Characterization of candidate genes in inflammatory bowel disease-associated risk loci.

GWAS have linked SNPs to risk of inflammatory bowel disease (IBD), but a systematic characterization of disease-associated genes has been lacking. Prior studies utilized microarrays that did not capture many genes encoded within risk loci or defined expression quantitative trait loci (eQTLs) using peripheral blood, which is not the target tissue in IBD. To address these gaps, we sought to characterize the expression of IBD-associated risk genes in disease-relevant tissues and in the setting of active IBD. Terminal ileal (TI) and colonic mucosal tissues were obtained from patients with Crohn's disease or ulcerative colitis and from healthy controls. We developed a NanoString code set to profile 678 genes within IBD risk loci. A subset of patients and controls were genotyped for IBD-associated risk SNPs. Analyses included differential expression and variance analysis, weighted gene coexpression network analysis, and eQTL analysis. We identified 116 genes that discriminate between healthy TI and colon samples and uncovered patterns in variance of gene expression that highlight heterogeneity of disease. We identified 107 coexpressed gene pairs for which transcriptional regulation is either conserved or reversed in an inflammation-independent or -dependent manner. We demonstrate that on average approximately 60% of disease-associated genes are differentially expressed in inflamed tissue. Last, we identified eQTLs with either genotype-only effects on expression or an interaction effect between genotype and inflammation. Our data reinforce tissue specificity of expression in disease-associated candidate genes, highlight genes and gene pairs that are regulated in disease-relevant tissue and inflammation, and provide a foundation to advance the understanding of IBD pathogenesis.

Mechanisms of Pediatric Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), including Crohn disease and ulcerative colitis, is characterized by chronic intestinal inflammation due to a complex interaction of genetic determinants, disruption of mucosal barriers, aberrant inflammatory signals, loss of tolerance, and environmental triggers. Importantly, the incidence of pediatric IBD is rising, particularly in children younger than 10 years. In this review, we discuss the clinical presentation of these patients and highlight environmental exposures that may affect disease risk, particularly among people with a background genetic risk. With regard to both children and adults, we review advancements in understanding the intestinal epithelium, the mucosal immune system, and the resident microbiota, describing how dysfunction at any level can lead to diseases like IBD. We conclude with future directions for applying advances in IBD genetics to better understand pathogenesis and develop therapeutics targeting key pathogenic nodes.

Diagnostic and Therapeutic Yield of Endoscopy in Patients with Elevated INR and Gastrointestinal Bleeding.

BACKGROUND: Gastrointestinal bleeding is a well-known risk of systemic anticoagulation. However, bleeding in the setting of supratherapeutic anticoagulation may have a milder natural history than unprovoked bleeding. It is a common clinical gestalt that endoscopy is common, but bleeding source identification or intervention is uncommon, yet few data exist to inform this clinical impression. Consequently, we sought to examine our institutional experience with gastrointestinal bleeding in the setting of supratherapeutic international normalized ratio (INR) with the aim of identifying predictors of endoscopically identifiable lesions, interventions, and outcomes. METHODS: A retrospective review was conducted at a tertiary referral academic medical center to identify patients presenting with gastrointestinal bleeding in the setting of warfarin and a supratherapeutic INR (>3.5) who underwent an endoscopic procedure. Relevant clinical covariates, endoscopic findings, need for intervention, and outcomes were collected by review of the medical record. Logistic regression adjusting for potential confounders identified predictors of endoscopically significant lesions as well as intervention and outcomes. RESULTS: A total of 134 patients with INR 3.5 or greater (mean 5.5, range 3.5-17.1) presented with symptoms of gastrointestinal bleeding, most commonly as melena or symptomatic anemia. Antiplatelet agents were used by 54% of patients, and 60% of patients were on concomitant acid suppression on admission. Procedures included esophagogastroduodenoscopy (upper endoscopy; EGD) (n = 128), colonoscopy (n = 73), and video capsule endoscopy (n = 32). Active bleeding at first EGD or colonoscopy was found in only 19 patients (18%), with endoscopic intervention in only 26 patients (25%). At a critical threshold of INR 7.5 at presentation, the likelihood of finding an endoscopically significant lesion fell to <20%. On multivariate logistic regression, concomitant antiplatelet therapy (odds ratio [OR] 2.59; 95% confidence interval [CI], 1.13-5.94), timing of EGD within 12 hours of presentation (OR 3.71; 95% CI, 1.05-13.08), and INR level (OR 0.79; 95% CI, 0.64-0.98) were the only significant independent predictors of identifying a source of bleeding. A risk score incorporating these covariates performed modestly in identifying risk of significant finding on EGD (area under the curve 0.68). We found no association between identification of a significant lesion at EGD and future readmission for gastrointestinal bleeding. CONCLUSION: This study demonstrates that the relationship between INR elevation and identification of a bleeding source or endoscopic intervention at EGD are indeed antiparallel. Concomitant antiplatelet therapy increases the likelihood of bleeding source identification and intervention, as does EGD within 12 hours of presentation. However, regardless of source identification or endoscopic intervention, important clinical outcomes were unchanged, suggesting that decisions about endoscopy should be made on a case-by-case basis, particularly in patients with INR > 7.5. Future prospective studies on appropriate indications and timing of endoscopy in such patients are warranted.

