks post-infection, from the lungs of vaccinated and unvaccinated mice. Experimental groups were evaluated using a 2-way ANOVA statistical test. Sample size (n)= 5 mice/group in all treatment groups. (F) Venn diagram highlighting the number of common and unique differentially expressed genes (DEGs) from each pairwise comparison between BCG vaccinated + infected, only infected and uninfected controls (G) z-scaled expression of the DEGs corresponding to the significantly enriched Gene Ontology Biological processes obtained using gene set co-regulation analysis of the DEGs (H) highlights the enriched KEGG pathways based on gene set co-regulation analysis of the DEGs. Within the plotGesecaTable() function, z-scores are first calculated for each normalised gene expression across the dataset. For each GO BP/KEGG pathway, the expression of its corresponding genes are summed up per sample and scaled and defined within the range of −2 to 2.

Uncontrolled neutrophilia, huge Mtb burden, severe disease pathology and death in IFNγ−/− mice is mediated by high IL-17

(A) Experimental design and timeline. C57BL/6 mice (WT and IFNγ−/−) were infected with H37Rv, 100 CFU, via the aerosol route. Mice from both groups were euthanized at 4,7- and 10 weeks post-infection time-point. Lung and spleen tissues were harvested and processed for diverse assays, as shown in the study design. (B) H&E-stained lung sections of WT and IFNγ−/− mice at 10X magnification, showing severe histopathology in the latter group of mice. (C) Lung IFA images from the same group of mice showing the extensive infiltration of PMNs (Ly6G+ cells stained in red colour) in Mtb-infected IFNγ−/− mice compared to WT mice. (D) Representative FACS dot plot showing the percentage of PMNs in the lungs of Mtb-infected WT and IFNγ−/− mice (population is gated on CD45+ cells). Percentage of PMNs in the lungs of Mtb-infected WT and IFNγ−/− mice (population is gated on CD45+ cells) is shown at the right. (E) Mtb burden in the PMNs, sorted from the lung of Mtb infected WT and IFNγ−/− (plotted as CFU/104 sorted cells) at 4-,7- and 10 weeks post-infection. Experimental groups were evaluated using a 2-way ANOVA statistical test. Sample size (n)= 5-6 mice/group in both the groups. Luminex data: Quantification of several cytokines and chemokines from the lung supernatant of uninfected and Mtb-infected WT and IFNγ−/− mice. (F) Cytokines and chemokines that are upregulated in Mtb-infected IFNγ−/− mice compared to WT mice (G) Cytokines and chemokines that are downregulated in Mtb-infected IFNγ−/− mice compared to WT mice. Data was evaluated using a 2-way ANOVA statistical test. Sample size (n)= 5-6 mice/group in all the groups.

PMNs are one of the major sources of IL-17 in the lungs of Mtb-infected mice.

(A) Levels of IL-23 in the lung supernatant of uninfected and Mtb-infected WT and IFNγ−/− mice. (B) PGE2 levels in the lungs of Mtb-infected IFNγ−/− mice compared to uninfected control. An unpaired student’s t-test was applied to calculate the significance between the two groups. (C) Lung cells from Mtb-infected WT mice were stained with different antibodies and assayed through flow cytometry to elucidate the cellular source for elevated IL-17. FACS gating strategy showing PMNs (Ly6G+ CD11b+ cells)) as the dominant producer of IL-17 in the lungs of Mtb-infected mice compared to Ly6G− CD11b− cells. (D) Number of Ly6G+ IL-17+ granulocytes in the lungs of uninfected and Mtb-infected mice. (E) IFA images of the lungs of Mtb-infected WT mice at 6 weeks post-infection time-point showing the co-localization of CD4+ T cells (shown in red colour) with IL-17 (shown in green colour). (F) IFA images of the lungs of Mtb-infected WT mice at 6 weeks post-infection time-point showing the co-localization of Ly6G+ Gra (shown in red colour) with IL-17 (shown in green colour). (G) IFA images of the lungs of Mtb-infected IFNγ−/− mice at 6 weeks post-infection time-point showing the co-localization of PMNs (Ly6G+ cells shown in red colour) and IL-17 (shown in green colour). (H) Co-localization of PMNs (CD66b+ cells shown in red colour) and IL-17 (shown in green colour) in the gut granuloma section of intestinal TB (ITB) patients.

Celecoxib treatment with IL-17 neutralization reverses IL-17-dependent neutrophilia and controls TB disease in IFNγ−/− mice.

