Interleukin-22 orchestrates a pathological endoplasmic reticulum stress response transcriptional programme in colonic epithelial cells

Abstract

Objective: The functional role of interleukin-22 (IL22) in chronic inflammation is controversial, and mechanistic insights into how it regulates target tissue are lacking. In this study, we evaluated the functional role of IL22 in chronic colitis and probed mechanisms of IL22-mediated regulation of colonic epithelial cells.

Design: To investigate the functional role of IL22 in chronic colitis and how it regulates colonic epithelial cells, we employed a three-dimentional mini-gut epithelial organoid system, in vivo disease models and transcriptomic datasets in human IBD.

Results: As well as inducing transcriptional modules implicated in antimicrobial responses, IL22 also coordinated an endoplasmic reticulum (ER) stress response transcriptional programme in colonic epithelial cells. In the colon of patients with active colonic Crohn's disease (CD), there was enrichment of IL22-responsive transcriptional modules and ER stress response modules. Strikingly, in an IL22-dependent model of chronic colitis, targeting IL22 alleviated colonic epithelial ER stress and attenuated colitis. Pharmacological modulation of the ER stress response similarly impacted the severity of colitis. In patients with colonic CD, antibody blockade of IL12p40, which simultaneously blocks IL12 and IL23, the key upstream regulator of IL22 production, alleviated the colonic epithelial ER stress response.

Conclusions: Our data challenge perceptions of IL22 as a predominantly beneficial cytokine in IBD and provide novel insights into the molecular mechanisms of IL22-mediated pathogenicity in chronic colitis. Targeting IL22-regulated pathways and alleviating colonic epithelial ER stress may represent promising therapeutic strategies in patients with colitis.

Trial registration number: NCT02749630.

Keywords: ER stress; Interleukin 22; inflammatory bowel disease.

Figure 1

IL22 induces an ER stress/unfolded protein response transcriptional module in colonic epithelial cells. (A) Heat map demonstrating pathway specific transcript expression in murine colonoids treated with (+IL22, n=3) or without (control, n=3) recombinant IL22. Mouse gene 2.0 ST array platform (affymetrix). (B) GSEA evaluating enrichment of ER stress response transcriptional module in IL22 treated colonoids. A core set of colonic epithelial-specific ER stress genes was defined by analysing significantly differentially expressed (p<0.05 and absolute value of the log2 fold change >±2) transcripts in colonoids treated with tunicamycin (n=3) or medium alone (n=3). (C) Expression of ER stress response transcripts in IL22 treated WT and Il22ra1^−/− colonoids (RNA-seq dataset ERR247358-ERR247389, Pham et al, 2014). (D) Enrichment analysis for ER stress-related functional annotation groups (GO biological processes) in IL22-treated colonoids from dataset ERR247358-ERR247389. (E) Microarray analysis of core ER stress response transcripts in colonoids treated with tunicamycin (n=3), tunicamycin+IL22 (n=3) or untreated (control, n=3). (F) Real-time PCR quantification of ER stress transcripts in colonoids treated with IL22 (n=11), IL17A (n=6) and IL22+IL17A (n=6) and unexposed controls. *P<0.01. (G) Immunoblot and densitometry quantification (H) detecting GRP78 protein expression in colonoids treated with different cytokines. *P<0.026, one tailed t test. ER, endoplasmic reticulum; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis; IL22, interleukin-22.

Figure 2

IL22 and IL17A promote ER stress and intestinal epithelial apoptosis. (A) Transcript expression in IL22-treated colonoids from our dataset^a and Pham et al ERR247358-ERR247389^b. (B) Panther analysis of pathways activated in IL22-treated colonoids. (C) MTT assay demonstrating colonic epithelial cell viability after treatment with IL22, IL22+IL17A, or tunicamycin, versus untreated colonoids. *P<0.02, **P<0.0001. (D) In vivo model of intestinal epithelial apoptosis showing representative immunohistochemistry (caspase 3 immunoreactivity) and statistical analyses (E and F) of intestinal sections harvested at different time points following pretreatment with either IL22, IL22+IL17A, or PBS, prior to administration of TNFα. *P<0.005. IL22, interleukin-22.

Figure 3

IL22/ER stress axis is functionally important in chronic colitis. (A) Volcano plot (fold change vs p value) showing gene expression in the colon of Rag2^−/− (n=3) and TRUC (n=3) mice, microarray analysis (MouseWG-6 v2.0 expression BeadChip, Illumina). Transcripts annotated in red are among the top 20 most highly upregulated genes in IL22-treated colonoids. (B) Representative immunohistochemistry (IRE1α immunoreactivity) in distal colon of villin-cre Atg16l1^fl/fl mice and TRUC mice. (C) Western blot of distal colon segments from TRUC and Rag2^−/− mice probed with anti-GRP78. Corresponding densitometry plots are shown in online supplementary figure 7. (D) Representative histology (H&E) and histology scores of the distal colon of TRUC mice administered 4-PBA in drinking water (n=8) or water alone (n=8). P<0.025. (E) Colon micrograph (H&E stain), and colitis score of distal colon of TRUC mice treated with anti-IL22 mAb (n=6) or control antibody (n=10). P<0.001. (F) Colitis score of distal colon of TRUC (n=16) and TRUC Il22^−/− (n=10) mice. P<0.0001. (G) Western blot of distal colon segments from TRUC and TRUC Il22^−/− mice probed with anti-GRP78. Corresponding densitometry plots are shown in online supplementary figure 7. (H) Real-time PCR quantifying ER stress transcripts in the distal colon of TRUC mice (n=9), TRUC Il22^−/− mice (n=5) and TRUC mice treated with anti-IL22 (n=6). *P<0.05, **P<0.02, ***P<0.001. 4-PBA, 4-phenylbutryic acid; IL22, interleukin-22.