Body Mass Index, Genetic Susceptibility, and Risk of Complications Among Individuals with Crohn's Disease.

BACKGROUND: Obesity is associated with systemic and intestine-specific inflammation and alterations in gut microbiota, which in turn impact mucosal immunity. Nonetheless, a specific role of obesity and its interaction with genetics in the progression of Crohn's disease (CD) is unclear. METHODS: We conducted a cross-sectional study of patients with CD enrolled in Prospective Registry in Inflammatory Bowel Disease Study at Massachusetts General Hospital (PRISM). Information on diagnosis of CD and its complications were collected and confirmed through review of medical records. A genetic risk score was calculated using previously reported single-nucleotide polymorphisms-associated genome-wide with CD susceptibility. We used logistic regression to estimate the effect of body mass index (BMI) and its interaction with genetic risk on risk of CD complications. RESULTS: Among 846 patients with CD, 350 required surgery, 242 with penetrating disease, 182 with stricturing disease, and 226 with perianal disease. There were no associations between obesity (BMI ≥ 30 kg/m2) and risk of perianal disease, stricturing disease, or surgery. Compared with normal-weight individuals with BMI < 25 kg/m2, obesity was associated with lower risk of penetrating disease (odds ratio [OR = 0.56; 95% confidence interval [CI], 0.31-0.99). This association persists among a subgroup of participants with available BMI before development of penetrating disease (OR = 0.40; 95% CI, 0.16-0.88). There were no interactions between BMI and genetic risk score on risk of CD complications (all P interaction > 0.28). CONCLUSIONS: Our data suggest that obesity does not negatively impact long-term progression of CD, even after accounting for genetic predisposition.

Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense.

A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1ß production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1-dependent antibacterial autophagy or IL-1ß production. These findings demonstrate how the T300A polymorphism leads to cell type- and pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.

Selective modulation of autophagy, innate immunity, and adaptive immunity by small molecules.

Autophagy is an evolutionarily conserved catabolic process that directs cytoplasmic proteins, organelles and microbes to lysosomes for degradation. Autophagy acts at the intersection of pathways involved in cellular stress, host defense, and modulation of inflammatory and immune responses; however, the details of how the autophagy network intersects with these processes remain largely undefined. Given the role of autophagy in several human diseases, it is important to determine the extent to which modulators of autophagy also modify inflammatory or immune pathways and whether it is possible to modulate a subset of these pathways selectively. Here, we identify small-molecule inducers of basal autophagy (including several FDA-approved drugs) and characterize their effects on IL-1ß production, autophagic engulfment and killing of intracellular bacteria, and development of Treg, TH17, and TH1 subsets from naïve T cells. Autophagy inducers with distinct, selective activity profiles were identified that reveal the functional architecture of connections between autophagy, and innate and adaptive immunity. In macrophages from mice bearing a conditional deletion of the essential autophagy gene Atg16L1, the small molecules inhibit IL-1ß production to varying degrees suggesting that individual compounds may possess both autophagy-dependent and autophagy-independent activity on immune pathways. The small molecule autophagy inducers constitute useful probes to test the contributions of autophagy-related pathways in diseases marked by impaired autophagy or elevated IL-1ß and to test novel therapeutic hypotheses.

Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection.

BACKGROUND & AIMS: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. METHODS: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. RESULTS: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3ß (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. CONCLUSIONS: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.

The microbiota and inflammatory bowel disease: insights from animal models.

