Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

The stepwise assembly of the neonatal virome is modulated by breastfeeding.

The gut of healthy human neonates is usually devoid of viruses at birth, but quickly becomes colonized, which-in some cases-leads to gastrointestinal disorders1-4. Here we show that the assembly of the viral community in neonates takes place in distinct steps. Fluorescent staining of virus-like particles purified from infant meconium or early stool samples shows few or no particles, but by one month of life particle numbers increase to 109 per gram, and these numbers seem to persist throughout life5-7. We investigated the origin of these viral populations using shotgun metagenomic sequencing of virus-enriched preparations and whole microbial communities, followed by targeted microbiological analyses. Results indicate that, early after birth, pioneer bacteria colonize the infant gut and by one month prophages induced from these bacteria provide the predominant population of virus-like particles. By four months of life, identifiable viruses that replicate in human cells become more prominent. Multiple human viruses were more abundant in stool samples from babies who were exclusively fed on formula milk compared with those fed partially or fully on breast milk, paralleling reports that breast milk can be protective against viral infections8-10. Bacteriophage populations also differed depending on whether or not the infant was breastfed. We show that the colonization of the infant gut is stepwise, first mainly by temperate bacteriophages induced from pioneer bacteria, and later by viruses that replicate in human cells; this second phase is modulated by breastfeeding.

Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2.

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (Treg) cells4-8, and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain Treg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce Treg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1ß. Macrophages in the small intestine produce IL-1ß, and activation of this pathway involves MYD88- and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining Treg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of Treg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1ß-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine.

Stratification of risk of progression to colectomy in ulcerative colitis via measured and predicted gene expression.

An important goal of clinical genomics is to be able to estimate the risk of adverse disease outcomes. Between 5% and 10% of individuals with ulcerative colitis (UC) require colectomy within 5 years of diagnosis, but polygenic risk scores (PRSs) utilizing findings from genome-wide association studies (GWASs) are unable to provide meaningful prediction of this adverse status. By contrast, in Crohn disease, gene expression profiling of GWAS-significant genes does provide some stratification of risk of progression to complicated disease in the form of a transcriptional risk score (TRS). Here, we demonstrate that a measured TRS based on bulk rectal gene expression in the PROTECT inception cohort study has a positive predictive value approaching 50% for colectomy. Single-cell profiling demonstrates that the genes are active in multiple diverse cell types from both the epithelial and immune compartments. Expression quantitative trait locus (QTL) analysis identifies genes with differential effects at baseline and week 52 follow-up, but for the most part, differential expression associated with colectomy risk is independent of local genetic regulation. Nevertheless, a predicted polygenic transcriptional risk score (PPTRS) derived by summation of transcriptome-wide association study (TWAS) effects identifies UC-affected individuals at 5-fold elevated risk of colectomy with data from the UK Biobank population cohort studies, independently replicated in an NIDDK-IBDGC dataset. Prediction of gene expression from relatively small transcriptome datasets can thus be used in conjunction with TWASs for stratification of risk of disease complications.

Whole-genome sequencing of African Americans implicates differential genetic architecture in inflammatory bowel disease.

Whether or not populations diverge with respect to the genetic contribution to risk of specific complex diseases is relevant to understanding the evolution of susceptibility and origins of health disparities. Here, we describe a large-scale whole-genome sequencing study of inflammatory bowel disease encompassing 1,774 affected individuals and 1,644 healthy control Americans with African ancestry (African Americans). Although no new loci for inflammatory bowel disease are discovered at genome-wide significance levels, we identify numerous instances of differential effect sizes in combination with divergent allele frequencies. For example, the major effect at PTGER4 fine maps to a single credible interval of 22 SNPs corresponding to one of four independent associations at the locus in European ancestry individuals but with an elevated odds ratio for Crohn disease in African Americans. A rare variant aggregate analysis implicates Ca2+-binding neuro-immunomodulator CALB2 in ulcerative colitis. Highly significant overall overlap of common variant risk for inflammatory bowel disease susceptibility between individuals with African and European ancestries was observed, with 41 of 241 previously known lead variants replicated and overall correlations in effect sizes of 0.68 for combined inflammatory bowel disease. Nevertheless, subtle differences influence the performance of polygenic risk scores, and we show that ancestry-appropriate weights significantly improve polygenic prediction in the highest percentiles of risk. The median amount of variance explained per locus remains the same in African and European cohorts, providing evidence for compensation of effect sizes as allele frequencies diverge, as expected under a highly polygenic model of disease.

