Association of a functional variant in the Wnt co-receptor LRP6 with early onset ileal Crohn's disease.
ABSTRACT: Ileal Crohn's Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these ?-defensins are exclusively produced in Paneth cells (PCs) at small intestinal crypt bases. Different ileal CD-associated genes including NOD2, ATG16L1, and recently the ?-catenin-dependant Wnt transcription factor TCF7L2 have been linked to impaired PC antimicrobial function. The Wnt pathway influences gut mucosal homeostasis and PC maturation, besides directly controlling HD-5/6 gene expression. The herein reported candidate gene study focuses on another crucial Wnt factor, the co-receptor low density lipoprotein receptor-related protein 6 (LRP6). We analysed exonic single nucleotide polymorphisms (SNPs) in a large cohort (Oxford: n?=?1,893) and prospectively tested 2 additional European sample sets (Leuven: n?=?688, Vienna: n?=?1,628). We revealed an association of a non-synonymous SNP (rs2302685; Ile1062Val) with early onset ileal CD (OR 1.8; p?=?0.00034; for homozygous carriers: OR 4.1; p?=?0.00004) and additionally with penetrating ileal CD behaviour (OR 1.3; p?=?0.00917). In contrast, it was not linked to adult onset ileal CD, colonic CD, or ulcerative colitis. Since the rare variant is known to impair LRP6 activity, we investigated its role in patient mucosa. Overall, LRP6 mRNA was diminished in patients independently from the genotype. Analysing the mRNA levels of PC product in biopsies from genotyped individuals (15 controls, 32 ileal, and 12 exclusively colonic CD), we found particularly low defensin levels in ileal CD patients who were carrying the variant. In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection. Taken together, we identified LRP6 as a new candidate gene in ileal CD. Impairments in Wnt signalling and Paneth cell biology seem to represent pathophysiological hallmarks in small intestinal inflammation and should therefore be considered as interesting targets for new therapeutic approaches.
Project description:Reduced expression of Paneth cell antimicrobial alpha-defensins, human defensin (HD)-5 and -6, characterizes Crohn's disease (CD) of the ileum. TCF-4 (also named TCF7L2), a Wnt signalling pathway transcription factor, orchestrates Paneth cell differentiation, directly regulates the expression of HD-5 and -6, and was previously associated with the decrease of these antimicrobial peptides in a subset of ileal CD. To investigate a potential genetic association of TCF-4 with ileal CD, we sequenced 2.1 kb of the 5' flanking region of TCF-4 in a small group of ileal CD patients and controls (n = 10 each). We identified eight single nucleotide polymorphisms (SNPs), of which three (rs3814570, rs10885394, rs10885395) were in linkage disequilibrium and found more frequently in patients; one (rs3814570) was thereby located in a predicted regulatory region. We carried out high-throughput analysis of this SNP in three cohorts of inflammatory bowel disease (IBD) patients and controls. Overall 1399 healthy individuals, 785 ulcerative colitis (UC) patients, 225 CD patients with colonic disease only and 784 CD patients with ileal involvement were used to determine frequency distributions. We found an association of rs3814570 with ileal CD but neither with colonic CD or UC, in a combined analysis (allele positivity: OR 1.27, 95% CI 1.07 to 1.52, p = 0.00737), which was the strongest in ileal CD patients with stricturing behaviour (allele frequency: OR 1.32, 95% CI 1.08 to1.62, p = 0.00686) or an additional involvement of the upper GIT (allele frequency: OR 1.38, 95% CI 1.03 to1.84, p = 0.02882). The newly identified genetic association of TCF-4 with ileal CD provides evidence that the decrease in Paneth cell alpha-defensins is a primary factor in disease pathogenesis.
Project description:Crohn's disease (CD) is associated with a multitude of genetic defects, many of which likely affect Paneth cell function. Paneth cells reside in the small intestine and produce antimicrobial peptides essential for the host barrier, principally human α-defensin 5 (HD5) and HD6. Patients with CD of the ileum are characterized by reduced constitutive expression of these peptides and, accordingly, compromised antimicrobial barrier function. Here, we present a previously unidentified regulatory mechanism of Paneth cell defensins. Using cultures of human ileal tissue, we showed that the secretome of peripheral blood mononuclear cells (PBMCs) from healthy controls restored the attenuated Paneth cell α-defensin expression characteristic of patients with ileal CD. Analysis of the Wnt pathway in both cultured biopsies and intestinal epithelial cells implicated Wnt ligands driving the PBMC effect, whereas various tested cytokines were ineffective. We further detected another defect in patients with ileal CD, because the PBMC secretomes derived from patients with CD were unable to restore the reduced HD5/HD6 expression. Accordingly, analysis of PBMC subtypes showed that monocytes of patients with CD express significantly lower levels of canonical Wnt ligands, including Wnt3, Wnt3a, Wnt1, and wntless Wnt ligand secretion mediator (Evi/Wls). These studies reveal an important cross-talk between bone marrow-derived cells and epithelial secretory Paneth cells. Defective Paneth cell-mediated innate immunity due to inadequate Wnt ligand stimulation by monocytes provides an additional mechanism in CD. Because defects of Paneth cell function stemming from various etiologies are overcome by Wnt ligands, this mechanism is a potential therapeutic target for this disease.
