Fig. 5: Virus RNA in monocytic macrophages leads to dose-dependent activation of pro-inflammatory cytokines by TLR signaling.

a Feature plots of entry factor expression in Uniform manifold approximation and projection (UMAP) projection. Coloration indicates expression values of indicated genes. b–e Detection of viral RNA by in situ-hybridization. Labeled are supposed endothelium (b, hash), bronchial epithelial cells (b, arrowhead), AT1 (c, arrowhead) and AT2 (c, arrow). (d, inset), macrophages containing viral RNA and cell debris (arrowhead), and an example of high levels of viral RNA without cell debris in the inset (arrow). For b–e red signals viral RNA and blue hemalaun counterstain. Time points: b, c from 2 dpi, d from 3 dpi, e staining control. Bars: b, d, e = 50 µm, c = 100 µm, Inset in d = 20 µm. Micrographs representative of n = 6 per time point pi. f Cells in the UMAP projection are colored by amount of viral RNA (log10 transformed percentage of viral RNA per cell), along with overview of identified cell types in lungs. g Dotplot of cytokine expression in monocytic macrophages containing viral RNA compared to those without viral RNA. Coloration and point size indicate log2 fold change and adjusted (adj.) p-value for each time point 2, 3, and 5 dpi. Adjusted (adj.) p-values were calculated by DEseq2 using Benjamini–Hochberg corrections of two-sided Wald test p-values. h, i Bar- and boxplots of Isg15 (h) and Cxcl10 (i) gene expression in AT2 and monocytic macrophages along, comparing cells containing viral RNA to those without for 2, 3, and 5 dpi. Barplot shows percentage of cells positive for respective gene. Boxplots show gene expression levels in cells positive for respective gene, j Bar- and boxplots of Cxcl10 in monocytic macrophages and fraction of Cxcl10 positive cells for each time point pi and naive animals, with cells grouped by increasing virus levels for 2, 3, and 5 dpi. h–j Barplots, data display means ± SD, n = 3 animals per time point. Significance levels calculated using two-sided generalized linear mixed-effects models. For box plots, the middle line in the boxplot displays the median, the box indicates the first and third quartile, whiskers the 1.5 interquartile range (IQR), cell gene expression data derived from n = 3 animals per time point two-sided Wilcoxon rank-sum test on all cells (i.e., not only the ones expressing the gene) for boxplots. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.0001. See “Methods” for details. AT1 and AT2: alveolar epithelial cell type 1 and 2, DC: dendritic cells, NK, natural killer cells; d0: day 0 = naive, d14: 14 dpi. Exact p-values in order of appearance in (h) upper panel: ^∗p = 0.0338; lower panel: ^∗∗p = 0.0093; ^∗p = 0.014; ^∗∗∗p < 0.0001; ^∗∗∗p < 0.0001; ^∗∗∗p < 0.0001; i upper panel: ^∗p = 0.0270; ^∗∗∗p < 0.0001; ^∗∗∗p < 0.0001; ^∗∗∗p < 0.0001; lower panel: ^∗∗∗p < 0.0001; ^∗∗∗p < 0.0001; ^∗∗∗p < 0.0001; j upper panel: ^∗∗∗p < 0.0001; ^∗∗p = 0.0004; ^∗∗p = 0.0022; ^∗p = 0.0131; ^∗∗∗p < 0.0001; ^∗∗∗p < 0.0001; ^∗∗p = 0.0003; ^∗∗p = 0.0033; ^∗∗∗p < 0.0001; lower panel: ^∗∗∗p < 0.0001; ^∗∗p = 0.0015; ^∗∗p = 0.0064; ^∗∗p = 0.0034; ^∗∗∗p < 0.0001; ^∗∗∗p < 0.0001; ^∗∗∗p < 0.0001.