Extended Data Fig. 6: Circulating CSF1 is unchanged in hypoxic ALI and exogenous CSF1 improves injury outcomes altering the CD64^hiSiglecF⁻ Mϕ phenotype.

Serum MCSF (CSF1) from LPS-challenged mice housed in normoxia (N) or hypoxia (H) for 24 hours (a) or 5 days (b) was measured. (c) BAL CXCL1 measured in LPS-challenged mice housed in hypoxia for 5 days and treated with PBS or CSF1-Fc (H CSF1). (d) weight loss from baseline in hypoxic LPS-induced ALI treated with PBS (H PBS) or CSF1-Fc (H CSF1). (e) Lung monocyte numbers, (f) arbitrary sickness scores, (g) BAL protein and (h) LDH activity (as measured by NADH) measured at 48 hours in mice with virally-induced ALI housed in hypoxia and treated with PBS or CSF1-Fc (H CSF1). (i) Baseline blood chimerism (proportion of donor cells relative to host) of circulating monocytes in lung-protected chimeras prior to ALI induction and chimerism of monocytes based on Ly6C expression post-LPS. (j) Lung cDC1 (gated on Alive CD45⁺Lin-CD64⁻CD11c⁺Cd103⁺) chimerism and counts and (k) cDC2 (gated on Alive CD45⁺Lin-CD64⁻CD11c⁺Cd103⁻ CD11b⁺) chimerism and counts. Chimerism relative to blood monocyte chimerism. (l) Il10 expression was measured by NanoString platform analysis in MHC-lung macrophages from LPS-challenged mice housed in hypoxia for 5 days and treated with PBS and compared to Lyve1+MHCII- of LPS-challenged mice, housed in hypoxia and treated with CSF1-Fc. (m) Representative dot plots of lung digests showing gating strategy for identification of the different monocyte and macrophage populations in the lung gated on Live Singles CD45⁺Ly6G⁻ cells, and including Lyve1⁺MHC-CD64^hiSiglecF⁻Mϕ, and associated APC FMO control including Lyve1⁺MHC-CD64^hiSiglecF⁻Mϕ, and associated APC FMO control. Ifnar^−/− (KO) mice were nebulised with LPS and treated with PBS (KO PBS) or CSF1-Fc (KO CSF1). Mice were sacrificed on day 5 and (n) blood monocyte and (o) lung Ly6C⁺ monocytes were quantified by flow cytometry. c, d Mean±SD, e-h mean±SEM. c-h, j, k 2 pooled experiments, l representative of 3 experiments, n, o representative of 2 experiments. Statistical testing e-h, n, o unpaired two-sided t-test, k, one-way ANOVA with Tukey’s multiple comparisons test.