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.

This study assessed the seroprevalence of anti-Anisakis simplex antibodies in Norwegian patients with inflammatory bowel disease (IBD), specifically ulcerative colitis (UC) and Crohn’s disease (CD), compared with healthy controls. Associations between anti-A. simplex antibody positivity and clinical or laboratory parameters in IBD were also explored. A total of 86 UC patients, 68 CD patients, and 41 healthy controls were prospectively enrolled from four Norwegian hospitals (2013–2022). Diagnosis and disease activity were established using standard clinical, endoscopic, and biomarker criteria. Serum samples were analyzed for total Ig, IgG, IgM, IgA, and IgE antibodies against A. simplex and Pseudoterranova decipiens using ELISA. Anti-A. simplex IgG seroprevalence was 4.9% in controls and 3.2% in IBD (3.5% UC, 2.9% CD). IgM seroprevalence was 0% in all groups. IgA seroprevalence was higher in IBD (16.2%) than controls (4.9%), with 14.0% in UC and 19.1% in CD. IgE seroprevalence was low across all groups. Smoking correlated with lower antibody levels and higher surgery rates. In UC, higher anti-A. simplex IgG and IgE levels were associated with milder disease and better prognosis. Anti-TNFα and azathioprine treatments were linked to higher anti-A. simplex IgA. Norwegian UC and CD patients had significantly higher anti-A. simplex total Ig and IgA seroprevalence than healthy controls, indicating increased exposure or immune response. Anti-A. simplex IgG and IgE may serve as markers of clinical activity in UC. Further research is warranted to clarify the clinical significance of these findings.

BACKGROUND:There is growing evidence of the role of the mycobiome in inflammatory bowel disease (IBD). Variations within phenotypes and activity and with prognosis have been poorly studied. METHODS:A total of 111 individuals were prospectively enrolled: 89 IBD patients (52 ulcerative colitis and 37 Crohn's disease [CD]) and 22 healthy individuals. Disease characteristics were collected and a fecal calprotectin >100 μg/mg was considered indicative of activity. A subset of patients was followed for 6 ± 2 years. Disease course was designated as either complicated or uncomplicated based on the need of intensified medication and/or surgery. ITS sequencing was performed targeting the ITS1 region. RESULTS:We found lower Ascomycota/Basidiomycota ratio in IBD. Patients showed a marked increase in Candida dublinensis and Ca albicans and were depleted of Aspergillus rubrobrunneus and Penicillium brevicompactum (P ≤ .001) Saccharomyces was predominant in total colitis and Penicillium in proctitis. Several Penicillium species were depleted in total colitis vs proctitis. Ileal CD patients were enriched in Debaromyces hansenii and depleted of Ca tropicalis (P ≤ .001). Ca albicans was overrepresented in inflammatory (B1) vs fibrostenosing (B2) CD. Ca dublinensis was more abundant in active patients and correlated positively with fecal calprotectin and neutrophil gelatinase-associated lipocalin, while S pastorianus correlated inversely with activity. Ca sake was associated with complicated disease and increased abundance of Cryptococcus carnescens with the need for surgery in CD. CONCLUSIONS:This study shows important differences in the mycobiome in IBD and within phenotypes. Selected fungal species were associated with complicated disease and the need of surgery in CD. This work adds to our understanding of the role of fungi in IBD, with potential clinical implications.

BackgroundThe incidence of inflammatory bowel disease (IBD) is increasing. The prevalence of overweight and obesity is increasing in parallel with IBD and could contribute to IBD development. The aim of this study was to assess the relationship between weight change and the risk for IBD.MethodsData gathered from 55,896 adult participants in the three first population-based Tr & oslash;ndelag Health Studies (HUNT1-3), Norway, performed in 1984-2008 was used. The exposure was change in body mass index between two HUNT studies. The outcome was a new IBD diagnosis recorded during a ten-year follow-up period after the exposure assessment. The risk of IBD by weight change was assessed by Cox regression analyses reporting hazard ratios (HRs) and 95% confidence intervals (CIs), adjusted for sex, age, and smoking status.ResultsThere were 334 new cases of ulcerative colitis (UC) and 54 of Crohn's disease (CD). Weight loss decreased the risk of a new UC diagnosis by 38% (adjusted HR 0.62, 95% CI 0.39-0.97) and seemed to double the risk of getting a new CD diagnosis (adjusted HR 2.01, 95% CI 0.91-4.46). Weight gain was not associated with a new diagnosis of neither UC (adjusted HR 1.00, 95% CI 0.78-1.26) nor CD (adjusted HR 1.08, 95% CI 0.56-2.08).ConclusionIn this study, weight loss was associated with decreased risk of UC. However, no associations were seen between weight gain and the risk of UC or CD, suggesting that the increasing weight in the general population cannot explain the increasing incidence of IBD.

