Figure 1. IL-33 Promotes TNF-α Secretion and TNFR2 Expression on ILC2s.

(A) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 μg rmIL-33 or PBS.

(B) On day 4, BAL fluid was collected, and supernatant TNF-α was measured by ELISA.

(C) Number of TNF-α-producing CD45⁺ lung cells on day 4, cultured for 4 h with GolgiPlug.

(D–F) Representative flow cytometry plots in naive (D) and activated (E) lungs of CD45⁺ TNF-α⁺ cells backgated for AMs (CD45⁺ SiglecF⁺ CD11b⁻, CD11c⁺, and F4/80⁺) and SiglecF⁻CD11b⁺ and SiglecF⁻CD11b⁻ populations and (F) corresponding quantitation presented as mean frequency ± SEM.

(G) AMs were FACS sorted from PBS- or rmIL-33-challenged mice on day 4 and cultured for 24 h in vitro (10⁶ AMs/well), and supernatant TNF-α was measured by ELISA.

(H and I) Gating strategy for murine ILC2s showing (H) lineage⁻ staining and (I) lineage⁻CD45⁺ CD127⁺ ST2⁺ GATA-3^hi ILC2s.

(J) nILC2s were FACS sorted from naive BALB/cByJ mice and cultured (50 × 10⁴/mL) ex vivo in the presence of rmIL-2 (10 ng/mL), rmIL-7 (10 ng/mL), and rmIL-33 (50 ng/mL) for the indicated times. nILC2s were gated as lineage⁻ CD45⁺ ST2⁺ CD127⁺.

(K and L) Representative flow cytometry plot of TNFR1 (K) and TNFR2 (L) expression and corresponding quantitation, presented as Mean Fluorescence Intensity (MFI) ± SEM.

(M) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 μg rmIL-33 or PBS.

(N and O) Representative flow cytometry plot of lung ILC2 expression of TNFR1 (N) and TNFR2 (O) expression on day 4 and corresponding quantitation, presented as MFI ± SEM.

Data are representative of 4 individual experiments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. See also Figure S1.