Fig. 5: Neonatal neutrophil-derived 12-HETE programmes AM proliferation.

a, Pulmonary levels of 12(S)-HETE in PND1, PND3 and adult WT mice (left to right, n = 10, 8 or 8 per group). b, Scheme (left) and BAL AM numbers (right) in 12-HETE-treated Alox15^−/− pups that were left to age until adulthood (n = 5 per group). c, Left, BrdU⁺ AMs after GM-CSF culture (n = 5 (Uns.) or 11, 10 or 12 (GM-CSF) fields of view (FOVs) per group). Right, basal Cdkn1a expression in AMs (n = 3 biological replicates per group). d, BAL PGE₂ levels (left) and pulmonary viral loads (right) at day 3 after IAV infection (50 p.f.u.) (n = 4 per group). e, Left, quantification of ALOX15 expression in CD45⁺ or CD45⁻ lung cells at various ages (n = 4 mice per group). MFI, mean fluorescence intensity. Right, ALOX15⁺ CD45⁺ cells were further gated using CD11b, Ly6C and Ly6G in PND1 lungs (n = 8 mice per group). f, Postnatal neutrophil depletion in WT mice using anti-Ly6G (left) and quantification (right) of BAL AMs in animals that were left to age until adulthood (n = 6 (isotype) or 5 (anti-Ly6G) mice per group). g, Left, BrdU⁺ AMs after GM-CSF culture (n = 3 (Uns.) or 5 (GM-CSF) fields of view per group). Right, basal Cdkn1a and Cdkn2a expression in AMs (n = 3 biological replicates per group). h, BAL PGE₂ levels (left) and pulmonary viral loads (right) at day 3 after IAV infection (50 p.f.u.) (n = 4 mice per group). i, Lungs were isolated from PND1 or adult (6–8 weeks) WT mice and stained for Ly6G (neutrophils, red), CD11c (AMs, blue) and CD31 (endothelial cells, green). j, Quantification of macrophages and neutrophils (PND1: n = 6, Adults: n = 11). k, Mean distance between neutrophils and macrophages (PND1: n = 45, Adult: n = 43). l, Left, generation of neutrophil-specific Alox15^lox/loxMrp8^cre mouse model. Right, BAL AM numbers in adult mice (n = 4 per group). m, Left, BrdU⁺ AMs after GM-CSF culture (n = 4 (Uns.) or 6 (GM-CSF) fields of view). Right, basal Cdkn1a expression (n = 3 biological replicates per group). n, Representative uniform manifold approximation and projection (UMAP) plots of Gpr31b and Ltb4r2 expression by single-cell RNA-seq in PND1 lungs. o, BAL AM numbers in adult WT and Ltb4r2^−/− mice (n = 4 per group). p, BrdU⁺ AMs after GM-CSF culture (left; n = 3 (Uns.) or 6 (GM-CSF) fields of view per group) and basal Cdkn1a expression (right; n = 3 biological replicates per group) in adult WT and Ltb4r2^−/− AMs. q, Postnatal inhibition of LTB4R2 signalling using LY255283 in WT mice (left) and BAL AM numbers in adult mice (right) (n = 7 per group). r, BrdU⁺ AMs after GM-CSF culture (left; n = 3 (Uns.) or 5 (GM-CSF) fields of view per group) and basal expression of Cdkn1a (right; n = 3 biological replicates per group) in adult AMs. Data are presented as the mean ± s.e.m. and are pooled from one (d,h,n), two (a,b,e,f,l,o,q) or three (c,g,i–k,m,p,r) independent experiments or representative of two (c,g,m,p,r (left)) independent experiments and were analysed using two-tailed unpaired t-test (e–h,k–m,o–r), one-way ANOVA followed by Tukey’s multiple comparisons test (a–d) or two-way ANOVA followed by Tukey’s (c) or Sidak’s (e,g,j,m,p,r) multiple comparisons test. The models in b, f, l and q were created using BioRender (https://biorender.com).

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