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Ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory intestinal diseases that show a perplexing heterogeneity in manifestations and response to treatment. The molecular basis for this heterogeneity remains uncharacterized. We applied single-cell RNA sequencing and CosMx™ Spatial Molecular Imaging to human colon and found the highest diversity in cellular composition in the myeloid compartment of UC and CD patients. Besides resident macrophage subsets (M0 and M2), patients showed a variety of activated macrophages including classical (M1 CXCL5 and M1 ACOD1) and new inflammation-dependent alternative (IDA) macrophages. In addition, we captured intestinal neutrophils in three transcriptional states. Subepithelial IDA macrophages expressedNRG1, which promotes epithelial differentiation. In contrast,NRG1lowIDA macrophages were expanded within the submucosa and in granulomas, in proximity to abundant inflammatory fibroblasts, which we suggest may promote macrophage activation. We conclude that macrophages sense and respond to unique tissue microenvironments, potentially contributing to patient-to-patient heterogeneity.

Background Janus kinase (JAK) inhibition shows promise for treatment of patients with moderate to severe Crohn’s disease. We aimed to provide mechanistic insights into the JAK1-selective inhibitor upadacitinib through a transcriptomics substudy on biopsies from patients with Crohn's disease from CELEST. Methods Seventy-four patients consented to this optional substudy. Ileal and colonic biopsies were collected during endoscopy at screening and week 12 or 16. RNA isolated from 226 samples was analyzed by RNAseq, with additional qPCR analysis. Additional biopsies from patients with Crohn's disease receiving anti-tumor necrosis factor (anti-TNF; n = 34) and healthy controls (n = 10) were used for qPCR. Single-cell RNAseq public profiles were used to evaluate treatment effects on specific cellular subsets, associations with endoscopic improvement, and indirect comparisons with the anti-TNF-treated cohort. Results In involved areas of mucosa with endoscopic remission after upadacitinib treatment, 1156 and 76 protein-coding genes were significantly regulated (false discovery rate < 0.05) at week 12/16 in colonic and ileal biopsies, respectively (60 overlapped), compared with baseline. Upadacitinib did not significantly affect transcriptomes of noninvolved intestinal areas. CELEST patients (mostly anti-TNF-refractory) showed baseline differences in gene expression compared with a separate cohort of biologic-naïve patients. Notably, upadacitinib reversed overexpression of inflammatory fibroblast and interferon-γ effector signature markers. Conclusions Upadacitinib modulates inflammatory pathways in mucosal lesions of patients with anti-TNF-refractory Crohn's disease, including inflammatory fibroblast and interferon-γ-expressing cytotoxic T cell compartments. This substudy is the first to describe the molecular response to JAK1 inhibition in inflammatory bowel disease and differential effects relative to anti-TNF treatment. (Clinical trial identifier: NCT02365649)

Ulcerative colitis (UC) is characterized by a chronic overproduction of proinflammatory cytokines. During an acute phase, the endoplasmic reticulum (ER) is overloaded and the protein folding process is impaired, a condition named ER stress. This state induces a response (unfolded protein response (UPR)), initiated by the activation of IRE1/Xbp-1, PERK/eIF2α, and ATF6 pathways, which has previously been linked to intestinal inflammation in experimental models. ER stress and UPR activation trigger the activation of proinflammatory, autophagy, and apoptosis genes, in addition to promoting protein degradation. Therefore, the goal of this study was to evaluate the activation of ER stress and UPR in colonic mucosa of UC patients. Patient and Methods. Transcriptional analysis of ER stress- and UPR-related genes was performed by qPCR from intestinal mucosa of patients with UC. We also performed in situ hybridization (ISH) and immunohistochemistry (IHQ) of PERK/eIF2α and IRE1/Xbp-1 pathways and UPR-related chaperones. Results. We first evaluated inflammatory genes via qPCR, and we observed that all analyzed proinflammatory transcripts were upregulated in UC patients. ISH and IHQ images showed that ER stress is activated via PERK/eIF2α and IRE1/Xbp-1 pathways not only in intestinal epithelial cells but also in cells of the lamina propria of UC colonic mucosa. Transcriptional analysis confirmed that EIF2AK3 was upregulated in UC patients. UPR-related genes, such as ATF3, STC2, and DDIT3, along with the chaperones and cochaperones DNAJC3, CALR, HSP90B1, and HSPA5, were also upregulated in UC patients. In addition, we observed that proapoptotic and autophagy genes (Bax and ATG6L1, respectively) were also upregulated. Conclusion. Our results suggest that ER stress and UPR are indeed activated in UC patients and this may contribute to the chronic inflammatory process seen in UC. The increased apoptosis and autophagy markers further support the activation of these findings once they are activated to counterbalance tissue damage. These findings provide new insights into the molecular mechanisms that maintain UC activity and open new possibilities to attenuate intestinal inflammation.

