Ly6G+ granulocytes-derived IL-17 limits protective host responses and promotes tuberculosis pathogenesis
- Cellular Immunology Group, International Centre for Genetic Engineering and Biotechnology, India
- National Institute of Immunology, India
- Department of Pathology, All India Institute of Medical Sciences, India
- Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore
- Infectious Diseases Labs (ID labs), Agency for Science, Technology and Research (A*STAR), Singapore
- Department of Immunology, ICMR-National Institute for Research in Tuberculosis, India
- Prof. M. Viswanathan Diabetes Research Center, India
- Department of Medicine, University of Massachusetts Medical School, United States
- NIH-International Center of Excellence in Research, India
- Laboratory of Parasitic Diseases, NIAID-NIH, United States
- Centre for Cellular and Molecular Biology, India
- Lee Kong Chian School of Medicine, Nanyang Technological University (NTU), Singapore
Figures
Figure 1 with 1 supplement
PMNs harbors substantial amounts 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-, and 12 weeks post-infection time point. A two-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 color) and Mtb antigen Ag85B (shown in green color). (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, and 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, and 12 weeks post-infection, from the lungs of vaccinated and unvaccinated mice. Experimental groups were evaluated using a two-way ANOVA statistical test. Sample size (n)=5 mice/group in all treatment groups. This BCG vaccination experiment was carried out independently in two distinct experimental replicates. (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 normalized 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.
Figure 1—figure supplement 1
PMNs harbor a substantial amount of Mtb.
(A) Schematic representation of the study design and experimental timeline. C57BL/6 mice were infected with H37Rv, and at 4, 8, and 12 weeks post-infection, mice were sacrificed for various studies. Bacterial burden in (B) Lung and (C) Spleen of infected mice at 4, 8, and 12 weeks post-Mtb infection. (D) Bar graphs showing the numbers of sorted lung cells (PMNs, macrophages, and MSCs), normalized to per 1 million of total acquired cells at 4, 8, and 12 weeks post-infection. (E) Representative absolute CFU count present inside total sorted PMNs, macrophages, and MSCs from the lung. Experimental groups were evaluated using a two-way ANOVA and one-way ANOVA statistical test, respectively. Sample size (n)=5 mice/group in all mice groups. (F) Time course of the lung and spleen bacterial burden in unvaccinated and BCG-vaccinated groups. An unpaired student’s t-test was applied to compare both experimental groups at different time points. (G) Representative H&E-stained lung sections showed less pathology in the BCG-vaccinated group compared to the unvaccinated group. (H) Quantification of the sorted cells normalized to per 1 million of total acquired cells from the lungs of vaccinated and unvaccinated mice. Experimental groups were evaluated using a two-way ANOVA statistical test, n=5 mice/group in both groups.
Figure 2 with 1 supplement
Uncontrolled neutrophilia, huge Mtb burden, severe disease pathology, and death in ifng-/- mice is mediated by high IL-17.
(A) Experimental design and timeline. C57BL/6 mice (WT and ifng-/-) 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 ifng-/- mice at ×10 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 color) in Mtb-infected ifng-/- mice compared to WT mice. (D) Representative FACS dot plot showing the percentage of PMNs in the lungs of Mtb-infected WT and ifng-/- mice (population is gated on CD45+ cells). Percentage of PMNs in the lungs of Mtb-infected WT and ifng-/- 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 ifng-/-, plotted as CFU/104 sorted cells at 4, 7, and 10 weeks post-infection. Experimental groups were evaluated using a two-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 ifng-/- mice. (F) Cytokines and chemokines that are upregulated in Mtb-infected ifng-/- mice compared to WT mice (G) Cytokines and chemokines that are downregulated in Mtb-infected ifng-/- mice compared to WT mice. Data were evaluated using a two-way ANOVA statistical test. Sample size (n)=5–6 mice/group in all the groups. This experiment was performed only once as a single experimental replicate.
Figure 2—figure supplement 1
Contribution of neutrophilia to the severity of TB disease.
