Fig. 4: Alox15^−/− AMs have dysregulated responses to sterile inflammation and viral infection.

a, WT or Alox15^−/− AMs were transferred (intratracheal (i.t.)) into Csf2rb^−/− mice, and BAL protein levels were determined 6 weeks after transfer (left to right, n = 6, 11, 9 or 9 per group). b, CXCL1 production by WT or Alox15^−/− AMs after stimulation with LPS (100 ng ml^–1) (n = 4 biological replicates per group). c–f, LPS (20 µg) was delivered intranasally to adult WT and Alox15^−/− mice. c, BAL CXCL1 levels (left to right, n = 3, 11 or 7 (WT) or 3, 10 or 8 (Alox15^−/−) mice per time point). d, Pulmonary pathology after LPS treatment. Scale bar, 100 µm. e, BAL protein levels (left to right, n = 3, 11 or 7 (WT) or 3, 9 or 7 (Alox15^−/−) mice per time point). f, BAL neutrophil numbers (left to right, n = 3, 11 or 7 (WT) or 3, 10 or 8 (Alox15^−/−) mice per time point). g,h, Intravital microscopy following 2 h of intravenous LPS (20 µg) treatment. g, Intravascular neutrophil (cyan) aggregation (top) and cluster formation quantified (bottom) by defining contiguous objects and measuring the areas (blue, low; red, high area clusters). Vascular endothelium is red. h, Clusters were classified as large (≥3,000 µm²) (n = 5 (WT) or 17 (Alox15^−/−) cells), medium (500–2,999 µm²) (n = 105 (WT) or 66 (Alox15^−/−) cells) or small or individual neutrophils (500 µm²) (n = 16 (WT) or 161 (Alox15^−/−) cells). n = 3 per group with 3 fields of view per mouse. i,j, Scheme (i) and quantification (j) of PGE₂ production by WT and Alox15^−/− AMs 4 h after IAV infection (multiplicity of infection of 1) (n = 3 biological replicates per group). k, PGE₂ production by BAL AMs from IAV-infected mice (day 1, 50 p.f.u.) after 4 h of culture (n = 5 (WT) or 4 (Alox15^−/−)). l–n, WT and Alox15^−/− mice were infected with IAV (50 p.f.u.). l, BAL levels of PGE₂ (left to right, n = 6, 9, 9 or 8 (WT) or 5, 7, 9 or 7 (Alox15^−/−) per time point). m, IFNβ (left to right, n = 3, 7, 9 or 8 (WT) or 3, 6, 10 or 7 (Alox15^−/−) per time point). n, Pulmonary viral loads (n = 4 (day 3) or 3 (day 6) mice per group). o, Survival of WT (n = 15) and Alox15^−/− (n = 16) animals infected with IAV (90 p.f.u.). p, WT AMs were transferred into Alox15^−/− mice. Animals were infected 2 h after transfer with IAV (50 p.f.u.). Pulmonary viral loads were determined 3 days after infection (left to right, n = 8, 7 or 6 per group). q, Pulmonary viral loads in IAV-infected (50 p.f.u.) WT and Alox15^−/− mice (treated or with or without mPGES1 inhibitor) (n = 5 per group). r,s, Scheme (r) and survival of Alox15^+/+ (n = 20) and Alox15^−/− (n = 17) K18-hACE2 mice infected with SARS-CoV-2. Data are presented as the mean ± s.e.m. and are from one experiment (k,q), pooled from two (a,c,e,f,m,p,s), three (g,h,j,l,o) or four (b) independent experiments, or representative of two (n) independent experiments or six biological replicates (d). Data were analysed using two-tailed unpaired t-test (k), two-tailed Mann–Whitney test (h), one-way ANOVA followed by Tukey’s multiple comparisons test (a,p,q), two-way ANOVA followed by Sidak’s multiple comparisons test (b,c,e,f,j,l–n) or log-rank test (o,s). The models in a, I, p and r were created using BioRender (https://biorender.com).

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