Fig 1: IL-18-Akt-Tcf4 signalling upregulates Lgr5+ stem cells.a–c Flow cytometry analysis of CD24-/low ileum crypts, isolated from the indicated uninfected or AIEC-infected mice at d6, for Lgr5+ stem cells. An isotype antibody was included to indicate the specificity of the Lgr5 antibody. d Immunofluorescence analysis of ileum crypts for Olfm4+ stem cells in the indicated uninfected or AIEC-infected mice at d6. Crypt base columnar (CBC, outlined with a solid line) stem cell and the above transit-amplifying (TA, outlined with a dashed line) compartments are indicated. e Flow cytometry analysis of IL-18-stimulated ileum organoids, derived from the indicated mice, for Lgr5+ stem cells. f Chromatin immunoprecipitation (ChIP) and PCR analyses of IL-22 or IL-18-stimulated wild-type ileum organoids for the binding of Tcf4 to the mouse Lgr5 promoter regions (P1~P6). Fold induction is calculated based on PCR signal strength of Tcf4-bound Lgr5 promoter P1 region before and after IL-22 or IL-18 stimulation. g Western blot and flow cytometry analyses of IL-18/Akt inhibitor-stimulated CMT93 cells for Akt phosphorylation and Lgr5. Quantification and the ratio (pAkt to total Akt, Lgr5 to ß-actin) of protein bands are indicated. h ChIP and PCR analyses of IL-18/Akt inhibitor-stimulated CMT93 cells for the binding of Tcf4 to the mouse Lgr5 promoter regions (P1 and P5). Fold induction is calculated based on PCR signal strength of Tcf4-bound Lgr5 promoter P1 region before and after stimulation. Each symbol in bar graphs represents a well of CMT93 cell culture (g), an ileum crypt sample (a–c) or organoid culture (e, f) derived from one mouse. Data shown are representative (d, f–h) or combined (a–c, e) results from two independent reproducible experiments. Statistical significance is indicated using unpaired two-tailed t test (a–c), One-way ANOVA with Sidak’s multiple comparisons test (f, g), or Two-way ANOVA with Tukey’s multiple comparisons test (e). Data are presented as mean ± SD. Source data are provided as a Source Data file.
Fig 2: IL-18 is a bona fide regulator of Paneth cells during AIEC host defence.a Quantitative real-time PCR and ELISA analyses of ileum crypts or tissues for IL-18 in uninfected or AIEC-infected mice. b Quantitative real-time PCR analysis of IL-18-stimulated ileum organoids, derived from the indicated mice, for anti-microbial peptides. c Flow cytometry analysis of IL-18-stimulated ileum organoids, derived from the indicated mice, for CD24+ Lysozyme+ Paneth cells. d Flow cytometry analysis of ileum crypts, derived from IL-22 or IL-18-injected mice, for phospho-Stat3 (pStat3) in the indicated epithelial subsets. Mean fluorescence intensity (MFI) of pStat3 is indicated. e Fresh crypts were stimulated with IL-22, IL-18, or both, to induce the release of anti-microbial peptides (AMP). The AMP-containing supernatants were then incubated with live AIEC and the percentage of bacterial killing was determined by the CFU assay after normalized to unstimulated crypts. f Immunofluorescence analysis of ileum crypts (outlined with a dashed line) for Lysozyme+ Paneth cells at the crypt base in the indicated mice. g Flow cytometry analysis of ileum crypts, derived from the indicated mice, for CD24+ Lysozyme+ Paneth cells. h Colony-forming unit (CFU) of AIEC was analyzed in feces and intestinal tissues isolated from the indicated mice at d6 post infection. i Flow cytometry analysis of PMA/Ionomycin (P/I)-stimulated ileum lamina propria cells for intracellular IFN? in CD4+ or CD8+ T cells in AIEC-infected mice. j CFU of AIEC was analyzed in feces and intestinal tissues isolated from the indicated PBS or IL-18-injected mice at d6 post infection. k Immunofluorescence analysis of ileum crypts for Lysozyme+ Paneth cells at the crypt base in the indicated IL-18-injected mice at d6 post AIEC infection. l Flow cytometry analysis of ileum crypts, derived from the indicated IL-18-injected mice, for CD24+ Lysozyme+ Paneth cells. m Quantitative real-time PCR analysis of ileum crypts from the indicated IL-18-injected mice, for Paneth cell marker Lysozyme and Cryptdin. Each symbol in bar graphs represents an ileum crypt sample (a, d, e, g, l, m), tissue sample (a, h, i, j), or organoid culture (b, c), derived from one mouse. Data shown are representative (f, i, k) or combined (a–e, g, h, j, l, m) results from two independent reproducible experiments. Figures 2f and 4c were performd in the same experiment. Statistical significance is indicated using unpaired two-tailed t test (g, h), One-way ANOVA with Sidak’s multiple comparisons test (a, d, e), or Two-way ANOVA with Tukey’s multiple comparisons test (b, c, i, j, l, m). Data are presented as mean ± SD. Source data are provided as a Source Data file.
