eQTL in diseased colon tissue identifies novel target genes associated with IBD.

Genome-wide association studies (GWAS) have identified over 300 loci associated with the inflammatory bowel diseases (IBD), but putative causal genes for most are unknown. We conducted the largest disease-focused expression quantitative trait loci (eQTL) analysis using colon tissue from 252 IBD patients to determine genetic effects on gene expression and potential contribution to IBD. Combined with two non-IBD colon eQTL studies, we identified 194 potential target genes for 108 GWAS loci. eQTL in IBD tissue were enriched for IBD GWAS loci colocalizations, provided novel evidence for IBD-associated genes such as ABO and TNFRSF14, and identified additional target genes compared to non-IBD tissue eQTL. IBD-associated eQTL unique to diseased tissue had distinct regulatory and functional characteristics with increased effect sizes. Together, these highlight the importance of eQTL studies in diseased tissue for understanding functional consequences of genetic variants, and elucidating molecular mechanisms and regulation of key genes involved in IBD.

TNF Promoter Hypomethylation Is Associated With Mucosal Inflammation in IBD and Anti-TNF Response.

Background and Aims: Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the tumor necrosis factor (TNF) promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and nonresponders. Methods: We obtained mucosal biopsies from 200 participants (133 IBDs and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 nonresponders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. Results: TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated intestinal epithelial cells from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF nonresponders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed 2 missense variants in DNA methyltransferase 1, 1 of which had reduced function in vivo. Conclusion: Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.

Post-operative Crohn's Disease Recurrence and Infectious Complications: A Transcriptomic Analysis.

BACKGROUND: Crohn's disease (CD) is a chronic inflammatory condition affecting the gastrointestinal tract, characterized by complications such as strictures, fistulas, and neoplasia. Despite medical advancements, a significant number of patients with Crohn's disease require surgery, and many experience post-operative complications and recurrence. Previous studies have analyzed gene expression to study recurrence and post-operative complications independently. This study aims to identify overlapping differentially expressed genes and pathways for recurrence and post-operative complications. METHODS: A dataset including 45 patients with Crohn's disease, including gene expression from ileum and colon tissue, endoscopic recurrence, and intra-abdominal septic complications was analyzed. Gene set enrichment analysis was used to identify gene pathways associated with the outcomes. Finally, a multi-variable logistic regression model was created to assess whether gene pathways were independently associated with both outcomes. RESULTS: In ileum tissue, several inflammatory pathways, including interferon alpha and gamma response were upregulated in patients with endoscopic recurrence and intra-abdominal septic complications. In addition, there was upregulation of the epithelial mesenchymal transition pathway. In colon tissue, metabolic processes, such as myogenesis and oxidative phosphorylation were downregulated in both outcomes. In a multivariate model, downregulation of myogenesis in colon tissue was significantly associated with both endoscopic recurrence and intra-abdominal septic complications. CONCLUSION: These findings shed light on the underlying biology of these outcomes and suggest potential biomarkers or therapeutic targets to reduce their occurrence. Further validation and multi-institutional studies are warranted to confirm these results and improve post-operative outcomes for patients with Crohn's disease.

CoVar: A generalizable machine learning approach to identify the coordinated regulators driving variational gene expression.

Network inference is used to model transcriptional, signaling, and metabolic interactions among genes, proteins, and metabolites that identify biological pathways influencing disease pathogenesis. Advances in machine learning (ML)-based inference models exhibit the predictive capabilities of capturing latent patterns in genomic data. Such models are emerging as an alternative to the statistical models identifying causative factors driving complex diseases. We present CoVar, an ML-based framework that builds upon the properties of existing inference models, to find the central genes driving perturbed gene expression across biological states. Unlike differentially expressed genes (DEGs) that capture changes in individual gene expression across conditions, CoVar focuses on identifying variational genes that undergo changes in their expression network interaction profiles, providing insights into changes in the regulatory dynamics, such as in disease pathogenesis. Subsequently, it finds core genes from among the nearest neighbors of these variational genes, which are central to the variational activity and influence the coordinated regulatory processes underlying the observed changes in gene expression. Through the analysis of simulated as well as yeast expression data perturbed by the deletion of the mitochondrial genome, we show that CoVar captures the intrinsic variationality and modularity in the expression data, identifying key driver genes not found through existing differential analysis methodologies.

TNF promoter hypomethylation is associated with mucosal inflammation in IBD and anti-TNF response.