WT and IFNγ -/-mice 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 in Figure 2A. (A) Lung pictures from Mtb-infected WT and ifng-/- mice showing the disease pathology. (B) Enumeration of lung and spleen bacterial burden in WT and ifng-/- mice, 4, 7, and 10 weeks post-infection, n=5–6 mice per experimental group. A two-way ANOVA statistical test was applied to compare lung and spleen CFU all in WT and ifng-/- at different time points post-infection. (C) Survival curve of WT and ifng-/- following Mtb infection. Both strains of mice were infected with 100–200 CFU of H37Rv through an aerosol route followed by incubation till their natural death. The time of death of each mouse was noted and plotted for the survival analysis (n=12 mice/group). (D) Count of PMNs, normalized to 1 million of total acquired cells, in the lungs of infected WT and ifng-/- mice at 4, 7, and 10 weeks post-infection. n=5–6 mice per experimental group. Two-way ANOVA statistical test was applied to compare groups at different time points post-infection (E) Luminex data: A panel of 36 cytokines and chemokines was quantified from the lung supernatant of uninfected and Mtb-infected WT and ifng-/- mice. All the upregulated and downregulated cytokines and chemokines were compared pairwise between two treatment groups and plotted in the form of a heat map. (F) Lung cells from uninfected WT mice were stained with different antibodies and assayed through flow cytometry to elucidate the cellular source and levels of IL-17 in uninfected mice. Plots showing pooled samples from three mice. (G) Number of CD11b-Ly6G- IL-17+ granulocytes in the lungs of uninfected and Mtb-infected mice.
Figure 3
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 ifng-/- mice. (B) PGE2 levels in the lungs of Mtb-infected ifng-/- 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) and Ly6G- CD11b- cells showing IL-17 positivity in the lungs of Mtb-infected mice. (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 color) with IL-17 (shown in green color). The quantification of the CD4+ cells, IL-17+ cells, and double-positive IL-17+ CD4+ cells is presented as the proportion of DAPI+ cells. (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 color) with IL-17 (shown in green color). The quantification of the Ly6G+ cells, IL-17+ cells, and double-positive IL-17+ Ly6G+ cells is presented as the proportion of DAPI+ cells. (G) IFA images of the lungs of Mtb-infected ifng-/- mice at 6 weeks post-infection time point showing the co-localization of PMNs (Ly6G+ cells shown in red color) and IL-17 (shown in green color). The quantification of the Ly6G+ cells, IL-17+ cells, and double-positive IL-17+ Ly6G+ cells is presented as the proportion of DAPI+ cells. (H) Co-localization of PMNs (CD66b+ cells shown in red color) and IL-17 (shown in green color) in the gut granuloma section of intestinal TB (ITB) patients. The quantification of the CD66b+ cells, IL-17+ cells, and double-positive IL-17+ CD66b+ cells is presented as the proportion of DAPI+ cells.
Figure 4 with 1 supplement
Celecoxib treatment with IL-17 neutralization reverses IL-17-dependent neutrophilia and controls TB disease in ifng-/- 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 (100 µg/mouse), and the other arm received an isotype control antibody (100 µg/mouse) intraperitoneally twice in a week, starting a day before Mtb infection. After 3 weeks of infection, both groups of mice were further divided into two groups, one that received celecoxib (50 mg/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 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). This experiment was performed only once as a single experimental replicate. (F) Survival curve of the group of Mtb-infected ifng-/- 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 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). This experiment was performed only once as a single experimental replicate. (G) H&E-stained lung sections and granuloma scoring showing lung pathology from all four 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 color) and IL-17 (shown in green color). (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 the isotype control group.
Figure 4—figure supplement 1
IL-17 neutralization synergizes with COX-2 inhibition and controls TB disease in ifng-/- mice.
Study plan (as shown in Figure 4A): ifng-/- 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 (100 µg/mouse), and the other arm received an isotype control antibody (100 µg/mouse) intraperitoneally twice in a week, starting a day before Mtb infection. After 3 weeks of infection, both groups of mice were further divided into two groups, one that received celecoxib (50 mg/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. (A) Lung IL-17 levels in all four mice treatment groups as explained above along with uninfected control. (B) %Th17 cells (gated on CD4+ T cells) in the lungs of mice from the same treatment groups described above. The statistical significance of the treatment groups was assessed by applying a one-way ANOVA test. n=3–4 mice/group.