Development and Validation of the TUMMY-UC: A Patient-Reported Outcome for Pediatric Ulcerative Colitis.

BACKGROUND & AIMS: The TUMMY-UC is a patient-reported outcome measure for pediatric ulcerative colitis (UC) with an observer-reported outcome version for children aged <8 years. It includes eight items selected by concept elicitation interviews. We aimed to finalize the TUMMY-UC by cognitive interviews (stage 2) and to evaluate the index for its psychometric properties (stage 3). METHODS: The TUMMY-UC items were first finalized during 129 cognitive debriefing interviews. Then, in a prospective, multicenter validation study, 84 children who underwent colonoscopy or provided stool for calprotectin completed the TUMMY-UC and various measures of disease activity. Assessments were repeated after 7 and 21 days for evaluating reliability and responsiveness. RESULTS: During stage 2, the items were formatted with identical structure to ensure conceptual equivalence and weighted based on ranking of importance. In stage 3, the TUMMY-UC total score had excellent reliability in repeated assessments (intraclass correlation coefficient, 0.90; 95% confidence interval, 0.84-0.94). It also had moderate to strong correlations with all constructs of disease activity: r = 0.70 with UC endoscopic index of severity, r = 0.63 with the IMPACT-III questionnaire, r = 0.43 with calprotectin, r = 0.80 with the Pediatric Ulcerative Colitis Activity Index, r = 0.75 with global assessment of disease activity, and r = 0.46 with C-reactive protein (all P < .015). The index had excellent discrimination of disease activity, with a score of <9 defining remission (area under the receiver operating characteristic curve, 0.95; 95% confidence interval, 0.93-0.99). The ΔTUMMY-UC showed high responsiveness and differentiated well between children who experienced changed from those with no change. CONCLUSIONS: The TUMMY-UC, constructed from patient-reported outcome and observer-reported outcome versions, is a reliable, valid and responsive index that can be now used in practice and clinical trials.

Progression of Pediatric Crohn's Disease Is Associated With Anti-Tumor Necrosis Factor Timing and Body Mass Index Z-Score Normalization.

BACKGROUND & AIMS: The evolution of complicated pediatric Crohn's disease (CD) in the era of anti-tumor necrosis factor (aTNF) therapy continues to be described. Because CD progresses from inflammatory to stricturing (B2) and penetrating (B3) disease behaviors in a subset of patients, we aimed to understand the risk of developing complicated disease behavior or undergoing surgery in relation to aTNF timing and body mass index z-score (BMIz) normalization. METHODS: Multicenter, 5-year longitudinal data from 1075 newly diagnosed CD patients were analyzed. Descriptive statistics, univariate and stepwise multivariate Cox proportional hazard regression (CPHR), and log-rank analyses were performed for risk of surgery and complicated disease behaviors. Differential gene expression from ileal bulk RNA sequencing was correlated with outcomes. RESULTS: Stricturing complications had the largest increase: from 2.98% to 10.60% over 5 years. Multivariate CPHR showed aTNF exposure within 3 months from diagnosis (hazard ratio [HR], 0.33; 95% CI, 0.15-0.71) and baseline L2 disease (HR, 0.29; 95% CI, 0.09-0.92) to be associated with reduced B1 to B2 progression. For children with a low BMIz at diagnosis (n = 294), multivariate CPHR showed BMIz normalization within 6 months of diagnosis (HR, 0.47; 95% CI, 0.26-0.85) and 5-aminosalicyclic acid exposure (HR, 0.32; 95% CI, 0.13-0.81) were associated with a decreased risk for surgery while B2 (HR, 4.20; 95% CI, 1.66-10.65) and B2+B3 (HR, 8.24; 95% CI, 1.08-62.83) at diagnosis increased surgery risk. Patients without BMIz normalization were enriched for genes in cytokine production and inflammation. CONCLUSIONS: aTNF exposure up to 3 months from diagnosis may reduce B2 progression. In addition, lack of BMIz normalization within 6 months of diagnosis is associated with increased surgery risk and a proinflammatory transcriptomic profile.