Background and AimsVedolizumab is an anti-α4β7 antibody approved for the treatment of ulcerative colitis [UC]. Although it is assumed that vedolizumab blocks intestinal homing of lymphocytes, its effects on different intestinal cell populations are not fully stablished. In order to establish the unique mechanisms of action of vedolizumab in UC patients, we compared its effects to those induced by anti-tumour necrosis factor [TNF].MethodsPatients with active UC [endoscopic Mayo score >1] starting vedolizumab [n = 33] or anti-TNF [n = 45] and controls [n = 22] were included. Colon biopsies [at weeks 0, 14 and 46] and blood samples [at weeks 0, 2, 6, 14, 30 and 46] were used for cell phenotyping, transcriptional analysis [qPCR], and to measure receptor occupancy.ResultsVedolizumab, in contrast to anti-TNF, significantly reduced the proportion of α4β7+ cells within intestinal T subsets while preserving the percentage of α4β7+ plasma cells. The marked decrease in α4β7 did not change the percentage of colonic αEβ7+ cells [at 46 weeks]. Both vedolizumab and anti-TNF significantly downregulated inflammation-related genes in the colon of responders [Mayo score < 2]. Moreover, both treatments significantly decreased the percentage of intestinal, but not blood, total lymphocytes [T and plasma cells], as well as the proportion of α4β1+ cells within intestinal T lymphocytes.ConclusionsOur data show that while vedolizumab and anti-TNF block two unrelated targets, they induce remarkably similar effects. On the other hand, vedolizumab’s unique mechanism of action relies on blocking intestinal trafficking of α4β7 T cells, despite effectively binding to B and plasma cells that express α4β7.

Over the last decades, Adherent-Invasive Escherichia coli (AIEC) has been linked to the pathogenesis of Crohn’s Disease. AIEC’s characteristics, as well as its interaction with the gut immune system and its role in intestinal epithelial barrier dysfunction, have been extensively studied. Nevertheless, the currently available techniques to investigate the cross-talk between this pathogen and intestinal epithelial cells (IECs) are based on the infection of immortalized cell lines. Despite their many advantages, cell lines cannot reproduce the conditions in tissues, nor do they reflect interindividual variability or gut location-specific traits. In that sense, the use of human primary cultures, either healthy or diseased, offers a system that can overcome all of these limitations. Here, we developed a new infection model by using freshly isolated human IECs. For the first time, we generated and infected monolayer cultures derived from human colonic organoids to study the mechanisms and effects of AIEC adherence and invasion on primary human epithelial cells. To establish the optimal conditions for AIEC invasion studies in human primary organoid-derived epithelial monolayers, we designed an infection-kinetics study to assess the infection dynamics at different time points, as well as with two multiplicities of infection (MOI). Overall, this method provides a model for the study of host response to AIEC infections, as well as for the understanding of the molecular mechanisms involved in adhesion, invasion and intracellular replication. Therefore, it represents a promising tool for elucidating the cross-talk between AIEC and the intestinal epithelium in healthy and diseased tissues AM was supported by the Programa Estatal de Investigación, Desarrollo e Innovación del Ministerio de Economía, Industria y Competitividad, co-funded by the European Social Fund (ESF) under grant agreement number FPI BES-2016-076642. ID is funded by the Horizon 2020 Framework Programme (EU for Research and Innovation H2020), Grant 720905. QB-R was supported by the Programa d’Ajuts per a Investigadors en Formació de la Universitat de Girona – IFUdG 2019/21. AS is funded by the Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD) and Grant RTI2018- 096946-B-I00 from the Ministerio de Ciencia, Innovación y Universidades, Spain. MM-M is funded by the Spanish Ministry of Economy and Competitiveness through project SAF2017-82261-P, which is co-funded by the European Regional Development Fund

