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The gastrointestinal tract is a multi-organ system crucial for efficient nutrient uptake and barrier immunity. Advances in genomics and a surge in gastrointestinal diseases 1,2 has fuelled efforts to catalogue cells constituting gastrointestinal tissues in health and disease 3 . Here we present systematic integration of 25 single-cell RNA sequencing datasets spanning the entire healthy gastrointestinal tract in development and in adulthood. We uniformly processed 385 samples from 189 healthy controls using a newly developed automated quality control approach (scAutoQC), leading to a healthy reference atlas with approximately 1.1 million cells and 136 fine-grained cell states. We anchor 12 gastrointestinal disease datasets spanning gastrointestinal cancers, coeliac disease, ulcerative colitis and Crohn's disease to this reference. Utilizing this 1.6 million cell resource (gutcellatlas.org), we discover epithelial cell metaplasia originating from stem cells in intestinal inflammatory diseases with transcriptional similarity to cells found in pyloric and Brunner's glands. Although previously linked to mucosal healing 4 , we now implicate pyloric gland metaplastic cells in inflammation through recruitment of immune cells including T cells and neutrophils. Overall, we describe inflammation-induced changes in stem cells that alter mucosal tissue architecture and promote further inflammation, a concept applicable to other tissues and diseases., Competing Interests: Competing interests: S.A.T. is a scientific advisory board member of ForeSite Labs, OMass Therapeutics, a co-founder and equity holder of TransitionBio and EnsoCell Therapeutics, a non-executive director of 10x Genomics and a part-time employee of GlaxoSmithKline. R.E. is an equity holder in EnsoCell. P.K. has consulted for AstraZeneca, UCB, Biomunex and Infinitopes. N.M.P reports consulting fees from Infinitopes. J.S.-R. reports funding from GSK, Pfizer and Sanofi and fees/honoraria from Travere Therapeutics, Stadapharm, Astex, Owkin, Pfizer, Moderna and Grunenthal. A.S. is the recipient of research grants from Roche-Genentech, Abbvie, GSK, Scipher Medicine, Pfizer, Alimentiv, Boehringer Ingelheim and Agomab and has received consulting fees from Genentech, GSK, Pfizer, HotSpot Therapeutics, Alimentiv, Agomab, Goodgut and Orikine. R.E. and S.A.T are inventors on the patent GB2412853.0 filed in the UK, some components of which are related to this work. All other authors declare no competing interests., (© 2024. The Author(s).)

Background and Aim: Autologous haematopoietic stem cell transplantation [AHSCT] is a therapeutic option for refractory Crohn's disease [CD]. However, high adverse event rates related to chemotherapy toxicity and immunosuppression limit its applicability. This study aims to evaluate AHSCT's safety and efficacy using a cyclophosphamide [Cy]-free mobilisation regimen., Methods: A prospective, observational study included 14 refractory CD patients undergoing AHSCT between June 2017 and October 2022. The protocol involved outpatient mobilisation with G-CSF 12-16 μg/kg/daily for 5 days, and optional Plerixafor 240 μg/d [1-2 doses] if the CD34 + cell count target was unmet. Standard conditioning with Cy and anti-thymocyte globulin was administered. Clinical, endoscopic, and radiological assessments were conducted at baseline and during follow-up., Results: All patients achieved successful outpatient mobilisation [seven patients needed Plerixafor] and underwent transplantation. Median follow-up was 106 weeks (interquartile range [IQR] 52-348). No mobilisation-related serious adverse events [SAEs] or CD worsening occurred. Clinical and endoscopic remission rates were 71% and 41.7% at 26 weeks, 64% and 25% at 52 weeks, and 71% and 16.7% at the last follow-up, respectively. The percentage of patients who restarted CD therapy for clinical relapse and/or endoscopic/radiological activity was 14% at 26 weeks, 57% at 52 weeks, and 86% at the last follow-up, respectively. Peripheral blood cell populations and antibody levels post-AHSCT were comparable to Cy-based mobilisation., Conclusions: Cy-free mobilisation is safe and feasible in refractory CD patients undergoing AHSCT. Although relapse occurs in a significant proportion of patients, clinical and endoscopic responses are achieved upon CD-specific therapy reintroduction., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Ulcerative colitis and Crohn's disease are chronic inflammatory intestinal diseases with perplexing heterogeneity in disease manifestation and response to treatment. While the molecular basis for this heterogeneity remains uncharacterized, single-cell technologies allow us to explore the transcriptional states within tissues at an unprecedented resolution which could further understanding of these complex diseases. Here, we apply single-cell RNA-sequencing to human inflamed intestine and show that the largest differences among patients are present within the myeloid compartment including macrophages and neutrophils. Using spatial transcriptomics in human tissue at single-cell resolution (CosMx Spatial Molecular Imaging) we spatially localize each of the macrophage and neutrophil subsets identified by single-cell RNA-sequencing and unravel further macrophage diversity based on their tissue localization. Finally, single-cell RNA-sequencing combined with single-cell spatial analysis reveals a strong communication network involving macrophages and inflammatory fibroblasts. Our data sheds light on the cellular complexity of these diseases and points towards the myeloid and stromal compartments as important cellular subsets for understanding patient-to-patient heterogeneity., (© 2023. The Author(s).)