One-shot distributed algorithms for addressing heterogeneity in competing risks data across clinical sites.

OBJECTIVE: To characterize the interplay between multiple medical conditions across sites and account for the heterogeneity in patient population characteristics across sites within a distributed research network, we develop a one-shot algorithm that can efficiently utilize summary-level data from various institutions. By applying our proposed algorithm to a large pediatric cohort across four national Children's hospitals, we replicated a recently published prospective cohort, the RISK study, and quantified the impact of the risk factors associated with the penetrating or stricturing behaviors of pediatric Crohn's disease (PCD). METHODS: In this study, we introduce the ODACoRH algorithm, a one-shot distributed algorithm designed for the competing risks model with heterogeneity. Our approach considers the variability in baseline hazard functions of multiple endpoints of interest across different sites. To accomplish this, we build a surrogate likelihood function by combining patient-level data from the local site with aggregated data from other external sites. We validated our method through extensive simulation studies and replication of the RISK study to investigate the impact of risk factors on the PCD for adolescents and children from four children's hospitals within the PEDSnet, A National Pediatric Learning Health System. To evaluate our ODACoRH algorithm, we compared results from the ODACoRH algorithms with those from meta-analysis as well as those derived from the pooled data. RESULTS: The ODACoRH algorithm had the smallest relative bias to the gold standard method (-0.2%), outperforming the meta-analysis method (-11.4%). In the PCD association study, the estimated subdistribution hazard ratios obtained through the ODACoRH algorithms are identical on par with the results derived from pooled data, which demonstrates the high reliability of our federated learning algorithms. From a clinical standpoint, the identified risk factors for PCD align well with the RISK study published in the Lancet in 2017 and other published studies, supporting the validity of our findings. CONCLUSION: With the ODACoRH algorithm, we demonstrate the capability of effectively integrating data from multiple sites in a decentralized data setting while accounting for between-site heterogeneity. Importantly, our study reveals several crucial clinical risk factors for PCD that merit further investigations.

Learning Longitudinal Patterns and Subtypes of Pediatric Crohn Disease Treated With Infliximab via Trajectory Cluster Analysis.

BACKGROUND: The objective of this study is to identify subgroups of pediatric Crohn disease (CD) who had differential responses to the infliximab treatment through trajectory cluster analysis of disease activity using data from electronic health records. METHODS: We conducted a retrospective study of 295 pediatric patients with CD who had been treated with infliximab for a minimum of one year at the Center for Inflammatory Bowel Disease at The Children's Hospital of Philadelphia between January 2010 and December 2017. The evolution of disease was described, and subgroups of patients were identified using trajectory analysis of longitudinal data of C-reactive protein (CRP). We compared patient characteristics, biomarker for disease activity, and long-term surgical outcomes across subgroups. Cox regression models were used to evaluate the added value of the subgroup classification to baseline phenotype and location in prediction of long-term surgical outcomes. RESULTS: We identified three subgroups of patients with differential relapse-and-remission profiles (n = 33, 65 and 197 from subgroup 1 to 3), which represented patients with a higher risk of infliximab non-response, with infliximab response but with occasional disease flares, and patients with long-term response. Patients with the best treatment response had a significantly lower frequency of complicated disease phenotypes (P = 0.01), including perianal involvement (P = 0.05), lower baseline CRP (P < 0.01) and calprotectin (P = 0.01), and lowest risk of IBD-related gastrointestinal surgery within 10 years of starting treatment (P < 0.01). CONCLUSIONS: Readily available longitudinal data from electronic health records can be leveraged to provide deeper characterization of treatment response in pediatric CD.

Outcomes and Predictors of Sustained Remission After Drug Withdrawal in Pediatric Crohn Disease.