Inflammatory bowel disease (IBD) is thought to result from a dysregulated immune response to intestinal microbial flora in individuals with genetic predisposition(s). Genome-wide association studies (GWAS) in human IBD have identified more than 150 associated loci, some of which are key players in innate immunity and bacterial handling, reflecting the importance of the microbiota in disease pathogenesis. In fact, the presence of a microbial flora is not only crucial to the development of a normal murine immune system but also critical for the development of disease in the majority of animal models of IBD. Although animal models do not perfectly recapitulate human IBD, they have led to the discovery of important concepts in IBD pathogenesis, such as the central role of microbiota in disease development and perpetuation. Many genetically susceptible models do not develop colitis when raised in a germ-free or Helicobacter-free environment. In fact, disease in most models can be attenuated or completely abolished with antibiotic treatment. Moreover, an interplay between intestinal microbiota and mucosal immune activation is suggested by the presence of serum antibodies against the Cbir1 flagellin, an immunodominant antigen that activates TLR5, in certain models of spontaneous colitis as well as in human patients. Furthermore, T cells reactive to Cbir1 are able to induce disease in recipient mice upon adoptive cell transfer, demonstrating the pro-inflammatory properties of certain bacterial products. In fact, it has been shown that transfer of certain intestinal bacteria from a specific genetically altered mouse model with spontaneous colitis can induce disease in wild-type mice upon co-housing or direct feeding. These observations demonstrate the pathogenic potential of intestinal microbiota in IBD. However, intestinal bacteria are not always maladaptive in mucosal homeostasis. Both Bacteroides fragilis and Clostridium species promote the number and function of a certain regulatory T cell subset in the colon leading to protection against murine colitis. In fact, normal development of regulatory cells and epithelial cell integrity are abolished in the absence of an intestinal flora, suggestive of the need for certain microbial components to induce beneficial anti-inflammatory mechanisms. All in all, altered immune responses to microbes play a crucial role in IBD pathogenesis. However, certain components of the microbiota are also likely critical for normal development of regulatory mechanisms that contribute to mucosal homeostasis. Findings in animal models highlight the concept that IBD is a disease that results from the interplay of genetics and microbial/environmental factors.

The Mayo Dysphagia Questionnaire-30: documentation of reliability and validity of a tool for interventional trials in adults with esophageal disease.

The aim of this study was to develop the Mayo Dysphagia Questionnaire-30 Day (MDQ-30), a tool to measure esophageal dysphagia, by adapting items from validated instruments for use in clinical trials, and assess its feasibility, reproducibility, and concurrent validity. Outpatients referred to endoscopy for dysphagia or seen in a specialty clinic were recruited. Feasibility testing was done to identify problematic items. Reproducibility was measured by test-retest format. Concurrent validity reflects agreement between information gathered in a structured interview versus the patients' written responses. The MDQ-30, a 28-item instrument, took 10 min (range = 5-30 min) to complete. Four hundred thirty-one outpatients [210 (49%) men; mean age = 61 years] participated. Overall, most concurrent validity kappa values for dysphagia were very good to excellent with a median of 0.78 (min 0.28, max 0.95). The majority of reproducibility kappa values for dysphagia were moderate to excellent with a median kappa value of 0.66 (min 0.07, max 1.0). Overall, concurrent validity and reproducibility kappa values for gastroesophageal reflux disease (GERD) symptoms were 0.81 (95% CI = 0.72, 0.91) and 0.66 (95% CI = 0.55, 0.77), respectively. Individual item percent agreement was generally very good to excellent. Internal consistency was excellent. We conclude that the MDQ-30 is an easy-to-complete tool to evaluate reliably dysphagia symptoms over the last 30 days.

Diagnostic ionizing radiation exposure in a population-based cohort of patients with inflammatory bowel disease.

OBJECTIVE: For diagnosis, assessing disease activity, complications and extraintestinal manifestations, and monitoring response to therapy, patients with inflammatory bowel disease undergo many radiological studies employing ionizing radiation. However, the extent of radiation exposure in these patients is unknown. METHODS: A population-based inception cohort of 215 patients with inflammatory bowel disease from Olmsted County, Minnesota, diagnosed between 1990 and 2001, was identified. The total effective dose of diagnostic ionizing radiation was estimated for each patient. Linear regression was used to assess the median total effective dose since symptom onset. RESULTS: The number of patients with Crohn's disease and ulcerative colitis was 103 and 112, with a mean age at diagnosis of 38.6 and 39.4 yr, respectively. Mean follow-up was 8.9 yr for Crohn's disease and 9.0 yr for ulcerative colitis. Median total effective dose for Crohn's disease was 26.6 millisieverts (mSv) (range, 0-279) versus 10.5 mSv (range, 0-251) for ulcerative colitis (P < 0.001). Computed tomography accounted for 51% and 40% of total effective dose, respectively. Patients with Crohn's disease had 2.46 times higher total effective dose than ulcerative colitis patients (P= 0.001), adjusting for duration of disease. CONCLUSIONS: Annualizing our data, the radiation exposure in the inflammatory bowel disease population was equivalent to the average annual background radiation dose from naturally occurring sources in the U.S. (3.0 mSv). However, a subset of patients had substantially higher doses. The development of imaging management guidelines to minimize radiation dose, dose-reduction techniques in computed tomography, and faster, more robust magnetic resonance techniques are warranted.