Fig.Figure 6(A) Expression relative to GAPDH of pro-inflammatory cytokines (TNFA, IL1B, IL6), interferon genes (IFNB, IFNL1, IFNL3) and interferon stimulating genes (IP10, ISG15) in the AT and endothelial layer of infected LoCs at 1 dpi reconstituted without macrophages (n=3 biological replicates for IFNB, IFNL1 and IFNL3 in the AT layer, n=4 biological replicates for all other data). (B) Fold-change in the markers in (A) relative to uninfected controls (n=3 biological replicates) (data in Appendix Fig. S12) at the same timepoint. The bar represents the mean, and the error bars the standard deviation. ( Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001. (C) Enzyme-linked immunosorbent assay (ELISA) measurements for IL-6 in the vascular effluent for LoCs reconstituted with and without macrophages (at least n=2 biological replicates at each timepoint). Uninfected controls for each condition are labelled with a 'C' and grey bars, and LoCs with Tocilizumab administration are indicated by ^. The height of the bars represents the mean, the median is represented by a solid line, and the error bars represent the standard deviation. Each dot represents the mean of two technical replicates. Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001. (D) Expression of IL6R and ADAM17 relative to GAPDH in the AT and endothelial layer of infected LoCs at 1 dpi (n=3 biological replicates for the AT layer and n=4 biological replicates for endothelial layer). (E) Fold-change in expression of IL6 and ADAM17 in the epithelial and endothelial layers of infected LoCs at 1 dpi relative to uninfected controls (n=3 biological replicates for both layers). ( Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001. (F-I) 3D views of representative 232 x 232 μm2 fields of view of the AT (F, H) and endothelial layer (G, I) from infected LoCs reconstituted with macrophages at 1 (F, G) and 3 dpi (H, I). IL6 mRNA, and nuclear staining are indicated with the chartreuse and electric indigo LUTs, respectively. (J) Quantification of IL6 expression in ATs and endothelial cells from a pair of otherwise identical LoCs (n=2 biological replicates) analyzed at 1 and 3 dpi, respectively. Plots show the total number of spots normalized by the number of cells in 4-6 fields of view (technical replicates) detected using RNAscope assay using identical imaging conditions for all chips. Bars represent the mean value, the solid line represents the median, and error bars represent the standard deviation. Data from uninfected controls for 3 days at air-liquid interface is indicated by 'C'. Data information: The bar represents the mean, and the error bars the standard deviation. P-values are calculated using a Kruskal-Wallis One-Way ANOVA Test, * represents p≤0.05, ** represents p≤0.01, ** represents p≤0.001. (K-N) Zooms corresponding to the regions in (I) highlighted with white (K) and yellow boxes with solid (L) and dashed lines (M, N) respectively. In these panels, orf1abantisense RNA (pink) and S RNA (amber) are also shown. The panels show examples of cells with similar levels of viral infection but with no (K), intermediate (L) and high levels (M, N) of IL6 expression.