Alan Huett

Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis

We present a genome-wide association study of ileal Crohn disease and two independent replication studies that identify several new regions of association to Crohn disease. Specifically, in addition to the previously established CARD15 and IL23R associations, we identified strong and significantly replicated associations (combined P < 10−10) with an intergenic region on 10q21.1 and a coding variant in ATG16L1, the latter of which was also recently reported by another group. We also report strong associations with independent replication to variation in the genomic regions encoding PHOX2B, NCF4 and a predicted gene on 16q24.1 (FAM92B). Finally, we demonstrate that ATG16L1 is expressed in intestinal epithelial cell lines and that functional knockdown of this gene abrogates autophagy of Salmonella typhimurium. Together, these findings suggest that autophagy and host cell responses to intracellular microbes are involved in the pathogenesis of Crohn disease.

Deletion polymorphism upstream of IRGM associated with altered IRGM expression and Crohn's disease

Following recent success in genome-wide association studies, a critical focus of human genetics is to understand how genetic variation at implicated loci influences cellular and disease processes. Crohns disease (CD) is associated with SNPs around IRGM, but coding-sequence variation has been excluded as a source of this association. We identified a common, 20-kb deletion polymorphism, immediately upstream of IRGM and in perfect linkage disequilibrium (r2 = 1.0) with the most strongly CD-associated SNP, that causes IRGM to segregate in the population with two distinct upstream sequences. The deletion (CD risk) and reference (CD protective) haplotypes of IRGM showed distinct expression patterns. Manipulation of IRGM expression levels modulated cellular autophagy of internalized bacteria, a process implicated in CD. These results suggest that the CD association at IRGM arises from an alteration in IRGM regulation that affects the efficacy of autophagy and identify a common deletion polymorphism as a likely causal variant.

Impaired Autophagy of an Intracellular Pathogen Induced by a Crohn's Disease Associated ATG16L1 Variant

The genetic risk factors predisposing individuals to the development of inflammatory bowel disease are beginning to be deciphered by genome-wide association studies. Surprisingly, these new data point towards a critical role of autophagy in the pathogenesis of Crohns disease. A single common coding variant in the autophagy protein ATG16L1 predisposes individuals to the development of Crohns disease: while ATG16L1 encoding threonine at amino acid position 300 (ATG16L1*300T) confers protection, ATG16L1 encoding for alanine instead of threonine (ATG16L1*300A, also known as T300A) mediates risk towards the development of Crohns disease. Here we report that, in human epithelial cells, the Crohns disease-associated ATG16L1 coding variant shows impairment in the capture of internalized Salmonella within autophagosomes. Thus, we propose that the association of ATG16L1*300A with increased risk of Crohns disease is due to impaired bacterial handling and lowered rates of bacterial capture by autophagy.

Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly.

Ileal lesions in Crohns disease (CD) patients are colonized by pathogenic adherent‐invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome‐wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD‐associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3‐positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non‐pathogenic, environmental, commensal, or pathogenic strains involved in gastro enteritis. Together these findings demonstrate a central role for autophagy restraining Adherent‐Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation.

Organ‐specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium

Citrobacter rodentium belongs to a family of human and animal enteric pathogens that includes the clinically significant enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens use attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract. In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity, dynamics of colonization and clearance of mice by C. rodentium in situ and in real time. The bioluminescent C. rodentium derivative, strain ICC180, expresses the luxCDABE operon from the entemopathogenic nematode symbiont Photorhabdus luminescens and light levels accurately reflect bacterial numbers both in vitro and in vivo. We have demonstrated that primary colonization of the mouse by C. rodentium takes place within the caecum, specifically within the specialized patch of lymphoid tissue known as the caecal patch. Following colonization of the caecum C. rodentium established a colonic infection. Clearance of C. rodentium ICC180 parallels the colonization dynamics, i.e. the caecum was first to be cleared followed by the colon. A bioluminescent eae (encoding the outer membrane adhesin intimin) C. rodentium mutant failed to establish long‐term colonization, although low levels of bacteria could be recovered for up to 3 days post challenge from the caecum.

Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity

In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for “nucleic acid-binding” function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)–dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.

Crohn disease: A current perspective on genetics, autophagy and immunity

Crohn disease (CD) is a chronic and debilitating inflammatory condition of the gastrointestinal tract.1 Prevalence in western populations is 100–150/100,000 and somewhat higher in Ashkenazi Jews. Peak incidence is in early adult life, although any age can be affected and a majority of affected individuals progress to relapsing and chronic disease. Medical treatments rely significantly on empirical corticosteroid therapy and immunosuppression, and intestinal resectional surgery is frequently required. Thus, 80% of patients with CD come to surgery for refractory disease or complications. It is hoped that an improved understanding of pathogenic mechanisms, for example by studying the genetic basis of CD and other forms of inflammatory bowel diseases (IBD), will lead to improved therapies and possibly preventative strategies in individuals identified as being at risk.

