Extended Data Fig. 4: Rspondin3 regulates lung interstitial macrophage phenotype transition following acute lung injury.

a, Gene expression profiling performed by qPCR to evaluate the expression of macrophage marker genes, anti-inflammatory and pro-inflammatory genes in sorted IM and AM from WT and Rspo3^EC-/- mice lungs under baseline conditions and endotoxemia. Heatmap shows the fold change of gene levels as normalized to WT IM at basal conditions (Data are representative of three independent experiments, n=3 samples per group); b, Lung myeloid populations analyzed by CyTOF in WT and Rspo3^EC-/- mice subjected to bleomycin induced acute lung injury and basal conditions (Data are representative of three independent experiments with five mice per group). Graphs show the mean ± s.d, with each dot representing an individual mouse. Statistical significance was determined by two-way ANOVA with Sidak’s multiple comparisons test using GraphPad Prism with individual P values (left to right) are: IM (*P=0.0487, ****P<0.0001), AM (ns P<0.3554, ns P<0.9932), neutrophils (ns P=0.9329, ns P=0.9798), eosinophils (ns P=0.9557, ns P=0.7925), Ly6C⁺ Mo (ns P=0.8486, ns P=0.9777), Ly6C^- Mo (ns P=0.9040, ns P=0.9863), CD103⁺ DC (ns P=0.9526, ns P=0.7602), plasmacytoid DC (ns P=0.9410, ns P=0.7104), CD11b⁺ DC (ns P=0.9613, ns P=0.7995); c, Levels of the anti-inflammatory markers (CD206, CD301, arginase 1, IL-10) and pro-inflammatory markers (CD86, CD80, iNOS, TNF) in IM in WT and Rspo3^EC-/- mice under baseline conditions and post bleomycin injury for 5 days as measured by CyTOF (Data are representative of three independent experiments with n=5 mice per group). Graphs show the mean ± s.d, with each dot representing an individual mouse. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism with individual P values (left to right) are: CD206 (ns P=0.7843, ****P<0.0001, ns P>0.9999, ns P=0.9642), CD301 (ns P=0.5538, ****P<0.0001, ns P>0.9999, ns P>0.9999), arginase 1 (ns P=0.3438, ****P<0.0001, ns P=0.9857, ns P=0.9963), IL-10 (ns P=0.6175, ****P<0.0001, ns P=0.9926, ns P=0.9502), CD86 (ns P=0.0553, ****P<0.0001, ns P=0.9178, ns P=0.9983), CD80 (ns P=0.0882, ****P<0.0001, ns P=0.9978, ns P=0.9970), iNOS (ns P=0.1009, ****P<0.0001, ns P=0.9991, ns P=0.7604), TNF (ns P=0.0736, ****P<0.0001, ns P=0.9997, ns P=0.9979); d, Representative overlaid CyTOF histograms showing the CyTOF signal intensity of the anti-inflammatory markers (CD206, CD301, arginase 1, IL-10) and pro-inflammatory markers (CD86, CD80, TNF, iNOS) in IM and AM in WT and Rspo3^EC-/- mice at baseline conditions or 5 days after bleomycin i.t. for data in (e).

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