Fig. 3. BMDM2 as opposed to BMDM1 exert proliferative and barrier-protective effects on alveolar epithelial cells in vitro and in vivo.

Bar graphs indicate the percentage of apoptotic (a, Annexin V+) (*p = 0.038, **p = 0.0023, ***p = 0.0005), and proliferating (b, Ki67+) (**p = 0.008, ***p = 0.0018) control (n = 3; 5) or IAV-infected (n = 6; 7) (24 h) primary murine AECs that were co-cultured with BMDM1 (n = 3; 6) or BMDM2 (n = 3; 4) (flow-sorted from BALF of infected mice) for further 24 h. c Schematic representation of BALO-BMDM co-culture assay setup. d Quantification of organoid numbers (*p = 0.041) and e diameter (µm) (**p = 0.0099, ***p = 0.0002, ****p < 0.0001) of BALO after 7D of co-culture, in control experiments (n = 7) (no BMDM) and in co-culture with BMDM1 (n = 9) or BMDM2 (n = 9). f Representative whole-well picture of organoids after 21D in culture. Images were obtained using EVOS FL microscope. Scale bars indicate 50 µm. g Schematic representation of experimental setup of BMDM intrapulmonary transfer into IAV-infected Ccr2^-/- mice referring to data in (h)–(j). The figure was created with BioRender. h Percentage of apoptotic AEC (CD31/45^negEpCam⁺Annexin V⁺⁾ analyzed by FACS. Groups: no cells (n = 6), BMDM1 (n = 6), BMDM2 (n = 10) (*p = 0.049, **p = 0.0012). i Assessment of lung barrier permeability by FITC fluorescence in BALF of Ccr2^-/- recipient mice after intravenous application of FITC-labeled albumin, ratios of BALF to serum fluorescence are given as arbitrary units. Groups: no cells (n = 4), BMDM1 (n = 6), BMDM2 (n = 6) (**p = 0.0079, ***p = 0.0053). j Percentage of proliferating AEC II (CD31/45^negEpCam⁺T1α^negKi67⁺) analyzed by flow cytometry. BMDM1 and BMDM2 were flow-sorted from BALF of IAV-infected WT mice at D7 and D21, respectively for experiments in (a)–(j). Groups: no cells (n = 4), BMDM1 (n = 10), BMDM2 (n = 10) (***p < 0.0001, *p = 0.0236). Bar graphs are representative of three independent experiments showing means ± SEM and individual data points. Statistical significance was calculated using one-Way ANOVA and Tukey’s post-hoc tests except in (d) where Dunnett’s post-test was used, and (e), (i) where Brown Forsythe and Welch ANOVA followed by Games–Howell’s test was used. In (d) and (e), single data points represent means of organoid numbers and diameters per well. BMDM bone marrow-derived macrophage, ACE alveolar epithelial cell, BALO bronchoalveolar lung organoid, D day. Source data are provided as a Source Data file.