Fig 3: IL-22 links IL-18 for IFN? and Paneth cell responses during AIEC infection.a Colony-forming unit (CFU) of AIEC was analyzed in feces and intestinal tissues isolated from the indicated PBS or IL-18-injected mice at day-6 (d6) post infection. b Flow cytometry analysis of PMA/Ionomycin-stimulated ileum lamina propria cells for intracellular IFN? in CD4+ or CD8+ T cells in the indicated PBS or IL-18-injected mice. c Immunofluorescence analysis of ileum crypts for Lysozyme+ Paneth cells at the crypt base in the indicated mice at d6 post infection. d CFU of AIEC was analyzed in feces and intestinal tissues isolated from the indicated PBS or IL-22-injected mice at d6 post infection. e Flow cytometry analysis of PMA/Ionomycin-stimulated ileum lamina propria cells for intracellular IFN? in CD4+ or CD8+ T cells in the indicated PBS or IL-22-injected mice. f Immunofluorescence analysis of ileum crypts for Lysozyme+ Paneth cells at the crypt base in the indicated mice at d6 post infection. Each symbol in bar graphs represents an intestinal tissue sample (a, b, d, e) isolated from one mouse. Data shown are representative (c–f) or combined (a, b) results from two independent reproducible experiments. Statistical significance is indicated using Two-way ANOVA with Tukey’s multiple comparisons test (a, b, d, e). Data are presented as mean ± SD. Source data are provided as a Source Data file.
Fig 4: Differential role of IL-22 and IL-18 in organoid culture.a Flow cytometry analysis of IL-22 or IL-18-stimulated ileum organoids, derived from the indicated mice, for Ki67+ proliferating cells. Mean fluorescence intensity (MFI) of Ki67+ organoids is shown. b Immunofluorescence analysis of ileum crypts for Ki67+ proliferating cells in the indicated naïve or AIEC-infected mice. Crypt base columnar (CBC, outlined with a solid line) stem cell and the above transit-amplifying (TA, outlined with a dashed line) compartments are indicated. c Flow cytometry analysis of ileum crypts for CD24-/low Ki67+ proliferating cells in the indicated AIEC-infected mice. d Quantitative real-time PCR analysis of IL-22 or IL-18-stimulated ileum organoids, derived from the indicated mice, for stem cell marker Lgr5, Ascl2, and Olfm4. e Quantification of the number of buds per organoid and size in IL-22 or IL-18-stimulated ileum organoids, derived from the indicated mice. “n” indicates the number of images taken from organoids derived from four mice in each group. f Quantification of the number of buds in IL-18-stimulated wild-type ileum organoids. “n” indicates the number of images taken from organoids derived from three mice per group. g Quantitative real-time PCR analysis of IL-22-stimulated ileum organoids, derived from the indicated mice, for stem cell marker Lgr5, Ascl2, and Olfm4. h Quantification of the number of buds per organoid and size in IL-22-stimulated ileum organoids, derived from the indicated mice. “n” indicates the number of images taken from organoids derived from four mice per group. Each symbol in bar graphs represents an ileum crypt sample (c) or organoid culture (a, d, g), derived from one mouse. Data shown are representative (b) or combined (a, c–h) results from two independent reproducible experiments. Statistical significance is indicated using unpaired two-tailed t test (c), One-way ANOVA with Sidak’s multiple comparisons test (f), or Two-way ANOVA with Tukey’s multiple comparisons test (a, d, e, g, h). Data are presented as mean ± SD. Source data are provided as a Source Data file.
Fig 5: IL-22 transcriptionally induces epithelial IL-18 via phospho-Stat3Y705.a Colony-forming unit (CFU) of AIEC was analyzed in feces and intestinal tissues isolated from the indicated mice at day-8 (d8) post infection. b Flow cytometry analysis of PMA/Ionomycin (P/I)-stimulated ileum lamina propria cells for intracellular IFN? in CD4+ or CD8+ T cells in AIEC-infected mice. c Quantitative real-time PCR analysis of ileum crypts from the indicated mice for Il-18 mRNA levels. d Western blot analysis of ileum crypts from the indicated mice for IL-18. Quantification and the ratio (IL-18 to ß-actin) of protein bands are indicated. e Flow cytometry analysis of IL-22-stimulated ileum organoids for intracellular IL-18. f Quantitative real-time PCR analysis of IL-22 or IL-18-stimulated ileum organoids, derived from the indicated mice, for Il-18 mRNA levels. g Flow cytometry analysis of naïve wild-type ileum crypts for IL-22R or IL-18R expression levels in the indicated epithelial subsets. h Chromatin immunoprecipitation (ChIP) and PCR analyses of IL-22-stimulated ileum crypts for the binding of phospho-Stat3Y705 to the mouse Il-18 promoter regions (P1~P5). Fold induction is calculated based on PCR signal strength of pStat3-bound Il-18 promoter region (P3,P4,P5) before and after IL-22 stimulation. Each symbol in bar graphs represents an ileum crypt sample (c, d, g, h), tissue sample (a, b), or organoid culture (e, f), derived from one mouse. Data shown are representative (b, d, g, h) or combined (a, c, e, f) results from two independent reproducible experiments. Statistical significance is indicated using unpaired two-tailed t test (a, c, d, e, h), One-way ANOVA with Sidak’s multiple comparisons test (g), or Two-way ANOVA with Tukey’s multiple comparisons test (b, f). Data are presented as mean ± SD. Source data are provided as a Source Data file.
Supplier Page from BioLegend for Recombinant Mouse IL-22 (carrier-free)