Figure 3

(a) Lung alveolar macrophage numbers (CD45⁺/CD11c⁺/Siglec-F⁺ cells) and (b) interstitial macrophage numbers (CD45⁺/CD11b⁺/CD64⁺ cells) in digested right lung lobes after naphthalene-induced epithelial injury (200mg/kg) analysed by flow cytometry of lung digests in Panx1+^/+ and Panx1^-/- mice (n=3-5 corn oil; n=5-9 post-naphthalene). (c) Schema of experimental protocol for lung macrophage depletion in Panx1+^/+ mice, achieved by intratracheal (i.t.) administration of 100μL clodronate liposomes to deplete alveolar macrophages, and PLX5622 containing chow (1200mg/kg feed) ad libitum for 3d prior to experimentation with additional PLX5622 (65mg/kg) given by oral gavage (on days 0,1,2,3&4 of naphthalene administration) to deplete interstitial macrophages. (d) Confirmation of macrophage depletion after epithelial injury and (e) In vivo epithelial injury-induced proliferation analysed by EdU incorporation (EdU 1.5mg/mouse given intraperitoneally (i.p.) on days 4&4.5) with or without macrophage depletion in response to high dose (200mg/kg, n=3) or intermediate dose (160mg/kg n=8) naphthalene, assessed by t-test. (f) Representative H&E staining of lung tissue sections at day 7 after naphthalene injury (200mg/kg) with or without macrophage depletion, with injured and repairing airway epithelium highlighted by blue arrows. Grey scale bars, 50um. (g) In vivo epithelial injury-induced proliferation analysed by EdU incorporation with alveolar macrophage depletion (by clodronate liposomes) or interstitial macrophage depletion (by PLX5622) in response to naphthalene (160mg/kg, n=4/group, one experiment, assessed by one-way ANOVA with Holm-Šídák's multiple comparisons test). (h) Macrophage ablation achieved using the csf1ra:NfsB line (NfsB) and metronidazole (met) treatment (NfsB⁺/met^-, NfsB^-/met⁺ and NfsB⁺/met⁺ groups; macrophage depletion only with NfsB⁺/met⁺) with macrophage ablation confirmed in NfsB⁺/met⁺ fish by fluorescent microscopy (mCherry⁺ macrophages pseudocoloured in magenta) scale bar 100um. (i) Defective tailfin regeneration in macrophage-deficient zebrafish (NfsB⁺/met⁺ group) (n=14, assessed by one-way ANOVA with Holm-Šídák's multiple comparisons test). *p<0.05, **p<0.01 *** p<0.001, ****p<0.0001.