Tuberculosis susceptibility in genetically diverse mice reveals functional diversity of neutrophils
- Department of Plant and Microbial Biology, University of California, Berkeley, United States
- Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, United States
- School of Public Health, Division of Infectious Disease and Vaccinology, University of California, Berkeley, United States
- Department of Integrative Biology, University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
Mouse lines from different geographical regions show variable susceptibility to Mtb infection.
(A) Mice were collected from five different sites across the Americas and inbred to generate individual genetic lines from each location. Map obtained from https://simplemaps.com/resources/svg-world. (B) Bacterial burden in the lungs of the mice derived from the different locations at 21 days post-infection with aerosolized Mtb. Comparison of the susceptibility among genetically distinct Manaus lines (C) or Saratoga lines (D) infected with aerosolized Mtb. The average inoculum dose is 400 CFU/mouse. CFU data was normalized to CFU enumeration on day 1 to correct for variation in inoculum dose between experimental days. EDM: Edmonton, SAR: Saratoga Springs, TUC: Tucson, GAI: Gainesville, MAN: Manaus. Data represent 2–6 independent experiments for each mouse line with 6–34 mice per line. Each dot represents a mouse. The p values were determined using a Kruskal–Wallis ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 1—figure supplement 1
Variation in susceptibility to infection in wild mice does not depend on mouse weight or sex.
CFU in the lungs of mice from (A) Tucson, (B) Gainesville, and (C) Edmonton at 21 days post-infection were enumerated by CFU. (D) Body weight of adult male (M) and female (F) mice at age 7 weeks of age. (E) Spearman correlation analysis of CFU in the lungs versus weight at 7 weeks. (F, G) CFU data from the lungs of mice at 21 days post-infection broken down by both sex and genotype. CFU data was normalized to CFU enumeration on day 1 to correct for variation in inoculum dose between experimental days. Data represent 2–3 independent experiments, except for a single experiment for the TUCC, EDMA, EDMC, and EDMD lines. Each dot represents a mouse. *p < 0.05, **p < 0.01, ***p < 0.001 by Mann–Whitney U test.
Figure 2 with 3 supplements
Genetic diversity translates into variability in bacterial burden and immune cells in the lung during Mtb infection.
Cellular percentages were enumerated in the lungs using flow cytometry at 21 days post-infection with Mtb between and within mouse lines from different locales (A–E). The correlation between immune cell type (as shown in A–E) and CFU in the lung is depicted for individual mice (F–M). Data represent 2–6 independent experiments for each mouse line with 6–34 mice per line. The p value was determined using two-tailed Spearman correlations.
Figure 2—figure supplement 1
Cell percentages from flow cytometry-based analysis in the lungs of infected wild mice relative to B6.
Percentages of live cell populations were enumerated at 21 days post-infection with Mtb. Each circle represents an individual mouse. Data is representative of two to six independent experiments for each line. The p values were determined using a Kruskal–Wallis ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 2—figure supplement 2
The proportion of IFN-γ producing CD4 T cells in the lung does not correlate with bacterial burden.
(A) Gating strategy for individual cell types in the lung. All cell populations were gated on live cells. CD4 and CD8 cell populations were identified from CD3+ cells, followed by IFN-γ staining. Neutrophils were identified as CD11b+Ly6G+ and macrophages/monocytes as CD11b+Ly6G−. (B) Spearman correlation of CFU versus CD4+IFN-γ+ cells in the lungs of B6 or SARA, SARB, and SARC mouse lines infected with Mtb enumerated at 21 days post-infection. Data represent at least 2 independent experiments per line. The p value was determined using two-tailed Spearman correlations.
Figure 2—figure supplement 3
Immune cell composition in the lungs of infected mice.
Spearman correlations between bacterial burden and presence of neutrophils (CD11b+Ly6G+) in the lung of infected B6 (A), Saratoga (B), and Manaus (C) mice. (D–G) UMAP plots of single cells isolated from the lungs of Mtb-infected mice at 21 days post-infection. (D) B6, (E) MANA, (F) MANB, and (G) MANC.
Figure 3
Intrinsic control of infection and response to IFN-γ is not impaired in macrophages from wild-derived mice.