OBJECTIVES: The primary aim of this study was to determine the proportion of pediatric Crohn disease (CD) subjects in sustained drug-free remission 52 weeks after stopping pharmacological therapy. We also aimed to explore the effects of the Crohn Disease Exclusion Diet (CDED) and microbiome composition on remission. METHODS: We performed a prospective study following 18 CD patients ages 13-21 years in deep clinical remission withdrawing from immunomodulator (n = 7) or anti-TNFα (n = 11) monotherapy at two tertiary care centers. Stool for calprotectin and microbiome analyses was collected over 52 weeks. Participants followed either the CDED or free diet after drug withdrawal. The primary endpoint was sustained relapse-free drug-free remission (calprotectin <250 µg/g) at 52 weeks. RESULTS: Seventeen participants were followed through 52 weeks with 11 (64.7%) in sustained remission. There was no improvement in remission among participants following the CDED (5/9; 55.6%), P = 0.63. By 104 weeks, only 8 (47.1 %) participants remained off immunosuppressive therapies. Analysis of shotgun metagenomic sequence data revealed that taxonomic and gene function abundance in the gut microbiome was relatively stable for participants in remission and relapse. However, a predictive model incorporating gut microbial gene pathway abundance for amino sugar/nucleotide sugar metabolism and galactose metabolism from baseline samples predicted relapse at 52 weeks with 80% accuracy. CONCLUSIONS: After withdrawal of immunomodulator or anti-TNFα monotherapy among a small cohort of pediatric CD subjects in deep remission, nearly 65% sustained remission at 52 weeks. Baseline microbiome alterations predicted relapse. Large prospective studies are needed to better understand outcomes after treatment de-escalation.

Venous Thromboembolism in Pediatric Inflammatory Bowel Disease: A Case-Control Study.

OBJECTIVES: Inflammatory bowel disease (IBD) is associated with increased risk of venous thromboembolism (VTE). Despite this recognized risk, there are limited data and no anticoagulation guidelines for hospitalized pediatric IBD patients. The objectives of this study were to characterize pediatric IBD patients with VTE and determine risk factors. METHODS: This was a nested case-control study comparing hospitalized children with IBD diagnosed with VTE to those without VTE over a decade at a large referral center. Standard descriptive statistics were used to describe the VTE group. Multivariable conditional logistic regression was used to assess risk factors. RESULTS: Twenty-three cases were identified. Central venous catheter (CVC) presence (odds ratio [OR] 77.9; 95% confidence interval [CI]: 6.9--880.6; P < 0.001) and steroid use (OR 12.7; 95% CI: 1.3--126.4; P = 0.012) were independent risk factors. Median age at VTE was 17 years (interquartile range [IQR] 13.5--18.2), and in 48%, VTE was the indication for admission. Median duration of anticoagulation was 3.8 months (IQR 2.3--7.6), and there were no major bleeding events for patients on anticoagulation. There were no patients with known sequelae from VTE, though 22% had severe VTE that required interventions. CONCLUSIONS: Pediatric patients with IBD are at risk for VTE, although the absolute risk remains relatively low. The safety and efficacy of pharmacologic thromboprophylaxis needs to be further evaluated in this population with attention to risk factors, such as steroid use and presence of CVC.

Designing clinical trials in paediatric inflammatory bowel diseases: a PIBDnet commentary.

INTRODUCTION: The optimal trial design for assessing novel therapies in paediatric IBD (PIBD) is a subject of intense ongoing global discussions and debate among the different stakeholders. However, there is a consensus that the current situation in which most medications used in children with IBD are prescribed as off-label without sufficient paediatric data is unacceptable. Shortening the time lag between adult and paediatric approval of drugs is of the upmost importance. In this position paper we aimed to provide guidance from the global clinical research network (Pediatric Inflammatory Bowel Disease Network, PIBDnet) for designing clinical trials in PIBD in order to facilitate drug approval for children. METHODS: A writing group has been established by PIBDnet and topics were assigned to different members. After an iterative process of revisions among the writing group and one face-to-face meeting, all statements have reached consensus of >80% as defined a priori. Next, all core members of PIBDnet voted on the statements, reaching consensus of >80% on all statements. Comments from the members were incorporated in the text. RESULTS: The commentary includes 18 statements for guiding data extrapolation from adults, eligibility criteria to PIBD trials, use of placebo, dosing, endpoints and recommendations for feasible trials. Controversial issues have been highlighted in the text. CONCLUSION: The viewpoints expressed in this paper could assist planning clinical trials in PIBD which are both of high quality and ethical, while remaining pragmatic.