Background and Aims Ulcerative colitis [UC] is a chronic inflammatory disease of the colon. Colonoscopy remains the gold standard for evaluating disease activity, as clinical symptoms are not sufficiently accurate. The aim of this study is to identify new accurate non-invasive biomarkers based on whole-blood transcriptomics that can predict mucosal lesions and response to treatment in UC patients. Methods Whole-blood samples were collected for a total of 152 UC patients at endoscopy. Blood RNA from 25 UC individuals and 20 controls was analysed using microarrays. Genes that correlated with endoscopic activity were validated using real-time polymerase chain reaction in an independent group of 111 UC patients, and a prediction model for mucosal lesions was evaluated. Responsiveness to treatment was assessed in a longitudinal cohort of 16 UC patients who started anti-tumour necrosis factor [TNF] therapy and were followed up for 14 weeks. Results Microarray analysis identified 122 genes significantly altered in the blood of endoscopically active UC patients. A significant correlation with the degree of endoscopic activity was observed in several genes, including HP, CD177, GPR84, and S100A12. Using HP as a predictor of endoscopic disease activity, an accuracy of 67.3% was observed, compared with 52.4%, 45.2%, and 30.3% for C-reactive protein, erythrocyte sedimentation rate, and platelet count, respectively. Finally, at 14 weeks of treatment, response to anti-TNF therapy induced alterations in blood HP, CD177, GPR84, and S100A12 transcripts that correlated with changes in endoscopic activity. Conclusions Transcriptional changes in UC patients are sensitive to endoscopic improvement and appear to be an effective tool to monitor patients over time.

Ferrer-Picón et al. show that in active IBD patients, tumor necrosis factor alpha affects the response of the intestinal epithelium to bacteria-derived butyrate. This observation raises questions about the beneficial effects of butyrate supplementation during active inflammation., Background Butyrate-producing gut bacteria are reduced in patients with active inflammatory bowel disease (IBD), supporting the hypothesis that butyrate supplementation may be beneficial in this setting. Nonetheless, earlier studies suggest that the oxidation of butyrate in IBD patients is altered. We propose that inflammation may decrease epithelial butyrate consumption. Methods Non-IBD controls and IBD patients were recruited for the study. Stool samples were used for short-chain fatty acid and bacterial butyryl CoA:acetate CoA-transferase quantification. Colonic biopsies and ex vivo differentiated epithelial organoids (d-EpOCs) treated with butyrate and/or tumor necrosis factor alpha (TNFα) were used for analyzing the expression of transporters MCT1 and ABCG2, metabolic enzyme ACADS, and butyrate receptor GPR43, and for butyrate metabolism and consumption assays. Results We observed that lower stool content of butyrate-producing bacteria in active IBD patients did not correlate with decreased butyrate concentrations. Indeed, the intestinal epithelial expression of MCT1, ABCG2, ACADS, and GPR43 was altered in active IBD patients. Nonetheless, d-EpOCs derived from IBD patients showed SLC16A1 (gene encoding for MCT1 protein), ABCG2, ACADS, and GPR43 expression levels comparable to controls. Moreover, IBD- and non-IBD-derived d-EpOCs responded similarly to butyrate, as assessed by transcriptional regulation. TNFα significantly altered SLC16A1, ABCG2, and GPR43 transcription in d-EpOCs, mimicking the expression profile observed in biopsies from active IBD patients and resulting in reduced butyrate consumption. Conclusions We provide evidence that the response to butyrate is not intrinsically altered in IBD patients. However, TNFα renders the epithelium less responsive to this metabolite, defeating the purpose of butyrate supplementation during active inflammation.

Background Anti-tumour necrosis factor α (TNFα) therapy effectively induces and maintains remission in Crohn9s disease (CD). Up to 40% of patients, however, fail to respond to anti-TNFα. Objective To identify the mechanisms underlying the persistence of mucosal lesions in patients who fail to respond to anti-TNFα therapy. Design An observational study based on whole-genome transcriptional analysis was carried out using intestinal biopsy specimens from patients with CD receiving (n=12) or not (n=10) anti-TNFα therapy. The transcriptional signature of responders was compared with that of non-responders after anti-TNFα therapy. Controls with non-inflammatory bowel disease (non-IBD) (n=17) were used for comparisons. Genes of interest were validated by real-time RT-PCR in an independent cohort of patients with CD receiving (n=17) or not (n=16) anti-TNFα and non-IBD controls (n=7). Results We confirmed that response to anti-TNFα is accompanied by significant regulation of a large number of genes, including IL1B, S100A8, CXCL1, which correlated with endoscopic activity. Remarkably, patients who failed to respond to anti-TNFα showed a mixed signature, maintaining increased expression of IL1B, IL17A and S100A8, while showing significant modulation of other genes commonly upregulated in active CD, including IL6 and IL23p19. Conclusions Our results show that anti-TNFα therapy significantly downregulates a subset of inflammatory genes even in patients who fail to achieve endoscopic remission, suggesting that these genes may not be dominant in driving inflammation in non-responders. On the other hand, we identified IL1B and IL17A as genes that remained altered in non-responders, pointing to potentially more relevant targets for modulating mucosal damage in refractory patients.

