Fig 2: IL-36γ is an upstream amplifier in mouse macrophages and fibroblasts.a Relative mRNA amounts of Il36r in naive mouse bone marrow-derived macrophages (BMDMs, n = 6) in response to no stimulation (−) or stimulation with GM-CSF and IL-36γ. b CXCL1 protein concentrations in supernatant of BMDMs (n = 4) after no stimulation (−), or stimulation with IL-36αβγ. IL-1α, IL-1β alone, or in combination with GM-CSF and TGFβ c, d Relative mRNA amounts of Il36g, Cxcl1, Il1a, Il1b, Il1r1, Il1rn, and Il36rn in BMDMs (n = 4) (c) and primary mouse fibroblasts (n = 4) (d) after no stimulation (−) or stimulation with IL-36αβγ, IL-1α, IL-1β alone, or in combination with GM-CSF and TGF-β. e Relative mRNA amounts in primary mouse fibroblasts (n = 4) of Il36r after no stimulation (−) or stimulation with IL-36αβγ or GM-CSF and TGF-β. Shown are the mean values ± SEM of biological replicates. a–d *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs all other groups by one-way ANOVA and Tukey’s correction. e *P ≤ 0.05, vs untreated by t test.

Fig 3: IL-36γ is an upstream inflammatory driver in mouse neutrophils and alveolar macrophages.a Neutrophil and macrophage counts and myeloperoxidase concentration in the bronchoalveolar lavage fluid (BALF) from room air (RA) exposed (WT n = 9; Il36r−/− n = 6) and 3-week cigarette smoke (CS) exposed mice (WT n = 10; Il36r−/− n = 10). b–d Neutrophil and macrophage counts, CXCL1, IL-1α, IL-1β, and GM-CSF protein concentrations in BALF from untreated (n = 6) and IL-36γ-exposed mice (intratracheal instillation) (n = 7). e, f Cxcl1, Il1a, Il1b, and Il36g mRNA expression in either e naive mouse alveolar macrophages (pooled n = 15 mice) and stimulated in vitro with no cytokines (−), IL-36γ, or IL-1α/IL-1β or f in mouse bone marrow-derived neutrophils (from n = 4 mice) in vitro stimulated with no cytokines (−), IL-36γ, GM-CSF, or IL-36γ+GM-CSF. g Il36r and Il1r mRNA expression in mouse bone marrow-derived neutrophils (from n = 4 mice) in vitro stimulated with no cytokines (−), IL-36γ, GM-CSF, and IL-36γ+GM-CSF. a, e, f *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs all other groups by one-way ANOVA and Tukey’s correction. b, c, d, g **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs untreated by t test. e AMs were pooled from 15 mice, data are presented as (mean ± SEM) of technical triplicates.

Fig 4: Neutrophils are a source of IL-36γ in acute lung injury.a UMAP representation of the cell populations identified in BAL from LPS-exposed mice. Cell identity is indicated by the color code. Relative frequency of the cell types is shown in the bar plot using the same color code. b Violin plots display normalized expression levels of Il36g (Il1f9), Il1a, Il1b, and Cxcl1 across the different cell populations. c Relative mRNA amounts of the same genes used in b after 4 h in vitro LPS stimulation of naive mouse alveolar macrophages (AM) and naive mouse bone marrow-derived neutrophils; depicted are mean values ± SEM of biological duplicates (each biological duplicate represents pooled AMs from 4 mice) and n = 8 from the neutrophils analyzed by one-way ANOVA. d Visualization of normalized IL-36g (Il1f9) expression levels per single cell. e mRNA expression of IL-36g after LPS stimulation in vitro of human neutrophils, MDMs (depicted are mean values ± SME of technical triplicates from one representative of three to four experiments) and human fibroblasts n = 4. Data represents the depicted mean ± SEM of biological replicates; ***P ≤ 0.001, ****P ≤ 0.0001 vs untreated by t test.

Fig 5: Small airway epithelial cells as the main cellular source of GMCSF.a GM-CSF, CXCL1, IL-36γ, IL-1β, and IL-1α protein concentrations and GM-CSF, CXCL1, IL36G, IL1A, and IL1B mRNA expression of lung epithelial basal cells from healthy donor stimulated with poly(I:C) and LPS (depicted are mean values ± SEM of technical triplicates from one representative of three experiments). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 vs all other groups by one-way ANOVA and Tukey’s correction.