Anti-TNF therapy is central for treating ulcerative colitis, yet ∼40% are primary non-responders. To find pre-treatment signatures distinguishing anti-TNF non-responders from responders, we did serum cytokine profiling and laser microdissection transcriptomics on paired uninflamed and inflamed colonic epithelium and lamina propria from patients matched at baseline but differing in endoscopic outcome. Although inflammation drove more epithelial changes, the signatures separating non-responders from responders were found in uninflamed lamina propria, with elevated cell-cycle genes and a co-expression network enriched for humoral immune genes consistently higher expressed in non-responders. Cellular deconvolution localized this network to IgA plasma cells, confirmed by tissue staining, and the association was validated by pseudobulk analysis of an independent single-cell atlas. Baseline epithelial signatures or serum cytokines did not differ significantly between groups. These findings point to TNF-independent humoral activity already present in uninflamed mucosa as a candidate contributor to non-response, with implications for prediction and alternative therapies.

Abstract Ulcerative colitis (UC) is characterized by cytokine-driven inflammation and barrier disruption in the colon, making epithelial dysfunction central to disease pathology. Intestinal epithelial organoids (IEOs) preserve donor-specific genetics and architecture, offering a promising model, but their ability to replicate patient-specific epithelial inflammation remains undetermined. To directly compare in vivo epithelial transcriptional states with defined cytokine-induced responses in vitro, we analyzed patient-matched transcriptomics from laser microdissected inflamed and uninflamed colonic epithelium and IEOs derived from uninflamed biopsies of the same UC patients. IEOs were stimulated with UC-relevant cytokines (TNF, IFNγ, IFNλ1, or TNF + IFNγ) or a cytokine cocktail (TNF, IL17, IL1β, IL22, Poly(I:C), IFNγ). Key inflammatory genes were validated by immunoblotting and immunostaining. Cytokine-stimulated IEOs recapitulated key in vivo epithelial inflammation, including interferon signaling, antigen presentation, and unfolded protein response pathways. Among the tested conditions, TNF + IFNγ combination and the cytokine cocktail most closely replicated UC epithelial inflammation, with concordance for over 350 UC-relevant genes and protein-level validation of IRF1, ERAP2, NOS2, DUOX2 confirmed patient-dependent expression between inflamed epithelium and cytokine-stimulated IEOs. Our study shows that cytokine-stimulated IEOs provide a robust, personalized platform for modeling epithelial inflammation, enabling discovery of epithelial-specific disease mechanisms and therapeutic targets.

[This corrects the article DOI: 10.1371/journal.pone.0265189.].

Restoration of the intestinal epithelial barrier is crucial for achieving mucosal healing, the therapeutic goal for inflammatory bowel disease (IBD). During homeostasis, epithelial renewal is maintained by crypt stem cells and progenitors that cease to divide as they differentiate into mature colonocytes. Inflammation is a major effector of mucosal damage in IBD and has been found to affect epithelial stemness, regeneration and cellular functions. However, the impact of immune cell-modulating IBD drugs on epithelial homeostasis and repair is poorly understood. It is likely that these drugs will have distinct mechanisms of action (MOA) in intestinal epithelium relevant for homeostasis that will vary among patients. We investigated cellular effects of pan-Janus Kinase (JAK) inhibitor tofacitinib and the corticosteroid budesonide on uninflamed and TNF + Poly(I:C) stimulated human colon organoids (colonoids) from healthy donors and IBD-patients. Our findings reveal that although both tofacitinib and budesonide exhibit anti-inflammatory effects, tofacitinib increased colonoid size and proliferation during differentiation, and promoted epithelial stemness. In contrast, budesonide decreased colonoid size and showed no consistent effect on proliferation or stemness. Our study demonstrates the value of employing human colonoids to investigate how IBD drugs affect intestinal epithelial cells and inter-individual variations relevant to mucosal healing and personalized IBD treatment.

