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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Nat Immunol. 2020 Aug 24;21(11):1430–1443. doi: 10.1038/s41590-020-0764-8

Extended Data Fig. 3: Rspondin3 regulates lung interstitial macrophage phenotype transition following acute lung injury.

Extended Data Fig. 3:

(a) Absolute cell number of lung myeloid cells including neutrophils, eosinophils, Ly6C+ Mo, Ly6C- Mo, CD103+ DC, plasmacytoid DC and CD11b+ DC in WT and Rspo3EC−/− mice with or without rRspondin3 i.v. under baseline conditions and post sublethal LPS challenge for 24h or 48h 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: neutrophils (ns P>0.9999, ns P=0.9995, ns P=0.9991, ****P<0.0001, ****P<0.0001, ****P<0.0001, ns P=0.9864, ns P=0.2420, ns P=0.6898), eosinophils (ns P>0.9710, ns P>0.9789, ns P>0.9865), Ly6C+ Mo (ns P>0.9933, ****P<0.0001, ns P=0.8936, ns P=0.7754, ns P>0.9878), Ly6C− Mo (ns P>0.9942, ****P<0.0001, ns P=0.9059, ns P=0.7988, ns P>0.9807), CD103+ DC (ns P>0.9676, ns P>0.1488, ns P>0.8806), plasmacytoid DC (ns P>0.9445, ns P>0.2290, ns P>0.9786), CD11b+ DC (ns P>0.9377, ns P>0.2463, ns P>0.9941); (b) Levels of anti-inflammatory markers (CD206, CD301, RELMα, Arginase 1, IL-10) and pro-inflammatory markers (CD86, CD80, iNOS, TNF, CD69) in lung IM in WT and Rspo3EC−/− mice with or without rRspondin3 i.v. under basal and post sublethal LPS challenge for 24h or 48h measured by CyTOF (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 Tukey’s multiple comparisons test using GraphPad Prism with individual P values (left to right) are: CD206 (ns P>0.4229, ****P<0.0001, ****P<0.0001, **P=0.0095, ****P<0.0001, ****P<0.0001, ****P<0.0001), CD301 (ns P>0.2041, ***P=0.0002, **P=0.0049, *P=0.0439, ****P<0.0001, ****P<0.0001, ****P<0.0001), RELMα (ns P=0.4547, **P=0.0023, ns P=0.6850, **P=0.0028, ****P<0.0001, ns P=0.1014, ****P<0.0001, ****P<0.0001, ****P<0.0001), Arginase 1 (ns P=0.1315, ns P=0.0640, ns P=0.2260, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001), IL-10 (ns P=0.0847, ns P=0.5465, ns P=0.3525, **P=0.0037, *P=0.0448, *P=0.0217, ****P<0.0001, ****P<0.0001, ****P<0.0001), CD86 (ns P>0.7231, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001), CD80 (ns P>0.9697, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001), iNOS (ns P>0.9568, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001), TNF (ns P>0.9977, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001), CD69 (ns P>0.9790, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, **P=.0015, ****P<0.0001). (c) Heatmap for the levels of anti-inflammatory markers (CD206, CD301, RELMα, Arginase 1, IL-10) and pro-inflammatory markers (CD86, CD80, iNOS, TNF, CD69) in lung AM in WT and Rspo3EC−/− mice with or without rRspondin3 i.v. under baseline conditions and post sublethal LPS challenge for 24h or 48h as measured by CyTOF (Data are representative of three independent experiments with n = 5 mice per group, shown as fold change by the mean CyTOF signal intensity normalized to Control group); (d) Wnt signaling activities in mice lung myeloid populations determined by CyTOF using Wnt reporter mice (TCF/Lef:H2B-GFP transgenic mice) with or without rRspondin3 i.v. (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: ns P>0.9999, ns P=0.9301, ns P>0.9999, ****P<0.0001, ns P=0.9992, ns P>0.9999, ns P>0.9999, ns P>0.9999, ns P>0.9999.