Ril 33

WT mice received PPE or PBS by intratracheal instillation on day 0. The mice received an intraperitoneal injection of 1000 ng of rIL-33 (R&D Systems) on days 0 and 3. Control mice received PBS at the same time points. Lung function and lung histology were evaluated on day 21. In some experiments, IL33^−/− mice received an intraperitoneal injection of 1000 ng rIL-33 on day 0 following intratracheal instillation of PPE or PBS. Control mice received PBS at the same time point. BAL was performed on day 2, and the levels of HGF and VEGF in BAL fluid were measured.

Wild‐type (WT) C57BL/6 mice were obtained from the Shanghai Laboratory Animal Center (Shanghai, China). Panx1‐knockout mice were littermates that were used in our previous study.⁹ All the animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86‐23 revised 1985). The mice were housed in the animal facility of the Institut Pasteur of Shanghai and received standard animal management. To eliminate potential differences caused by sample selection, all the experiments were conducted with 6‐ to 8‐week‐old male mice. Liver injury was established by an intraperitoneal injection of LPS (Escherichia coli 0111:B5; Sigma–Aldrich, St. Louis, MO, USA) dissolved in phosphate‐buffered saline (PBS) at doses of 10, 15, or 30 mg/kg body weight. Adeno‐associated viruses (AAVs) encoding short hairpin RNA (shRNA) targeting P2x7 or Panx1 (GenePharma, Shanghai, China) were injected via the hydrodynamic tail vein. Some mice were pretreated with a single intraperitoneal injection of PBS vehicle or rIL‐33 (2 μg/mouse) (R&D Systems, Minneapolis, MN, USA) 12 h prior to LPS administration.

Mice were anesthetized by isoflurane inhalation followed by a single i.n. administration of 50 µl of A. alternata extract (12.5 µg; Greer Laboratories) or recombinant IL-33 (rIL-33, 1 µg; house-made [Cayrol et al., 2018 (link)]), and/or recombinant TL1A (rTL1A, 5 µg; R&D Systems) in PBS. Lungs and BAL fluids were collected at different time points (15 min, 1 h, 3 h, 6 h, 14 h, 24 h, 48 h) after the single i.n. treatment with A. alternata or cytokines for flow cytometry, Western blot, qPCR, and ELISA analyses. In some experiments, mice were treated by a single i.n. administration of A. alternata extract (12.5 µg), coadministrated with two doses (10 µg i.n. at t-12 h and 10 µg i.n. at t0) of the function-blocking anti-TL1A mAb L4G6 (Fang et al., 2008 (link)) (L4G6, # EMI006; Kerafast) or its isotype control (Armenian hamster IgG, clone PIP, #BE0260, RRID: AB_2687739; BioXCell) (Fig. 8, C–H), or the function-blocking anti-IL-2 mAb JES6-1A12 (# 503706, RRID: AB_11150775; Biolegend) or its isotype control (rat IgG2a, clone 2A3, # BE0089, RRID: AB_1107769; BioXCell) (Fig. S5 G). For in vivo imaging of endogenous IL-9^high ILC2s (Fig. 5, K and L), 100 µl of rIL-33 (1 µg) in PBS was injected i.p. for 6 consecutive days to expand lung ILC2s (Fig. S5 F), before a single i.n. administration of rIL-33 (1 µg) and/or rTL1A (5 µg) in PBS.

As described by Bruce et al., 8–12 week-old B6 mice were given 0.4 μg recombinant mouse IL-17E/IL-25 per day (Biolegend, #587306) by intra-peritoneal injection for 4 days [14 (link)]. On day 5, cells were isolated from the mesenteric lymph nodes and processed using Hank’s Balanced Salt Solution with 2% newborn bovine serum. ILC2s were isolated by sorting for Lin⁻Thy1.2⁺ cells. The lineage cocktail included anti-mouse FcεRI, anti-mouse B220, anti-mouse CD19, anti-mouse Mac1, anti-mouse Gr1, anti-mouse CD11c, anti-mouse NK1.1, anti-mouse Ter-119, anti-mouse CD3, anti-mouse CD8a, anti-mouse CD5, anti-mouse TCRβ, and anti-mouse TCRγδ antibodies and Thy1.2 was stained using antibodies conjugated with fluorescein isothiocyanate. Cells were cultured for 7 days in complete media with rIL-7 and rIL-33 (10 ng/ml) (R&D Systems and eBioscience, respectively), with the media replenished every 2 days.