Abstract Background ERAP2 is an aminopeptidase involved in antigen processing and presentation, and harbor genetic variants linked to several inflammatory diseases such as Inflammatory Bowel Disease (IBD). The lack of an ERAP2 gene homologue in mice has hampered functional studies, and most human studies have focused on cells of hematopoietic origin. Using an IBD biobank as vantage point, this study explores how genetic variation in ERAP2 affects gene expression in human-derived epithelial organoids upon proinflammatory stimulation. Methods An IBD patient cohort was genotyped with regards to two single nucleotide polymorphisms (SNP) (rs2910686/rs2248374) associated with ERAP2 expression levels, and we examined the correlation between colon gene expression and genotype, specifically aiming to establish a relationship with ERAP2 expression proficiency. Human-derived colon organoids (colonoids) with known ERAP2 genotype were established and used to explore differences in whole genome gene expression between ERAP2-deficient (n = 4) and -proficient (n = 4) donors upon pro-inflammatory encounter. Results When taking rs2910686 genotype into account, ERAP2 gene expression is upregulated in the inflamed colon of IBD patients. Colonoids upregulate ERAP2 upon IFNɣ stimulation, and ERAP2 expression proficiency is dependent on rs2910686 genotype. Colonoid genotyping confirms that mechanisms independent of the frequently studied SNP rs2248374 can cause ERAP2-deficiency. A total of 586 genes involved in various molecular mechanisms are differentially expressed between ERAP2 proficient- and deficient colonoids upon proinflammatory stimulation, including genes encoding proteins with the following molecular function: catalytic activity (AOC1, CPE, ANPEP and MEP1A), regulator activity (TNFSF9, MDK, GDF15, ILR6A, LGALS3 and FLNA), transmembrane transporter activity (SLC40A1 and SLC5A1), and extracellular matrix structural constituents (FGL2, HMCN2, and MUC17). Conclusions ERAP2 is upregulated in the inflamed IBD colon mucosa, and expression proficiency is highly correlated with genotype of rs2910686. While the SNP rs2248374 is commonly used to determine ERAP2 expressional proficiency, our data confirms that mechanisms independent of this SNP can lead to ERAP2 deficiency. Our data demonstrates that epithelial ERAP2 presence affects the inflammatory response in colonoids, suggesting a pleiotropic role of ERAP2 beyond MHC class I antigen processing.

Background The epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to <1% oxygen). We hypothesize that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of colonoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia and compare growth, differentiation, and immunological responses at 2% and 20% oxygen. Methods Growth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data. Results Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks. Conclusions Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.

In recent years it has become apparent that the epithelium is highly involved in inflammatory bowel disease (IBD) pathophysiology. The majority of gene expression studies of IBD are generated from heterogeneous biopsies, providing no distinction between immune cells, the epithelium and other mucosal cells. By using laser capture microdissection (LCM) coupled with RNA sequencing, we aimed to characterize the expressional changes of the isolated colonic epithelial monolayer from ulcerative colitis (UC) and Crohn’s disease (CD) patients compared to healthy controls (HC). The analysis identified 3706 genes as differentially expressed between active IBD epithelium and HC. Weighted gene co-expression network analysis was used to stratify genes into modules, which were subsequently characterized using enrichment analysis. Our data show a distinct upregulation of the antigen presentation machinery during inflammation, including major histocompatibility complex class II molecules (e.g. HLA-DPA1, HLA-DPB1, HLA-DRA) and key transcription factors/activators (STAT1, IRF1, CIITA). We also see an epithelial downregulation of retinoic acid-responsive nuclear receptors (RARA, RARB, RXRA), but upregulation of retinoid-metabolizing enzymes (RDH11, ALDH1A2, ALDH1A3), which together suggest a perturbation of epithelial vitamin A signaling during active IBD. Lastly, we identified a cluster of stress-related genes, including activator protein 1 components JUNB and ATF3, as significantly upregulated in active UC but not in CD, revealing an interesting aspect of IBD heterogeneity. The results represent a unique resource for enhanced understanding of epithelial involvement in IBD inflammation and is a valuable tool for further studies on these processes.

