Robert Häsler

A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1.

We performed a genome-wide association study of 19,779 nonsynonymous SNPs in 735 individuals with Crohn disease and 368 controls. A total of 7,159 of these SNPs were informative. We followed up on all 72 SNPs with P ≤ 0.01 with an allele-based disease association test in 380 independent Crohn disease trios, 498 Crohn disease singleton cases and 1,032 controls. Disease association of rs2241880 in the autophagy-related 16-like 1 gene (ATG16L1) was replicated in these samples (P = 4.0 × 10−8) and confirmed in a UK case-control sample (P = 0.0004). By haplotype and regression analysis, we found that marker rs2241880, a coding SNP (T300A), carries virtually all the disease risk exerted by the ATG16L1 locus. The ATG16L1 gene encodes a protein in the autophagosome pathway that processes intracellular bacteria. We found a statistically significant interaction with respect to Crohn disease risk between rs2241880 and the established CARD15 susceptibility variants (P = 0.039). Together with the lack of association between rs2241880 and ulcerative colitis (P > 0.4), these data suggest that the underlying biological process may be specific to Crohn disease.

Twin Study Indicates Loss of Interaction Between Microbiota and Mucosa of Patients With Ulcerative Colitis

BACKGROUND & AIMS Interactions between genetic and environmental factors are believed to be involved in onset and initiation of inflammatory bowel disease. We analyzed the interaction between gastrointestinal mucosal microbiota and host genes in twin pairs discordant for ulcerative colitis (UC) to study the functional interaction between microbiota and mucosal epithelium. METHODS Biopsy were collected from sigmoid colon of UC patients and their healthy twins (discordant twin pairs) and from twins without UC. Microbiota profiles were determined from analysis of 16S ribosomal DNA libraries; messenger RNA profiles were determined by microarray analysis. RESULTS Patients with UC had dysbiotic microbiota, characterized by less bacterial diversity and more Actinobacteria and Proteobacteria than that of their healthy siblings; healthy siblings from discordant twins had more bacteria from the Lachnospiraceae and Ruminococcaceae families than twins who were both healthy. In twins who were both healthy, 34 mucosal transcripts correlated with bacterial genera, whereas only 25 and 11 correlated with bacteria genera in healthy individuals and their twins with UC, respectively. Transcripts related to oxidative and immune responses were differentially expressed between patients with UC and their healthy twins. CONCLUSIONS The transcriptional profile of the mucosa appears to interact with the colonic microbiota; this interaction appears to be lost in colon of patients with UC. Bacterial functions, such as butyrate production, might affect mucosal gene expression. Patients with UC had different gene expression profiles and lower levels of biodiversity than their healthy twins, as well as unusual aerobic bacteria. Patients with UC had lower percentages of potentially protective bacterial species than their healthy twins.

Nod2 is essential for temporal development of intestinal microbial communities

Objective The mammalian commensal gut microbiota is highly diverse and displays an individual-specific composition determined by host genotype and environmental factors. The temporal development of host–microbial homeostasis in the digestive tract is recognised as a major function of the immune system. However, the underlying cellular and molecular mechanisms are just beginning to come to light. Nucleotide-binding, oligomerisation domain 2 (NOD2) recognises bacterial muramyl dipeptide and is regarded as a pivotal sensor molecule of the intestinal barrier. The aim of this study was to investigate its influence on the development and composition of the intestinal microbiota using a Nod2-deficient mouse model. Methods The dynamics of faecal and ileal microbial composition were investigated in Nod2+/+and Nod2−/− mice on a C57BL/6J background. We assessed microbial diversity and composition using 16S ribosomal RNA gene-based clone library sequencing and high throughput pyrosequencing and quantified the observed changes by real-time PCR. Changes in the major bacterial phyla were investigated in human samples by quantitative real-time PCR. Results We found that adult Nod2-deficient mice display a substantially altered microbial community structure and a significantly elevated bacterial load in their faeces and terminal ileum compared to their wild-type counterparts. Interestingly, we demonstrate that these findings are also present in weaning mice, indicating a profound influence of Nod2 on the early development and composition of the intestinal microbiota. We demonstrate that NOD2 genotypes also influence the microbial composition in humans. Conclusions Our results point to an essential role of Nod2 for the temporal development and composition of the host microbiota, both in mice and in humans, which may contribute to the complex role of NOD2 for the aetiopathogenesis of Crohns disease.