(A) Study plan. IFNγ−/−C57BL/6 mice infected with H37Rv at a dose of 100 CFU via the aerosol route. One arm of the Mtb-infected mice received an αIL-17 neutralizing antibody (100µg/mouse), and the other arm received an isotype control antibody (100µg/mouse) intraperitoneally twice in a week, starting a day before Mtb infection. After 3 weeks of infection, both the groups of mice were further divided into two groups, one that received celecoxib (50mg/kg, orally) for 3 weeks and the other that received vehicle control (DMSO here). Mice from all four groups of mice were euthanized at 6 weeks post Mtb infection, and their lungs and spleen were harvested, and serum was stored for ELISA. (B) PMN’s count, normalized to 1 million total acquired cells, in the lungs of infected mice in all four treatment groups at 6 weeks post-infection. (C) CFU (normalized to per 104 sorted cells) inside PMNs sorted out from the lungs of infected mice at 6 weeks post-infection sacrifice. Bacterial burden in lung (D) and spleen (E) of Mtb infected mice from described treatment groups at 6 weeks post-infection time-point sacrifice. The statistical significance of the treatment groups was assessed by applying a one-way ANOVA test. (n= 3-4 mice/group). (F) Survival curve of the group of Mtb infected IFNγ−/− mice that received αIL-17 neutralizing antibody, celecoxib alone or together, along with the untreated control group and infected WT mice group. Both mice strains were infected with 100 CFU of H37Rv through an aerosol route. One arm of the Mtb-infected mice received an αIL-17 neutralizing antibody, and the other arm received an isotype control antibody twice a week, starting a day before the Mtb infection. After 3 weeks of infection, both the groups of mice were further divided into two groups, one that received celecoxib for 3 weeks and the other that received vehicle control (DMSO). Mice were incubated till their natural death. The time of death of each mouse was noted and plotted for survival analysis (n= 7 mice/group). (G) H&E-stained lung sections and granuloma scoring showing lung pathology from all 4 treatment groups. (H) Lung IFA images from the above-stated treatment groups of mice show the colocalization (yellow) of PMNs (Ly6G+ cells shown in red colour) and IL-17 (shown in green colour). (I) Representative FACS dot plot showing the percentage of PMNs in the lungs of Mtb-infected mice in all four above stated treatment groups (population is gated on CD45+ cells). This also shows the presence of a distinct CD11bmid Ly6Ghi population in isotype control group.

RORγt inhibition brings down Mtb burden and augments BCG vaccine efficacy in WT mice.

(A) IL-17 levels in the lung homogenates of uninfected and Mtb-infected WT mice. Unpaired student’s t-test was applied to calculate significance between the two groups. Sample size (n)= 5-6 mice/group (B) Study plan. C57BL/6 mice were infected with H37Rv at a 100-200 CFU dose via the aerosol route. At 3 weeks post-infection, treatment with celecoxib and SR 2211 (inverse agonist for RORγt) was started for further 3 weeks. Celecoxib (50mg/kg) was administered orally once a day, and SR 2211 (20mg/kg) was administered intraperitoneally thrice in a week. At 6 weeks post-infection, mice from the treatment and untreated control groups were sacrificed, lung and spleen were harvested for CFU, histopathology and FACS analysis, and lung homogenates were stored for ELISA. (C) PGE2 levels in the lung homogenates of uninfected and Mtb-infected WT mice at 4- and 12-weeks post infection time-point. Unpaired student’s t-test was applied to calculate significance between the two groups at each time point. Sample size (n)= 3-4 mice/group. (D) Levels of IL-17 in the lung homogenates of all four-treatment groups of mice. The statistical significance of the treatment groups was assessed by applying a one-way ANOVA test, n=3-4 mice/group. (E) Representative FACS dot plot showing the percentage of PMNs in the lungs of Mtb-infected mice in all four above stated treatment groups (population is gated on CD45+ cells). Mycobacterial burden in the lung (F) and spleen (G), in the above-stated treatment groups of mice. (H) Lung PMN’s Mtb burden normalized to per 104 sorted cells in all four treatment groups of mice. The statistical significance of the treatment groups was assessed by applying a one-way ANOVA test, n=5 mice/group. (I) Lung IFA images from the above-stated treated group of mice show the colocalization of PMNs (Ly6G+ cells shown in red colour) and IL-17 (shown in green colour).

Targeting IL-17 via COX2 inhibition augments BCG efficacy by lowering Ly6G+Gra-resident Mtb.

(A) Study plan. C57BL/6 mice were vaccinated intradermally with 106 BCG bacilli. After 8 weeks, vaccinated and unvaccinated mice were subjected to aerosol infection with H37Rv (100 CFU). 4 weeks post Mtb infection, mice received celecoxib (50mg/kg, orally) treatment for 4 and 8 weeks in with and without BCG vaccination group keeping the untreated arm as control. At 4,8,12 weeks post-infection, mice were euthanized, and lung and spleen were harvested. (B) Enumeration of lung and spleen mycobacterial burden in all four treatment groups (Control, BCG-vaccinated, celecoxib-treated and BCG-vaccinated celecoxib-treated) at 8- and 12-weeks post-infection. (C) Mtb burden in individual sorted cells (PMNs, Macrophages and MSCs), plotted as CFU/104 sorted cells at 12 weeks post-infection time point, from the lungs of all 4 treatment groups of mice. 2-way ANOVA statistical test was applied to compare all 4 treatment groups at different time points. Sample size (n) = 5 mice/group in all treatment groups. (D) H&E-stained lung sections of Control, BCG-vaccinated, celecoxib-treated and BCG-vaccinated celecoxib-treated group at 10X magnification. (E) Granuloma scoring of the same treatment groups depicting histopathology.

High IL-17 precipitates adverse treatment outcomes in pulmonary TB patients.

(A) Pulmonary TB (PTB) patients-cohort study design: It includes 133 subjects that were cured of TB after the full standard ATT regime and 68 subjects that have failed the standard ATT regime (independent of drug resistance) or have experienced recurrence of symptoms. At the time of recruitment in the cohort study, the baseline blood levels of IL17, neutrophils, total lymphocytes in both the treatment groups were measured. (B) blood IL17 levels, (C) blood neutrophils level and (D) blood neutrophil/total lymphocyte ratio in treatment failure and treatment success cases of pulmonary TB patients. Unpaired student’s t-test was applied to calculate significance. (E) Schematic showing the therapeutic potential of COX-2 and RORγt inhibitor in TB by targeting IL-17-PMN axis.