BACKGROUND:Whereas the exact aetiology of microscopic colitis [MC] remains unknown, a dysregulated immune response to luminal factors or medications is the most accepted pathogenesis hypothesis.METHODS:We conducted a systematic review of the pathogenesis of MC. We applied the Joanna Briggs Institute methodologies and the PRISMA statement for the reporting of systematic reviews [PROSPERO Trial Identifier: CRD42020145008]. Populations, Exposure of interest, and Outcome [PEO] questions were used to explore the following topics in MC: 1] intestinal luminal factors; 2] autoimmunity; 3] innate immunity; 4] adaptive immunity; 5] extracellular matrix; 6] genetic risk factors; and 7] mechanism of diarrhoea. A search was done in PubMed, Embase, and Web of Science up to February 2020. A narrative description was performed explaining the findings for each aspect of MC aetiopathogenesis.RESULTS:Thirty-eight documents provided evidence for PEO1, 100 for PEO2, 72 for PEO3 and 4, 38 for PEO5, 20 for PEO6, and 23 for PEO7. The majority of documents were cohorts, case reports, and case series, with a few case-control and some experimental studies. Consistency among data provided by different studies was considered to support pathogenetic hypotheses. MC is a multifactorial disease believed to involve innate and adaptive immune responses to luminal factors, genetic risk, autoimmunity, and extracellular matrix alterations, all contributing by varied mechanisms to watery diarrhoea.CONCLUSIONS:This is the first systematic review on the aetiology of MC supporting the notion that MC is a multifactorial disease. However, high-profile studies are lacking, and most evidence derives from small heterogeneous studies.

Major Histocompatibility Complex (MHC)-I and -II genes are upregulated in intestinal epithelial cells (IECs) during active inflammatory bowel diseases (IBD), but little is known about how IBD-relevant pro-inflammatory signals and IBD drugs can regulate their expression. We have previously shown that the synthetic analog of double-stranded RNA (dsRNA) Polyinosinic:polycytidylic acid (Poly(I:C)), induces interferon stimulated genes (ISGs) in colon organoids (colonoids). These ISGs may be involved in the induction of antigen presentation. In the present study, we applied colonoids derived from non-IBD controls and ulcerative colitis patients to identify induction and effects of IBD-drugs on antigen presentation in IECs in the context of Tumor Necrosis Factor (TNF)-driven inflammation. By RNA sequencing, we show that a combination of TNF and Poly(I:C) strongly induced antigen-presentation gene signatures in colonoids, including expression of MHC-II genes. MHC-I and -II protein expression was confirmed by immunoblotting and immunofluorescence. TNF+Poly(I:C)-dependent upregulation of MHC-II expression was associated with increased expression of Janus Kinases JAK1/2 as well as increased activation of transcription factor Signal transducer and activator of transcription 1 ( STAT1 ). Accordingly, pre-treatment of colonoids with IBD-approved pan-Janus Kinase (JAK) inhibitor Tofacitinib led to the downregulation of TNF+Poly(I:C)-dependent MHC-II expression associated with the abrogation of STAT1 activation. Pre-treatment with corticosteroid Budesonide, commonly used in IBD, did not alter MHC-II expression. Collectively, our results identify a regulatory role for IBD-relevant pro-inflammatory signals on MHC-II expression that is influenced by Tofacitinib.

INTRODUCTION:Microscopic colitis is a chronic inflammatory bowel disease characterised by normal or almost normal endoscopic appearance of the colon, chronic watery, nonbloody diarrhoea and distinct histological abnormalities, which identify three histological subtypes, the collagenous colitis, the lymphocytic colitis and the incomplete microscopic colitis. With ongoing uncertainties and new developments in the clinical management of microscopic colitis, there is a need for evidence-based guidelines to improve the medical care of patients suffering from this disorder.METHODS:Guidelines were developed by members from the European Microscopic Colitis Group and United European Gastroenterology in accordance with the Appraisal of Guidelines for Research and Evaluation II instrument. Following a systematic literature review, the Grading of Recommendations Assessment, Development and Evaluation methodology was used to assess the certainty of the evidence. Statements and recommendations were developed by working groups consisting of gastroenterologists, pathologists and basic scientists, and voted upon using the Delphi method.RESULTS:These guidelines provide information on epidemiology and risk factors of microscopic colitis, as well as evidence-based statements and recommendations on diagnostic criteria and treatment options, including oral budesonide, bile acid binders, immunomodulators and biologics. Recommendations on the clinical management of microscopic colitis are provided based on evidence, expert opinion and best clinical practice.CONCLUSION:These guidelines may support clinicians worldwide to improve the clinical management of patients with microscopic colitis.