Figure 5 with 1 supplement
RORγt inhibition reduces mycobacterial burden and neutrophil infiltration in the lungs of Mtb-infected mice.
(A) IL-17 levels in the lung homogenates of uninfected and Mtb-infected WT mice. An 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 a further 3 weeks. Celecoxib (50 mg/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 PMNs 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 color) and IL-17 (shown in green color). The IL-17+ Ly6G+ (double-positive) cells were quantified and represented as a percentage of DAPI+ cells in all four treatment groups. This experiment was performed only once as a single experimental replicate.
Figure 5—figure supplement 1
IL-17 production inhibition is key to limiting TB severity in WT mice.
The experimental plan is shown in Figure 5(B). 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 a further 3 weeks. Celecoxib was administered orally once a day, and SR 2211 was administered intraperitoneally thrice a week. At 6 weeks post-infection, mice from the treatment and untreated control groups were sacrificed, and lung and spleen were harvested for CFU, histopathology, and FACS analysis. (A) Lung PMN count normalized to 1 million of total acquired cells in the above-stated treatment groups of mice. The statistical significance of the treatment groups was assessed by applying a one-way ANOVA test, n=5 mice/group. (B) H&E-stained lung sections showing pathology in SR2211+Celecoxib treatment group. (C) Granuloma scoring depicting lung pathology in four treatment groups of mice. n=5 mice per treatment group.
Figure 6
Targeting IL-17 via COX2 inhibition and RORγt 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 (50 mg/kg, orally) treatment for 4 and 8 weeks in with and without BCG vaccination group, keeping the untreated arm as control. At 4, 8, and 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 four treatment groups of mice. A two-way ANOVA statistical test was applied to compare all four 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 ×10 magnification. (E) Granuloma scoring of the same treatment groups depicting histopathology. This experiment was independently conducted in three separate experimental replicates (F) 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). At 4 weeks post-Mtb infection, mice were administered SR2211 (20 mg/kg) intraperitoneally three times per week for 3 weeks, in both BCG-vaccinated and unvaccinated groups, with untreated mice serving as vehicle control. At 7 weeks post-infection, mice were euthanized, and lung and spleen were harvested. (G) The Mtb burden in sorted PMNs was assessed at 7 weeks post-infection and expressed as CFU per 10⁴ sorted cells from the lungs of all four treatment groups (Control, BCG-vaccinated, SR2211-treated, and BCG-vaccinated+SR2211-treated). (H) Lung mycobacterial burden was also quantified across all four groups at the same time point. A two-way ANOVA was performed to compare the groups. Sample size (n)=6 mice/group in all treatment groups. This experiment was performed only once as a single experimental replicate.
Figure 7
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 IL-17, neutrophils, and total lymphocytes in both the treatment groups were measured. (B) Blood IL-17 levels, (C) blood neutrophil levels, 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.
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Additional files
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Supplementary file 1
Differentially expressed genes (DEGs) in PMNs identified between each pairwise comparison.
- https://cdn.elifesciences.org/articles/100966/elife-100966-supp1-v1.xlsx
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Supplementary file 2
Differentially regulated genes in Ly6G+Gra from unvaccinated, uninfected versus BCG-vaccinated, uninfected animals.
- https://cdn.elifesciences.org/articles/100966/elife-100966-supp2-v1.xlsx
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Supplementary file 3
Gene-set co-regulation analysis of the DEGs with GO-BP in PMNs among the Uninfected control group, Mtb-infected group, and BCG-vaccinated Mtb-infected group.
- https://cdn.elifesciences.org/articles/100966/elife-100966-supp3-v1.xlsx
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Supplementary file 4
Gene-set co-regulation analysis of the DEGs with KEGG pathways in PMNs among the Uninfected control group, Mtb-infected group, and BCG-vaccinated Mtb-infected group.
- https://cdn.elifesciences.org/articles/100966/elife-100966-supp4-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/100966/elife-100966-mdarchecklist1-v1.docx
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Ly6G+ granulocytes-derived IL-17 limits protective host responses and promotes tuberculosis pathogenesis
eLife 13:RP100966.
https://doi.org/10.7554/eLife.100966.3
