Fig 1.
Type I interferon signaling is protective in the absence of interleukin-1 signaling in rpoB-H445Y Mycobacterium tuberculosis infection.
C57Bl/6 (B6), Ifnar1-/-, Il1r1-/-, and Ifnar1-/- Il1r1-/- [double knockout (DKO)] mice were aerosol infected with a low dose of wt or rpoB-H445Y Mtb. Lung Mtb burden was evaluated at 14- and 30-days following A) wt or B) rpoB-H445Y Mtb infection. C) Formalin-fixed, paraffin-embedded (FFPE) lung sections were stained with hematoxylin and eosin (H&E), and the total inflammatory areas were quantified after rpoB-H445Y Mtb infection at 30 days post-infection (dpi). D) Using flow cytometry, the total numbers of lung MHCIIhi RMs and mDCs were determined at 30 dpi after infection with rpoB-H445Y Mtb. The data shown represent the means ± SD of seven to ten mice per experiment. The data were evaluated for normality using the Shapiro-Wilk Test and passed (p-value > 0.05). Two-way ANOVA was used for A and B, and One-way ANOVA was used for C and D, with Tukey’s multiple comparisons test. Significant differences are indicated with asterisks (*, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤ 0.001; ****, p-value ≤ 0.0001) by appropriate statistical tests. One of three independent experiments shown.
Fig 2.
Type I IFN signaling signatures are common across wt and rpoB-H445Y Mtb infections.
C57Bl/6 (B6), Ifnar1-/-, Il1r1-/-, and Ifnar-/- Il1r1-/- [double knockout (DKO)] mice were aerosol infected with a low dose of wt or rpoB-H445Y Mtb and sacrificed at 14 dpi. RNA was extracted from homogenized lung tissue and sequenced. Heatmaps displaying average expression levels of top differentially expressed genes (DEGs) across mouse genotypes after A) wt or B) rpoB-H445Y Mtb infection with annotated pathways (four to six mice per group). Significant DEGs related to the corresponding reactome pathways are visualized using heatmaps(|log2FC| >0, adjusted p-value < 0.05). Average expression (normalized) of selected genes on log2 scale are shown.
Fig 3.
Type I IFN producers are CD11b+ myeloid cells, while LysM+ cells respond to type I IFN following rpoB-H445Y Mtb infection.
IFNβ-YFP mice were aerosol infected with rpoB-H445Y Mtb and sacrificed at the indicated timepoints along with uninfected controls. A) Representative histograms for various subsets [Monocytes (left), Recruited Macrophages (RMs) (middle left), Neutrophils (middle right), and Alveolar Macrophages (AMs) (right)] depicting YFP expression by intracellular antibody staining (green) compared to controls [uninfected IFNβ-YFP mice stained with intracellular antibody (orange), infected IFNβ-YFP mice stained with intracellular isotype antibody (blue), and infected B6 non-reporter mice stained with intracellular antibody (red)]. B) The frequencies of YFP+ cells were determined by flow cytometry following sacrifice of rpoB-H445Y Mtb-infected IFNβ-YFP mice at 7, 14, and 28 dpi, along with uninfected controls. C) Experimental scheme for the LysM-Cre Ifnar1fl/fl mice with the purple arrow indicating aerosol infection with rpoB-H445Y Mtb and the pink arrow indicating administration of a-IL1R or isotype antibodies i.p. D) Lung Mtb burden was determined at 28 dpi. E) LysM-Cre+ Ifnar1fl/fl (semi-closed green circles) and Cd11c-Cre+ Ifnar1fl/fl mice (semi-closed orange circles), along with littermate controls (open blue circles), were infected with rpoB-H445Y Mtb, and bacterial burden was determined in the lungs at 28 dpi. The data shown represent the means ± SD of three to eight mice per experiment. The data were evaluated for normality using the Shapiro-Wilk Test and passed (p-value > 0.05). Two-way ANOVA and One-way ANOVA, with Tukey’s multiple comparisons tests, were used for B and D-E, respectively. Significant differences are indicated with asterisks (*, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤ 0.001; ****, p-value ≤ 0.0001) by appropriate statistical tests. One-way ANOVA. Two independent experiments shown.
Fig 4.
NO production restricts intracellular rpoB-H445Y Mtb growth in macrophages.
Bone marrow-derived macrophages (BMDMs) from B6, Ifnar1-/-, Il1r1-/-, DKO mice were infected with either A) wt Mtb or B) rpoB-H445Y Mtb, and Mtb CFU was determined at 6 dpi. C) The concentration of nitrite in the supernatants of wt or rpoB-H445Y Mtb infected BMDMs was determined, along with uninfected controls. D) B6, Il1r1-/-, Ifnar-/- and DKO BMDMs were infected with rpoB-H445Y Mtb and then treated with the indicated concentrations of an NO scavenger, Carboxy-PTIO (C-PTIO). Bacterial burden was determined on 6 dpi. Fold change in Mtb growth was determined relative to untreated and infected BMDMs. E) B6 and Nos2-/- BMDMs were infected with rpoB-H445Y Mtb, and Mtb CFU was determined at 6 dpi. The data shown represent the means ± SD of three to four biological replicates per experiment. The data were evaluated for normality using the Shapiro-Wilk Test and passed (p-value > 0.05). One-way ANOVA and Two-way ANOVA, with Tukey’s multiple comparisons tests, were used for A-B and C-D, respectively. Unpaired two-tailed student’s t-test was used for E. Significant differences are indicated with asterisks (*, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤ 0.001; ****, p-value ≤ 0.0001) by appropriate statistical tests. One of three independent experiments shown.