Clinical and biological predictors of response to standardised paediatric colitis therapy (PROTECT): a multicentre inception cohort study.

BACKGROUND: Lack of evidence-based outcomes data leads to uncertainty in developing treatment regimens in children who are newly diagnosed with ulcerative colitis. We hypothesised that pretreatment clinical, transcriptomic, and microbial factors predict disease course. METHODS: In this inception cohort study, we recruited paediatric patients aged 4-17 years with newly diagnosed ulcerative colitis from 29 centres in the USA and Canada. Patients initially received standardised mesalazine or corticosteroids, with pre-established criteria for escalation to immunomodulators (ie, thiopurines) or anti-tumor necrosis factor-α (TNFα) therapy. We used RNA sequencing to define rectal gene expression before treatment, and 16S sequencing to characterise rectal and faecal microbiota. The primary outcome was week 52 corticosteroid-free remission with no therapy beyond mesalazine. We assessed factors associated with the primary outcome using logistic regression models of the per-protocol population. This study is registered with ClinicalTrials.gov, number NCT01536535. FINDINGS: Between July 10, 2012, and April 21, 2015, of 467 patients recruited, 428 started medical therapy, of whom 400 (93%) were evaluable at 52 weeks and 386 (90%) completed the study period with no protocol violations. 150 (38%) of 400 participants achieved week 52 corticosteroid-free remission, of whom 147 (98%) were taking mesalazine and three (2%) were taking no medication. 74 (19%) of 400 were escalated to immunomodulators alone, 123 (31%) anti-TNFα therapy, and 25 (6%) colectomy. Low baseline clinical severity, high baseline haemoglobin, and week 4 clinical remission were associated with achieving week 52 corticosteroid-free remission (n=386, logistic model area under the curve [AUC] 0·70, 95% CI 0·65-0·75; specificity 77%, 95% CI 71-82). Baseline severity and remission by week 4 were validated in an independent cohort of 274 paediatric patients with newly diagnosed ulcerative colitis. After adjusting for clinical predictors, an antimicrobial peptide gene signature (odds ratio [OR] 0·57, 95% CI 0·39-0·81; p=0·002) and abundance of Ruminococcaceae (OR 1·43, 1·02-2·00; p=0·04), and Sutterella (OR 0·81, 0·65-1·00; p=0·05) were independently associated with week 52 corticosteroid-free remission. INTERPRETATION: Our findings support the utility of initial clinical activity and treatment response by 4 weeks to predict week 52 corticosteroid-free remission with mesalazine alone in children who are newly diagnosed with ulcerative colitis. The development of personalised clinical and biological signatures holds the promise of informing ulcerative colitis therapeutic decisions. FUNDING: US National Institutes of Health.

Lifetime Economic Burden of Crohn's Disease and Ulcerative Colitis by Age at Diagnosis.

BACKGROUND & AIMS: Understanding the burden of Crohn's disease (CD) and ulcerative colitis (UC) is important for measuring treatment value. We estimated lifetime health care costs incurred by patients with CD or UC by age at diagnosis. METHODS: We collected data from 78,620 patients with CD, 85,755 with UC, and propensity score-matched control subjects from the Truven Health MarketScan insurance claims databases (2008‒2015). Total medical (inpatient, outpatient) and pharmacy costs were captured. Cost variations over a lifetime were estimated in cost-state Markov models by age at diagnosis, adjusted to 2016 U.S. dollars and discounted at 3% per annum. We measured lifetime total and lifetime incremental cost (the difference between costs of CD or UC patients vs matched controls). RESULTS: For CD, the lifetime incremental cost was $707,711 among patients who received their diagnosis at 0‒11 years, and $177,614 for patients 70 years or older, averaging $416,352 for a diagnosis at any age. Lifetime total cost was $622,056, consisting of outpatient ($273,056), inpatient ($164,298), pharmacy ($163,722), and emergency room (ER) ($20,979) costs. For UC, the lifetime incremental cost was $369,955 among patients who received their diagnosis at 0‒11 years, and $132,396 for individuals 70 years or older, averaging $230,102 for a diagnosis at any age. Lifetime total cost was $405,496, consisting of outpatient ($163,670), inpatient ($123,190), pharmacy ($105,142), and ER ($13,493) costs. Therefore, the prevalent populations of patients with CD or UC in the United States in 2016 are expected to incur lifetime total costs of $498 billion and $377 billion, respectively. CONCLUSIONS: Using a Markov model, we estimated lifetime costs for patients with CD or UC to exceed previously published estimates. Individuals who receive a diagnosis of CD or UC at an early age (younger than 11 years) incur the highest lifetime cost burden. Advancing management strategies may significantly improve patient outcomes and reduce lifetime health care spending.

