Type I IFN-inducible gene expression and NETs in lungs of C57BL/6 and C3HeB/FeJ mice. (a) Dot plots showing expression of individual representative ISGs or a 37-gene type I IFN-response signature (Kotov et al., 2023) in myeloid cell populations in scRNA-seq data. (b–g) Analysis of lung multiplex immunofluorescence in HN878-infected mice with and without IFNAR blockade in the experiments described in Figs. 8 and 9. (b and c) Numbers of total CD4+ T cells in whole left lungs normalized for tissue area. (d) Abundance of CD4+ T cells in lung lesions expressed relative to that in non-lesional lung. Statistical analysis: unpaired t test, with Welch’s correction applied in b. (e) Representative images showing all merged channels for NET staining (top) and CitH3 and DAPI alone (bottom) in C3HeB/FeJ mice at 20 days after infection. Scale bar = 50 μm. (f) Quantification of CitH3 NET staining relative to Ly6G staining in lung lesions. Statistical analysis: unpaired t test. (g) Percentage of lung lesions with low (CitH3/Ly6G < 0.2), intermediate (CitH3/Ly6G 0.2–0.4), or high (CitH3/Ly6G > 0.4) NET burden. Data shown are means ± standard error. Statistical analysis shown is Dirichlet-multinomial regression analysis of the effect of αIFNAR treatment on lesion NET status. Symbols indicate significant differences in the proportion of NET-low lesions. Data in b–g are from single experiments with N = 5 mice per group and are representative of two independent experiments. (h) DESeq2-normalized expression values of genes of interest as identified in Fig. 10 in whole lungs early in infection with HN878. Data shown are from a single bulk RNA-seq experiment with N = 5 mice per group and represent individual replicate mice as points with lines at the mean. Statistical analysis: two-way ANOVA with Holm–Sidak post hoc test. (i) Dot plot showing expression of Slfn4 in scRNA-seq clusters. Data in a and i are from a single scRNA-seq experiment, and plots show combined data from cells from N = 3 mice per group. Circle sizes represent the abundance of cells expressing the gene, as a percentage of either cells in the cluster (a) or within total cells (i). Circle color is proportional to the mean expression of the gene within all cells in the cluster. Actual P values are shown or: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.
Figure S5.

Type I IFN-inducible gene expression and NETs in lungs of C57BL/6 and C3HeB/FeJ mice. (a) Dot plots showing expression of individual representative ISGs or a 37-gene type I IFN-response signature (Kotov et al., 2023) in myeloid cell populations in scRNA-seq data. (b–g) Analysis of lung multiplex immunofluorescence in HN878-infected mice with and without IFNAR blockade in the experiments described in Figs. 8 and 9. (b and c) Numbers of total CD4+ T cells in whole left lungs normalized for tissue area. (d) Abundance of CD4+ T cells in lung lesions expressed relative to that in non-lesional lung. Statistical analysis: unpaired t test, with Welch’s correction applied in b. (e) Representative images showing all merged channels for NET staining (top) and CitH3 and DAPI alone (bottom) in C3HeB/FeJ mice at 20 days after infection. Scale bar = 50 μm. (f) Quantification of CitH3 NET staining relative to Ly6G staining in lung lesions. Statistical analysis: unpaired t test. (g) Percentage of lung lesions with low (CitH3/Ly6G < 0.2), intermediate (CitH3/Ly6G 0.2–0.4), or high (CitH3/Ly6G > 0.4) NET burden. Data shown are means ± standard error. Statistical analysis shown is Dirichlet-multinomial regression analysis of the effect of αIFNAR treatment on lesion NET status. Symbols indicate significant differences in the proportion of NET-low lesions. Data in b–g are from single experiments with N = 5 mice per group and are representative of two independent experiments. (h) DESeq2-normalized expression values of genes of interest as identified in Fig. 10 in whole lungs early in infection with HN878. Data shown are from a single bulk RNA-seq experiment with N = 5 mice per group and represent individual replicate mice as points with lines at the mean. Statistical analysis: two-way ANOVA with Holm–Sidak post hoc test. (i) Dot plot showing expression of Slfn4 in scRNA-seq clusters. Data in a and i are from a single scRNA-seq experiment, and plots show combined data from cells from N = 3 mice per group. Circle sizes represent the abundance of cells expressing the gene, as a percentage of either cells in the cluster (a) or within total cells (i). Circle color is proportional to the mean expression of the gene within all cells in the cluster. Actual P values are shown or: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

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