Background and aims: Inflammatory Bowel Diseases (IBD) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the TNF promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and non-responders. Methods: We obtained mucosal biopsies from 200 participants (133 IBD and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 non-responders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. Results: TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated IECs from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF non-responders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed two missense variants in DNMT1, one of which had reduced function in vivo. Conclusions: Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.

Aberrant miR-29 is a predictive feature of severe phenotypes in pediatric Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory gut disorder. Molecular mechanisms underlying the clinical heterogeneity of CD remain poorly understood. MicroRNAs (miRNAs) are important regulators of gut physiology, and several have been implicated in the pathogenesis of adult CD. However, there is a dearth of large-scale miRNA studies for pediatric CD. We hypothesized that specific miRNAs uniquely mark pediatric CD. We performed small RNA-Seq of patient-matched colon and ileum biopsies from treatment-naive pediatric patients with CD (n = 169) and a control cohort (n = 108). Comprehensive miRNA analysis revealed 58 miRNAs altered in pediatric CD. Notably, multinomial logistic regression analysis revealed that index levels of ileal miR-29 are strongly predictive of severe inflammation and stricturing. Transcriptomic analyses of transgenic mice overexpressing miR-29 show a significant reduction of the tight junction protein gene Pmp22 and classic Paneth cell markers. The dramatic loss of Paneth cells was confirmed by histologic assays. Moreover, we found that pediatric patients with CD with elevated miR-29 exhibit significantly lower Paneth cell counts, increased inflammation scores, and reduced levels of PMP22. These findings strongly indicate that miR-29 upregulation is a distinguishing feature of pediatric CD, highly predictive of severe phenotypes, and associated with inflammation and Paneth cell loss.

Linking gene expression to clinical outcomes in pediatric Crohn's disease using machine learning.

Pediatric Crohn's disease (CD) is characterized by a severe disease course with frequent complications. We sought to apply machine learning-based models to predict risk of developing future complications in pediatric CD using ileal and colonic gene expression. Gene expression data was generated from 101 formalin-fixed, paraffin-embedded (FFPE) ileal and colonic biopsies obtained from treatment-naïve CD patients and controls. Clinical outcomes including development of strictures or fistulas and progression to surgery were analyzed using differential expression and modeled using machine learning. Differential expression analysis revealed downregulation of pathways related to inflammation and extra-cellular matrix production in patients with strictures. Machine learning-based models were able to incorporate colonic gene expression and clinical characteristics to predict outcomes with high accuracy. Models showed an area under the receiver operating characteristic curve (AUROC) of 0.84 for strictures, 0.83 for remission, and 0.75 for surgery. Genes with potential prognostic importance for strictures (REG1A, MMP3, and DUOX2) were not identified in single gene differential analysis but were found to have strong contributions to predictive models. Our findings in FFPE tissue support the importance of colonic gene expression and the potential for machine learning-based models in predicting outcomes for pediatric CD.

Cutting Edge: Hypoxia Sensing by the Histone Demethylase UTX (KDM6A) Limits Colitogenic CD4+ T Cells in Mucosal Inflammation.

Hypoxia is a hallmark of inflammatory conditions (e.g., inflammatory bowel disease [IBD]), and adaptive responses have consequently evolved to protect against hypoxia-associated tissue injury. Because augmenting hypoxia-induced protective responses is a promising therapeutic approach for IBD, a more complete understanding of these pathways is needed. Recent work has demonstrated that the histone demethylase UTX is oxygen-sensitive, but its role in IBD is unclear. In this study, we show that hypoxia-induced deactivation of UTX downregulates T cell responses in mucosal inflammation. Hypoxia results in decreased T cell proinflammatory cytokine production and increased immunosuppressive regulatory T cells, and these findings are recapitulated by UTX deficiency. Hypoxia leads to T cell accumulation of H3K27me3 histone modifications, suggesting that hypoxia impairs UTX's histone demethylase activity to dampen T cell colitogenic activity. Finally, T cell-specific UTX deletion ameliorates colonic inflammation in an IBD mouse model, implicating UTX's oxygen-sensitive demethylase activity in counteracting hypoxic inflammation.

Hypoxia-sensing by the Histone Demethylase UTX ( KDM6A ) Controls Colitogenic CD4 + T cell Fate and Mucosal Inflammation.

