Fig 2: IL33 promotes expression of REG3γ in both murine and human IEC. (A) MSIE cells (n = 3 wells/group) were treated with a series of doses of IL33 for 12 hours. The expression of REG3γ, β-defensin-1, -3, and -4 was determined by qRT-PCR, and normalized against glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (GAPDH). (B) MSIE cells (n = 3 wells/group) were treated with 10 ng/mL of IL33. The expression of REG3γ was determined at different time points from 0 hours to 24 hours by qRT-PCR and normalized against GAPDH. (C) WT enteroids (n = 3 wells/group) were treated with IL33 (20 ng/mL) for 48 hours, and REG3γ expression was determined by qRT-PCR and normalized against GAPDH. (D) ST2 knockout (ST2 KO) and IL33 knockout (IL33 KO) MSIE cells (n = 4 wells/group), generated by CRISPR, were treated with 10 ng/mL of IL33 together with WT MSIE cells. The expression of REG3γ was determined by qRT-PCR and normalized against GAPDH. (E) MSIE cells (n = 3 wells/group) were treated with 10 ng/mL of IL17 or IL22 in the presence or absence of 10 ng/mL of IL33 for 12 hours. The expression of REG3γ was determined by qRT-PCR and normalized against GAPDH. (F) WT (n = 4) and IL33-/- mice (n = 4) were administered intraperitoneally with IL17 (1 μg/mouse) daily or IL22 (2 μg/mouse) every other day for 10 days. The IEC expression of REG3γ was determined by qRT-PCR and normalized against GAPDH. (G) Human HT-29 cells (n = 4 wells/group) were treated with a series of doses of human IL33 for 12 hours. The expression of REG3α was determined by qRT-PCR, and normalized against GAPDH. Significance was calculated using 1-way ANOVA test (A, B, D–G), or Student t test (C). *P < .05; **P < .01. Data are represented as means ± standard deviation. One representative of 3–5 experiments with similar results.

Fig 3: 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 4: IL-22 promotes mucin secretion via IL-18 during AIEC infection.a Flow cytometry analysis of ileum epithelial cells, isolated from the indicated naïve mice, for Muc2+ Goblet cells. b, c Immunofluorescence analysis of ileum villi for Muc2+ Goblet cells in the indicated naïve or AIEC-infected mice at d6 (n = 3 in each group). Mucin in the lumen after infection is also detected and indicated by yellow arrowheads. Quantification of Muc2+ Goblet cells per villus is indicated. d Quantitative real-time PCR analysis of IL-22/IL-18-stimulated ileum organoids, derived from the indicated mice, for Goblet cell marker Muc2. e Graphic illustration of integrated and differential regulation of barrier function and epithelial subsets by IL-22 and IL-18 during AIEC host defence. IL-22 initiates an IL-18-dependent response circuit which boosts innate immunity and activates adaptive immunity. Each symbol in bar graphs represents an ileum sample (a) or organoid culture (d) derived from one mouse. Data shown are representative (b, c) or combined (a, d) results from two independent reproducible experiments. Statistical significance is indicated using unpaired two-tailed t test (a) or Two-way ANOVA with Tukey’s multiple comparisons test (b, d). 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.