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–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 3: Liver group 2 innate lymphoid cell (ILC2)-derived IL-13 directly suppresses gluconeogenesis in hepatocytes.a Blood glucose levels in wild-type (wt) mice treated with phosphate-buffered saline (PBS) or IL-13 as measured by the pyruvate tolerance test (PTT) (n = 8 per group). b, c Il13 expression levels in liver tissue (normalized to L32) (b) and serum IL-13 concentrations (c) in wt, nude, and NOD/Scid/Il2R?null (NSG) mice intraperitoneally (i.p.). injected with PBS or recombinant IL-33 (rIL-33) for 5 consecutive days (wt rIL-33[-]: n = 8, wt rIL-33[+]: n = 9, nude rIL33[-]: n = 9, nude rIL33[+]: n = 10, NSG rIL-33[-] n = 6, and NSG rIL-33[+]: n = 6 in b; wt rIL-33[-]: n = 4, wt rIL-33[+]: n = 4, nude rIL33[-]: n = 4, nude rIL33[+]: n = 4, NSG rIL-33[-] n = 6, and NSG rIL-33[+]: n = 5 in c). d, e Fasting blood glucose (d) and blood glucose levels as measured by PTT (e) in Il13+/-, Il13+/- and Il13-/- mice after 5 days of rIL-33 injection (Il13+/+rIL-33[-]: n = 5, Il13+/+rIL-33[+]: n = 5, Il13+/-rIL-33[-]: n = 7, Il13+/-rIL-33[+]: n = 4, Il13-/- rIL-33[-]: n = 9 and Il13-/- rIL-33[+]: n = 7 in (d), Il13+/+rIL-33[-]: n = 7, Il13+/+rIL-33[+]: n = 5, Il13+/-: n = 4, Il13+/-rIL-33: n = 7 and Il13+/-rIL-33: n = 6 in e). f Schema of the experiment. PBS, liver ILC2s from Il13+/- mice or liver ILC2s from Il13-/- mice were injected intravenously into NSG mice. rIL-33 was i.p. injected for 5 consecutive days, and fasting blood glucose levels were measured after 3 and 5 days. g Fasting blood glucose levels in NSG mice without ILC2 transfer and with Il-13+/-, Il-13+/- or Il-13-/- liver ILC2 transfer at days 3 and 5 (n = 3). h Experimental schema for coculture and subsequent real-time quantitative PCR (RT–qPCR) and metabolome analysis. After 6 h of coculture with ILC2s, the hepatocytes were used for RT–qPCR and metabolomic analysis. i RT–qPCR analysis of gluconeogenesis-related genes in primary hepatocytes cocultured with ILC2s (Ct: control hepatocytes, +ILC2: hepatocytes cocultured with ILC2s, + ILC2 + anti-IL13 Ab: hepatocytes cocultured with ILC2s in the presence of an anti-IL-13 antibody; n = 3 per group). j, k, l Metabolomic analysis of primary hepatocytes after coculture with ILC2s (Ct: control hepatocytes, +ILC2s: hepatocytes cocultured with ILC2s; n = 6 per group). j Amino acid metabolites categorized into the pyruvic acid pathway, acetoacetate pathway, 2-oxoglutaric acid pathway and others. k Metabolites in the gluconeogenesis-related pathway. (G6P; glucose 6-phosphate, F6P; fructose 6-phosphate, F1,6P; fructose 1,6-diphosphate, G3P; glyceraldehyde 3-phosphate, 3-PG; 3-phosphoglyceric acid, PEP; phosphoenolpyruvic acid, OAA; oxaloacetate, Pklr; pyruvate Kinase L/R, Pcx; pyruvate carboxylase). l Metabolites in the tricarboxylic acid (TCA) cycle. 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 4: 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.

Fig 5: IL-33 suppresses gluconeogenesis in hepatocytes, especially in the cluster that most significantly interacts with liver group 2 innate lymphoid cells (ILC2).a Schema of the experiment. Hepatocytes were isolated from phosphate-buffered saline (PBS)- and recombinant IL-33 (rIL-33)-injected mice, and then subjected to scRNA-seq analysis. b scRNA-seq data (n = 11,327 single cells from the liver) for PBS and rIL-33-treated livers, shown as nonlinear representations of the top 50 principal components; the cells are colored based on uniform manifold approximation and projection (UMAP)-based cell clusters. c Dot plot showing the expression of zonation markers (Glul and Cdh1, left) and differentially expressed genes (DEG, right) in each hepatocyte cluster (n = 10,186 hepatocytes). d, e Interaction analysis using CellPhoneDB. Interaction heatmap plotting the total number of receptor and ligand interactions for the indicated cell clusters (d). Dot plot showing the results for the IL13-IL-13R interactions with the corresponding P-values and mean expression levels of IL-13 in ILC2s and IL13R in hepatocytes (e). f Representative image of immunofluorescence of PCK1-positive hepatocytes and ILC2s (CD3E-KLRG1 + ). PCK1: magenta, CD3E: red, KLRG1: green, nucleus (DAPI): blue. In total, 24 locations were observed in 5 independent mice with IL33 treatment. g Enriched genes upstream of the DEGs identified between the PBS and rIL-33 groups. h The dot plot shows the expression of genes downstream of Hnf4a, including G6pc and Pck1, in all hepatocytes. i UMAP plots (upper panel) and violin plots (lower panel) showing G6pc and Pck1 expression in all hepatocytes. j Volcano plots showing the DEGs between the PBS and IL-33 groups in Hepatocyte4 cells. Upregulated (red) and downregulated (blue) genes in the rIL-33-treated group. k Dot plot showing IL13 receptor subunit (Il13ra1, Il13ra2 and Il4ra) and Stat3/6 expression. The expression levels of Il13ra1 and Stat3 were elevated in Hepatocyte4. l STAT3 binding to the upstream regions of the G6pc and Pck1 genes was assessed by chromatin immunoprecipitation (ChIP) with quantitative PCR (qPCR) (n = 3 per group). m Primary hepatocytes were transfected with small interfering RNAs (siRNAs), and 48 h later, they were subjected to RT–qPCR for G6pc or Pck1 (n = 3 per group). Unpaired one-sided Student’s t-test. *P < 0.05; **P < 0.01; † < 10-10; †† < 10-20; ††† < 10-30.