Fig. 2. AAM resident airway macrophages (P3) are essential for bronchiolar epithelial regeneration.

a Schematic for clodronate (CL)-mediated macrophage depletion during naphthalene treatment (NA + macrophage depletion). NA-treated macrophage-depleted mice were then either given no cells or intratracheally adopted with GFP⁺ P2 or P3 cells (NA + P3/AAM adoptive transfer). b Levels of the Scgb1a1 mRNA in total lung homogenates from NA-treated mice ± macrophage depletion. c Assessment of club cell regeneration after NA-induced injury in lung tissue of NA-treated WT mice (Control) or NA-injected mice treated with clodronate liposomes (Depleted) or AAM adoptively transferred into depleted mice (see Fig. 2a). CCSP immunofluorescence staining was performed at d35. Graph on the right represents CCSP quantification in lung tissue sections, expressed as percentage of fluorescence within bronchioles (150–400 µm diameter). d Total BAL cells subdivided into P1–P3 gates as defined in (Fig. 1f) determined by counting and flow cytometry from NA-treated mice ± macrophage depletion. e Representative flow plots illustrating the percentage of adoptively transferred GFP⁺ P2 cells that switched into CD11c⁺ SiglecF⁺ cells after 1 week in the lungs of depleted mice. Graph on the right represents the quantification of CD11c⁺ SiglecF⁺ GFP⁺ cells. Data are from 6 to 10 (b), 3 (c), 6 (d), and 3 to 7 (e) mice, obtained in three independent experiments and represented as mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001 between macrophage-depleted and NA-treated WT mice using one-way ANOVA, Bonferroni post-test. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 between AAM adoptively transferred and macrophage-depleted WT mice using one-way ANOVA, Bonferroni post-test. Scale bar in c = 50 µm.