Background & Aims Crohn's disease (CD) has been associated with an altered immune response to commensal microbiota, mostly based on increased seroreactivity to microbial proteins. Although T cells are believed to contribute to the development of CD, little is known about the antigens involved. We investigated the antigen-specificity of T cells isolated from patients with CD. Methods We isolated peripheral blood mononuclear cells from 65 patients with CD and 45 healthy individuals (controls). We investigated T-cell reactivity to commensal microbial antigens using proliferation assays (based on thymidine incorporation and carboxyfluorescein succinimidyl ester dilution). Gene expression patterns were determined using microarray and real-time polymerase chain reaction analyses. Cytokines, chemokines, and antibodies were measured by enzyme-linked immunosorbent assay, flow cytometry, or multiplex cytokine assays. Intestinal crypts were obtained from surgical resection specimens of 7 individuals without inflammatory bowel disease. We examined the effects of commensal-specific CD4 + T cells on primary intestinal epithelial cells from these samples. Results The bacterial proteins FlaX, A4-fla2, and YidX increased proliferation of CD4 + T cells isolated from peripheral blood of patients with CD compared with controls. In blood samples from controls, CD4 + T cells specific for FlaX, A4-fla2, or YidX had a T-helper (Th)1 phenotype; a larger proportion of CD4 + T cells specific for these proteins in patients with CD had a Th17 phenotype or produced Th1 and Th17 cytokines. When supernatants collected from commensal-specific CD4 + T cells from patients with CD were applied to healthy intestinal epithelial cells, the epithelial cells increased the expression of the chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL8 and the CC chemokine ligand 20 (CCL20). Conclusions A larger proportion of commensal-specific CD4 + T cells from patients with CD have a Th17 phenotype or produce Th1 and Th17 cytokines, compared with T cells from controls; this might contribute to intestinal inflammation in patients with CD. These cells might be targeted for treatment of CD. The transcriptional data of commensal-specific CD4 + T cells from healthy individuals and CD patients have been deposited in the Gene Expression Omnibus at the National Center for Biotechnology Information (accession no: GSE70469).

Objective Ulcerative colitis (UC) is a chronic condition characterised by the relapsing inflammation despite previous endoscopic and histological healing. Our objective was to identify the molecular signature associated with UC remission. Design We performed whole-genome transcriptional analysis of colonic biopsies from patients with histologically active and inactive UC, and non-inflammatory bowel disease (non-IBD) controls. Real-time reverse transcriptase-PCR and immunostaining were used for validating selected genes in independent cohorts of patients. Results Microarray analysis (n=43) demonstrates that UC patients in remission present an intestinal transcriptional signature that significantly differs from that of non-IBD controls and active patients. Fifty-four selected genes were validated in an independent cohort of patients (n=30). Twenty-nine of these genes were significantly regulated in UC-in-remission subjects compared with non-IBD controls, including a large number of epithelial cell-expressed genes such as REG4, S100P, SERPINB5, SLC16A1, DEFB1, AQP3 and AQP8, which modulate epithelial cell growth, sensitivity to apoptosis and immune function. Expression of inflammation-related genes such as REG1A and IL8 returned to control levels during remission. REG4, S100P, SERPINB5 and REG1A protein expression was confirmed by immunohistochemistry (n=23). Conclusions Analysis of the gene signature associated with remission allowed us to unravel pathways permanently deregulated in UC despite histological recovery. Given the strong link between the regulation of some of these genes and the growth and dissemination of gastrointestinal cancers, we believe their aberrant expression in UC may provide a mechanism for epithelial hyper-proliferation and, in the context of malignant transformation, for tumour growth.

Background: Clinical trials in ulcerative colitis (UC) rely on certain parameters to evaluate responses that are highly subjective or of low sensitivity. Here, using a select group of genes, we tested the accuracy of gene expression analysis as a biomarker of clinical, endoscopic, and histologic improvements. Methods: Intestinal biopsies were obtained from UC patients included in two cohorts. Cohort 1 was used to select for genes whose expression was modulated in active (vs. inactive) UC. Cohort 2 included patients recruited in a phase II study receiving placebo, mesalazine, or dersalazine sodium for 4 weeks. The expression of 44 genes identified in Cohort 1 was assessed at weeks 0 and 4, and was then correlated with biomarkers, as well as with clinical, endoscopic, and histologic scores. Results: Significant changes in the expression of 31 of the 44 genes tested were detected in Cohort 2 at week 4. Gene expression (ΔCt) significantly correlated with the total Mayo score, C-reactive protein (CRP), and fecal calprotectin. The number of genes significantly regulated at week 4 was highly associated with histologic and endoscopic responses. Logistic regression analysis identified four separate genes (IFITM1, ITGB2, IL1R2, IL2RA) whose relative change was independently associated with endoscopic remission with high specificity and sensitivity. Conclusions: Change in the expression of a select set of genes can serve as an early biomarker, one with high specificity and sensitivity to clinical, endoscopic, and histologic responses. This could represent a new tool for identifying early response to treatment in mild to moderately active UC patients. (Inflamm Bowel Dis 2012;)