BACKGROUND AND AIMS:The pathophysiology of the inflammatory bowel disease collagenous colitis (CC) is poorly described. Our aim was to use RNA sequencing of mucosal samples from patients with active CC, CC in remission, refractory CC, ulcerative colitis (UC), and control subjects to gain insight into CC pathophysiology, identify genetic signatures linked to CC, and uncover potentially druggable disease pathways. METHODS:We performed whole transcriptome sequencing of CC samples from patients before and during treatment with the corticosteroid drug budesonide, CC steroid-refractory patients, UC patients, and healthy control subjects (n = 9-13). Bulk mucosa and laser-captured microdissected intestinal epithelial cell (IEC) gene expression were analyzed by gene set enrichment and gene set variation analyses to identify significant pathways and cells, respectively, altered in CC. Leading genes and cells were validated using reverse-transcription quantitative polymerase chain reaction or immunohistochemistry. RESULTS:We identified an activation of the adaptive immune response to bacteria and viruses in active CC that could be mediated by dendritic cells. Moreover, IECs display hyperproliferation and increased antigen presentation in active CC. Further analysis revealed that genes related to the immune response (DUOX2, PLA2G2A, CXCL9), DNA transcription (CTR9), protein processing (JOSD1, URI1), and ion transport (SLC9A3) remained dysregulated even after budesonide-induced remission. Budesonide-refractory CC patients fail to restore normal gene expression, and displayed a transcriptomic profile close to UC. CONCLUSIONS:Our study confirmed the implication of innate and adaptive immune responses in CC, governed by IECs and dendritic cells, respectively, and identified ongoing epithelial damage. Refractory CC could share pathomechanisms with UC.

Ulcerative colitis is characterized by relapsing and remitting colonic mucosal inflammation. During the early stages of viral infection, innate immune defenses are activated, leading to the rapid release of cytokines and the subsequent initiation of downstream responses including inflammation. Previously, intestinal viruses were thought to be either detrimental or neutral to the host. However, persisting viruses may have a role as resident commensals and confer protective immunity during inflammation. On the other hand, the dysregulation of gut mucosal immune responses to viruses can trigger excessive, pathogenic inflammation. The purpose of this review is to discuss virus-induced innate immune responses that are at play in ulcerative colitis.

Abstract Background Gene expression analyses on IBD tissue are dominated by analyses of biopsy homogenates that are highly heterogeneous due to a difference in cell populations when contrasting inflamed vs. healthy mucosa. We wished to characterise the inflammatory activity of the isolated epithelial monolayer (EM), avoiding the infiltrating inflammatory cells, thereby form a comprehensive understanding of the epithelium’s role in IBD pathobiology. Methods Total RNA was isolated from laser capture microdissected (LCM) colon EM, and sequencing libraries were prepared using TruSeq RNA access kit (Illumina, CA, USA). Gene cluster analysis (WGCNA) was used to stratify the expression data in genetic modules. Following this, we characterised the modules correlated to IBD status, using Metacore pathway analysis, in order to suggest what underlying biological processes contribute to the observed correlation of expression data. Finally, we used immunohistochemistry to verify the protein expression pattern of key players in the suggested pathways, thereby confirming EM involvement in these processes during inflammation. Results Biopsies from active (a) IBD (7 UCa, 5 CDa) and from un-inflamed (u) IBD and healthy control (7 UCu, 5 CDu and 6 HC) were included in the analysis. 6386 genes were differentially expressed (adjusted p-value (pval.) <0.05) in EM from active inflamed colon vs. un-inflamed (Figure 1). Six gene modules showed a significant correlation between inflammatory status and gene expression (Figure 2). Of note was a broad, significant up-regulation of MHC class II presentation machinery, e.g. STAT1 (log2 fold change (lfc) 0.93, p < 0.001), CIITA (lfc 2.57, p < 0.001) and HLA-DRB1 (lfc 3.69, p < 0.001), and a down-regulation unique to epithelial cells of the retinoic acid receptors RARA (lfc −0.79, p < 0.001) and RXRA (log fc −0.56, p < 0.001). The most significantly up-regulated pathway was ‘Immune Response’, induction of antigen presentation machinery by IFN-γ (FDR 2.5 × 10–18), while ‘RXR-dependent regulation of lipid metabolism’ (FDR 2.6 × 10–8) was the most significantly down-regulated pathway. Conclusion Our analyses suggest that the involvement of the EM in IBD inflammation goes far beyond acting as a barrier. The largest genetic modules are dominated by genes important for inflammatory regulation, suggesting that the EM is much more involved in these processes than what is commonly understood. This study lends support to further analyses of interplay between epithelial cells and immune cells, particularly in a pro-inflammatory environment.

