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 < 0.005, ***p < 0.0001, ****p < 0.0001.

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.

Intrinsic control of infection and response to IFN-γ is not impaired in macrophages from wild-derived mice.

(A) 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 hours 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 hours post infection with reporter cells lines. Data are a representative of 2 independent experiments. The p values were determined using a Kruskal-Wallis ANOVA. **p < 0.005, ****p < 0.0001.

Diverse populations of neutrophils are present during infection of Manaus mice.

(A) Bacterial burden in the 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 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. (G,H) 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 a representative of 3 independent (MANC) or 2 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 4 mice.

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 neutrophils signature according to neutrophil developmental stage.

Expression of neutrophil markers across multiple cell lineages in MANC mice.

(A) heat map 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.