Figure 4

Local induction of ER stress reinstates colitis in TRUC Il22^−/− mice. (A) Histological appearance (H&E stain) and colitis score of the distal colon, and (B) colon mass of TRUC mice treated with intrarectal tunicamycin (n=8) or vehicle control (n=9). *P<0.01. Bar charts depict mean and SEM in graphs showing dots, each dot represents an individual mouse.

Figure 5

Increased expression of IL22 and IL22-regulated transcriptional modules in active colitis. (A) Serum IL22 concentration in healthy control (HC; n=29) and patients with CD from UNITI1 (n=191) and UNITI2 (n=205) trial programmes. *P<0.0005, **p<0.0001). (B) IL22 responsive transcript GSVA enrichment scores of the 20 mostly highly upregulated IL22 responsive transcripts in patients with CD (n=162) from the UNITI trial programme (at baseline prior to randomisation to placebo or ustekinumab) or HC subjects (n=23). Each dot represents an individual patient. Line depicts median. Gene expression data for the UNITI cohort were quantified using the Affymetrix Hg U133 PM array. *P<0.0003. (C) GSVA showing IL22 responsive transcript enrichment scores in colonic biopsies from patients with active (Mayo endoscopy subscore 2–3) or quiescent (Mayo endoscopy subscore 0–1) UC, CD or HCs (gene expression microarray datasets GSE50971 and GSE16879). (C–E) Correlation between IL22 enrichment score (GSVA) in rectal biopsies of patients with CD, with faecal calprotectin (C), faecal lactoferrin (D) and (E) regional SES-CD (ie, endoscopic severity of mucosal disease at same site mucosal biopsy was sampled from). Displayed is baseline rectum gene expression from UNITI-1 subpopulation with colon involvement. Faecal calprotectin and lactoferrin were log 2 transformed data. (F) PCA of baseline rectum gene expression of IL22 responsive genes in UNITI-1 segregates CD patients with and without baseline ulceration. (G) A multivariant model using IL22-responsive transcripts to predict endoscopic activity by SES-CD in UNITI cohort, with correlation of predicted and actual SES-CD score in the training set using baseline rectal biopsies in UNITI-1. (H) Coefficients of determination of the training and testing datasets in UNITI-1 and genes used in the predictive model. *Predicted SES-CD at left colon; ˆGenes sorted by selection order. bL, baseline; CD, Crohn’s disease; GSVA, Gene Set Variation Analysis; IL22, interleukin-22; R², coefficient of determination; SES-CD, Simple Endoscopic Score – Crohn’s Disease; Wk8, week 8.

Figure 6

Colonic epithelial cell-specific ER stress response transcriptional module is enriched in IBD patients with active colitis. (A) Diagrammatic representation of how our colonic epithelial-specific ER stress response transcriptional signature was derived. (B) ER stress response GSVA enrichment scores in colonic biopsies from CD patients from the UNITI trial programme (at baseline prior to randomisation to placebo or ustekinumab) or healthy control subjects. Each dot represents an individual patient. line depicts median. *P<0.0001 (CD vs control). (C) ER stress response GSVA enrichment scores in colonic biopsies from UC, CD and healthy control (HC) patients from an independent dataset (GSE16879). *p<0.0005, **p<0.0001. (D) heat MAP depicting transcript level changes using unsupervised hierarchical clustering of the ER stress transcriptional module in patients with colonic CD and healthy, non-inflammatory control subjects (GEO59071). (E) Correlation between ER stress GSVA enrichment score and faecal lactoferrin concentration (log2) transformed data and (F) regional SES-CD. Displayed is baseline rectum gene expression from UNITI-1 subpopulation with colonic involvement. (G) Correlation between IL22 GSVA enrichment score and epithelial cell-specific ER stress GSVA enrichment score. CD, Crohn’s disease; DEGs, differentially expressed genes; ER, endoplasmic reticulum; GO, Gene Ontology; GSVA, Gene Set Variation Analysis; IL22, interleukin-22; SES-CD, Simple Endoscopic Score – Crohn’s Disease.

Figure 7

Blockade of the IL12/IL23 axis with ustekinumab alleviates colonic ER stress in CD patients with active colitis. (A) quantification of transcripts encoding XBP1 and GRP78 (log2 transformed expression intensity) in colonic biopsies of patients with CD in UNITI-1 randomised maintenance population post-treatment with maintenance placebo or maintenance ustekinumab (pooled data of ustekinumab 90 mg SC every 12 weeks and ustekinumab 90 mg SC every 8 weeks at week 44 comparing to week 0. (B) GSVA enrichment scores of ER stress signatures in the UNITI-2 pooled (randomised and non-randomised) maintenance population who received placebo or ustekinumab 90 mg every 8 weeks maintenance therapy until week 44 comparing with week 0). Gene expression data for the UNITI cohort were quantified using the Affymetrix Hg U133 PM array. CD, Crohn’s disease; ER, endoplasmic reticulum; IL22, interleukin-22; GSVA, Gene Set Variation Analysis.