Abstract Background Collagenous colitis (CC) is a common inflammatory bowel disease that exhibits chronic watery diarrhoea. Treatment with the glucocorticoid budesonide is effective to induce and maintain clinical remission in most cases; however, few patients do not respond to treatment. To distinguish between different CC subgroups on a molecular level, we performed genome-wide RNA sequencing (RNAseq) analysis on mucosal samples. Methods We collected colonic biopsies from healthy controls (Hc) and CC patients with active disease. Additionally, we obtained matched samples from budesonide-responsive patients (clinical remission) under treatment with budesonide (9 mg/day, 8 weeks), and biopsies from budesonide-refractory patients (n = 9 patients/group). Ulcerative colitis (UC) samples were included as a separate control. Total RNA was isolated for RNAseq. Whole-genome expression data were analysed by principal component analysis (PCA) and differential gene expression (DGE) using R software. The Benjamini–Hochberg FDR method adjusted p values, considering <0.05 as statistically significant. Gene-set enrichment analysis (GSEA) was performed using GSEA software and visualised in Cytoscape. Results were validated in tissue samples from the same patients by immunohistochemistry (IHC). Results PCA of all samples identified three principal components which separated sample groups into distinct clusters of gene expression, explaining 31% of the transcriptional variation. PCA clearly demarcated UC samples from Hc and CC. Mucosal samples from budesonide-refractory patients exhibited a discrete RNA expression profile that was distinct from all other groups. Moreover, PCA of budesonide-responsive persons also separated active from treated CC. Subsequent DGE analysis revealed differential expression of 395 genes in patients with active CC compared with Hc, and that these genes were mainly involved in immune response, cell cycle and apoptosis, according to GSEA. Using IHC, we confirmed the impaired proliferation and immune response on the protein level. In samples from budesonide-treated patients, 75 genes were differentially regulated compared with controls. A paired comparison of matched samples from CC patients before and after induction of remission showed an expression change of 337 genes, mainly involved in DNA recognition. Conclusion CC is a transcriptionally homogeneous disease that can be characterised by differential gene expression. Unique gene expression profiles describe patient sub-populations, which could define budesonide-refractory CC as a distinct disease entity. Further study of the transcriptional landscape of CC may reveal pathogenic mechanisms and therapeutic targets for this common, debilitating inflammatory bowel disease.

Abstract Background The nuclear receptor peroxisome proliferation-activated receptor γ (PPARγ) harbours anti-inflammatory effects. There is evidence that PPARγ mediates the effect of 5-aminosalicylic acid (5-ASA). 5-ASA is the first-line drug in ulcerative colitis (UC, while its use in Crohn`s disease (CD) is debated and not recommended according to guidelines. We hypothesise that the inconsistent therapeutic effect of 5-ASA in Crohn’s disease is caused by different expression of PPARγ between the large and small intestine. Methods Levels of PPARγ mRNA were measured by RNASeq in mucosal biopsies from (1) active/inactive ileal CD (n = 14/17) and healthy controls (n = 9) and (2) colonic biopsies from patients with active/inactive UC (n = 24/24) and active/inactive CD (n = 24/21) and healthy controls (n = 20). Subsets of ileal and colonic biopsies were examined by western blot, immunohistochemistry (IHC) and in situ hybridisation (ISH). The effects of 5-ASA on PPARγ expression and TNF/IL17/polyI:C induced cytokine release were examined in the colonic cell-line HT29 and primary human IECs (colonoids) using RNASeq and ELISA in. Results PPARγ mRNA was strongly downregulated in colonic biopsies from inflamed mucosa of UC (log2 = −1.65, fold change 0.319 (p < 0.001)) and CD (log2 = −1.3, fold change 0.406 (p < 0.001)) compared with healthy controls. In ileal biopsies from CD and controls, PPARγ mRNA was not differentially expressed between inflamed and non-inflamed or healthy mucosa. These findings were confirmed by ISH in a subset of biopsies. Western blot analysis and IHC revealed almost undetectable levels of PPARγ protein in ileum, in the colon however, PPARγ was strongly expressed in healthy controls and inactive IBD; with significantly lower levels in active IBD. PPARγ was downregulated by TNF, TNF+IL17 and TNF + poly(I:C) in colonoids derived from IBD-patients (n = 3) and non-IBD controls (n = 3). 5-ASA attenuated the release of TNF-induced proinflammatory cytokines such as CXCL1, CXCL8 and CXCL10 from both HT29 cells and colonoids. This effect was reversed by the PPARγ antagonist GW9662. Conclusion 5-ASA harbours anti-inflammatory effects on intestinal epithelial cells mediated by epithelial PPARγ. We suggest that the previously observed lack of effect of 5-ASA in CD is related to differences in PPARγ expression in small intestine vs. colon. These results suggest that patients with Crohn’s colitis may benefit from 5-ASA similarly to UC patients and challenge the current view on use of 5-ASA in CD.