[Background]: Th1 and Th17 cells have been implicated in Crohn's disease (CD) pathophysiology and may play a role in disease persistence. [Aim]: To determine Th1 and Th17 responses in intestine and peripheral blood of early (2 years) CD patients. [Methods]: Cytokine mRNA in intestinal biopsies was determined by RT-PCR. Cytokine concentration in culture was measured by ELISA and cytokine-producing cells were identified by intracellular staining. [Results]: The inflamed mucosa showed significantly increased IL-17 mRNA levels compared with non-inflamed areas, both in early and late CD patients. However, only patients with late (n = 12), but not early (n = 9), active disease showed increased IL-17 production, as well as a significantly higher percentage of IL-17+CD4+ cells in blood, compared with controls (n = 12) or patients in remission (n = 13). Moreover, cultured peripheral CD4 + cells from late active CD patients presented significantly higher percentages of IL-17+, IL-22+ and IFN-γ+ and a significantly increased production of IL-17 and IL-22, but not IFN-γ+. [Conclusions]: Increased IL-17 gene transcription is common to early and late CD mucosa. However, exacerbated Th17 responses in the peripheral blood appear only in late disease. We propose that this population may constitute a mechanism of perpetuating the disease. © 2010 Blackwell Publishing Ltd., This work was funded by grants SAF2005⁄03755 and BFU2008-02683⁄BFI to A.S. and SAF2006⁄03074 to J.P. from the Ministerio de Ciencia e Innovación, Spain. M.V. is the recipient of a FPI fellowship (SAF2005⁄03755) from the Ministerio de Ciencia e Innovación, Spain.

et al., Loss of tolerance toward commensal bacteria has been invoked as a mechanism for Crohn's disease. IL-10 is a key anti-inflammatory cytokine that plays a role in induction and maintenance of tolerance. The aim of this study is to determine IL-10 production in response to bacterial components in Crohn's disease patients, who were classified according to their phenotypes as stricturing, penetrating, or inflammatory. Peripheral blood was obtained from Crohn's disease patients and healthy controls. Cytokine production was measured in whole blood cultures, isolated CD4(+) cells, and monocyte-derived dendritic cells (MDDCs). Under unstimulated conditions, IL-10, but not IL-12, was down-regulated significantly in blood cultures of patients with severe phenotypes, compared with inflammatory, nonpenetrating, nonstricturing Crohn's disease patients. In response to LPS, IL-10 was up-regulated more significantly in patients with no fistulae or fibrosis. Study of IL-10 production by isolated cell subsets showed that DCs, but not CD4(+) T cells, from penetrating Crohn's disease produced significantly less IL-10 in response to LPS. Differences were not associated with the 1082A/G polymorphism in the IL-10 gene promoter. We show a defect in IL-10 production in whole blood cell cultures and MDDCs in patients with severe forms of Crohn's disease. This defect in IL-10 production by a group of Crohn's disease patients may represent a mechanism mediating more severe manifestations of the disease. We propose that treatment with IL-10 or IL-10-inducing therapies could be of particular benefit to these group of patients, This work was supported by Grant SAF2005-03755 from the Ministerio de Educación y Ciencia, Spain.

BACKGROUND: Crohn's disease (CD) is a chronic inflammatory disease with a relapsing course. Its clinical treatment has greatly improved with the introduction of antibodies such as anti-tumor necrosis factor (TNF)-α. Nonetheless, over time up to 40% of patients become unresponsive to anti-TNF-α treatment. AIM: To identify the molecular mechanisms underlying the persistence of mucosal lesions in patients who do not respond to anti-TNF-α therapy. METHODS: Whole genome-transcriptional analysis was carried out using intestinal biopsies from CD patients before and after anti-TNF-α therapy. The transcriptional signature of responders was compared to that of non-responders after anti-TNF-α therapy. Non-inflammatory bowel disease (non-IBD) controls were also used for comparisons. Genes of interest were validated by real-time RT-PCR in an independent cohort of individuals. For statistical analysis, the non-parametric Wilcoxon test was performed and the Benjamini-Hochberg procedure was used for multiple testing corrections. A p-value of