Hypoxia is a feature of inflammatory conditions [e.g., inflammatory bowel disease (IBD)] and can exacerbate tissue damage in these diseases. To counteract hypoxia's deleterious effects, adaptive responses have evolved which protect against hypoxia-associated tissue injury. To date, much attention has focused on hypoxia-activated HIF (hypoxia-inducible factor) transcription factors in these responses. However, recent work has identified epigenetic regulators that are also oxygen-sensitive, but their role in adaptation to hypoxic inflammation is currently unclear. Here, we show that the oxygen-sensing epigenetic regulator UTX is a critical modulator of colitis severity. Unlike HIF transcription factors that act on gut epithelial cells, UTX functions in colitis through its effects on immune cells. Hypoxia results in decreased CD4 + T cell IFN-γ production and increased CD4 + regulatory T cells, and these findings are recapitulated by T cell-specific UTX deficiency. Hypoxia impairs the histone demethylase activity of UTX, and loss of UTX function leads to accumulation of repressive H3K27me3 epigenetic marks at IL12/STAT4 pathway genes ( Il12rb2, Tbx21, and Ifng ). In a colitis mouse model, T cell-specific UTX deletion ameliorates colonic inflammation, protects against weight loss, and increases survival. Together these findings implicate UTX's oxygen-sensitive histone demethylase activity in mediating protective, hypoxia-induced pathways in colitis.

CoVar: A generalizable machine learning approach to identify the coordinated regulators driving variational gene expression.

Network inference is used to model transcriptional, signaling, and metabolic interactions among genes, proteins, and metabolites that identify biological pathways influencing disease pathogenesis. Advances in machine learning (ML)-based inference models exhibit the predictive capabilities of capturing latent patterns in genomic data. Such models are emerging as an alternative to the statistical models identifying causative factors driving complex diseases. We present CoVar, an inference framework that builds upon the properties of existing inference models, to find the central genes driving perturbed gene expression across biological states. We leverage ML-based network inference to find networks that capture the strength of regulatory interactions. Our model first pinpoints a subset of genes, termed variational, whose expression variabilities typify the differences in network connectivity between the control and perturbed data. Variational genes, by being differentially expressed themselves or possessing differentially expressed neighbor genes, capture gene expression variability. CoVar then creates subnetworks comprising variational genes and their strongly connected neighbor genes and identifies core genes central to these subnetworks that influence the bulk of the variational activity. Through the analysis of yeast expression data perturbed by the deletion of the mitochondrial genome, we show that CoVar identifies key genes not found through independent differential expression analysis.

BET Protein Inhibition Regulates Macrophage Chromatin Accessibility and Microbiota-Dependent Colitis.

Introduction: In colitis, macrophage functionality is altered compared to normal homeostatic conditions. Loss of IL-10 signaling results in an inappropriate chronic inflammatory response to bacterial stimulation. It remains unknown if inhibition of bromodomain and extra-terminal domain (BET) proteins alters usage of DNA regulatory elements responsible for driving inflammatory gene expression. We determined if the BET inhibitor, (+)-JQ1, could suppress inflammatory activation of macrophages in Il10-/- mice. Methods: We performed ATAC-seq and RNA-seq on Il10-/- bone marrow-derived macrophages (BMDMs) cultured in the presence and absence of lipopolysaccharide (LPS) with and without treatment with (+)-JQ1 and evaluated changes in chromatin accessibility and gene expression. Germ-free Il10-/- mice were treated with (+)-JQ1, colonized with fecal slurries and underwent histological and molecular evaluation 14-days post colonization. Results: Treatment with (+)-JQ1 suppressed LPS-induced changes in chromatin at distal regulatory elements associated with inflammatory genes, particularly in regions that contain motifs for AP-1 and IRF transcription factors. This resulted in attenuation of inflammatory gene expression. Treatment with (+)-JQ1 in vivo resulted in a mild reduction in colitis severity as compared with vehicle-treated mice. Conclusion: We identified the mechanism of action associated with a new class of compounds that may mitigate aberrant macrophage responses to bacteria in colitis.

Single-Cell Analysis Reveals Unexpected Cellular Changes and Transposon Expression Signatures in the Colonic Epithelium of Treatment-Naïve Adult Crohn's Disease Patients.

