Journal of Crohn's and Colitis

BackgroundFatigue is commonly identified by IBD patients as major issue that affects their wellbeing. This presentation, however, is complex, multifactorial and mired in clinical heterogeneity. Aims/MethodsWe prospectively captured patient reported outcomes (PROs) from 2 current IBD biomarker studies in Scotland with [~]100 clinical metadata points; and an international dataset (that includes non-IBD healthy controls) using CUCQ32, a validated IBD questionnaire to generate a contemporaneous dataset of fatigue and overall wellbeing (2021-2024) and utilized 6 different machine learning (ML) approaches to predict IBD-associated fatigue and patterns that may aid future stratification to human mechanistic and clinical studies. ResultsIn 2 970 responses from 2 290 participants, CUCQ32 were higher in active IBD vs. remission; and in remission, higher than in non-IBD controls (both p<0.0001). CUCQ32-specific fatigue score significantly correlated to all CUCQ32 components (p=2.9 x 10-28 to 3.2 x 10-147). During active IBD, patients had significantly more fatigue days compared to those in remission and non-IBD controls (medians 14 vs. 7 vs. 4 [out of 14 days]; both p<0.0001). We determine a threshold of [&ge;]10/14 days of fatigue as clinically relevant - Fatiguehigh. Overall, 72.8% (863/1185), 45.0% (408/906) and 13.7% (46/355) responses in active, remission and non-IBD controls were in Fatiguehigh. Using train-validate-test steps, we incorporated all available metadata to generate ML-models to predict Fatiguehigh. The 6 ML models performed similarly (all 6 models AUC of [~]0.70). SHapley Additive exPlanations (SHAP) analysis revealed that each algorithm places different importance on variables with seasonality, biologic drug levels, BMI and gender identified as factors. ML prediction of Fatiguehigh in patients in biochemical remission (CRP<5 mg/l and calprotectin <250g/g) was more challenging with AUC of 0.66-0.61. ConclusionWe provide a comprehensive patient involvement-ML-pathway to predict IBD-associated fatigue. Our data suggests a large hidden pathobiological component and current work is in progress to integrate deep molecular data and build a clinical-scientific ML model as a step towards better understanding of IBD-associated fatigue.

Background and aimsThe IBD-Character consortium has recruited large internationally based inception cohorts of treatment-naive inflammatory bowel disease (IBD) patients, providing a unique resource to derive a simple transcriptome signature in the field of prognostication. MethodsThe discovery cohort (n=160) was recruited in Norway, Sweden and Spain. The replication inception cohort from the United Kingdom (n=97) was followed-up for a mean ({+/-}SD) of 350{+/-}228 days. Treatment escalation was formally defined as the need for a biologic agent, ciclosporin and/or surgery, instituted for disease flare after initial remission, or colectomy during the index admission for ulcerative colitis. Whole blood RNA was subject to paired-end sequencing. In the discovery cohort a simple procedure was applied, which exploited differences of transcript ratios. The ten top performing ratios were tested using Cox regression models in the validation cohort. ResultsNewly diagnosed IBD patients with high CACNA1E/LRRC42 expression ratio had an increased risk of treatment intensification (validation cohort: HR=19.3, 95%CI 2.6-143.9, p=0.000005; AUC 0.76, 95%CI 0.66-0.86). In 51 patients with CRP < 3.5 mg/L, CACNA1E/LRRC42 still predicted escalation (HR=10.4; 95%CI 1.2-86.5, p=0.007). The second best performing transcript ratio was CACNA1E/CEACAM21 yielding a HR of 10.9 (95%CI 2.5-46.7, p=0.00002) and an AUC of 0.76 (95%CI 0.65-0.86) in the validation cohort. ConclusionTranscriptomic profiling of an IBD inception cohort identified gene expression ratios CACNA1E/LRRC42 and CACNA1E/CEACAM21 as prognostic biomarkers. These were validated in a replication cohort as strongly associated with short- and long-term risk of treatment intensification and may provide valuable information in clinical decision-making.

