Fig 2: Itk-deficient mice have late defects in ILC2 function.(a) Outline of 14day papain-induced sensitization. (b) Numbers of ILC2+, IL-9- and IL-13-ILC2+ producing cells in lungs are shown from cells harvested at day 14 from female mice challenged with papain. (c) Outline of papain sensitization of female mice for 4 days. (d) Numbers of ILC2+ and IL-9-producing ILC2+ cells from mice treated with papain on day 4. (e) Outline of IL-33-induced sensitization for 4 days. (f) Numbers of ILC2+ cells in lungs harvested at day 4 from mice challenged with IL-33. (g) ILC2+ cells were sorted from IL-33-treated mice, cultured with 10 μg ml−1 of the indicated cytokines for 3 days and IL-9, IL-5 and IL-13 was measured in the culture supernatants (IL-9 and IL-5 have the same scale for Y axis). Experiments were performed at least twice using six female mice for papain-treated or IL-33 groups and four mice for PBS controls. (b,d,f) Mean±s.e.m. from one representative experiment, *P<0.05, **P<0.01 using Mann–Whitney test.

Fig 3: scRNA-seq reveals that liver group 2 innate lymphoid cells (ILC2) highly express Il13, which may contribute to the blood glucose-lowering effect of IL-33.a–i Liver and lung ILC2s were sorted from wild-type BALB/c mice intraperitoneally (i.p.) injected with phosphate-buffered saline (PBS) (control) or recombinant IL-33 (rIL-33) for 5 days. ILC2s from each of the four groups were profiled by droplet-based scRNA-seq. a, b The scRNA-seq data (n = 14,026 single ILC2s) across all four groups of ILC2s are shown as nonlinear representations of the top 50 principal components; the cells are colored according to uniform manifold approximation and projection (UMAP)-based clusters (a) or according to treatment and tissue type (b). Subclustering of ILC2s was performed with a resolution of 0.5. c Proportions of the clusters within each group as defined by treatment condition and tissue source. d UMAP plots showing the expression of the innate lymphoid cell (ILC) markers Gata3 and Il1rl1 in all ILC2 samples. e Dot plot showing the differentially expressed genes (DEG) in each cluster as defined by the FindAllMarkers function. f Volcano plots showing the DEGs between PBS-treated liver and rIL-33-treated liver samples (left) or between rIL-33-treated liver and rIL-33-treated lung samples (right). g Expression levels of representative Gata3 downstream genes, as grouped by treatment and tissue. h Gating for Lin-Thy1+CD127+ST2+ ILC2s (left) and frequencies and fluorescence intensities of IL-13 in liver and lung ILC2s from PBS- or rIL-33-treated mice (right) (n = 3 per group). i Mean fluorescence intensity (MFI) of IL-13 in ILC2s in each group (mean ± SD, n = 3). Unpaired one-sided Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: ILCs Are Required for the Development of Pericarditis, and ILC2s Are Sufficient to Induce Eosinophilic Infiltration to the Heart(A) Representative images of H&E-stained heart sections of WT (left), Rag2-/- (center), and Rag2-/- Il2rg-/- (right) mice. Bars: 100 µm.(B) Severity of pericarditis was scored on H&E-stained heart sections.(C) Total number of heart-infiltrating CD45+ leukocytes was determined by flow cytometry.(D and E) Number of (D) ILC2s and (E) eosinophils in the hearts.(F) Representative flow cytometry plots of ILC2s found in the hearts of naive Rag2-/-Il2rg-/- mice and Rag2-/-Il2rg-/- mice injected with media or ILC2s followed by IL-33 treatment.(G) Number of heart-infiltrating CD45.2+ leukocytes.(H) Representative flow cytometry plots of CD45.2+ cells. Gates show the frequency of CD11b+SiglecF+ eosinophils in the hearts of naive Rag2-/-Il2rg-/- mice and Rag2-/-Il2rg-/- mice injected with media or ILC2s followed by IL-33 treatment.(I) Number of eosinophils in the hearts.Both male and female mice were used in each group in (B). Data are representative of two independent experiments and displayed as the mean. Kruskal-Wallis H test (B) or one-way ANOVA followed by Tukey’s post hoc test (C–E, G, and I) was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001.See also Figure S4.

Fig 5: IRAK-M KO and PIN1 KO dramatically reduce lung inflammation upon IL-33 treatment in vivo. a Representative H&E, PAS staining of lung sections from various treated mice, as well as BALF cytospin from the treated mice stained with Giemsa stain (n = 5). Scale bar = 50 µm. b Histopathology score- All H&E and PAS histological samples were examined and scored in a blind manner by the core facility pathologist (n = 5). c ELISA measurements of IL-33, -5, -13 and IL-4 in the BALF of the mice treated with PBS or IL-33. d RNA was obtained from the whole lung tissue and analyzed for the relative expression of Il6, Csf3, Cxcl2 and Ccl5 by qRT-PCR. e CD11c+ CD11b+ CD205+ cells were monitored after PBS or IL-33 challenge in the indicated mice (n = 3). In WT mice there was a 3-fold induction (P < 0.05) in CD11c+ CD11b+ CD205+ cells upon IL-33 treatment. PIN1 KO mice showed a more moderate increase and IRAK-M KO mice showed non significant (NS) increase in the CD11c+ CD11b+ CD205+ cells upon IL-33 treatment. The data were analyzed by a Student’s two-tailed t test and the values are reported as mean ± standard errors of the means (SEM). *- statistical significance (P < 0.05), **- significance (P < 0.01)