BACKGROUND & AIMS: The intestinal barrier comprises a monolayer of specialized intestinal epithelial cells (IECs) that are critical in maintaining mucosal homeostasis. Dysfunction within various IEC fractions can alter intestinal permeability in a genetically susceptible host, resulting in a chronic and debilitating condition known as Crohn's disease (CD). Defining the molecular changes in each IEC type in CD will contribute to an improved understanding of the pathogenic processes and the identification of cell type-specific therapeutic targets. We performed, at single-cell resolution, a direct comparison of the colonic epithelial cellular and molecular landscape between treatment-naïve adult CD and non-inflammatory bowel disease control patients. METHODS: Colonic epithelial-enriched, single-cell sequencing from treatment-naïve adult CD and non-inflammatory bowel disease patients was investigated to identify disease-induced differences in IEC types. RESULTS: Our analysis showed that in CD patients there is a significant skew in the colonic epithelial cellular distribution away from canonical LGR5+ stem cells, located at the crypt bottom, and toward one specific subtype of mature colonocytes, located at the crypt top. Further analysis showed unique changes to gene expression programs in every major cell type, including a previously undescribed suppression in CD of most enteroendocrine driver genes as well as L-cell markers including GCG. We also dissect an incompletely understood SPIB+ cell cluster, revealing at least 4 subclusters that likely represent different stages of a maturational trajectory. One of these SPIB+ subclusters expresses crypt-top colonocyte markers and is up-regulated significantly in CD, whereas another subcluster strongly expresses and stains positive for lysozyme (albeit no other canonical Paneth cell marker), which surprisingly is greatly reduced in expression in CD. In addition, we also discovered transposable element markers of colonic epithelial cell types as well as transposable element families that are altered significantly in CD in a cell type-specific manner. Finally, through integration with data from genome-wide association studies, we show that genes implicated in CD risk show heretofore unknown cell type-specific patterns of aberrant expression in CD, providing unprecedented insight into the potential biological functions of these genes. CONCLUSIONS: Single-cell analysis shows a number of unexpected cellular and molecular features, including transposable element expression signatures, in the colonic epithelium of treatment-naïve adult CD.

Biochemical and rheological analysis of human colonic culture mucus reveals similarity to gut mucus.

The goal of this project was to validate the functional relevance and utility of mucus produced by an in vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, noninflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle-tracking microrheology, multiangle laser light scattering, refractometry, and immunohistochemical imaging. An air-liquid interface culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight percent (wt%) of solids over 1 week (1.3 ± 0.5% at 2 days vs. 2.4 ± 0.3% at 7 days). The full range of mucus concentrations (0.9-3.3%) observed during culture was comparable to that displayed by ex vivo mucus (1.3-1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in vitro and ex vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. Particle-tracking microrheology showed higher complex viscosities for ex vivo mucus samples than in vitro mucus which was explained by a greater fraction of water present in in vitro mucus than ex vivo, i.e., in vitro mucus is more heterogeneous than ex vivo. Refractometry, multiangle laser light scattering, and immunostaining showed increased mucus complex size in ex vivo mucus compared with in vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex vivo mucus collection. The air-liquid interface culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus.

A machine learning approach identifies 5-ASA and ulcerative colitis as being linked with higher COVID-19 mortality in patients with IBD.

Inflammatory bowel diseases (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammation within the gastrointestinal tract. IBD patient conditions and treatments, such as with immunosuppressants, may result in a higher risk of viral and bacterial infection and more severe outcomes of infections. The effect of the clinical and demographic factors on the prognosis of COVID-19 among IBD patients is still a significant area of investigation. The lack of available data on a large set of COVID-19 infected IBD patients has hindered progress. To circumvent this lack of large patient data, we present a random sampling approach to generate clinical COVID-19 outcomes (outpatient management, hospitalized and recovered, and hospitalized and deceased) on 20,000 IBD patients modeled on reported summary statistics obtained from the Surveillance Epidemiology of Coronavirus Under Research Exclusion (SECURE-IBD), an international database to monitor and report on outcomes of COVID-19 occurring in IBD patients. We apply machine learning approaches to perform a comprehensive analysis of the primary and secondary covariates to predict COVID-19 outcome in IBD patients. Our analysis reveals that age, medication usage and the number of comorbidities are the primary covariates, while IBD severity, smoking history, gender and IBD subtype (CD or UC) are key secondary features. In particular, elderly male patients with ulcerative colitis, several preexisting conditions, and who smoke comprise a highly vulnerable IBD population. Moreover, treatment with 5-ASAs (sulfasalazine/mesalamine) shows a high association with COVID-19/IBD mortality. Supervised machine learning that considers age, number of comorbidities and medication usage can predict COVID-19/IBD outcomes with approximately 70% accuracy. We explore the challenge of drawing demographic inferences from existing COVID-19/IBD data. Overall, there are fewer IBD case reports from US states with poor health ranking hindering these analyses. Generation of patient characteristics based on known summary statistics allows for increased power to detect IBD factors leading to variable COVID-19 outcomes. There is under-reporting of COVID-19 in IBD patients from US states with poor health ranking, underpinning the perils of using the repository to derive demographic information.