BackgroundCrohns disease (CD) and ulcerative colitis (UC) are highly heterogeneous, dynamic and unpredictable, with a marked disconnect between symptoms and intestinal inflammation. ObjectiveWe aimed to describe latent disease heterogeneity in IBD by identifying longitudinal faecal calprotectin (FC) and CRP patterns. DesignIn this longitudinal study, patient-level measurements of FC and CRP in two European cohorts were modelled. Latent class mixed models were used to cluster individuals with similar longitudinal profiles. Associations between cluster assignment and baseline characteristics were quantified using multinomial logistic regression. Differences in advanced therapy use across clusters were explored. Finally, we considered the overlap between FC and CRP clusters. ResultsWe included 1036 patients in the FC discovery analysis (Lothian) with a total of 10545 FC observations (median 9 per subject, IQR 6-13), and 7880 patients in the replication (Denmark). The CRP discovery analysis consisted of 1838 patients with 49364 measurements (median 20 per subject; IQR 10-36), with 10041 patients in the replication cohort. Eight distinct clusters of inflammatory behaviour over time were identified in the FC and CRP analysis. Similar patterns were observed in the replication cohort. The clusters included rapid remitters, delayed remitters, remitting-relapsing disease, and non-remitters The highest use of early advanced therapy was in the group with the lowest overall inflammation. There was broadly poor agreement between FC and CRP cluster assignment. ConclusionDistinct patterns of inflammatory behaviour over time are evident in patients with IBD. These data pave the way for a deeper understanding of disease heterogeneity in IBD.

Background and AimsThe 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 Crohns disease (CD) and non-inflammatory bowel disease (non-IBD) controls. We hypothesized that the colonic expression levels of ACE2 impacts CD course. MethodsWe 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. ResultsColonic 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). ConclusionIncreased 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.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease for which the vast majority of the approved therapies target the immune system. We aimed to identify consistently dysregulated genes and pathways across independent UC transcriptomic cohorts, distinguishing constitutive from inflammation-dependent changes. We performed a random-effects meta-analysis of 14 microarray datasets from the Gene Expression Omnibus (972 mucosal biopsies, 9 platforms), comparing inflamed UC, uninflamed UC, and inflamed Crohns disease (CD) to controls, as well as UC to CD directly. The inflamed UC analysis revealed an upregulated inflammatory transcriptomic profile in UC, providing a rationale for the use of all approved anti-inflammatory therapies. In parallel, the predominant downregulated signal was metabolic, driven by PPARGC1A, PPARGC1B, and ES-RRA, indicating a coordinated, inflammation-dependent collapse. The uninflamed UC analysis revealed a separate set of potentially constitutive vulnerabilities -- fully suppressed glucuronidation, upregulated translation, complement priming, and altered iron export -- that are not downstream of the energy collapse. The metabolic deficit was more severe in UC than in CD, while immune pathways were shared. These findings suggest a two-layer model of UC pathology: a constitutive impairment of metabolic pathways that is further exacerbated by inflammation. The inflammation analysis reveals new targets for immune suppression while the constitutive analysis identifies targets for proactive intervention between flares directed at the metabolic deficiency.