Blood-Derived DNA Methylation Signatures of Crohn's Disease and Severity of Intestinal Inflammation.

BACKGROUND & AIMS: Crohn's disease is a relapsing and remitting inflammatory disorder with a variable clinical course. Although most patients present with an inflammatory phenotype (B1), approximately 20% of patients rapidly progress to complicated disease, which includes stricturing (B2), within 5 years. We analyzed DNA methylation patterns in blood samples of pediatric patients with Crohn's disease at diagnosis and later time points to identify changes that associate with and might contribute to disease development and progression. METHODS: We obtained blood samples from 164 pediatric patients (1-17 years old) with Crohn's disease (B1 or B2) who participated in a North American study and were followed for 5 years. Participants without intestinal inflammation or symptoms served as controls (n = 74). DNA methylation patterns were analyzed in samples collected at time of diagnosis and 1-3 years later at approximately 850,000 sites. We used genetic association and the concept of Mendelian randomization to identify changes in DNA methylation patterns that might contribute to the development of or result from Crohn's disease. RESULTS: We identified 1189 5'-cytosine-phosphate-guanosine-3' (CpG) sites that were differentially methylated between patients with Crohn's disease (at diagnosis) and controls. Methylation changes at these sites correlated with plasma levels of C-reactive protein. A comparison of methylation profiles of DNA collected at diagnosis of Crohn's disease vs during the follow-up period showed that, during treatment, alterations identified in methylation profiles at the time of diagnosis of Crohn's disease more closely resembled patterns observed in controls, irrespective of disease progression to B2. We identified methylation changes at 3 CpG sites that might contribute to the development of Crohn's disease. Most CpG methylation changes associated with Crohn's disease disappeared with treatment of inflammation and might be a result of Crohn's disease. CONCLUSIONS: Methylation patterns observed in blood samples from patients with Crohn's disease accompany acute inflammation; with treatment, these change to resemble methylation patterns observed in patients without intestinal inflammation. These findings indicate that Crohn's disease-associated patterns of DNA methylation observed in blood samples are a result of the inflammatory features of the disease and are less likely to contribute to disease development or progression.

Genetic variants and pathways implicated in a pediatric inflammatory bowel disease cohort.

In the United States, approximately 5% of individuals with inflammatory bowel disease (IBD) are younger than 20 years old. Studies of pediatric cohorts can provide unique insights into genetic architecture of IBD, which includes Crohn's disease (CD) and ulcerative colitis (UC). Large genome-wide association studies have found more than 200 IBD-associated loci but explain a minority of disease variance for CD and UC. We sought to characterize the contribution of rare variants to disease development, comparing exome sequencing of 368 pediatric IBD patients to publicly available exome sequencing (dbGaP) and aggregate frequency data (ExAC). Using dbGaP data, we performed logistic regression for common variants and optimal unified association tests (SKAT-O) for rare, likely-deleterious variants. We further compared rare variants to ExAC counts with Fisher's exact tests. We did pathway enrichment analysis on the most significant genes from each comparison. Many variants overlapped with known IBD-associated genes (e.g. NOD2). Rare variants were enriched in CD-associated loci (p = 0.009) and showed suggestive enrichment in neutrophil function genes (p = 0.05). Pathway enrichment implicated immune-related pathways, especially cell killing and apoptosis. Variants in extracellular matrix genes also emerged as an important theme in our analysis.