G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation.

Molecular danger signals attract neutrophilic granulocytes (polymorphonuclear leukocytes (PMNs)) to sites of infection. The G protein-coupled receptor (GPR) 43 recognizes propionate and butyrate and is abundantly expressed on PMNs. The functional role of GPR43 activation for in vivo orchestration of immune response is unclear. We examined dextrane sodium sulfate (DSS)-induced acute and chronic intestinal inflammatory response in wild-type and Gpr43-deficient mice. The severity of colonic inflammation was assessed by clinical signs, histological scoring, and cytokine production. Chemotaxis of wild-type and Gpr43-deficient PMNs was assessed through transwell cell chemotactic assay. A reduced invasion of PMNs and increased mortality due to septic complications were observed in acute DSS colitis. In chronic DSS colitis, Gpr43−/− animals showed diminished PMN intestinal migration, but protection against inflammatory tissue destruction. No significant difference in PMN migration and cytokine secretion was detected in a sterile inflammatory model. Ex vivo experiments show that GPR43-induced migration is dependent on activation of the protein kinase p38α, and that this signal acts in cooperation with the chemotactic cytokine keratinocyte chemoattractant. Interestingly, shedding of L-selectin in response to propionate and butyrate was compromised in Gpr43−/− mice. These results indicate a critical role for GPR43-mediated recruitment of PMNs in containing intestinal bacterial translocation, yet also emphasize the bipotential role of PMNs in mediating tissue destruction in chronic intestinal inflammation.

Colonic mucosa-associated microbiota is influenced by an interaction of Crohn disease and FUT2 (Secretor) genotype

The FUT2 (Secretor) gene is responsible for the presence of ABO histo-blood group antigens on the gastrointestinal mucosa and in bodily secretions. Individuals lacking a functional copy of FUT2 are known as “nonsecretors” and display an array of differences in susceptibility to infection and disease, including Crohn disease. To determine whether variation in resident microbial communities with respect to FUT2 genotype is a potential factor contributing to susceptibility, we performed 454-based community profiling of the intestinal microbiota in a panel of healthy subjects and Crohn disease patients and determined their genotype for the primary nonsecretor allele in Caucasian populations, W143X (G428A). Consistent with previous studies, we observe significant deviations in the microbial communities of individuals with Crohn disease. Furthermore, the FUT2 genotype explains substantial differences in community composition, diversity, and structure, and we identified several bacterial species displaying disease-by-genotype associations. These findings indicate that alterations in resident microbial communities may in part explain the variety of host susceptibilities surrounding nonsecretor status and that FUT2 is an important genetic factor influencing host–microbial diversity.

Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice.

The protease a disintegrin and metalloprotease (ADAM) 17 cleaves tumor necrosis factor (TNF), L-selectin, and epidermal growth factor receptor (EGF-R) ligands from the plasma membrane. ADAM17 is expressed in most tissues and is up-regulated during inflammation and cancer. ADAM17-deficient mice are not viable. Conditional ADAM17 knockout models demonstrated proinflammatory activities of ADAM17 in septic shock via shedding of TNF. We used a novel gene targeting strategy to generate mice with dramatically reduced ADAM17 levels in all tissues. The resulting mice called ADAM17ex/ex were viable, showed compromised shedding of ADAM17 substrates from the cell surface, and developed eye, heart, and skin defects as a consequence of impaired EGF-R signaling caused by failure of shedding of EGF-R ligands. Unexpectedly, although the intestine of unchallenged homozygous ADAM17ex/ex mice was normal, ADAM17ex/ex mice showed substantially increased susceptibility to inflammation in dextran sulfate sodium colitis. This was a result of impaired shedding of EGF-R ligands resulting in failure to phosphorylate STAT3 via the EGF-R and, consequently, in defective regeneration of epithelial cells and breakdown of the intestinal barrier. Besides regulating the systemic availability of the proinflammatory cytokine TNF, our results demonstrate that ADAM17 is needed for vital regenerative activities during the immune response. Thus, our mouse model will help investigate ADAM17 as a potential drug target.