Autophagy as an important process in gut homeostasis and Crohn’s disease pathogenesis

Crohns disease (CD) is a complex polygenic trait whereby multiple genetic and non-genetic risk factors contribute to disease susceptibility. Association testing is a statistical approach commonly used for identifying genetic risk factors for complex/multigenic disease, which typically compares the allele frequency of a selected marker, most often a bi-allelic single nucleotide polymorphism (SNP), for differences between patient and control populations. SNPs represent most of the common genetic variation, with an estimated 10 million SNPs found in the human genome.1 Although a powerful statistical approach, until recently the majority of association studies were limited to the examination of a small number of candidate genes, the selection of which will inevitably be biased by the current knowledge of disease pathogenesis. Following some key developments in our understanding of genetic variation within the human genome, as well as technological advances that have enabled affordable genotyping of 300 000–1 000 000 SNPs per study subject (and, therefore, ∼1 billion genotypes or more per genetic study), association testing can now be applied genome wide in order to search for genetic risk factors in an unbiased manner.2–4 In one of the first genome-wide association studies (GWAS), we and our colleagues of the NIDDK IBD Genetics Consortium tested approximately 300 000 SNPs in 1000 patients with CD and in 1000 healthy individuals, and identified association with variants in the autophagy-related 16-like 1 ( Saccharomyces cerevisiae ) or ATG16L1 gene.5 The ATG16 gene product is part of a multimeric protein complex that is essential for autophagy, a biological process that mediates the bulk degradation of cytoplasmic components in lysosomes and vacuoles.6 This recent GWAS specifically identified an associated SNP that encodes a non-synonymous amino acid change—an alanine to threonine substitution in exon 8 (also known as Ala197Thr)—in the human equivalent of the ATG16 gene. In all of the …

Impaired Immunity to Intestinal Bacterial Infection in Stromelysin-1 (Matrix Metalloproteinase-3)-Deficient Mice

Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. Matrix metalloproteinases (MMPs) have been shown to mediate matrix remodeling and cell migration during tissue injury and repair in the intestine. We have previously shown enhanced pathology in infected TNFRp55−/−, IL-12p40−/−, and IFN-γ−/− mice, and here we show that this is associated with an increase in stromelysin-1 (MMP3) transcripts in colonic tissues. We have therefore investigated the role of MMP3 in colonic mucosal hyperplasia and the local Th1 responses using MMP3−/− mice. In MMP3−/− mice, similar mucosal thickening was observed after infection as in wild-type (WT) mice. Colonic tissues from MMP3−/− mice showed a compensatory increase in the expression of other MMP transcripts, such as MMP7 and MMP12. However, MMP3−/− mice showed delayed clearance of bacteria and delayed appearance of CD4+ T lymphocytes into intestinal lamina propria. CSFE-labeled mesenteric lymph node CD4+ T lymphocytes from infected WT mice migrated in fewer numbers into the mesenteric lymph nodes and colon of MMP3−/− mice than into those of WT mice. These studies show that mucosal remodeling can occur in the absence of MMP3, but that MMP3 plays a role in the migration of CD4+ T lymphocytes to the intestinal mucosa.

Mucosal Vaccination against Serogroup B Meningococci: Induction of Bactericidal Antibodies and Cellular Immunity following Intranasal Immunization with NadA of Neisseria meningitidis and Mutants of Escherichia coli Heat-Labile Enterotoxin

ABSTRACT Conjugated polysaccharide vaccines protect against serogroup C meningococci. However, this approach cannot be applied to serogroup B, which is still a major cause of meningitis. We evaluated the immunogenicity of three surface-exposed proteins from serogroup B Neisseria meningitidis (App, NhhA, and NadA) identified during whole-genome sequencing. Mice were immunized intranasally with individual proteins in the presence of wild-type Escherichia coli heat-labile enterotoxin (LTwt), LTR72, a partially inactivated mutant, or LTK63, a completely nontoxic mutant, as the adjuvant. Each of the meningococcal proteins induced significant cellular responses; NhhA and NadA induced strong antibody responses, but only NadA induced bactericidal antibody when administered intranasally with mucosal adjuvants. In addition, immunoglobulin A and bactericidal antibodies were detected in the respiratory tract following intranasal delivery of NadA. Analysis of antigen-specific cytokine production by T cells from immunized mice revealed that intranasal immunization with NadA alone failed to generate detectable cellular immune responses. In contrast, LTK63, LTR72, and LTwt significantly augmented NadA-specific gamma interferon, interleukin-4 (IL-4), IL-5, and IL-10 production by spleen and lymph node cells, suggesting that both Th1 and Th2 cells were induced in vivo. The strongest cellular responses and highest bactericidal antibody titers were generated with LTR72 as the adjuvant. These findings demonstrate that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the antigen as well as the adjuvant and suggest that nasal delivery of NadA with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.