Clinical and Genomic Correlates of Neutrophil Reactive Oxygen Species Production in Pediatric Patients With Crohn's Disease.

BACKGROUND & AIMS: Individuals with monogenic disorders of phagocyte function develop chronic colitis that resembles Crohn's disease (CD). We tested for associations between mutations in genes encoding reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, neutrophil function, and phenotypes of CD in pediatric patients. METHODS: We performed whole-exome sequence analysis to identify mutations in genes encoding NADPH oxidases (such as CYBA, CYBB, NCF1, NCF2, NCF4, RAC1, and RAC2) using DNA from 543 pediatric patients with inflammatory bowel diseases. Blood samples were collected from an additional 129 pediatric patients with CD and 26 children without IBD (controls); we performed assays for neutrophil activation, reactive oxygen species (ROS) production, and bacteria uptake and killing. Whole-exome sequence analysis was performed using DNA from 46 of the children with CD to examine associations with NADPH gene mutations; RNA sequence analyses were performed using blood cells from 46 children with CD to test for variations in neutrophil gene expression associated with ROS production. RESULTS: We identified 26 missense mutations in CYBA, CYBB, NCF1, NCF2, and NCF4. Patients with CD who carried mutations in these genes were 3-fold more likely to have perianal disease (P = .0008) and stricturing complications (P = .002) than children with CD without these mutations. Among patients with CD with none of these mutations, 9% had undergone abdominal surgery; among patients with mutations in these NADPH oxidase genes, 31% had undergone abdominal surgery (P = .0004). A higher proportion of neutrophils from children with CD had low ROS production (47%) than from controls (15%) among the 129 patients tested for ROS (P = .002). Minor alleles of the NADPH genes were detected in 7% of children with CD whose neutrophils produced normal levels of ROS vs 38% of children whose neutrophils produced low levels of ROS (P = .009). Neutrophils that produced low levels of ROS had specific alterations in genes that regulate glucose metabolism and antimicrobial responses. CONCLUSIONS: We identified missense mutations in genes that encode NADPH oxidases in children with CD; these were associated with a more aggressive disease course and reduced ROS production by neutrophils from the patients.

Serum Protein Biomarkers of Fibrosis Aid in Risk Stratification of Future Stricturing Complications in Pediatric Crohn's Disease.

OBJECTIVES: Avoiding fibrostenotic complications is of paramount concern in the management of Crohn's disease (CD). We sought to investigate the association of candidate biomarkers of fibrosis collected at diagnosis with the future development of fibrostenotic CD. METHODS: Using the Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's Disease cohort, a multicenter prospective observational pediatric inception cohort, subjects with an inflammatory phenotype (B1) at diagnosis who later converted to a stricturing phenotype (B2) within 3 years were compared with those who remained B1. Serum collected at diagnosis underwent both parallel reaction monitoring-targeted proteomic analysis and conventional enzyme-linked immunosorbent assay for 10 candidate biomarkers of intestinal fibrosis. Cox proportional hazard regression was used for multivariable analysis of time-dependent outcomes. RESULTS: In 116 subjects 58 subjects with verified B1 phenotype at diagnosis who later converted to B2 disease were compared with 58 subjects who remained B1 over 3 years of follow-up. Extracellular matrix protein 1 (ECM1) levels in the upper quartile (hazard ratio [HR] 3.43, 95% confidence limit [CL] 1.33, 8.42) were associated with future fibrostenotic disease. ASCA IgA (HR 4.99, 95% CL 1.50, 16.68) and CBir levels (HR 5.19, 95% CL 1.83, 14.74) were also associated with future intestinal fibrostenosis, although ECM1 continued to demonstrate independent association with conversion to B2 even with adjustment for serologies in multivariable analysis (HR 5.33, 95% CL 1.29, 22.13). CONCLUSIONS: ECM1 and other biomarkers of fibrosis may aid in determining the risk of uncomplicated inflammatory disease converting to B2 stricturing phenotypes in children with CD. Prospective validation studies to verify test performance and optimize clinical utilization are needed before clinical implementation.

Prediction of complicated disease course for children newly diagnosed with Crohn's disease: a multicentre inception cohort study.