Pediatric Inflammatory Bowel Disease (IBD) is a chronic condition characterized by per-sistent intestinal inflammation in children and adolescents. Despite a rising global incidence, the underlying causes and optimal management strategies for pediatric IBD are still not fully understood. Compared to adult IBD, pediatric IBD frequently presents with distinct disease phenotypes, and is more commonly linked to rare monogenic variants that cause intestinal epithelial barrier dysfunction or affect the function of mucosal immune cells. While more than 100 genes have been associated with early-onset IBD, the roles of many of these genes in the intestinal epithelium and the mechanisms by which genetic variants contribute to disease remain poorly defined. Here we aimed to improve our understanding of intestinal epithelium dysfunction in early-onset IBD by conducting extensive molecular and cellular characterization to gain insights into patient-specific epithelial phenotypes and identify therapeutic targets. We generated intestinal epithelial organoids (IEOs) from 94 pediatric IBD patients, representing diverse clinical characteristics and including those with monogenic variants (BTK n=4, TTC7A n=3, IL10RA n=1, LRBA n=1, STXBP2 n=1, TTC37 n=1, TRNT1 n=1, PLCG2 n=1, DKC1 n=1, POLA1 n=1), and 46 non-IBD controls. This effort resulted in the largest RNA-seq dataset of pediatric IBD intestinal epithelial organoids to date, encompassing both baseline conditions and post-immunological stimulation, serving as a valuable resource for future research. We observed that IEOs effectively initiate inflammation upon stimulation with bacterial lysate, regardless of disease status, origin, or mutation status. Inflammatory stimulation triggered single-gene upregulation of IBD-linked SERPINA1 and LIFR across the IBD population compared to controls, suggesting their role in intestinal epithelial innate immune responses. However, co-expression network analysis showed no consistent transcriptional signatures across the entire IBD group at the systems level. Instead, differences emerged between controls and specific genotypes (TTC7A, STXBP2, LRBA), with STXBP2 and LRBA sharing an upregulated transcriptional response of IL-1 and SLC30-mediated zinc trafficking pathways. These findings underscore the potential of IEOs as a valuable model for studying IBD and offer key insights that could guide the development of targeted therapies for both monogenic and non-monogenic forms of IBD. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/659052v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@10a4d9org.highwire.dtl.DTLVardef@b6cdadorg.highwire.dtl.DTLVardef@179216dorg.highwire.dtl.DTLVardef@181a4e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackground and AimsC_ST_ABSThe progressive nature of Crohns disease is highly variable and hard to predict. In addition, symptoms correlate poorly with mucosal inflammation. There is therefore an urgent need to better characterise the heterogeneity of disease trajectories in Crohns disease by utilising objective markers of inflammation. We aimed to better understand this heterogeneity by clustering Crohns disease patients with similar longitudinal faecal calprotectin profiles. MethodsLatent class mixed models were used to model faecal calprotectin trajectories within five years of diagnosis and to cluster subjects. Information criteria, alluvial plots, and cluster trajectories were used to decide the optimal number of clusters. Chi-squared, Fishers exact test, and ANOVA were used to test for associations with variables commonly assessed at diagnosis. ResultsOur study cohort comprised of 365 patients with newly diagnosed Crohns disease and 2856 faecal calprotectin measurements taken within five years of diagnosis (median 7 per subject). Four distinct clusters were identified by characteristic calprotectin profiles: a cluster with consistently high faecal calprotectin and three clusters characterised by different downward longitudinal trends. Cluster membership was significantly associated with smoking (p = 0.015), upper gastrointestinal involvement (p < 0.001), and early biologic therapy (p < 0.001). ConclusionsOur analysis demonstrates a novel approach to characterising the heterogeneity of Crohns disease by using faecal calprotectin. The group profiles do not simply reflect different treatment regimes and do not mirror classical disease progression endpoints. We believe these profiles represent an entirely new way of classifying disease behaviour in Crohns disease.