Abstract Background Extraintestinal manifestations (EIM) are common in IBD and they affect importantly the quality of life of patients. Although the pathogenesis of EIM is still poorly understood, an altered immune response involving T cells has been proposed. In addition, the presence of EIM during disease course will impact the therapeutic choices with new biologics. In the present work we aim to characterize T cell populations -with focus on Uncoventional T cells- in IBD patients presenting EIM using advance flow cytometry and proteomics. Methods A total of 184 individuals (106 F/78 M) and were prospectively included: 149 IBD patients (84 UC/65 CD) and 25 healthy matched controls. Biologic samples including Peripheral Blood Mononuclear Cells (PBMC) and plasma were consecutively stored and classified in an established cross sectional IBD biobank at the Norwegian University of Science and Technology. Patient characteristics including age, sex, smoking status, current medication, Montreal Classification phenotype and inflammation biomarkers (CRP) and fecal Calprotectin were recorded. PBMC were stained with antibodies and acquired using BD FACS Symphony flow cytometer using automatic compensations and Multiplex ELISA for 65 proteins was performed using Immune Monitoring 65-Plex Human ProcartaPlex™ Panel at Singapore Immunology Network (SIgN), as previously described. Statistical analysis was performed using GraphPad Prism (San Diego, CA). p≤ 0.05 was considered statistically significant. Results Of the 184 IBD patients included, the presence or not of EIM was registered in 149 (mean age 37 +/-7.2; 86 female) of which 84 had UC and 65 CD. Of them, 21 (14.1%) presented EIM at inclusion. We analyzed the differences in all subsets of unconventional T cells: gammadelta T cells (VD1, VD2, VD3), MAIT cells, innate NKT cells and CD4/CD8 positive T cells between IBD patients with or without EIM (Fig1A). MAIT cells were selectively depleted in patients with EIM (p<0.01), while there was no statistically significant change in the ratios of other T cell subpopulations. We then studied the concentrations of plasma proteins between the two groups. We found a significant increase in IL-2, GM-CSF, IL-6, Eotaxin, Leukemia Inhibitory Factor Protein (LIF), TNF-RII and Monocyte Chemoattractant Protein-1 (MCP), all p<0.05 in patients with EIM (Fig1B). In order to address the potential use of MAIT as marker of EIM we constructed a ROC curve showing an AUC 0.752 (95% CI 0.639-0.865, p= 0.0002) Fig. 1C Conclusion MAIT cell depletion associated strongly with the presence of EIM and could be used as a clinical marker for EIM in IBD. A deeper understanding of its role in the pathogenesis of EIM, as well as an external validation as a marker, deserves further investigation.

There are many unanswered questions regarding responses to proinflammatory signals in intestinal epithelial cells (IECs). For example, chemokines secreted by IECs upon external stimuli play multifunctional roles in both homeostasis and during inflammation. Several chemokines are upregulated during active inflammatory bowel disease (IBD), which is associated with an increased influx of immune cells into the gut mucosa. Therefore, studies on how chemokines are regulated in the intestinal epithelium may identify putative treatment targets in IBD. More recently, patient-derived ex vivo models such as intestinal organoids have facilitated molecular analysis of epithelial alterations in IBD patients own cells. Here, we describe refined experimental protocols and methods for the generation and maintenance of IBD patient-derived colonic organoids (colonoids) culture. We also give detailed description of medium, and supplements needed for colonoid establishment, growth, and differentiation, including production of Wnt-3A and Rspondin1 enriched media. Further, we present protocols for RNA and protein isolation from human colonoids, and subsequent gene expression analysis and Western blotting for e.g., signal transduction studies. We also describe how to process colonoids for chemokine protein expression analysis such as immunostaining, confocal imaging, and detection of secreted chemokines by e.g., enzyme-linked immunosorbent assay (ELISA). As proof of principle, we give examples of how the chemoattractant CCL20 can be regulated and expressed in colonoids derived from IBD-patients and healthy controls upon ligands-driven inflammation.