Introduction: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease., Methods: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells., Results: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines., Conclusion: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2 , a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Martín-Adrados, Wculek, Fernández-Bravo, Torres-Ruiz, Valle-Noguera, Gomez-Sánchez, Hernández-Walias, Ferreira, Corraliza, Sancho, Esteban, Rodriguez-Perales, Cruz-Adalia, Nakaya, Salas, Bernardo, Campos-Martín, Martínez-Zamorano, Muñoz-López, Gómez del Moral, Cubero, Blumberg and Martínez-Naves.)

Background and Aims: Vedolizumab 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]., Methods: Patients 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., Results: Vedolizumab, 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., Conclusions: Our 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., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

Background: Personalized medicine requires finding relationships between variables that influence a patient's phenotype and predicting an outcome. Sparse generalized canonical correlation analysis identifies relationships between different groups of variables. This method requires establishing a model of the expected interaction between those variables. Describing these interactions is challenging when the relationship is unknown or when there is no pre-established hypothesis. Thus, our aim was to develop a method to find the relationships between microbiome and host transcriptome data and the relevant clinical variables in a complex disease, such as Crohn's disease., Results: We present here a method to identify interactions based on canonical correlation analysis. We show that the model is the most important factor to identify relationships between blocks using a dataset of Crohn's disease patients with longitudinal sampling. First the analysis was tested in two previously published datasets: a glioma and a Crohn's disease and ulcerative colitis dataset where we describe how to select the optimum parameters. Using such parameters, we analyzed our Crohn's disease data set. We selected the model with the highest inner average variance explained to identify relationships between transcriptome, gut microbiome and clinically relevant variables. Adding the clinically relevant variables improved the average variance explained by the model compared to multiple co-inertia analysis., Conclusions: The methodology described herein provides a general framework for identifying interactions between sets of omic data and clinically relevant variables. Following this method, we found genes and microorganisms that were related to each other independently of the model, while others were specific to the model used. Thus, model selection proved crucial to finding the existing relationships in multi-omics datasets., Competing Interests: We confirm that the following competing interests’ statement does not alter our adherence to PLOS ONE policies on sharing data and materials: I have read the journal’s policy and the authors of this manuscript have the following competing interests: JP has received consulting fees from AbbVie, Arena Pharmaceuticals, Boehringer Ingelheim, Celgene, Celltrion, Ferring, Genentech, Janssen, Merck, Sharp & Dohme (MSD), Oppilan, Pfizer, Progenity, Robarts, Roche, Takeda, Theravance, and TiGenix; speaker’s fees from AbbVie, Ferring, Janssen, MSD, Shire Pharmaceuticals, and Takeda; and research funding from AbbVie and MSD. AS reports research grants from Roche, Genentech, Boehringer Ingelheim and AbbVie, lecture fees from Roche, Boehringer Ingelheim and Pfizer, and consultancy fees from Genentech and GSK.

Gut microbial and metabolite alterations have been linked to the pathogenesis of inflammatory bowel diseases. Here we perform a multi-omics microbiome and metabolite analysis of a longitudinal cohort of Crohn's disease patients undergoing autologous hematopoietic stem cell transplantation, and investigational therapy that induces drug free remission in a subset of patients. Via comparison of patients who responded and maintained remission, responded but experienced disease relapse and patients who did not respond to therapy, we identify shared functional signatures that correlate with disease activity despite the variability of gut microbiota profiles at taxonomic level. These signatures reflect the disease state when transferred to gnotobiotic mice. Taken together, the integration of microbiome and metabolite profiles from human cohort and mice improves the predictive modelling of disease outcome, and allows the identification of a network of bacteria-metabolite interactions involving sulfur metabolism as a key mechanism linked to disease activity in Crohn's disease.

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., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation.)

