Fig 2: IL-36? cooperates with poly(I:C) on mouse macrophages and fibroblasts.a Relative mRNA amounts of Il36g, Cxcl1, Il1a, and Il1b in mouse neutrophils (n = 2–6) unstimulated with (-) or stimulated with IL-36aß?, poly(I:C), or the combination of IL-36aß? and poly(I:C) (depicted are mean values ± SEM of biological replicates). b Relative mRNA amounts of Il36g, Cxcl1, Il1a, Il1b, and Gmcsf in BMDMs (n = 4) and primary mouse fibroblasts (n = 4) (depicted are mean values ± SEM of biological replicates) unstimulated (-) or stimulated with IL-36aß?, poly(I:C), or the combination of IL-36aß? and poly(I:C). *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001 vs all other groups by one-way ANOVA and Tukey’s correction.

Fig 3: IL-36? is an upstream inflammatory driver in human neutrophils and small airway epithelial cells.a, b CXCL1, IL1A, IL1B, and IL36G mRNA expression in human peripheral blood-derived neutrophils (from n = 4 donors) in vitro stimulated with no cytokines (-), IL-36?, GM-CSF, or IL-36?+GM-CSF. b IL36R and IL1R mRNA expression in human peripheral blood-derived neutrophils (from n = 4 donors) neutrophils in vitro stimulated with no cytokines (-), IL-36?, GM-CSF, and IL-36?+GM-CSF. c, d CXCL1, GM-CSF, and IL1a protein concentrations and CXCL1, IL36G, GM-CSF, IL1A, and IL1B mRNA expression in small airway epithelial cells (SAEC) stimulated with either (-) or IL-36aß?. a, b *P = 0.05, **P = 0.01, ***P = 0.001, vs all other groups by one-way ANOVA and Tukey’s correction. c, d *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001 vs untreated by t test.

Fig 4: Small airway epithelial cells as the main cellular source of GMCSF.a GM-CSF, CXCL1, IL-36?, IL-1ß, and IL-1a 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.

Fig 5: IL-36? 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-1a, IL-1ß, and GM-CSF protein concentrations in BALF from untreated (n = 6) and IL-36?-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-36?, or IL-1a/IL-1ß or f in mouse bone marrow-derived neutrophils (from n = 4 mice) in vitro stimulated with no cytokines (-), IL-36?, GM-CSF, or IL-36?+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-36?, GM-CSF, and IL-36?+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.