Abstract Colon mucosae of ulcerative colitis (UC) and Crohn's disease (CD) display differences in the number and distribution of immune cells that are difficult to assess by eye. Deep learning‐based analysis on whole slide images (WSIs) allows extraction of complex quantitative data that can be used to uncover different inflammatory patterns. We aimed to explore the distribution of CD3 and γδ T cells in colon mucosal compartments in histologically inactive and active inflammatory bowel disease. By deep learning‐based segmentation and cell detection on WSIs from a well‐defined cohort of CD (n = 37), UC (n = 58), and healthy controls (HCs, n = 33), we quantified CD3 and γδ T cells within and beneath the epithelium and in lamina propria in proximal and distal colon mucosa, defined by the Nancy histological index. We found that inactive CD had significantly fewer intraepithelial γδ T cells than inactive UC, but higher total number of CD3 cells in all compartments than UC and HCs. Disease activity was associated with a massive loss of intraepithelial γδ T cells in UC, but not in CD. The total intraepithelial number of CD3 cells remained constant regardless of disease activity in both CD and UC. There were more mucosal CD3 and γδ T cells in proximal versus distal colon. Oral corticosteroids had an impact on γδ T cell numbers, while age, gender, and disease duration did not. Relative abundance of γδ T cells in mucosa and blood did not correlate. This study reveals significant differences in the total number of CD3 and γδ T cells in particularly the epithelial area between CD, UC, and HCs, and demonstrates useful application of deep segmentation to quantify cells in mucosal compartments.

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.

IntroductionFungal microbiota's involvement in the pathogenesis of Crohn's disease (CD) is incompletely understood. The terminal ileum is a predilection site both for primary involvement and recurrences of CD. We, therefore, assessed the mucosa-associated mycobiota in the inflamed and non-inflamed ileum in patients with CD. MethodsThe mucosa-associated mycobiota was assessed by ITS2 sequencing in a total of 168 biopsies sampled 5 and 15 cm proximal of the ileocecal valve or ileocolic anastomosis in 44 CD patients and 40 healthy controls (HC). CD patients with terminal ileitis, with endoscopic inflammation at 5 cm and normal mucosa at 15 cm and no history of upper CD involvement, were analyzed separately. The need for additional CD treatment the year following biopsy collection was recorded. ResultsCD patients had reduced mycobiota evenness, increased Basidiomycota/Ascomycota ratio, and reduced abundance of Chytridiomycota compared to HC. The mycobiota of CD patients were characterized by an expansion of Malassezia and a depletion of Saccharomyces, along with increased abundances of Candida albicans and Malassezia restricta. Malassezia was associated with the need for treatment escalation during follow-up. Current anti-TNF treatment was associated with lower abundances of Basidiomycota. The alpha diversity of the inflamed and proximal non-inflamed mucosa within the same patients was similar. However, the inflamed mucosa had a more dysbiotic composition with increased abundances of Candida sake and reduced abundances of Exophiala equina and Debaryomyces hansenii. ConclusionsThe ileal mucosa-associated mycobiota in CD patients is altered compared to HC. The mycobiota in the inflamed and proximal non-inflamed ileum within the same patients harbor structural differences which may play a role in the CD pathogenesis. Increased abundance of Malassezia was associated with an unfavorable disease course.

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.

Abstract Background There is growing evidence of the role of the fungal microbiota in the pathogenesis of IBD, but a definite pattern defining the mycobiome in IBD is currently lacking. In addition, the variations within the different phenotypes as well as the correlation between fungal composition and disease outcomes has been poorly characterized. Our aim is to evaluate the fungal composition within the different IBD phenotypes, the changes with disease activity and the association with disease outcomes. Methods This prospective study presents sequencing of the faecal fungi of 111 individuals: 89 IBD (52 UC/37 CD) and 22 HC. Demographics,medication,Montreal Classification and disease history was collected. A fCalpro>100 was considered active disease. A subset of IBD patients was followed clinically (6±2 y) and classified as either “Complicated” or “Uncomplicated” based on the need of intensified medication and/or surgery. Sequencing was done on amplicons targeting the ITS1 region of fungal DNA on a Illumina MiSeq sequencer. Chosen OTUs were compiled taxonomically.Data analysis was performed in R(phyloseq and deSeq2-packages). Results We found significant variations in the fungal microbiota composition in the different IBD phenotypes(Fig1) In UC, Saccharomyces was predominant in pancolitis (E3), while Penicillium was more abundant in proctitis(E1) and Galactomyces in left-side UC (E2)(Pan.A). At species level, Rhodotorula mucilaginosa was significantly depleted in E3 (p≤0.05)(Pan.B) In CD, Aspergillus was predominant in ileal affection(L1) while Candida and Rhodotorula genus were more abundant in L2-L3.Patients with structuring phenotype(B2) were depleted of Apergillus and Rodhotorula (Pan.A). At species level, ileal (L1) patients were depleted of Candida tropicalis and Penicillium kudriavzevii. B1 patients had a marked predominance of Candida albicans vs B2(p≤0.01)(Pan.B) The mycobiome correlation with disease activity is depicted (Pan.C). Candida dublinensis was overrepresented in activity, while Saccharomyces pastorianus was depleted(p≤0.01) The correlation of the mycobiome with disease outcomes is represented in Pan.D. Penicillium glabrum was significantly overrepresented in patients with a complicated course. The abundance of Candida tropicalis and D. nepalensis was correlated with the need for surgery. Conclusion This study shows important differences in the fungal microbiota composition in the different IBD phenotypes. Some species are clearly correlated with the activity of the disease and worse clinical outcomes suggesting a potential deleterious role.Candida tropicalis was associated with the need of surgery in CD. This in-depth characterization of the mycobiome serves as a significant addition to the understanding of the role of fungi in IBD.

