Extended Data Fig. 3: Intact PM and BMDM proliferation.
From: Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE
(a-b) Peritoneal macrophages (PM) were harvested from adult naïve WT and Alox15−/− mice and BrdU incorporation after 7 days pulse (a) or Ki67 expression (b) were determined by flow cytometry (n = 5/group). (c-d) Bone marrow-derived macrophages were generated from adult WT and Alox15−/− mice and were treated with BrdU (day 3 and 5 of differentiation). At day 6 BrdU incorporation (c) (n = 6/group) and Ki67 expression (d) (n = 9/group) were evaluated by flow cytometry. (e) Representative FACS plot and quantification of Ki67+ AM frequencies in the lungs of adult WT and Alox5−/− mice (n = 4/group). (f-g) AM kinetics following local depletion by clodronate liposomes. Adult WT (control: n = 3/6/9/timepoint, clodronate: n = 3/6/10/timepoint), Alox15−/− (control: n = 3/6/10 mice/timepoint, clodronate: n = 3/6/9/timepoint) or Ccr2−/− (n = 3/3/5/group/timepoint) mice were given control or clodronate liposomes intranasally (70 µl). AM populations were assessed in the airways at day 2 and 14 post-delivery. (f) Numbers of AM in the BAL at various days post-infection. (g) Frequency of Ki67+ AM in the BAL at day 14 (n = 5/group). (h) Representative FACS plot of Ki67+ CD45.1 BAL AM before adoptive transfer. (i) Representative FACS plots of AM in the lungs of Alox15−/− mice 2 and 8 weeks after WT CD45.1 AM transfer. (j) Quantification of WT CD45.1+ or Alox15−/− CD45.2+ AM post-adoptive transfer in ALOX15-deficient mice (n = 5/group). (k) BAL AM from adult WT and Alox15−/− mice were cultured with M-CSF for 3 days before cell growth was evaluated (n = 3 biological replicates/group). (l) PM isolated from adult WT and Alox15−/− mice were cultured with M-CSF+IL-4 for 3 days before cell growth was evaluated (n = 3 biological replicates/group). (m) BMDM generated from adult WT (n = 4 biological replicates) and Alox15−/− (n = 3 biological replicates) mice were cultured with M-CSF for 3 days before cell growth was evaluated. (n-r) WT and Alox15−/− AM from adult and PND3 mice were subjected to ATAC-Seq (n = 3/group). (n) Principal component analysis of WT versus KO adult and pup cells. (o) The number of differentially accessible peaks comparing ALOX15-deficient and WT cells using the cutoffs: p.adj < 0.05 and abs(log2FC) > 1. Upward bars indicate increased accessibility in the KO condition and downward bars indicate decreased accessibility. (p) Venn diagram showing the relative numbers of DA peaks in adults and pups. Circle size is proportional to the number of DA peaks. Out of 64 total peaks with p.adj < 0.05 and abs(log2FC) > 1 in pups, ~65% (42/64) overlapped with the DA peaks set in adults. (q) Correlation (pearson cor = 0.702, p < 2.2e-16) of normalized enrichment scores (NES) for all enriched pathways in adults and pups. Pink highlighted points are the top 10 pathways in both adults and pups. (r) Heatmap showing the gene set enrichment results specifically for reactome pathways related to proliferation. Shading is indicative of significance with darker red colors indicating lower p.adjusted values. Data are presented as mean ± s.e.m and are from one (e, g-j, n-r), or pooled from two (c, f) or three (d, k-m) or representative of two (a-b) independent experiments. Data were analyzed using two-way ANOVA followed by Tukey’s (f) or Sidak’s (g, j-k) multiple comparisons test.