Increased colonic expression of ACE2 associates with poor prognosis in Crohn's disease.

The host receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small intestine. Our aim was to study colonic ACE2 expression in Crohn's disease (CD) and non-inflammatory bowel disease (non-IBD) controls. We hypothesized that the colonic expression levels of ACE2 impacts CD course. We examined the expression of colonic ACE2 in 67 adult CD and 14 NIBD control patients using RNA-seq and quantitative (q) RT-PCR. We validated ACE2 protein expression and localization in formalin-fixed, paraffin-embedded matched colon and ileal tissues using immunohistochemistry. The impact of increased ACE2 expression in CD for the risk of surgery was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator. To provide critical support for the generality of our findings, we analyzed previously published RNA-seq data from two large independent cohorts of CD patients. Colonic ACE2 expression was significantly higher in a subset of adult CD patients which was defined as the ACE2-high CD subset. IHC in a sampling of ACE2-high CD patients confirmed high ACE2 protein expression in the colon and ileum compared to ACE2-low CD and NIBD patients. Notably, we found that ACE2-high CD patients are significantly more likely to undergo surgery within 5 years of CD diagnosis, and a Cox regression analysis found that high ACE2 levels is an independent risk factor for surgery (OR 2.17; 95% CI, 1.10-4.26; p = 0.025). Increased intestinal expression of ACE2 is associated with deteriorated clinical outcomes in CD patients. These data point to the need for molecular stratification that can impact CD disease-related outcomes.

RNF20 and RNF40 regulate vitamin D receptor-dependent signaling in inflammatory bowel disease.

Despite the identification of several genetic factors linked to increased susceptibility to inflammatory bowel disease (IBD), underlying molecular mechanisms remain to be elucidated in detail. The ubiquitin ligases RNF20 and RNF40 mediate the monoubiquitination of histone H2B at lysine 120 (H2Bub1) and were shown to play context-dependent roles in the development of inflammation. Here, we aimed to examine the function of the RNF20/RNF40/H2Bub1 axis in intestinal inflammation in IBD patients and mouse models. For this purpose, intestinal sections from IBD patients were immunohistochemically stained for H2Bub1. Rnf20 or Rnf40 were conditionally deleted in the mouse intestine and mice were monitored for inflammation-associated symptoms. Using mRNA-seq and chromatin immunoprecipitation (ChIP)-seq, we analyzed underlying molecular pathways in primary intestinal epithelial cells (IECs) isolated from these animals and confirmed these findings in IBD resection specimens using ChIP-seq.The majority (80%) of IBD patients displayed a loss of H2Bub1 levels in inflamed areas and the intestine-specific deletion of Rnf20 or Rnf40 resulted in spontaneous colorectal inflammation in mice. Consistently, deletion of Rnf20 or Rnf40 promoted IBD-associated gene expression programs, including deregulation of various IBD risk genes in these animals. Further analysis of murine IECs revealed that H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and VDR target genes is RNF20/40-dependent. Finally, these effects were confirmed in a subgroup of Crohn's disease patients which displayed epigenetic and expression changes in RNF20/40-dependent gene signatures. Our findings reveal that loss of H2B monoubiquitination promotes intestinal inflammation via decreased VDR activity thereby identifying RNF20 and RNF40 as critical regulators of IBD.

Increased Colonic Expression of ACE2 Associates with Poor Prognosis in Crohn's disease.

