Ex-vivo Incubation of FACS-Purified ILC2s

ILC2 were purified from the lungs of IL-33 treated mice, 24 h after last challenge, as described previously (31, 32). Lungs were finely chopped and digested in 1 mg/mL Collagenase IV (MP Biomedicals, LLC) and DNaseI (Roche) for 30 min at 37°C followed by passage through 70 uM filter and pelleted with 30% Percoll to remove debris. ILC2 were FACS purified with ARIA Fusion cell sorter (BD Biosciences) gated as Live CD45⁺ Lineage⁻ Thy1-2⁺ CD127⁺ ST2⁺ to >95% purity. 5 × 10³ cells/well were cultured ex-vivo in RPMI (Lonza) with 10% FCS, HEPES, L-Glutamine, B-mercaptoethanol for 2–3 days in the presence of 10 ng/mL rmIL-2, rmIL-7, and 10 ng/mL IL-33 as indicated (R&D systems). Sodium chloride, acetate, propionate, and butyrate (Sigma) were dissolved in PBS pH 7.4 and added to the indicated concentrations. For knockdown studies, 1 uM GATA3 in-vivo morpholinos 5′-TGGTCCGCAGTCACCTCCATGTCCT-3′ or 5′mismatch control 5′-TGcTCCcCAcTCACCTCgATcTCCT-3′ (Gene Tools, Philomath OR) were added as free uptake oligos 24 h before butyrate addition. For GPR109a knockdown studies, translation blocking morpholino 5′- CTAGAAAATGGTCTGACTTGCTCAT-3′ and 5′mismatch control 5′- CTTGTAAATCGTCTCACTTCCTCAT-3′.