BACKGROUND: Stricturing and penetrating complications account for substantial morbidity and health-care costs in paediatric and adult onset Crohn's disease. Validated models to predict risk for complications are not available, and the effect of treatment on risk is unknown. METHODS: We did a prospective inception cohort study of paediatric patients with newly diagnosed Crohn's disease at 28 sites in the USA and Canada. Genotypes, antimicrobial serologies, ileal gene expression, and ileal, rectal, and faecal microbiota were assessed. A competing-risk model for disease complications was derived and validated in independent groups. Propensity-score matching tested the effect of anti-tumour necrosis factor α (TNFα) therapy exposure within 90 days of diagnosis on complication risk. FINDINGS: Between Nov 1, 2008, and June 30, 2012, we enrolled 913 patients, 78 (9%) of whom experienced Crohn's disease complications. The validated competing-risk model included age, race, disease location, and antimicrobial serologies and provided a sensitivity of 66% (95% CI 51-82) and specificity of 63% (55-71), with a negative predictive value of 95% (94-97). Patients who received early anti-TNFα therapy were less likely to have penetrating complications (hazard ratio [HR] 0·30, 95% CI 0·10-0·89; p=0·0296) but not stricturing complication (1·13, 0·51-2·51; 0·76) than were those who did not receive early anti-TNFα therapy. Ruminococcus was implicated in stricturing complications and Veillonella in penetrating complications. Ileal genes controlling extracellular matrix production were upregulated at diagnosis, and this gene signature was associated with stricturing in the risk model (HR 1·70, 95% CI 1·12-2·57; p=0·0120). When this gene signature was included, the model's specificity improved to 71%. INTERPRETATION: Our findings support the usefulness of risk stratification of paediatric patients with Crohn's disease at diagnosis, and selection of anti-TNFα therapy. FUNDING: Crohn's and Colitis Foundation of America, Cincinnati Children's Hospital Research Foundation Digestive Health Center.

Infliximab Is Not Associated With Increased Risk of Malignancy or Hemophagocytic Lymphohistiocytosis in Pediatric Patients With Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Immunosuppressive therapy for inflammatory bowel disease (IBD) in pediatric patients is thought to increase the risk of malignancy and lymphoproliferative disorders, including hemophagocytic lymphohistiocytosis (HLH). We compared unadjusted incidence rates of malignancy and HLH in pediatric patients with IBD exposed to infliximab (IFX) with patients not exposed to biologics and calculated standardized incidence ratios (SIRs). METHODS: We collected and analyzed data from 5766 participants in a prospective study of long-term outcomes of pediatric patients with IBD (NCT00606346), from May 31, 2007 through June 30, 2016. Patients were 17 years old or younger and had Crohn's disease, ulcerative colitis, or IBD-unclassified with 24,543.0 patient-years of follow-up. We estimated incidence rates for malignancy and HLH as events/1000 patient-years of follow-up. We calculated age-, sex-, and race-adjusted SIRs, with 95% confidence intervals (CIs), using the Surveillance, Epidemiology, and End Results Program (SEER) database. RESULTS: Thirteen of the 15 patients who developed a malignancy and all 5 of the patients who developed HLH had been exposed to thiopurines; 10 patients with malignancy had also been exposed to a biologic agent. Unadjusted incidence rates showed no increased risk of malignancy (0.46/1000 patient-years) or HLH (0.0/1000 patient-years) in patients exposed to IFX as the only biologic vs those unexposed to biologics (malignancy: 1.12/1000 patient-years; HLH: 0.56/1000 patient-years). SIRs did not demonstrate an increased risk of malignancy among patients exposed to IFX (SIR, 1.69; 95% CI, 0.46-4.32) vs patients not exposed to a biologic agent (SIR, 2.17; 95% CI, 0.59-5.56), even when patients were stratified by thiopurine exposure. CONCLUSIONS: In determination of age-, sex-, and race-adjusted SIRs using data from a large clinical study and the SEER database, we found that IFX exposure did not associate with increased risk of malignancy or HLH in pediatric patients with IBD. Thiopurine exposure is an important precedent event for the development of malignancy or HLH in pediatric patients with IBD.