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.

Treatment of inflammatory bowel disease (IBD) is challenging, with a series of available drugs each helping only a fraction of patients. Patients may face time-consuming drug trials while the disease is active, thus there is an unmet need for biomarkers and assays to predict drug effect. It is well known that the intestinal epithelium is an important factor in disease pathogenesis, exhibiting physical, biochemical and immunologic driven barrier dysfunctions. One promising test system to study effects of existing or emerging IBD treatments targeting intestinal epithelial cells (IECs) is intestinal organoids (“mini-guts”). However, the fact that healthy intestinal epithelium is in a physiologically hypoxic state has largely been neglected, and studies with intestinal organoids are mainly performed at oxygen concentration of 20%. We hypothesized that lowering the incubator oxygen level from 20% to 2% would recapitulate better the in vivo physiological environment of colonic epithelial cells and enhance the translational value of intestinal organoids as a drug testing platform. In the present study we examine the effects of the key IBD cytokines and drug targets TNF/IL17 on human colonic organoids (colonoids) under atmospheric (20%) or reduced (2%) O2. We show that colonoids derived from both healthy controls and IBD-patients are viable and responsive to IBD-relevant cytokines at 2% oxygen. Because chemokine release is one of the important immunoregulatory traits of the epithelium that may be fine-tuned by IBD-drugs, we also examined chemokine expression and release at different oxygen concentrations. We show that chemokine responses to TNF/IL17 in organoids display similarities to inflamed epithelium in IBD-patients. However, inflammation-associated genes induced by TNF/IL17 were attenuated at low oxygen concentration. We detected substantial oxygen-dependent differences in gene expression in untreated as well as TNF/IL17 treated colonoids in all donors. Further, for some of the IBD-relevant cytokines differences between colonoids from healthy controls and IBD patients were more pronounced in 2% O2 than 20% O2. Our results strongly indicate that an oxygen concentration similar to the in vivo epithelial cell environment is of essence in experimental pharmacology.

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.

BACKGROUND:Microbiota is most likely essential in the pathogenesis of Crohn's disease (CD). Fecal diversion after ileocecal resection (ICR) protects against CD recurrence, whereas infusion of fecal content triggers inflammation. After ICR, the majority of patients experience endoscopic recurrence in the neoterminal ileum, and the ileal microbiome is of particular interest. We have assessed the mucosa-associated microbiome in the inflamed and noninflamed ileum in patients with CD.METHODS:Mucosa-associated microbiome was assessed by 16S rRNA sequencing of biopsies sampled 5 and 15 cm orally of the ileocecal valve or ileocolic anastomosis.RESULTS:Fifty-one CD patients and forty healthy controls (HCs) were included in the study. Twenty CD patients had terminal ileitis, with endoscopic inflammation at 5 cm, normal mucosa at 15 cm, and no history of upper CD involvement. Crohn's disease patients (n = 51) had lower alpha diversity and separated clearly from HC on beta diversity plots. Twenty-three bacterial taxa were differentially represented in CD patients vs HC; among these, Tyzzerella 4 was profoundly overrepresented in CD. The microbiome in the inflamed and proximal noninflamed ileal mucosa did not differ according to alpha diversity or beta diversity. Additionally, no bacterial taxa were differentially represented.CONCLUSIONS:The microbiome is similar in the inflamed and proximal noninflamed ileal mucosa within the same patients. Our results support the concept of CD-specific microbiota alterations and demonstrate that neither ileal sublocation nor endoscopic inflammation influence the mucosa-associated microbiome.

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.