Extended Data Fig. 10: Neonatal neutrophils program AM proliferative capacity.

(a) CD45⁺ cells were isolated from the lungs of PND1 WT and Alox15^−/− pups and subjected to scRNA-Sequencing. (b) Uniform Manifold Approximation and Projection (UMAP) embedding of integrated CD45⁺ cells from the lungs of WT and Alox15^−/− mice. Unsupervised clusters were labelled using CelliD⁵⁰ and the expression levels of canonical cell type markers. Neutrophils were further partitioned through label transfer from adult mouse neutrophil cell atlas to create a final assignment (left). A graph representation of the results from Milo differential abundance testing (right). Nodes are neighbourhoods, coloured by their log fold change between WT and Alox15^−/− cells. Non-DA neighbourhoods (FDR > 10%) are coloured white, and sizes correspond to the number of cells in a neighbourhood. There is a marked increase in the relative abundance of WT cells in the “Neutrophil-G5b” cluster compared to Alox15^−/− cells. (c) Beeswarm plot showing the distribution of log-fold change between WT and Alox15^−/− cells in neighborhoods containing cells from different cell type clusters. Blue refers to neighborhoods within particular cell types that are significantly enriched (FDR<5%) in WT compared to Alox15^−/− animals and brown neighborhoods enriched in Alox15^−/− animals relative to WT. (d) Cell type label frequencies from PanglaoDB predictions within finalized manual labeling clusters (vertical facets) and split by condition. Confirms finalized labels as consistent with automated predictions. (e-g) quantification of neutrophils (e), total (f) and Ki67⁺ (g) alveolar macrophages in the lungs of PND3 WT mice following isotype or anti-Ly6G treatment (Iso.: n = 8, α-Ly6G: n = 10). (h) Representative micrographs of BrdU staining in vitro after AM culture with GM-CSF. Bar = 50 µm. (i-j) Parenchymal neutrophils were determined in WT mice at various ages by intravascular and ex vivo CD45.2 staining. Representative FACS plots (i) and quantification (j) (n = 11/4/5/group). (k) Expression of various genes involved in proliferation in BAL AM from adult Alox15^lox/lox and Alox15^lox/lox MRP8^Cre (n = 3 biological replicates/group). The genes were previously identified in our bulk RNA-Seq dataset on whole body Alox15^−/− mice. (l) Representative micrographs of SA-β-galactosidase in BAL AM from Alox15^lox/lox and Alox15^lox/lox MRP8^Cre mice. Bar=10 µm. (m) Model of EP2 signaling inhibition with TG6-10-1 in pups (left) and BAL AM numbers in adult mice (right) (n = 5/6/8/group). (n) BrdU⁺ AM following GM-CSF culture in vitro (left) (Uns.: n = 3, GM-CSF: n = 5 fields of view/group) and basal Cdkn1a expression (right) (n = 3 biological replicates/group) in BAL AM from adult mice. (o-p) Adult WT mice were treated with LY255283 and adult Alox15^−/− mice were treated with TG6-10-1 or 12-HETE for three consecutive days. AM populations in the BAL were evaluated after 7 days resting period (o) (left, n = 5/group; right, WT: n = 5, Untreated: n = 5, 12-HETE: n = 5, TG6-10-1: n = 6) and BrdU⁺ AM after 3 days of culture with GM-CSF in vitro (p) (Uns.: n = 3, GM-CSF: n = 4 fields of view/group). Data are presented as mean ± s.e.m and are from one (a-d, o-p) or pooled from two (e-g, i-j, m) or three (k, n) independent experiments or representative of two (h, l, n, p) independent experiments. Data were analyzed using two-tailed unpaired t-test (e, g, k), one-way ANOVA followed by Tukey’s multiple comparison test (j, m-o) or two-way ANOVA followed by Sidak’s multiple comparisons test (n, p). The models in (a, m, q) were created using BioRender (https://biorender.com).

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