The genetic predisposition of Inflammatory bowel disease (IBD) in the germline is well-established, while the potential role of somatic mutations in IBD remains underexplored. We aim to identify somatic mutations in intestinal mucosa and study their potential role in IBD pathogenesis. We identified somatic mutations using 538 intestinal biopsies and 268 blood samples from 268 IBD patients and 51 intestinal biopsies from 19 non-IBD controls. Mutations were called from RNA-seq data and whole exome sequencing data using Mutect2 software. Pathogenicity of mutations was predicted using the Variant Interpretation Pipeline. Mutation frequency and patterns were compared between IBD and controls, and within IBD-subgroups, considering both inflammation status and tissue of origin. IBD-affected tissue exhibited a significantly higher burden of somatic mutations compared to controls (p=0.02), with a striking enrichment for predicted likely pathogenic or pathogenic (pLP-P) mutations (p=2.07e-09). Notably, within IBD biopsies, pLP-P mutations were enriched in very early onset-IBD (VEO-IBD) genes (p[&le;]2.2e-16) and primary immune deficiency (PID) genes (p[&le;]2.2e-16), showing a 5.5-fold increase compared to non-IBD. Inflamed tissue showed an increase in mutations in PID and IBD-GWAS associated genes. Additionally, we identified pathogenic mutations in many IBD genes, such as the stop-gained mutation (c.517C>T) in HPS1, and many pLP-P mutations across the JAK-STAT pathway. Our analysis reveals widespread accumulation of pLP-P somatic mutations in IBD-affected tissue compared to non-IBD controls, with significant enrichment in genes that cause monogenic VEO-IBD. These findings suggest that somatic mutations can contribute to the initiation and perpetuation of inflammation in IBD.

Clonal hematopoiesis of indeterminate potential (CHIP) is the presence of somatic mutations in myeloid and lymphoid malignancy genes in the blood cells of individuals without a hematologic malignancy. Inflammation is hypothesized to be a key mediator in the progression of CHIP to hematologic malignancy and patients with CHIP have a high prevalence of inflammatory diseases. This study aimed to identify the prevalence and characteristics of CHIP in patients with inflammatory bowel disease (IBD). We analyzed whole exome sequencing data from 587 Crohns disease (CD), 441 ulcerative colitis (UC), and 293 non-IBD controls to assess CHIP prevalence and used logistic regression to study associations with clinical outcomes. Older UC patients (age>45) harbored increased myeloid-CHIP mutations compared to younger patients (age[&le;]45) (p=0.01). Lymphoid-CHIP was more prevalent in older IBD patients (p=0.007). Young CD patients were found to have myeloid-CHIP with high-risk features. IBD patients with CHIP exhibited unique mutational profiles compared to controls. Steroid use was associated with increased CHIP (p=0.05), while anti-TNF therapy was associated with decreased myeloid-CHIP (p=0.03). Pathway enrichment analyses indicated overlap between CHIP genes, IBD phenotypes, and inflammatory pathways. Our findings underscore a connection between IBD and CHIP pathophysiology. Patients with IBD and CHIP had unique risk profiles especially among older UC patients and younger CD patients. These findings suggest distinct evolutionary pathways for CHIP in IBD and necessitate awareness among IBD providers and hematologists to identify patients potentially at risk for CHIP-related complications including malignancy, cardiovascular disease and acceleration of their inflammatory disease.

In peripheral blood, DNA methylation (DNAm) patterns in inflammatory bowel disease patients reflect inflammatory status rather than disease status. Here, we studied DNAm in diseased rectal mucosa from ulcerative colitis (UC) patients, conducting a cell-type-specific EWAS in epithelial, immune and fibroblast cells to understand DNAm changes across disease states, course, and clinical outcomes. At diagnosis, rectal mucosa in UC exhibited a lower proportion of epithelial cells and fibroblasts, and higher proportion of immune cells, along with 3504, 910, and 2279 altered DNAm sites as detected in our cell-specific EWAS, respectively. While treatment had significant effects on DNAm of immune cells, its effects on fibroblasts and epithelial cells were attenuated. Those requiring colectomy exhibited cell composition and DNAm patterns at follow-up more like disease onset than patients who did not require colectomy. Collectively DNAm and gene expression analysis suggest that targeting epithelial genes involved in barrier function may improve clinical outcomes.

