Figure 1.

BQ123 exhibited protective effects against Alternaria alternata-induced airway inflammation (A) Representative the mean fluorescence intensity (MFI) from human ILC2s of ETAR-expressing CD45⁺ Lin^- CD127⁺ CRTH2^- cells and CD45⁺ Lin^- CD127⁺ CRTH2⁺ cells (n = 5). (B) Statistical analysis of ETAR expression. (C) mRNA expression levels of human endothelin receptor A (Ednra) were evaluated; β-actin level was used for normalization, and the lowest expression level in Ednra-negative cells was artificially set to 1 (n = 3). Purified ILC2s from mouse lung and human PBMCs were cultured with rm/rh-IL-2, rm/rh-IL-7 and with or without rm/rh-IL-33 for 72 h. Then ETAR expression levels were analyzed by flow cytometry. Representative MFI (D, I) and statistical analysis (E, J) of ETAR expression were shown. (F) Representative results of flow cytometry MFI from mouse lung ILC2s of ETAR -expressing CD45⁺ Lin^- CD127⁺ CD90.2⁺ CD25⁺ ST2^- cells and CD45⁺ Lin^- CD127⁺ CD90.2⁺ CD25⁺ ST2⁺ cells (n=5). (G) Representative statistical analysis of ETAR expression. (H) mRNA expression levels of mouse Ednra were evaluated; β-actin level was used for normalization, and the lowest expression level in Ednra -negative cells was artificially set to 1 (n = 3). (K) Experimental scheme. Female C57BL/6J mice were intranasally challenged with A. alternata on days 1–4 and were sacrificed 24 h after the last challenge on day 5. (L–O) Representative hematoxylin and eosin (H&E) staining of lung sections (L) and inflammation scores (M), as well as the infiltrating cells (N) and airway epithelium thickness (O) were shown. Bars, 100μm. Absolute number of BALF (P), typical example of flow cytometry (left) and statistical results (right) both population and the absolute numbers of EOS in the bronchoalveolar lavage fluid (BALF) (Q) were indicated. (R) IL-5 and IL-13 levels in BALF were determined. (S–V) Representative results of flow cytometry, statistical analysis of the frequencies of ILC2s and absolute counts (S), IL-5⁺ IL-13⁺ ILC2s (T), Ki67⁺ ILC2s (U), and levels of GATA3 (V) in the lungs were shown. (W) The mRNA expression levels of ILC2-related target genes in lung tissues, including Il5, Il13, and Gata3, were evaluated; β-actin level was used for normalization, and the lowest expression level in the A. alternata + BQ123 group was artificially set to 1 (n = 3). Data are representative of two or three independent experiments (n = 6 for the A. alternata + PBS group; n = 6 for the A. alternata + BQ123 group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. In all panels, individual results and mean ± standard error of the mean (SEM) are shown; statistical significance was determined using a two-tailed unpaired Student’s t-test (B, C, E, G, H, J, M–U, W) or Mann-Whitney test (V).