(A) Schematic of Alternaria experimental model. Mice were treated i.n. with 25 μg Alternaria extract (Alt) or PBS (control) every other day for 9 days. Mice receiving Alt were either treated i.p. with DMSO/saline vehicle control (Alt/D) or GW4869 (Alt/G, 2–2.5 μg/g) daily for 9 days. Some mice were analyzed 1 hour following the last Alt dose; the remainder were analyzed 24 hours after the last dose. (B) Lung tissue qPCR for the 3 groups (n = 3), demonstrating increased Il33 and Smpd3 mRNA with Alt treatment. (C) IL-33 protein quantified by ELISA (normalized to total protein) for lung lysate (n = 3), intracellular fraction (pooled single-cell suspension, n = 3 each), and 1-hour (n = 3) or 24-hour (n = 3) bronchoalveolar lavage (BAL) samples. IL-33 was increased in the intracellular fraction and decreased in BAL with GW4869 treatment. (D) Exosome quantity measured by TRPS for pooled (n = 3) BAL samples with vesicle quantity, representative size distribution (with peak), and CD9 Western blot, reflecting the decrease in BAL vesicle quantity and corresponding CD9 staining with GW4869 treatment. (E) Tissue IL-33 immunofluorescence staining (red) demonstrating increased cytoplasmic IL-33 signal (insets) and expansion of IL-33⁺ parenchymal cells in Alt groups. DAPI counterstain was also used. Scale bar: 50 μm (gray); 10 μm (white). (F) Lung tissue Il13 and Il5 qPCR (n = 3) demonstrating induction with Alt treatment that was attenuated with GW4869. (G) Representative FACS plots for sorted lung innate lymphoid type 2 cells (ILC2, pooled samples, n = 3) and Il13 qPCR in sorted cells, demonstrating induction of ILC2 cells and IL13 expression with Alt treatment and concomitant reduction with GW4869 treatment. Data are shown as the mean ± SEM. Statistical analysis: 1-way ANOVA (B, C, and F); *P < 0.05, **P < 0.01, ***P < 0.001.