BackgroundVedolizumab, a gut-selective anti-integrin therapy, is effective in IBD, but response rates remain variable. Conventional clinical and biochemical markers, including C-reactive protein and faecal calprotectin, have limited predictive value. Although recent transcriptomic studies have implicated T-cell-related signatures in predicting vedolizumab response, these findings lack validation across independent cohorts. MethodsWe analyzed pre-treatment transcriptomic profiles from whole blood and T-cell subsets across five independent cohorts comprising 100 patients with UC and CD. The primary outcome was clinical response. Secondary outcomes included clinical and biochemical remission. ResultsAmong the 100 patients, 61 were responders and 39 non-responders, with no significant baseline clinical differences. Gene set enrichment analyses revealed downregulation of interferon alpha and gamma signalling in responders baseline blood samples, a finding validated across independent cohorts. Downregulated interferon signalling at baseline was also observed in patients who achieved clinical and biochemical remission. To build a predictive model, an adaptive elastic net logistic regression model was applied to baseline whole-blood RNA-sequencing data. The classifier achieved an AUC of 1.0 in training, 0.71-0.83 in UC validation cohorts, and 0.64-1.0 in CD cohorts. Reduced interferon signalling was observed across CD4{square} and CD8{square} T-cell subsets, including regulatory T cells, suggesting a broad immune signature rather than cell-type specificity. ConclusionsDownregulated interferon signalling in peripheral blood prior to treatment is a reproducible molecular signature predictive of vedolizumab response and biochemical remission. Whole-blood transcriptomics revealed a robust interferon-axis signal that predicted vedolizumab response across independent cohorts, with stronger performance in UC than CD. Given heterogeneous clinical endpoints and assessment windows, these data provide proof-of-concept that warrants validation with standardised, endoscopy-based outcomes.

Background and AimsEven in the absence of inflammation, persistent symptoms in Crohns disease (CD) are prevalent and negatively impact quality of life. We aimed to determine whether quiescent CD patients with persistent symptoms (qCD+symptoms) have changes in microbial structure and functional potential compared to those without symptoms (qCD-symptoms). MethodsWe performed a prospective multi-center observational study nested within the SPARC IBD study. CD patients were included if they had evidence of quiescent disease as defined by fecal calprotectin level < 150 mcg/g. Persistent symptoms were defined by the CD-PRO2 questionnaire. Active CD (aCD), diarrhea-predominant irritable bowel syndrome (IBS-D), and healthy controls (HC) were included as controls. Stool samples underwent whole genome shotgun metagenomic sequencing. ResultsA total of 424 patients were analyzed, including 39 qCD+symptoms, 274 qCD-symptoms, 21 aCD, 40 IBS-D, and 50 HC. Patients with qCD+symptoms had a less diverse microbiome, including significant reductions in Shannon diversity (P<.001) and significant differences in microbial community structure (P<.0001), compared with qCD-symptoms, IBS-D, and HC. Further, patients with qCD+symptoms showed significant enrichment of bacterial species that are normal inhabitants of the oral microbiome, including Klebsiella pneumoniae (q=.003) as well as depletion of important butyrate and indole producers, such as Eubacterium rectale (q=.001), Lachnospiraceae spp. (q<.0001), and Faecalibacterium prausnitzii (q<.0001), compared with qCD-symptoms. Finally, qCD+symptoms showed significant reductions in bacterial tnaA genes, which mediate tryptophan metabolism, as well as significant tnaA allelic variation, compared with qCD-symptoms. ConclusionThe microbiome in patients with qCD+symptoms show significant changes in diversity, community profile, and composition compared with qCD-symptoms. Future studies will focus on the functional significance of these changes. What You Need to KnowO_ST_ABSBackgroundC_ST_ABSPersistent symptoms in quiescent Crohns disease (CD) are prevalent and lead to worse outcomes. While changes in the microbial community have been implicated, the mechanisms by which altered microbiota may lead to qCD+symptoms remain unclear. FindingsQuiescent CD patients with persistent symptoms demonstrated significant differences in microbial diversity and composition compared to those without persistent symptoms. Specifically, quiescent CD patients with persistent symptoms were enriched in bacterial species that are normal inhabitants of the oral microbiome but depleted in important butyrate and indole producers compared to those without persistent symptoms. Implications for Patient CareAlterations in the gut microbiome may be a potential mediator of persistent symptoms in quiescent CD. Future studies will determine whether targeting these microbial changes may improve symptoms in quiescent CD.

