Rmil 1α

Myeloid differentiation factor-88^−/−(33 (link)), MyD88^flox/flox mice (kindly provided by Dr. Matthias Muller and Franco di Padova) were used to generate MyD88 x Acid/AQP5 cre mice, which lack MyD88 in alveolar epithelial type 1 cells (34 (link)) as described (6 (link)), IL-1α- and IL-1β-deficient mice were provided by Dr. Yoichiro Iwakura (35 (link)), IL-18^−/− from Jackson laboratory, and C57BL/6 littermate control (WT) mice were used for the study. Mice were housed and bred in pathogen-free animal facility at Transgenose Institute (TAAM-CNRS, UPS 44 under agreement D-45-234-6, 2014), Orleans, France. Mice were bred in a temperature controlled (23°C) facility with strict 12 h light/dark cycles and were given free access to food and water. Female mice (8–10-month-old) were used in this study. Animal experiments were performed with the approval of the French Institutional Ethical Committee under agreement CLE CCO 2015-1088.
To block IL-1α, we used an anti-mouse-IL-1α antibody (Clone ALF-161, eBioscience) and its isotype, armenian hamster IgG (clone eBio299Arm, eBioscience) by intra-peritoneal injection given at 200 μg/mouse, 12 h before ozone exposure.
Recombinant mouse IL-1α (rmIL-1α) (Biolegend 575004, 0.8 μg/mice) was instilled intratracheally (i.t.) 4 h after ozone exposure.

Isolation of primary epidermal keratinocytes was performed as described earlier (23 (link)). Briefly, disinfected tail skins of adult mice were digested overnight at 4˚C with trypsin (0.25%; w/o CaCl₂, Gibco, Thermo Fisher Scientific, USA). Detached epidermal cells were seeded overnight at 32°C, 5% CO₂ in minimum essential medium with Earle`s Balanced Salt Solution medium (Lonza, Switzerland) containing 0.2 mM CaCl₂ (Merck, Germany). Subsequently 1x10⁶ isolated cells were cultured on fibronectin (Roche, Switzerland)/rat tail collagen I (Becton Dickinson, Corning, USA)-coated six well-culture plates in Keratinocyte Growth Medium 2 with supplement mix (PromoCell, Germany) to the confluence of 70–80%. Culture and stimulation conditions in Keratinocyte Growth Medium 2 (PromoCell, Germany) are indicated in Supplementary Figure 1. Times of stimulations are indicated in figure legends. Reagents: flagellin from Salmonella typhimurium (100 ng/ml, InvivoGen, France), rm IL-1α, rm IL-17A, rm IL-17F, and rm TNFα (100 ng/ml, BioLegend, USA), S100A8 (8 (link)). Possible endotoxin contaminations of S100A8 proteins were evaluated by a sensor chromogenic LAL endotoxin assay (GenScript, USA) and were < 2 pg/µg protein.