(A) Bone marrow-derived macrophages (BMDM) isolated from indicated mouse lines were infected with Mtb and bacterial growth was determined at 4 days post-infection by enumerating CFU. (B) The presence of nitrite in the supernatant was determined by Griess assay at 48 hr post-infection. (C) IFN-γ-activated BMDM were infected with Mtb and CFU were measured on day 4 post-infection. Production of type I IFN (D) or IL-1 (E) was measured at 24 hr post-infection with reporter cell lines. Data are representative of two independent experiments. The p values were determined using a Kruskal–Wallis ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.
Figure 4 with 2 supplements
Diverse populations of neutrophils are present during infection of Manaus mice.
(A) Bacterial burden in the lung was assessed in Manaus mice at 25 days post-infection. (B, C) Percentages and absolute counts of CD11b+Ly6G+ cells in the lungs at 25 days post-infection were enumerated by flow cytometry. Percentage is relative to the total number of live cells. (D, E) MANC and MANB (F) mice were treated with neutrophil-depleting antibody (1A8) or isotype control (2A3) every other day starting on day 11 post Mtb infection. Bacterial burden and neutrophil numbers were quantified on day 25 post-infection. Immunofluorescent microscopy on fixed lung tissues from MANB (G) and MANC (H) mice at 21 days post-infection. Area quantification of Ly6G+ (I) or CD4+ (J) staining in lung sections. (K, L) UMAP clustering of neutrophils from scRNAseq data of live cells in the lung at 21 days post Mtb infection. Signature functional genes that are characteristic of each cluster are provided with color coding according to cluster. (M) Gene ontology enrichment analysis of pathways down- or upregulated in MANC neutrophils compared with other neutrophil genotypes shows enrichment of genes involved in neutrophil activation and metabolism. CFU and antibody depletion data are representative of three independent (MANC) or two independent (MANB) experiments. The p values were determined using Mann–Whitney U test (A, I, J) and Kruskal–Wallis ANOVA (B, C), *p < 0.05. scRNAseq data represent a single experiment with pooled tissues from four mice.
Figure 4—figure supplement 1
Immune cell composition in the lungs of infected mice.
(A–C) Gene expression analysis of lung neutrophils scored according to gene signatures identified by Xie et al., 2020.
Figure 4—figure supplement 2
T cell subsets and gene expression in wild-derived mice.
(A) UMAP analysis of CD4 T cell subsets in the lung from scRNAseq expression data of Manaus and B6 mice. (B) Heatmap of scRNAseq expression data of T cell clusters from individual Manaus lines and clusters from mixed mouse lines. Significance calculation is students t-test, p < 0.05.
Figure 5
Neutrophil signature of MANC mice is unique relative to other susceptible mouse lines.
(A) Comparison of gene expression between highly expressed genes in the MANC neutrophil clusters with expression in MANB (B), MANA (A), B6, and clusters of mixed genotype (M). (B) Comparison of MANC signature genes with expression of genes in the Sp140–/– susceptible mouse line. (C) MANC neutrophil signature according to neutrophil developmental stage.
Figure 6 with 1 supplement
Expression of neutrophil markers across multiple cell lineages in MANC mice.
(A) Heatmap of gene expression of markers of macrophage/monocyte subsets according to cluster and genotype. (B) Expression of indicated genes across all cell types comparing the B6, MANB, and MANC genotypes. (C) Violin plot of S100a9 expression according to cell type and genotype.
Figure 6—figure supplement 1
Gene expression across immune cells.
(A) Unsupervised clustering of CD11b+Ly6G− monocyte/macrophage-like cells in the lung based on scRNAseq gene expression color coded by cluster (A) or genotype (B). (C) Expression of indicated genes across immune cell types analyzed using Immunological Genome Project (ImmGen) MyGeneset application (http://rstats.immgen.org/MyGeneSet_New/index.html). Inflammatory monocytes (iMos); patrolling monocytes (pMos); stem cells (stem); B cells; T cells/NK T cells (NKT); γδ T cells (γδ); natural killer/innate lymphoid cells (NK/ILC); dendritic cells (DC); macrophages (Ma); monocytes (Mo); neutrophils (Neut); mast cells/basophils (M/B); stromal cells (S).
Additional files
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Supplementary file 1
Cellular expression markers used for scRNAseq cluster assignment following unbiased clustering.
- https://cdn.elifesciences.org/articles/102441/elife-102441-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/102441/elife-102441-mdarchecklist1-v1.docx
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Tuberculosis susceptibility in genetically diverse mice reveals functional diversity of neutrophils
eLife 13:RP102441.
https://doi.org/10.7554/eLife.102441.2
