Joanna M. Peloquin

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

Significance Although advances in human genetics have shaped our understanding of many complex diseases, little is known about the mechanism of action of alleles that influence disease. By using mice expressing a Crohn disease (CD)-associated risk polymorphism (Atg16L1 T300A), we show that Atg16L1 T300A-expressing mice demonstrate abnormalities in Paneth cells (similar to patients with the risk polymorphism) and goblet cells. We show that Atg16L1 T300A protein is more susceptible to caspase-mediated cleavage than WT autophagy related 16-like 1 (Atg16L1), resulting in decreased protein stability and effects on antibacterial autophagy and inflammatory cytokine production. We also identify interacting proteins that contribute to autophagy-dependent immune responses. Understanding how ATG16L1 T300A modulates autophagy-dependent immune responses sheds light on the mechanisms that underlie initiation and progression of CD. 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-1β 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-1β 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.

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 3β (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.

Leadership curriculum in undergraduate medical education: a study of student and faculty perspectives.

Background: Leaders in medicine have called for transformative changes in healthcare to address systems challenges and improve the health of the public. The purpose of this study was to elicit the perspectives of students, faculty physicians and administrators regarding the knowledge and competencies necessary in an undergraduate leadership curriculum. Methods: A mixed-methods study was conducted using focus group discussions and semi-structured interviews with faculty physicians and administrative leaders, as well as a written survey of medical student leaders. Results: Twenty-two faculties participated in focus groups and interviews; 21 medical students responded to the written survey. Participants identified emotional intelligence, confidence, humility and creativity as necessary qualities of leaders; and teamwork, communication, management and quality improvement as necessary knowledge and skills. Students perceived themselves as somewhat or fully competent in communication (90%), conflict resolution (70%) and time management (65%), but reported minimal or no knowledge or competence in coding and billing (100%), writing proposals (90%), managed care (85%) and investment principles (85%). Both faculty and students believed that experiential training was the most effective for teaching leadership skills. Conclusions: Study participants identified the necessary qualities, knowledge and skills to serve as goals for an undergraduate leadership curriculum. Future studies should address optimal methods of teaching and assessing leadership skills among medical students.

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-1β 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-1β 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-1β and to test novel therapeutic hypotheses.

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.

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.

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 Crohns 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.

Identification and Characterization of a Novel Association between Dietary Potassium and Risk of Crohn’s Disease and Ulcerative Colitis

Background Recent animal studies have identified that dietary salt intake may modify the risk and progression of autoimmune disorders through modulation of the IL-23/TH17 pathway, which is critical in the pathogenesis of ulcerative colitis (UC) and Crohn’s disease (CD). Methods We conducted a prospective study of U.S. women enrolled in the Nurses’ Health Study (NHS) and NHSII who provided detailed and validated information on diet and lifestyle beginning in 1984 in NHS and 1991 in NHSII. We confirmed incident cases of UC and CD reported through 2010 in NHS and 2011 in NHSII. We used Cox proportional hazards models to calculate hazard ratios and 95% confidence intervals. In a case–control study nested within these cohorts, we evaluated the interaction between single nucleotide polymorphisms (SNPs) in genes involved in TH17 pathway and dietary potassium on risk of CD and UC. In a cohort of healthy volunteers, we also assessed the effect of supplemental potassium on development of naïve and memory T cells, differentiated with TGFβ1 or TH17 conditions. Results Among a total of 194,711 women over a follow-up of 3,220,247 person-years, we documented 273 cases of CD and 335 cases of UC. Dietary intake of potassium (Ptrend = 0.005) but not sodium (Ptrend = 0.44) was inversely associated with risk of CD. Although, both dietary potassium and sodium were not significantly associated with risk of UC, there was a suggestion of an inverse association with dietary potassium (Ptrend = 0.08). The association of potassium with risk of CD and UC appeared to be modified by loci involved in the TH17 pathway that have previously been associated with susceptibility to CD, particularly SNP rs7657746 (IL21) (Pinteraction = 0.004 and 0.01, respectively). In vitro, potassium enhanced the expression of Foxp3 in both naïve and memory CD4+ T cells via activating Smad2/3 and inhibiting Smad7 in TH17 cells. Conclusion Dietary potassium is inversely associated with risk of CD with both in vitro and gene–environment interaction data suggesting a potential role for potassium in regulating immune tolerance through its effect on Tregs and TH17 pathway.

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 Crohns 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 Pinteraction > 0.28). Conclusions:Our data suggest that obesity does not negatively impact long-term progression of CD, even after accounting for genetic predisposition.

O-002 Genes in IBD-Associated Risk Loci Demonstrate Genotype-, Tissue-, and Inflammation-Specific Patterns of Expression in Terminal Ileum and Colon Mucosal Tissue.

Background:Genome-wide association studies have linked single nucleotide polymorphisms (SNPs) to risk of inflammatory bowel disease (IBD). Yet, the majority of IBD-associated risk SNPs tag non-coding regions of the genome, with more than 1000 genes encoded within the risk loci. In addition to ongoing fine mapping of risk loci and exome sequencing studies, the study of gene expression and characterization of expression quantitative trait loci (eQTL) help to refine candidate genes in risk loci. Because the genetic effects are likely to be disease context-specific, we examined gene expression in biopsies and resected tissue from uninflamed and inflamed terminal ileum (TI) and colon from genotyped patients with Crohn’s disease (CD) and ulcerative colitis (UC). Methods:We designed a custom probe set on the NanoString digital gene expression platform capturing 678 genes encoded within IBD-associated risk loci and profiled 1100 tissue samples (including 178 paired uninflamed and inflamed samples) from 593 patients and healthy controls. Patients were genotyped on the Illumina ImmunoChip array, and we performed focused analysis of IBD-associated risk loci. We developed and applied an analytical framework including principal components analysis, differential expression, machine learning algorithms, and eQTL analysis to characterize inflammation signatures for CD and UC (specific or common to CD and UC), gene-gene interactions, and significant SNP-gene associations. Results:Differential expression of a subset of our genes readily discriminated TI and colon tissues, with FAM55A, a gene encoded in a UC-specific risk loci, most highly differentially expressed (180–190 fold relative expression in the colon compared to TI). Genes with the lowest variance in expression across healthy controls were the same genes with the greatest variance in patients, suggesting that mechanisms exist to tightly regulate the expression of these genes in homeostasis and increased variance of expression in disease states may reflect a loss of such regulatory mechanisms. Unique and shared inflammatory signatures between CD and UC were identified, including genes that exhibit a continuous gradient of increasing expression from healthy controls to uninflamed and inflamed IBD tissues (e.g., SLC11A1, CCL11, CDH3). Examining patterns of gene co-expression, we found a network of 41 genes, enriched for B cell signaling, with conserved co-expression across TI and colon, CD and UC, and inflammation subtypes, highlighting possible novel interactions between a subset of risk genes, including SP140 and PTPRC. In addition to cis- and trans-eQTLs specific to CD and UC, paired uninflamed and inflamed tissues from the same patient allowed characterization of fold-change eQTLs, demonstrating how IBD-associated risk SNPs may contribute to risk of disease through the modulation of a gene expression change under inflammatory conditions. Conclusions:This study demonstrated colon/TI-, CD/UC-, and inflammation-specific expression of IBD-associated risk genes, and, for a subset of genes, genotype-specific patterns of expression. These data prioritize candidate genes for functional characterization in 60% of IBD-associated risk loci. This analytical framework has relevance for the study of genetic variation associated with other complex, polygenic autoimmune and inflammatory diseases, underscoring the value of study in disease-relevant tissues and in the context of active disease.