Abstract Background Neutrophil gelatinase-associated lipocalin (NGAL) is upregulated in the intestinal epithelium in inflammatory bowel disease and is a biomarker with sensitivity and specificity comparable to calprotectin. Microscopic colitis (MC) is a common cause of chronic, watery diarrhoea and represents an inflammatory bowel disease with unknown aetiology and pathogenesis. Diagnosis depends on histological evaluation of colonic biopsies. There is a need for non-invasive diagnostic tools, and this study examines the potential of NGAL as a biomarker in collagenous colitis (CC) as one of the two main histological forms of MC. Methods Gene expression of colonic biopsies (n = 9/group) from active CC, budesonide treated CC in clinical remission and healthy controls (HC) was explored by RNA-sequencing analysis. An extended set of colonic biopsies (n = 17–29/group) was also examined by immunohistochemistry (IHC) and in situ hybridisation (ISH). Faecal samples from the same patient groups, in addition to samples from patients with irritable bowel disease diarrhoea (IBS-D), were assayed for NGAL and calprotectin (Calpro) by ELISA. Results The NGAL gene, LCN2, was significantly upregulated in active CC vs. HC (log2 2.694, fold change 6.471) adjusted p-value 0.005) and in pairs of active CC vs. treated CC LCN2 was significantly downregulated (log2 0.345, fold change −1.536), adjusted p-value 0.04). Both immunohistochemical staining and in situ hybridisation identified increased NGAL expression localised to the mucosal epithelial cells in active CC, compared with an almost absent and scarce expression in HC and treated CC, respectively. There were great individual differences in faecal concentrations of NGAL particularly in the active CC group, but the NGAL concentrations were significantly increased compared with HC, IBS-D and treated CC. Conclusion NGAL is upregulated and located mainly to the colonic epithelium of active CC and reduced in clinical remission after budesonide treatment. This is also reflected in the faecal concentrations. We propose NGAL as a valuable biomarker in evaluating the inflammatory activity related to CC and a potential faecal biomarker discriminating CC from IBS-D.

BACKGROUND AND AIMS:Intestinal epithelial cells [IECs] secrete cytokines that recruit immune cells to the mucosa and regulate immune responses that drive inflammation in inflammatory bowel disease [IBD]. However, experiments in patient-derived IEC models are still scarce. Here, we aimed to investigate how innate immunity and IEC-specific pattern recognition receptor [PRR] signalling can be involved in an enhanced type I interferon [IFN] gene signature observed in colon epithelium of patients with active IBD, with a special focus on secreted ubiquitin-like protein ISG15.METHODS:Gene and protein expression in whole mucosa biopsies and in microdissected human colonic epithelial lining, in HT29 human intestinal epithelial cells and primary 3D colonoids treated with PRR-ligands and cytokines, were detected by transcriptomics, in situ hybridisation, immunohistochemistry, western blots, and enzyme-linked immunosorbent assay [ELISA]. Effects of IEC-secreted cytokines were examined in human peripheral blood mononuclear cells [PBMCs] by multiplex chemokine profiling and ELISA.RESULTS:The type I IFN gene signature in human mucosal biopsies was mimicked in Toll-like receptor TLR3 and to some extent tumour necrosis factor [TNF]-treated human IECs. In intestinal biopsies, ISG15 expression correlated with expression of the newly identified receptor for extracellular ISG15, LFA-1 integrin. ISG15 was expressed and secreted from HT29 cells and primary 3D colonoids through both JAK1-pSTAT-IRF9-dependent and independent pathways. In experiments using PBMCs, we show that ISG15 releases IBD-relevant proinflammatory cytokines such as CXCL1, CXCL5, CXCL8, CCL20, IL1, IL6, TNF, and IFNγ.CONCLUSIONS:ISG15 is secreted from primary IECs upon extracellular stimulation, and mucosal ISG15 emerges as an intriguing candidate for immunotherapy in IBD.