T cell clonal expansions are present in the inflamed mucosa of patients with Crohn's disease (CD) and may be implicated in postoperative recurrence after ileocolonic resection., Methods: T cell receptor (TCR) analysis was performed in 57 patients included in a prospective multicentre cohort. Endoscopic recurrence was defined by a Rutgeerts score >i0. DNA and mRNA were extracted from biopsies collected from the surgical specimen and endoscopy, and analysed by high throughput sequencing and microarray, respectively., Results: TCR repertoire in the mucosa of patients with CD displayed diverse clonal expansions. Active smokers at time of surgery had a significantly increased proportion of clonal expansions as compared with non-smokers (25.9%vs17.9%, p=0.02). The percentage of high frequency clones in the surgical specimen was significantly higher in patients with recurrence and correlated with postoperative endoscopic recurrence (area under the curve (AUC) 0.69, 95% CI 0.54 to 0.83). All patients with clonality above 26.8% (18/57) had an endoscopic recurrence. These patients with a high clonality were more frequently smokers than patients with a low clonality (61% vs 23%, p=0.005). The persistence of a similar TCR repertoire at postoperative endoscopy was associated with smoking and disease recurrence. Patients with high clonality showed increased expression of genes associated with CD8 T cells and reduced expression of inflammation-related genes. Expanded clones were found predominantly in the CD8 T cell compartment., Conclusion: Clonal T cell expansions are implicated in postoperative endoscopic recurrence. CD patients with increased proportion of clonal T cell expansions in the ileal mucosa represent a subgroup associated with smoking and where pathogenesis appears as T cell driven., Trial Registration Number: NCT03458195., Competing Interests: Competing interests: MA received honoraria from Abbvie, MSD, Janssen, Takeda, Pfizer, Novartis, Ferring, Tillots, Celgene and Genentech/Roche. SN received honoraria from MSD, Abbvie, Takeda, Janssen, HAC Pharma, Tillots, Ferring and Novartis. AB received honoraria from MSD, Abbvie, Ferring, Takeda, Vifor Pharma, Sanofi‐Aventis, Hospira and Janssen. BP received honoraria from AbbVie, MSD, Takeda, Janssen, Bioagaran and Ferring. MF received honoraria from AbbVie, MSD, Takeda, Janssen, Pfizer, Ferring and Boehringer. HS received unrestricted study grants: Danone, Biocodex and Enterome; board membership, consultancy, or lecture fees: Carenity, Abbvie, Astellas, Danone, Ferring, Mayoly Spindler, MSD, Novartis, Roche, Tillots, Enterome, Maat, BiomX, Biose, Novartis and Takeda; cofunder of Nextbiotix. XT received honoraria from Abbvie, MSD, Takeda, Ferring, Norgine and Janssen. PS received honoraria from Takeda, MSD, Biocodex, Ferring and Abbvie, and non-financial support from Takeda. CA, MN, HB, VC, AMC, AS, CS and NB declare no competing interest., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Aims: Recent studies have shown the efficacy of autologous haematopoietic stem cell transplantation [HSCT] in severely refractory Crohn's disease [CD] patients. HSCT is thought to eliminate auto-reactive cells; however, no specific studies of immune reconstitution in CD patients are available., Methods: We followed a group of CD patients [n = 18] receiving autologous HSCT, with 50% of them achieving endoscopic drug-free remission. To elucidate the mechanisms driving efficacy, we monitored changes after HSCT in blood and intestine immune-cell composition. CD patients [n = 22] receiving anti-tumour necrosis factor [TNF]-α were included for comparison., Results: Severe immune ablation followed by HSCT induced dramatic changes in both peripheral blood T and B cells in all patients regardless of the efficacy of the treatment. Endoscopic remission at week 52 following HSCT was associated with significant intestinal transcriptional changes. A comparison of the remission signature with that of anti-TNFα identified both common and unique genes in the HSCT-induced response. Based on deconvolution analysis of intestinal biopsy transcriptome data, we show that response to HSCT, but not to anti-TNFα, is associated with an expansion of naïve B-cells, as seen in blood, and a decrease in the memory resting T-cell content. As expected, endoscopic remission, in response to both HSCT and anti-TNFα, led to a significant reduction in intestinal neutrophil and M1 macrophage content., Conclusions: Peripheral blood immune remodelling after HSCT does not predict efficacy. In contrast, a profound intestinal T-cell depletion that is maintained long after transplant is associated with mucosal healing following HSCT, but not anti-TNFα., (© European Crohn’s and Colitis Organisation (ECCO) 2018.)

Background and Aims: Despite the negative results of blocking IL-17 in Crohn's disease (CD) patients, selective modulation of Th17-dependent responses warrants further study. Inhibition of retinoic acid-related orphan receptor gamma (RORγt), the master regulator of the Th17 signature, is currently being explored in inflammatory diseases. Our aim was to determine the effect of a novel oral RORγt antagonist (BI119) in human CD and on an experimental model of intestinal inflammation. Methods: 51 CD patients and 11 healthy subjects were included. The effects of BI119 were tested on microbial-stimulated peripheral blood mononuclear cells (PBMCs), intestinal crypts and biopsies from CD patients. The ability of BI119 to prevent colitis in vivo was assessed in the CD4 + CD45RB high T cell transfer model. Results: In bacterial antigen-stimulated PBMCs from CD patients, BI119 inhibits Th17-related genes and proteins, while upregulating Treg and preserving Th1 and Th2 signatures. Intestinal crypts cultured with supernatants from BI119-treated commensal-specific CD4 + T cells showed decreased expression of CXCL1, CXCL8 and CCL20 . BI119 significantly reduced IL17 and IL26 transcription in colonic and ileal CD biopsies and did not affect IL22 . BI119 has a more profound effect in ileal CD with additional significant downregulation of IL23R, CSF2, CXCL1, CXCL8 , and S100A8 , and upregulation of DEFA5 . BI119 significantly prevented development of clinical, macroscopic and molecular markers of colitis in the T-cell transfer model. Conclusions: BI119 modulated CD-relevant Th17 signatures, including downregulation of IL23R while preserving mucosa-associated IL-22 responses, and abrogated experimental colitis. Our results provide support to the use of RORγt antagonists as a novel therapy to CD treatment.