Background and AimsThe pathobiology of the non-destructive inflammatory bowel disease (IBD) lymphocytic colitis (LC) is poorly understood. Our aim was to define a LC-specific transcriptome to gain insight into LC pathology, identify genetic signatures uniquely linked to LC, and uncover potentially druggable disease pathways. MethodsWe performed whole mucosa bulk RNA-sequencing of LC and CC samples from patients with active disease, and healthy controls (n=4-10 per cohort). Differential gene expression was analyzed by gene-set enrichment and deconvolution analyses to identify pathologically relevant pathways and cells, respectively, altered in LC. Key findings were validated using reverse transcription quantitative PCR and/or immunohistochemistry. Finally, we compared our sequencing data to a previous cohort of ulcerative colitis and Crohns disease patients (n=4 per group) to distinguish non-destructive from classic IBD. ResultsThe LC-specific transcriptome was defined by a limited mucosal immune response against microbiota compared to CC and classic IBD samples. In contrast, we noted a distinct induction of regulatory non-coding RNA species in LC samples. Moreover, compared to CC, we observed decreased water channel and cell adhesion molecule gene expression, which was associated with reduced intestinal epithelial cell proliferation. ConclusionsWe conclude that LC is a pathomechanistically distinct disease that is characterized by a dampened immune response despite massive mucosal immune cell infiltration. Our results point to regulatory micro-RNAs as a potential disease-specific feature that may be amenable to therapeutic intervention.

BackgroundEmerging evidence suggests that the oral microbiome may contribute to aberrant gut immune responses in Inflammatory Bowel Disease (IBD). MethodsWe performed a comprehensive, harmonised analysis of aggregated oral microbiome 16S rRNA datasets across multiple cohorts. Data were processed using a unified bioinformatics pipeline including DADA2 for taxonomic assignment, PICRUSt2 for functional prediction, MaAsLin2 for multivariable modelling, and machine learning. ResultsAcross 25 studies (n = 1,136 IBD; n = 759 controls), meta-analysis showed significantly reduced oral microbial Shannon diversity in IBD (standardised mean difference -0.31, p = 0.007). Secondary bioinformatics analysis of six datasets plus in-house data confirmed this reduction (Shannon diversity; Hedges SMD = -0.372, p < 0.001), driven primarily by Crohns disease (CD). Beta diversity demonstrated global compositional shifts, with CD demonstrating greater divergence from controls than ulcerative colitis (UC). Multivariable modelling identified reproducible taxa enriched in IBD, including Corynebacterium, Serratia and Streptoccocus, while Porphyromonas and Ruminococcaceae.G1 were enriched in controls. Functional pathway prediction indicated reduced butyrate metabolism in IBD sub-types and increased aromatic amino acid and related metabolite degradation pathways. Machine learning classifiers achieved modest discrimination (mean AUC [~]0.67), supporting the potential of saliva-based microbiome profiling to study dysbiosis in IBD. ConclusionsThese findings demonstrate that the oral microbiome in IBD is characterised by reduced diversity and reproducible structural community reorganisation. Together, these data support a contributory role for the oral-gut axis in CD pathogenesis and provide a rationale for targeted mechanistic and longitudinal studies to define causal links between oral dysbiosis and intestinal inflammation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/26351936v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@57306corg.highwire.dtl.DTLVardef@2c0ef0org.highwire.dtl.DTLVardef@88b0b3org.highwire.dtl.DTLVardef@8ed62_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe prospective Kiel Inflammatory Bowel Disease (IBD) Family Cohort Study (KINDRED cohort) was initiated in 2013 to systematically and extensively collect data and biosamples from index IBD patients and their relatives (e.g., blood, stool), a population at high risk for IBD development. Regular follow-ups were conducted to collect updated health and lifestyle information, to obtain new biosamples, and to capture the incidence of IBD during development. By combining taxonomic and imputed functional microbial data collected at successive time points with extensive anthropometric, medical, nutritional, and social information, this study aimed to characterize the factors influencing the microbiota in health and disease via detailed ecological analyses. ResultsUsing two dysbiosis metrics (MD-index, GMHI) trained on the German KINDRED cohort, we identified strong and generalizable gradients within and across different IBD cohorts, which correspond strongly with IBD pathologies, physiological manifestations of inflammation (e.g., Bristol stool score, ASCA IgA/IgG), genetic risk for IBD, and general risk of disease onset. Anthropometric and medical factors influencing transit time strongly modify bacterial communities. Various Enterobacteriaceae (e.g., Klebsiella sp.) and opportunistic Clostridia pathogens (e.g., C. XIVa clostridioforme), characterize in combination with ectopic oral taxa (e.g. Veillonella sp., Cand. Saccharibacteria sp., Fusobacterium nucleatum) the distinct and chaotic IBD-specific communities. Functionally, amino acid metabolism and flagellar assembly are beneficial, while mucolytic functions are associated with IBD. ConclusionsOur findings demonstrate broad-scale ecological patterns which indicate drastic state transitions of communities into characteristically chaotic communities in IBD patients. These patterns appear to be universal across cohorts and influence physiological signs of inflammation, display high resilience, but show only little heritability/intrafamily transmission.

