Fig 1.
IL-22 regulates genes involved in intestinal immune defence responses.
WT organoids were treated with IL-22 (2 ng/ml) for 3 hours. RNA was isolated for RNAseq analysis. (A) Volcano plot showing fold change (log2) in gene expression of WT organoids treated with IL-22 (2 ng/ml) for 3 hours compared to untreated WT organoids, plotted against significance (−log10[p-value]). Red = significantly up-regulated gene (fold change > 2, p < 0.05), blue = significantly down-regulated genes (fold change < 0.5, p < 0.05). (B) Functional enrichment analysis (GO term: “Biological processes”) for genes up- or down-regulated in WT organoids treated with IL-22, compared to untreated WT organoids. Shown are the top 15 up-regulated (red) and 10 down-regulated pathways (blue). (C) Heatmaps displaying mRNA expression level of significantly regulated genes in WT organoids either not treated or treated with IL-22. Genes of interest were manually grouped by their main biological function. Numerical values for panel B are available in S1 Data and for panels A and C in S2 Table. GO, gene ontology; IL-22, interleukin-22; NT, not treated; RNAseq, RNA sequencing; WT, wild-type.
Fig 2.
APC-mutant intestinal epithelial cells are defective in IL-22 mediated gene regulation.
(A). WT and ApcMin/Min organoids were treated with IL-22 (2 ng/ml) for 3 hours. RNA was isolated for RNAseq analysis. Graph shows the fold change of genes induced by IL-22 in WT and ApcMin/Min organoids. The 11 genes shown were the only genes up-regulated by IL-22 in ApcMin/Min organoids (p < 0.1). (B) Expression of candidate up-regulated genes from RNAseq data were verified by RT-qPCR. Data shown are relative levels of mRNA for genes of interest relative to Tbp expression. At least 3 independent experiments were performed in each case. *p < 0.05, **p < 0.01, ***p < 0.001 on paired t test. (C). WT and ApcMin/Min organoids were treated with IL-22 (10 ng/ml) for 3, 6, or 24 hours, and RT-qPCR analysis was performed. Data show the relative expression of mRNA for Reg3g or Socs3 compared to Tbp. Three independent experiments were performed. *p < 0.05, **p < 0.01, paired t test. Numerical values for (A), (B), and (C) are available in S1 Data. APC, adenomatous polyposis coli; IL-22, interleukin-22; Reg3g, regenerated islet-derived protein 3 gamma; RNAseq, RNA sequencing; RT-qPCR, quantitative reverse transcription polymerase chain reaction; Socs3, Suppressor of cytokine signalling 3; Tbp, TATA box binding protein; WT, wild-type.
Fig 3.
Differences in IL-22–induced STAT3 phosphorylation in WT and ApcMin/Min organoids.
(A). WT and ApcMin/Min organoids were stimulated with IL-22 (10 ng/ml) for 0.5 hours. Organoids were fixed and immunostained with STAT3 antibodies (green), and nuclei were visualised with Hoechst (blue). (B). Immunoblots and corresponding quantification of pSTAT3 (Tyr705) in WT and ApcMin/Min organoids with or without IL-22 stimulation. Relative level is expressed as the ratio of pSTAT3 (Tyr705) to total STAT3 in each sample and normalised to the corresponding ratio in WT organoids treated with IL-22 in each experiment. (C) Dose response of pSTAT3 (Tyr705) in organoids treated with 0.4, 2, 10, or 50 ng/ml IL-22 for 0.5 hours. (D). Time course of pSTAT3 (Tyr705) in organoids stimulated with IL-22 (10 ng/ml) for 0.5, 1, or 3 hours. pSTAT3/STAT3 levels in panels C and D were quantified by immunoblotting and normalised to WT + IL-22 at 10 ng/ml IL-22 and at 0.5 hours, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA. Numerical values for (B), (C), and (D) can be found in S1 Data. Apc, adenomatous polyposis coli; IL-22, interleukin-22; pSTAT3, phosphorylated STAT3; STAT3, signal transducer and activator of transcription 3; Tyr705, Tyrosine 705; WT, wild-type.
Fig 4.
ApcMin/Min organoids lose expression of the IL-22 receptor.
(A) Amount of pSTAT3 (Tyr705)/STAT3 in WT and ApcMin/Min organoids stimulated with IL-22 (10 ng/ml) or hyper IL-6 (50 μM) for 0.5 hours, normalised to that in WT treated with IL-22 in each experiment. **p < 0.01, paired t test. (B) pSTAT1 (Tyr701)/STAT1 in WT and ApcMin/Min organoids untreated or stimulated with IL-22 or IFNα (1,000 U/ml) for 0.5 hours, normalised to that in WT cells treated with IFNα in each experiment. (C) RT-qPCR analysis of Il22ra1 expression in WT and ApcMin/Min organoids, and in tissue biopsies (in vivo) from WT intestines or polyps from ApcMin/+ mice. Organoids, n = 6, tissue, n = 3. *p < 0.05, two-tailed t test. (D) WT, ApcMin/+, or ApcMin/Min organoids were dissociated, and IL22RA1 expression was measured by flow cytometry. MFI of IL22RA1 staining from 3 biological replicates is plotted. “2nd only” reflects samples that were stained only with secondary antibody (i.e., not exposed to primary antibodies). **p < 0.01 on one-way ANOVA. (D) WT and ApcMin/Min organoids were fixed and immunostained with IL22RA1 antibodies (green), and nuclei were stained with Hoechst (blue). Numerical values for (A), (B), (C), and (D) are available in S1 Data. Apc, adenomatous polyposis coli; IFN, interferon; IL-22, interleukin-22; IL22RA1, interleukin 22 receptor subunit alpha 1; MFI, mean fluorescence intensity; pSTAT3, phosphorylated STAT3; STAT1/3, signal transducer and activator of transcription 1/3; Tyr705, Tyrosine 705; RT-qPCR, quantitative reverse transcription polymerase chain reaction; WT, wild-type.
