Fig 2: 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 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: Trajectory analysis reveals that AP-1 family member expression negatively correlates with Il13 expression, especially in liver group 2 innate lymphoid cells (ILC2s).a–g Analysis based on single-cell RNA sequencing (scRNA-seq) data for liver ILC2s and lung ILC2s from phosphate-buffered saline-treated or recombinant IL-33 (rIL-33)-treated wild-type BALB/c mice. a Fold-change in the gene expression of AP-1 family members between control liver ILC2s and rIL-33-treated liver ILC2s (left) and between liver ILC2s and lung ILC2s (right). b Real-time quantitative PCR (RT?qPCR) analysis of Il13 24 h after siRNA transfection of AP-1 family members (Jun, Fos, Fosb, Fosl2) in liver ILC2s (normalized to L32) (n = 3 per group). c RT?qPCR analysis of Il13 24 h after transfection with a pCMV vector encoding JunB into liver ILC2s (left) and 5 days after infection with a retrovirus encoding JunB in the ILC2 cell line ILC2/b6 (right) (n = 3 per group). d, e Trajectory analysis of liver ILC2s and lung ILC2s using Monocle3. The root is defined as control ILC2s. d Module analysis showed that AP-1 family members and some genes downstream of GATA3 (Il13 and Il5) were coregulated and strongly associated with the end cluster (cluster 3). e Trajectory analysis of lung ILC2s showed a different end cluster compared with liver ILC2s, which was not associated with AP-1 family members and GATA3 downstream genes (cluster 5). f Correlation matrix for AP-1 family members and Gata3 downstream genes based on the average gene expression of each cluster in liver ILC2s (left) and lung ILC2s (right). g Pseudotime plot showing the expression levels of Il13 and AP-1 family members along the pseudotime axis. h Graphical abstract for this study. IL-33 induces IL-13 expression in liver ILC2s. IL-13 directly suppresses gluconeogenesis in hepatocytes by inhibiting gluconeogenic enzymes, such as G6pc and Pck1, leading to reduced blood glucose levels. AP-1 family members bind to GATA3 and suppress IL-13 production in liver ILC2s. Unpaired one-sided Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. Each bar and its error bars represent the mean ± SD.

Fig 5: AP-1 family members bind to GATA3 in liver and lung group 2 innate lymphoid cells (ILC2) and suppress GATA3 function to induce Il13 expression.a Experimental schema. Sorted liver and lung ILC2s were cultured and then subjected to immunoprecipitation with an IgG control or anti-GATA3 antibody and then to liquid chromatography–mass spectrometry/mass spectrometry (LC –MS/MS) analysis. The Mascot score was evaluated, and GATA3-specific interacting proteins were identified. b List of GATA3-specific nuclear and epigenetic factors. Bubble plot and heatmap showing the functions and Gene Ontology (GO) terms of the listed proteins. c Experimental schematic of assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and GATA3 chromatin immunoprecipitation sequencing (ChIP-seq). Differentially accessible regions (DAR) between recombinant (rIL-33)- and phosphate-buffered saline (PBS)-treated ILC2s were classified into GATA3-bound (blue) and nonbound (orange) regions (middle). Enriched sequence motifs for the GATA3-bound DARs (right). d Representative tracks at an arbitrary locus (near Il13) illustrating signals in ATAC-seq (first and second tracks) and GATA3 ChIP-seq (third track) samples of liver ILC2s. e JunB binding to the upstream regions of the Il13 gene was assessed by ChIP with quantitative PCR (qPCR) (right) (n = 3 per group). The results show JunB binding motif1 (first and second column), motif2 (third), and nonbinding control regions far upstream of the Il13 gene (fourth and fifth). f The ILC2 cell line ILC2/b6 was infected with a retrovirus encoding FLAG-GATA3 and a retrovirus encoding JunB. Five days later, the extracts were subjected to immunoprecipition (IP) with an anti-FLAG (GATA3) antibody and then subjected to immunoblotting (IB) with an anti-JunB antibody (upper panel). The total cell lysates (input) were also subjected to IB in parallel (lower panel). These are representative figures of two independent experiments. g Mascot score and expression levels from single-cell RNA sequencing (scRNA-seq) data in liver ILC2s. h Experimental schema. Twenty-four hours after small interfering RNA (siRNA) transfection, liver ILC2s were subjected to real-time quantitative PCR (RT–qPCR) and RNA-seq analyses. i RT–qPCR of liver ILC2s transfected with various siRNAs. The expression of Il13 is shown (normalized to L32) (Ct si: n = 4, Gata3 si: n = 4, Junb si: n = 5, Pa2g4 si: n = 4, Irf8 si: n = 4, Calr si: n = 4, Jund si: n = 4, Runx1 si: n = 4, Runx3 si: n = 4). j Heatmap showing differentially expressed genes (DEG) in liver ILC2s transfected with Ct si, Gata3 si, Junb si, or Runx1 si. Unpaired one-sided Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. Each bar and its error bars represent the mean ± SD.