Background and aimsInflammatory 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. MethodsWe 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. ResultsTNF 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. ConclusionsOur 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.

Current clinical decision-making is hindered by the absence of predictive preclinical models that faithfully bridge molecular diversity to patient outcomes. Here, we apply the principle of abstraction--deriving essential features from human tissues to build next-generation new approach methodologies (NAMs) that transform patient-derived organoids (PDOs) into predictive vehicles for Crohns disease (CD). From our living biobank of adult stem cell-derived colonic PDOs, we previously defined two molecular CD subtypes: Immune-Deficient Infectious CD (IDICD) and Stress and Senescence-Induced Fibrostenotic CD (S2FCD), each defined by unique genomic, transcriptomic, and functional profiles with matched therapeutic vulnerabilities. In this study, we prospectively anchored PDO-derived molecular phenotypes to real-world clinical outcomes, revealing that S2FCD maps to baseline and progressive colonic disease activity, whereas IDICD tracks with prior ileocecal surgery, penetrating disease behavior, as well as baseline and progressive ileal disease activity. By abstracting NAMs from human tissues and cycling insights between small- n organoids and Phase 3-sized datasets, this framework recasts PDOs as dynamic, predictive platforms that capture the past, present, and future of disease behavior. Beyond oncology, this work establishes PDOs as vehicles for prospective clinical trial-like studies in inflammatory diseases and highlights colonic immune dysfunction as a potential driver of ileal CD. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/25325058v3_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@3b0544org.highwire.dtl.DTLVardef@d6d868org.highwire.dtl.DTLVardef@119a19corg.highwire.dtl.DTLVardef@1c11082_HPS_FORMAT_FIGEXP M_FIG C_FIG

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystemic disease of unknown aetiology that is characterised by disabling chronic fatigue and involves both the immune and gastrointestinal (GI) systems. Patients display alterations in GI microbiome with a significant proportion experiencing GI discomfort and pain and elevated blood biomarkers for altered intestinal permeability compared with healthy individuals. To investigate a possible GI origin of ME/CFS we designed a feasibility study to test the hypothesis that ME/CFS pathogenesis is a consequence of increased intestinal permeability that results in microbial translocation and a breakdown in immune tolerance leading to generation of antibodies reactive to indigenous intestinal microbes. Secretory IgA and serum IgG levels and reactivity to intestinal microbes were assessed in five pairs of severe ME/CFS patients and matched same-household healthy controls. For profiling serum IgG we developed IgG-Seq which combines flow-cytometry based bacterial cell sorting and metagenomics to detect mucosal IgG reactivity to the microbiome. We uncovered evidence for immune dysfunction in severe ME/CFS patients that was characterised by reduced capacity and reactivity of serum IgG to stool microbes, irrespective of their source. This study provides the rationale for additional studies in larger cohorts of ME/CFS patients to further explore immune-microbiome interactions.