Fig 5.
HDAC inhibition partially rescues STAT3-mediated gene transcription in ApcMin/Min organoids.
(A) RT-qPCR analysis of WT and ApcMin/Min organoids treated with IL-22 (10 ng/ml) or hyper IL-6 (50 μM) for 3 hours. Data show mRNA expression of STAT3 target genes, Reg3g or Socs3, relative to Tbp. (B) RT-qPCR analysis for WT and ApcMin/Min organoids treated with IL-22 (10 ng/ml) or IFNα (1,000 U/ml) for 3 hours. Data show the expression of STAT1 target genes, Adar and Usp18 relative to Tbp. At least 3 independent experiments were performed. *p < 0.05, **p < 0.01, paired t test. (C) Western blot analysis of proteins in WT and ApcMin/Min organoids. Expression of HDAC1 and HDAC2 was normalized to loading control, STAT3, in each sample and expressed relative to that in WT organoids. (D) ApcMin/+ mouse small intestine was fixed and stained with antibodies against HDAC1, HDAC2, and β-catenin. Nuclei were visualised with DAPI. ApcMin/Min small intestinal polyps (marked by yellow dotted line) were defined by diffuse and increased β-catenin staining. (E) WT and ApcMin/Min organoids were treated with NaBu (10 mM), TSA (50 nM), or VPA (1 mM) for 16 hours and then stimulated with IL-22 (10 ng/ml) for 3 hours. RT-qPCR was performed. Data show the expression of Reg3g, Reg3b, and Socs3 relative to the IL-22–stimulated control in each genotype. **p < 0.01, ***p < 0.005, Two-way ANOVA, with Sidak’s multiple comparison. At least 4 independent experiments were performed. Numerical values for (A), (B), (C), and (E) are available in S1 Data. Adar, Adenosine deaminase RNA specific; Apc, adenomatous polyposis coli; HDAC, histone deacetylase; IFN, interferon; IL-22, interleukin-22; NaBu, sodium butyrate; ns, not significant; RT-qPCR, quantitative reverse transcription polymerase chain reaction; Reg3g, regenerated islet-derived protein 3 gamma; Reg3b, regenerated islet-derived protein 3 beta; Socs3, Suppressor of cytokine signalling 3; STAT3, signal transducer and activator of transcription 3; TSA, Trichostatin A; Usp18, ubiquitin specific peptidase 18; VPA, valproic acid; WT, wild-type.
Fig 6.
Reduced IL-22 responses in cells in ApcMin/Min small intestinal polyps in vivo.
Mice aged between 88 and 95 days were injected i.p. with 1 μg IL-22 or PBS. (A) Small intestine was harvested after 1 hour and tissue stained with antibodies against pSTAT3 (Tyr705) and β-catenin. Nuclei were stained with DAPI. Asterisk indicate polyps. (B) Small intestine was harvested 24 hours after IL-22 injection. RNA was isolated, and RT-qPCR was performed. Data show the level of Reg3g mRNA relative to Tbp. *P < 0.05, two-way ANOVA. (C) Small intestine was harvested 24 hours after IL-22 injection, and IHC was performed using antibodies against RegIIIγ and β-catenin. Nuclei were stained with DAPI. Three independent experiments were performed. Number of mice used in total: WT = 3; ApcMin/+ = 6. Scale bar = 50 μm. (D) Mean fluorescence intensity for RegIIIγ in the epithelial area analysed using Fuji software. Each dot represents the mean fluorescence intensity in 1 confocal image. *p < 0.05, **p < 0.01, t test. Numerical values for (B) and (D) are available in S1 Data. β-cat, β-catenin; Apc, adenomatous polyposis coli; DAPI, 4',6-diamidine-2'-phenylindole dihydrochloride; IHC, immunohistochemistry; IL-22, interleukin-22; i.p., intraperitoneal; pSTAT3, phosphorylated signal transducer and activator of transcription 3; Reg3g or RegIIIγ, Regenerated islet-derived protein 3 gamma; RT-qPCR, quantitative reverse transcription polymerase chain reaction; Tyr705, Tyrosine 705; WT, wild type.
Fig 7.
IL-22 increases iNOS and DNA damage in ApcMin/+ organoids.
(A) Western blot of iNOS or γH2AX in WT organoids treated with IL-22 (10 ng/ml) for 24 or 48 hours. (B) Western blotting for iNOS or γH2AX in WT or ApcMin/+ organoids treated with IL-22 (10 ng/ml) for 48 hours. TBP was used as loading control. The ratio of γH2AX to TBP in each sample was normalised to untreated control in each experiment. At least 3 independent experiments were performed. *p < 0.05, paired t test. (C) ApcMin/+ mice aged 27–30 days were injected twice a week for 4 weeks with 1 μg IL-22 or PBS or were left untreated. Untreated and PBS-injected mice were pooled together for the “control” cohort (there was no difference in the tumour number in these two cohorts). *p < 0.05, unpaired t test. Numerical values for (A) and (B) are available in S1 Data. γH2AX, Phospho-Histone H2A.X (Serine 139); Apc, adenomatous polyposis coli; IL-22, interleukin-22; iNOS, inducible nitric oxide synthase; SI, small intestine; TBP, TATA box binding protein; WT, wild-type.