BACKGROUND AND AIMS:Diarrhoea is a common, debilitating symptom of gastrointestinal disorders. Pathomechanisms probably involve defects in trans-epithelial water transport, but the role of aquaporin [AQP] family water channels in diarrhoea-predominant diseases is unknown. We investigated the involvement of AQPs in the pathobiology of collagenous colitis [CC], which features chronic, watery diarrhoea despite overtly normal intestinal epithelial cells [IECs].METHODS:We assessed the expression of all AQP family members in mucosal samples of CC patients before and during treatment with the corticosteroid drug budesonide, steroid-refractory CC patients and healthy controls. Samples were analysed by genome-wide mRNA sequencing [RNA-seq] and quantitative real-time PCR [qPCR]. In some patients, we performed tissue microdissection followed by RNA-seq to explore the IEC-specific CC transcriptome. We determined changes in the protein levels of the lead candidates in IEC by confocal microscopy. Finally, we investigated the regulation of AQP expression by corticosteroids in model cell lines.RESULTS:Using qPCR and RNA-seq, we identified loss of AQP8 expression as a hallmark of active CC, which was reverted by budesonide treatment in steroid-responsive but not refractory patients. Consistently, decreased AQP8 mRNA and protein levels were observed in IECs of patients with active CC, and steroid drugs increased AQP8 expression in model IECs. Moreover, low APQ8 expression was strongly associated with higher stool frequency in CC patients.CONCLUSION:Down-regulation of epithelial AQP8 may impair water resorption in active CC, resulting in watery diarrhoea. Our results suggest that AQP8 is a potential drug target for the treatment of diarrhoeal disorders.

Neutrophil gelatinase-associated lipocalin (NGAL), also known as Lipocalin 2, is an antimicrobial protein, encoded by the gene LCN2, strongly upregulated in inflammatory bowel disease (IBD) and a promising biomarker for IBD. Here we demonstrate that NGAL is highly expressed in all parts of pyloric metaplasia, also known as the ulcer-associated cell lineage (UACL), a metaplastic cell lineage suggested to play a role in wound healing in Crohn's disease (CD). We further show NGAL expression in regenerative intestinal crypts and in undifferentiated patient-derived colonoids. This indicates that NGAL is important in the tissue regeneration process. The remarkable overexpression of NGAL in UACL led us to explore the pathobiology of these cells by transcriptome-wide RNA sequencing. This study is, to our knowledge, the first to characterize the UACL at this level. Biopsies with UACL and inflamed non-UACL epithelium from the terminal ileum of CD patients and epithelium from healthy controls were laser capture microdissected for RNA sequencing. Among the 180 genes differentially expressed between UACL and control epithelium, the ten most-upregulated genes specific for UACL were MUC5AC, PGC, MUC6, MUC5B, LCN2, POU2AF1, MUC1, SDC3, IGFBP5, and SLC7A5. PDX1 was among the most upregulated in both UACL and inflamed non-UACL epithelium. Immunohistochemistry and iDisco 3D visualization was used to characterize UACL histo-morphologically, and to validate protein expression of 11 selected differentially expressed genes. Among these genes, LCN2, NOTCH2, PHLDA1, IGFBP5, SDC3, BPIFB1, and RCN1 have previously not been linked to UACL. Gene expression results were analyzed for functional implications using MetaCore, showing that differentially expressed genes are enriched for genes involved in cell migration and motility, and for biomarkers of gastrointestinal neoplasia. These results support a role for UACL as part of the reepithelialization process during and after destructive intestinal inflammation. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.