BACKGROUND AND AIMS: The host receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small intestine. Our aim was to study colonic ACE2 expression in Crohn's disease (CD) and non-inflammatory bowel disease (non-IBD) controls. We hypothesized that the colonic expression levels of ACE2 impacts CD course. METHODS: We examined the expression of colon ACE2 using RNA-seq and quantitative (q) RT-PCR from 69 adult CD and 14 NIBD control patients. In a subset of this cohort we validated ACE2 protein expression and localization in formalin-fixed, paraffin-embedded matched colon and ileal tissues using immunohistochemistry. The impact of increased ACE2 expression in CD for the risk of surgery was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator. To provide critical support for the generality of our findings, we analyzed previously published RNA-seq data from two large independent cohorts of CD patients. RESULTS: Colonic ACE2 expression was significantly higher in a subset of adult CD patients (ACE2-high CD). IHC in a sampling of ACE2-high CD patients confirmed high ACE2 protein expression in the colon and ileum compared to ACE2-low CD and NIBD patients. Notably, we found that ACE2-high CD patients are significantly more likely to undergo surgery within 5 years of diagnosis, with a Cox regression analysis finding that high ACE2 levels is an independent risk factor (OR 2.18; 95%CI, 1.05-4.55; p=0.037). CONCLUSION: Increased intestinal expression of ACE2 is associated with deteriorated clinical outcomes in CD patients. These data point to the need for molecular stratification that may impact CD disease-related outcomes.

Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn's Disease.

BACKGROUND & AIMS: Intestinal epithelial cell (IEC) barrier dysfunction is critical to the development of Crohn's disease (CD). However, the mechanism is understudied. We recently reported increased microRNA-31-5p (miR-31-5p) expression in colonic IECs of CD patients, but downstream targets and functional consequences are unknown. METHODS: microRNA-31-5p target genes were identified by integrative analysis of RNA- and small RNA-sequencing data from colonic mucosa and confirmed by quantitative polymerase chain reaction in colonic IECs. Functional characterization of activin receptor-like kinase 1 (ACVRL1 or ALK1) in IECs was performed ex vivo using 2-dimensional cultured human primary colonic IECs. The impact of altered colonic ALK1 signaling in CD for the risk of surgery and endoscopic relapse was evaluated by a multivariate regression analysis and a Kaplan-Meier estimator. RESULTS: ALK1 was identified as a target of miR-31-5p in colonic IECs of CD patients and confirmed using a 3'-untranslated region reporter assay. Activation of ALK1 restricted the proliferation of colonic IECs in a 5-ethynyl-2-deoxyuridine proliferation assay and down-regulated the expression of stemness-related genes. Activated ALK1 signaling increased colonic IEC differentiation toward colonocytes. Down-regulated ALK1 signaling was associated with increased stemness and decreased colonocyte-specific marker expression in colonic IECs of CD patients compared with healthy controls. Activation of ALK1 enhanced epithelial barrier integrity in a transepithelial electrical resistance permeability assay. Lower colonic ALK1 expression was identified as an independent risk factor for surgery and was associated with a higher risk of endoscopic relapse in CD patients. CONCLUSIONS: Decreased colonic ALK1 disrupted colonic IEC barrier integrity and was associated with poor clinical outcomes in CD patients.

Crohn's Disease Differentially Affects Region-Specific Composition and Aerotolerance Profiles of Mucosally Adherent Bacteria.

BACKGROUND: The intestinal microbiota play a key role in the onset, progression, and recurrence of Crohn disease (CD). Most microbiome studies assay fecal material, which does not provide region-specific information on mucosally adherent bacteria that directly interact with host systems. Changes in luminal oxygen have been proposed as a contributor to CD dybiosis. METHODS: The authors generated 16S rRNA data using colonic and ileal mucosal bacteria from patients with CD and without inflammatory bowel disease. We developed profiles reflecting bacterial abundance within defined aerotolerance categories. Bacterial diversity, composition, and aerotolerance profiles were compared across intestinal regions and disease phenotypes. RESULTS: Bacterial diversity decreased in CD in both the ileum and the colon. Aerotolerance profiles significantly differed between intestinal segments in patients without inflammatory bowel disease, although both were dominated by obligate anaerobes, as expected. In CD, high relative levels of obligate anaerobes were maintained in the colon and increased in the ileum. Relative abundances of similar and distinct taxa were altered in colon and ileum. Notably, several obligate anaerobes, such as Bacteroides fragilis, dramatically increased in CD in one or both intestinal segments, although specific increasing taxa varied across patients. Increased abundance of taxa from the Proteobacteria phylum was found only in the ileum. Bacterial diversity was significantly reduced in resected tissues of patients who developed postoperative disease recurrence across 2 independent cohorts, with common lower abundance of bacteria from the Bacteroides, Streptococcus, and Blautia genera. CONCLUSIONS: Mucosally adherent bacteria in the colon and ileum show distinct alterations in CD that provide additional insights not revealed in fecal material.