BackgroundCrohns disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract characterized by high post-operative recurrence (POR) rates, reaching up to 90% within one year. Current clinical and endoscopic predictors show limited accuracy. ObjectiveThis study aimed to identify molecular mechanisms associated with POR at the time of surgery through integrated transcriptomic and bacteriomic analyses of ileal tissue. DesignIleal samples were obtained during surgery from 20 patients with CD and 10 inflammatory bowel disease-free controls, with an independent validation cohort of 49 patients with CD. POR was evaluated every six months using ileocolonoscopy and defined by Rutgeerts score. Host gene expression and tissue-associated microbiome profiles were integrated using correlation and pathway enrichment analyses to uncover host-microbe interactions linked to POR. ResultsIn the inflamed mucosa of patients who developed endoscopic POR, we identified a novel immune interaction involving the Porphyromonadaceae family, mainly Parabacteroides gordonii, which was slightly depleted. This depletion was associated with downregulation of epithelial barrier and tissue repair genes, including TFF1 and LSR, findings confirmed in the validation cohort. Porphyromonadaceae abundance positively correlated with short-chain fatty acid levels, particularly propionate. Additionally, omics integration revealed an association between Xanthomonadaceae and increased expression of ITGAM, a gene involved in neutrophil activation. ConclusionThese results highlight microbial-host gene interactions associated with POR. The pathogenic ITGAM-driven immune signature and the protective Porphyromonadaceae-TFF1-propionate axis supporting epithelial integrity may enable microbiome-informed prognostic tools and therapeutic strategies for CD POR. O_LIWhat is already known on this topic: Post-operative recurrence in Crohns disease is linked to microbial dysbiosis, particularly reduced diversity and expansion of Enterobacteriaceae. However, how microbial changes translate into host molecular mechanisms driving POR remains unclear. C_LIO_LIWhat this study adds: This prospective multi-omic study identifies a disrupted Porphyromonadaceae-SCFA-epithelial barrier axis and the participation of neutrophil responses in patients who develop POR at surgery time. C_LIO_LIHow this study might affect research, practice or policy: The findings provide mechanistic targets for microbiome-informed risk stratification and prevention of POR. They support development of microbial or metabolite-based interventions aimed at restoring epithelial barrier function after surgery. C_LI

Our understanding of human mucosal T cell clonotype distribution in health and disease has centered on immunodominant antigens. We performed single cell T cell receptor (TCR) and RNA sequencing as an untargeted approach to define distributions of T cell clonal groups in health and ulcerative colitis (UC) across 333,088 T cells in colon and peripheral blood. Healthy donor-specific TCR repertoires had limited blood-colon clonal sharing, which was highest in cytotoxic T effector memory (Tem) populations and lowest in regulatory T cells (Tregs), reflecting tissue-based compartmentalization. Within healthy colon, TCR repertoires showed high T cell clonal sharing independent of anatomic distance, associated with high intra-clonal phenotypic diversity. Colon cytotoxic and Th17 populations showed high dispersion across sites, while Tregs were compartmentalized. Clonal lineages dispersed across blood and colon upregulated trafficking markers, suggesting active movement between tissues, while those dispersed across colon sites upregulated residency markers, suggesting intra-colon repertoire sharing is mediated by long-term, slow moving clonal groups. In UC, Tregs were expanded across inflamed sites, and increased CD8 Tem clonal groups showed increased dispersion regardless of inflammation. These findings reveal principles of T cell clonal organization in the human colon during health and disease, identifying opposing patterns of clonal dispersion among Treg and Th17 clonal groups, high phenotypic diversity within dispersed clonal groups, and elevated cross-colon dispersion of CD8 Tem clonotypes in UC.