Figure 2.

(a) Identifying lung macrophages as well as other myeloid cells by mass cytometry (CyTOF): resident macrophages were identified by CD45⁺F4/80⁺Ly6G⁻Ly6C⁻CD64⁺MerTK⁺, IM and AM were further identified by CD11b⁺SiglecF⁻ and CD11b⁻SiglecF⁺ respectively; other myeloid populations were identified as: Ly6C⁺ monocyte (Ly6C⁺ Mo: CD45⁺Ly6G⁻Ly6C⁺CD11b⁺CD24⁻MHCII⁻SiglecF⁻CD206⁻), Ly6C⁻ monocyte (Ly6C⁻ Mo: CD45⁺Ly6G⁻Ly6C⁻CD11b⁺CD24⁻MHCII⁻SiglecF⁻CD206⁻), Neutrophils (CD45⁺Ly6G⁺CD11b⁺F4/80⁻), CD103⁺ DC (CD45⁺Ly6G⁻CD11c⁺CD11b⁻CD24⁺CD64⁻Ly6C⁻SiglecF⁻CD103⁺BST2⁻), plasmacytoid DC (CD45⁺Ly6G⁻CD11c⁺CD11b⁻CD24⁺CD64⁻Ly6C⁺SiglecF⁻CD103⁻BST2⁺), CD11b⁺ DC (CD45⁺Ly6G⁻CD11c⁺CD11b⁺CD24⁺CD64⁻Ly6C⁻SiglecF⁻CD103⁻BST2⁻), and Eosinophils (CD45⁺Ly6G⁻Ly6C⁻CD11b⁺CD24⁺SiglecF⁺CD11c⁻MHCII⁻CD64⁻); (b) Absolute cell number for lung IM and AM in WT and Rspo3^EC−/− mice with or without rRspondin3 i.v. (0.25 mg/kg) under basal conditions and following post-sublethal LPS challenge (12 mg/kg, i.p.) for 24h or 48h as 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: IM (ns P=0.3306, ns P=0.5741, ns P=0.2460, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001), AM (ns P=0.8041, ns P=0.9768, ns P=0.9902, ns P=0.9835, ns P=0.7590, ns P=0.9409, ns P=0.9998, ns P=0.9862, ns P=0.9844). (c) Heatmap showing levels of the anti-inflammatory markers (upper panel) and pro-inflammatory markers (lower panel) in lung IM in WT and Rspo3^EC−/− mice with or without rRspondin3 i.v. under basal and post-sublethal LPS challenge for 24h or 48h as measured by CyTOF (n = 5 mice per group with three independent repeats, shown as fold changes by the mean CyTOF signal intensity normalized to control group); (d) Lung vascular permeability was measured by using the albumin-Evans blue dye tracer (EBA) in WT and Rspo3^EC−/− mice with or without rRspondin3 i.v. under basal and post-sublethal LPS challenge for 24h or 48h (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: ns P=0.9997, ns P=0.9996, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001; (e) Myeloperoxidase (MPO) activity of flushed lung samples from WT and Rspo3^EC−/− mice with or without rRspondin3 i.v. under basal and post-sublethal LPS challenge for 24h or 48h (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: ns P=0.9774, ns P=0.9715, ****P<0.0001, ****P<0.0001, ****P<0.0001, ****P<0.0001, *P=0.0250, ****P<0.0001; (f) Survival curves for WT and Rspo3^EC−/− mice with or without rRspondin3 i.v. during endotoxemia conditions (n = 16 mice for each group).