DNA Methylation Analysis in Nonalcoholic Fatty Liver Disease Suggests Distinct Disease-Specific and Remodeling Signatures after Bariatric Surgery

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries. Liver samples from morbidly obese patients (n = 45) with all stages of NAFLD and controls (n = 18) were analyzed by array-based DNA methylation and mRNA expression profiling. NAFLD-specific expression and methylation differences were seen for nine genes coding for key enzymes in intermediate metabolism (including PC, ACLY, and PLCG1) and insulin/insulin-like signaling (including IGF1, IGFBP2, and PRKCE) and replicated by bisulfite pyrosequening (independent n = 39). Transcription factor binding sites at NAFLD-specific CpG sites were >1,000-fold enriched for ZNF274, PGC1A, and SREBP2. Intraindividual comparison of liver biopsies before and after bariatric surgery showed NAFLD-associated methylation changes to be partially reversible. Postbariatric and NAFLD-specific methylation signatures were clearly distinct both in gene ontology and transcription factor binding site analyses, with >400-fold enrichment of NRF1, HSF1, and ESRRA sites. Our findings provide an example of treatment-induced epigenetic organ remodeling in humans.

Genome-wide association study for ulcerative colitis identifies risk loci at 7q22 and 22q13 (IL17REL)

We performed a genome-wide association analysis of 1,897,764 SNPs in 1,043 German ulcerative colitis (UC) cases and 1,703 controls. We discovered new associations at chromosome 7q22 (rs7809799) and at chromosome 22q13 in IL17REL (rs5771069) and confirmed these associations in six replication panels (2,539 UC cases and 5,428 controls) from different regions of Europe (overall study sample Prs7809799 = 8.81 × 10−11 and Prs5771069 = 4.21 × 10−8, respectively).

The Nucleotide-Binding Oligomerization Domain-Like Receptor NLRC5 Is Involved in IFN-Dependent Antiviral Immune Responses

Nucleotide-binding oligomerization domain-like receptors (NLRs) are a group of intracellular proteins that mediate recognition of pathogen-associated molecular patterns or other cytosolic danger signals. Mutations in NLR genes have been linked to a variety of inflammatory diseases, underscoring their pivotal role in host defense and immunity. This report describes the genomic organization and regulation of the human NLR family member NLRC5 and aspects of cellular function of the encoded protein. We have analyzed the tissue-specific expression of NLRC5 and have characterized regulatory elements in the NLRC5 promoter region that are responsive to IFN-γ. We show that NLRC5 is upregulated in human fibroblasts postinfection with CMV and demonstrate the role of a JAK/STAT-mediated autocrine signaling loop involving IFN-γ. We demonstrate that overexpression and enforced oligomerization of NLRC5 protein results in activation of the IFN-responsive regulatory promoter elements IFN-γ activation sequence and IFN-specific response element and upregulation of antiviral target genes (e.g., IFN-α, OAS1, and PRKRIR). Finally, we demonstrate the effect of small interfering RNA-mediated knockdown of NLRC5 on a target gene level in the context of viral infection. We conclude that NLRC5 may represent a molecular switch of IFN-γ activation sequence/IFN-specific response element signaling pathways contributing to antiviral defense mechanisms.

Genome-wide association analysis identifies variation in vitamin D receptor and other host factors influencing the gut microbiota

Human gut microbiota is an important determinant for health and disease, and recent studies emphasize the numerous factors shaping its diversity. Here we performed a genome-wide association study (GWAS) of the gut microbiota using two cohorts from northern Germany totaling 1,812 individuals. Comprehensively controlling for diet and non-genetic parameters, we identify genome-wide significant associations for overall microbial variation and individual taxa at multiple genetic loci, including the VDR gene (encoding vitamin D receptor). We observe significant shifts in the microbiota of Vdr−/− mice relative to control mice and correlations between the microbiota and serum measurements of selected bile and fatty acids in humans, including known ligands and downstream metabolites of VDR. Genome-wide significant (P < 5 × 10−8) associations at multiple additional loci identify other important points of host–microbe intersection, notably several disease susceptibility genes and sterol metabolism pathway components. Non-genetic and genetic factors each account for approximately 10% of the variation in gut microbiota, whereby individual effects are relatively small.