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. 2022 Jan 19;219(3):e20211001. doi: 10.1084/jem.20211001

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© 2022 Liu et al.

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Figure S4. — The effect of Ang II on ILC2 is dependent on IL-33, and AT1 deficiency attenuates papain-induced inflammation. (A) The mean fluorescence intensities (MFIs) of AT1a were determined by flow cytometry in IL-33^−/− and their littermate controls. Data are from two independent experiments; n = 4/group. (B) AT1a mRNA relative expression was analyzed by qRT-PCR in indicated cells or kidney tissue (n = 4). (C) IF staining of AT1a, DAPI in lung tissues from IL-5–tdTomato mice after PBS or IL-33 challenge. Representative images are shown from three independent experiments. Red, IL-5 tdTomato; green, AT1a; blue, DAPI; scale bar, 20 µm; n = 3/group. Lung tissue from AT1a^−/− mice was the negative control. (D–F) IL-33^+/+ and IL-33^−/− mice were i.n. administered Ang II or PBS daily for 5 d. (D) MFI of KLRG1, ST2, and CD25 was analyzed by flow cytometry in IL-33^+/+ mice. (E) MFI of CD25 and ST2 were analyzed by flow cytometry in IL-33^−/− mice. (F) Alveolar macrophages (AMs) were determined by flow cytometry. (G) IL-5 and IL-13 concentrations were determined by ELISA after IL-25 and IL-25 plus Ang II treatment in the presence of IL-2 and IL-7 for 3 d. (H and I) Naive mice were i.n. challenged with Ang II and PBS for 5 consecutive days. Flow-cytometric analysis of the proportions and absolute numbers of macrophages (H) and DCs (I) in lungs. (J–N) PBS and papain were i.n. administered daily for 5 consecutive days in AT1a^+/+ and AT1a^−/− mice and data were analyzed 24 h after the last administration. (J) Flow cytometry plots of ILC2s from lung tissue. (K) Flow plots of lung ILC2 proliferation. (L) MFI of ST2 and KLRG1 on ILC2s. (M) CD4-derived IL-5 and IL-13 were analyzed by flow cytometry in AT1a^+/+ and AT1a^−/− mice after papain challenge. (N) Ratio of natural and inflammatory ILC2s in AT1a^+/+ and AT1a^+/+ mice lung tissue after papain challenge. (D–I) Two repeated independent experiments, and J–N are repeated three times. Numbers denote percentage of cells in each gate. Data show mean ± SEM by unpaired t test. *, P < 0.05, **, P < 0.01. Lin, lineage.