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: IL-33 induces IRAK-M phosphorylation and binding to PIN1. a GST PIN1 pulldown assay with DC2.4 cell extracts either non-treated or treated with IL-33 (100 ng/ml) or LPS (100ng/ml) for 1 h. The GST-PIN1 bound proteins were eluted using reduced gluthatione and probed for IRAK-M. In the lower panel Coomassie blue staining of the blot shows equal amounts of GST or GST-PIN1 that were used for pull-down. b GST-PIN1 pulldown of DC2.4 cell extracts either treated or not with IL-33, followed by treatment in the absence or presence of calf intestinal alkaline phosphatase (CIP) for 30 min at room temperature before subjecting to GST-PIN1 pulldown. c DC2.4 cells were labeled with 10 μCi/ml {γ-32 P}ATP for 3 h. The cells were washed with fresh medium and treated with 100 ng/ml IL-33 for the indicated times prior to IRAK-M immunoprecipitation. d DC2.4 cells stably expressing IRAK-M were treated with IL-33 and at the indicated time points were subjected to CO-IP using anti-PIN1 antibody and blotted for IRAK-M. e HEK293 cells were transfected with different IRAK-M constructs expressing the N’ terminal domain (aa1-220), the middle portion of the protein (aa220-440) or the C’ terminal domain (aa 440-630), and then treated with IL-33, followed by CO-IP for PIN1. f DC2.4 cells stably expressing IRAK-M were treated with IL-33 and subjected to GST or GST-PIN1 pull-down. The bound proteins were eluted and subjected to IP using IRAK-M antibody. g LC-MS/MS analysis shows phosphorylation of IRAK-M at position Ser110. h WT IRAK-M or its mutants lacking the death domain (IRAK-M ∆DD), lacking the kinase domain (IRAK-M ∆KD), IRAK-M S110A or IRAK-M S467A (where these serine residues were mutated to alanine) were expressed in HEK293 cells, followed by CO-IP for GFP after IL-33 treatment. i IRAK-M, S110E or P111A stably expressing DC2.4 cells were stimulated with IL-33 and PIN1 interaction was monitored by CO-IP experiment as indicated. j HEK293 cells were co-expressed with IRAK-M and GFP, GFP-PIN1, GFP-WW domain or GFP-PPIase domain and, then treated with IL-33 before CO-IP for GFP. k HEK293 cells were co-expressed with IRAK-M and WT PIN1, PIN1 mutant W34A, or PIN1 mutant K63A and then were treated with IL-33 before CO-IP for GFP

Fig 4: 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 scRNA-seq data (n = 31,186 single immune cells) for phosphate-buffered saline- and recombinant IL-33 (rIL-33)-treated liver or lung ILC2s, shown as nonlinear representations of the top 50 principal components; the cells are colored based on cell type. b Cell numbers of the clusters within each group, as defined by the treatment condition and tissue. c Differentially expressed genes in each cell type, as defined by the FindAllMarkers function. d Uniform manifold approximation and projection (UMAP) plots of all immune cells showing the expression of Gata3 downstream genes (Il13, IL5, Areg, and Arg1). e Violin plot showing the expression of Il13 and Gata3 in each cluster. f Dot plot showing the expression of Gata3 downstream genes in each cell type.

Fig 5: IRAK-M is necessary for Il6, Csf3, Cxcl2 and Ccl5 expression in dendritic cells upon IL-33 induction. a Western blot shows IRAK-M levels in shIRAK-M expressing DC2.4 cells. Right panel shows heat map showing expression levels of different genes according to the affymetrix gene expression profiling analysis. b qRT-PCR analysis for expression of the indicated genes normalized to actin. The numbers over bars (95, 55 and 27) indicate the folds of induction of target genes in the presence vs absence of IL-33 in the sh IRAK-M group. c Quantification of IL-6 release measured by ELISA. d IRAK-M, IRAK-M S110E or IRAK-M P111A stably expressing DC2.4 cells were either treated or not with IL-33. Also as indicated, some cells were pretreated for 3 days with 5 μM or 10 μM of ATRA before IL-33 induction. e Relative gene expression of Il6, Csf3, Cxcl2 and Ccl5 in the different samples as in d normalized to actin. f IRAK-M was stably expressed in pLKO or TET on shPin1 expressing cells, followed by induction with IL-33, before western blot to examine protein expression of IRAK-M, PIN1 and tubulin as well as g qRT-PCR to determine the relative gene expression for Il6, Csf3, Cxcl2 and Ccl5 in the different samples. 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)