Project description:The pathogenesis of Crohn's disease (CD), an idiopathic inflammatory bowel disease, is attributed, in part, to intestinal bacteria that may initiate and perpetuate mucosal inflammation in genetically susceptible individuals. Paneth cells (PC) are the major source of antimicrobial peptides in the small intestine, including human alpha-defensins HD5 and HD6. We tested the hypothesis that reduced expression of PC alpha-defensins compromises mucosal host defenses and predisposes patients to CD of the ileum. We report that patients with CD of the ileum have reduced antibacterial activity in their intestinal mucosal extracts. These specimens also showed decreased expression of PC alpha-defensins, whereas the expression of eight other PC products either remained unchanged or increased when compared with controls. The specific decrease of alpha-defensins was independent of the degree of inflammation in the specimens and was not observed in either CD of the colon, ulcerative colitis, or pouchitis. The functional consequence of alpha-defensin expression levels was examined by using a transgenic mouse model, where we found changes in HD5 expression levels, comparable to those observed in CD, had a pronounced impact on the luminal microbiota. Thus, the specific deficiency of PC defensins that characterizes ileal CD may compromise innate immune defenses of the ileal mucosa and initiate and/or perpetuate this disease.
Project description:Paneth cells (PCs) are located at the base of small intestinal crypts and secrete the ?-defensins, human ?-defensin 5 (HD-5) and human ?-defensin 6 (HD-6) in response to bacterial, cholinergic and other stimuli. The ?-defensins are broad-spectrum microbicides that play critical roles in controlling gut microbiota and maintaining intestinal homeostasis. Inflammatory bowel disease, including ulcerative colitis and Crohn's disease (CD), is a complicated autoimmune disorder. The pathogenesis of CD involves genetic factors, environmental factors and microflora. Surprisingly, with regard to genetic factors, many susceptible genes and pathogenic pathways of CD, including nucleotide-binding oligomerization domain 2 (NOD2), autophagy-related 16-like 1 (ATG16L1), immunity-related guanosine triphosphatase family M (IRGM), wingless-related integration site (Wnt), leucine-rich repeat kinase 2 (LRRK2), histone deacetylases (HDACs), caspase-8 (Casp8) and X-box-binding protein-1 (XBP1), are relevant to PCs. As the underlying mechanisms are being unravelled, PCs are identified as the central element of CD pathogenesis, integrating factors among microbiota, intestinal epithelial barrier dysfunction and the immune system. In the present review, we demonstrate how these genes and pathways regulate CD pathogenesis via their action on PCs and what treatment modalities can be applied to deal with these PC-mediated pathogenic processes.
Project description:Paneth cells, specialized secretory epithelial cells of the small intestine, play a pivotal role in host defense and regulation of microbiota by producing antimicrobial peptides especially-but not only-the human α-defensin 5 (HD5) and HD6. In small intestinal Crohn's disease (CD) which is an entity of inflammatory bowel diseases, the expression of HD5 and HD6 is specifically compromised leading to a disturbed barrier and change in the microbial community. Different genetically driven but also non-genetic defects associated with small intestinal CD affect different lines of antimicrobial Paneth cell functions. In this review, we focus on the mechanisms and the crosstalk of Paneth cells and bone marrow-derived cells and highlight recent studies about the role of the Wnt signaling pathway in this connection of ileal CD. In summary, different lines of investigations led by us but also now numerous other groups support and reconfirm the proposed classification of this disease entity as Paneth's disease.
Project description:<h4>Objective</h4>Reduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn's disease (CD). PCs reside in proximity to Lgr5<sup>+</sup> intestinal stem cells (ISC) and mitochondria are critical for ISC-renewal and differentiation. Here, we characterise ISC and PC appearance under inflammatory conditions and describe the role of mitochondrial function for ISC niche-maintenance.<h4>Design</h4>Ileal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60<sup>?/?ISC</sup>) and CD-like ileitis (TNF<sup>?ARE</sup>), and intestinal organoids were used to characterise PCs and ISCs in relation to mitochondrial function.<h4>Results</h4>In patients with CD and TNF<sup>?ARE</sup> mice, inflammation correlated with reduced numbers of Lysozyme-positive granules in PCs and decreased <i>Lgr5</i> expression in crypt regions. Disease-associated changes in PC and ISC appearance persisted in non-inflamed tissue regions of patients with CD and predicted the risk of disease recurrence after surgical resection. ISC-specific deletion of Hsp60 and inhibition of mitochondrial respiration linked mitochondrial function to the aberrant PC phenotype. Consistent with reduced stemness in vivo, crypts from inflamed TNF<sup>?ARE</sup> mice fail to grow into organoids ex vivo. Dichloroacetate-mediated inhibition of glycolysis, forcing cells to shift to mitochondrial respiration, improved ISC niche function and rescued the ability of TNF<sup>?ARE</sup> mice-derived crypts to form organoids.<h4>Conclusion</h4>We provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.
Project description:Ileal Crohn's disease (CD) arising from the alteration of intestinal homeostasis is characterized by two features, namely a decrease in Paneth cell-produced antimicrobial peptides that play a key role in maintaining this balance and an increase in NOD2, an intracellular sensor. Although mutations in NOD2 are highly correlated with the incidence of CD, the physiological role of NOD2 in intestinal immunity remains elusive. Here, we show that NOD2 can down-regulate the expression of human enteric antimicrobial peptides during differentiation of the Paneth cell lineage. This finding, which links the decrease of human enteric antimicrobial peptides to increased NOD2 in ileal CD patients, provides a new view into the pathogenesis of ileal CD.
Project description:Intelectins (intestinal lectins) are highly conserved across chordate evolution and have been implicated in various human diseases, including Crohn's disease (CD). The human genome encodes two intelectin genes, intelectin-1 (ITLN1) and intelectin-2 (ITLN2). Other than its high sequence similarity with ITLN1, little is known about ITLN2. To address this void in knowledge, we report that ITLN2 exhibits discrete, yet notable differences from ITLN1 in primary structure, including a unique amino terminus, as well as changes in amino acid residues associated with the glycan-binding activity of ITLN1. We identified that ITLN2 is a highly abundant Paneth cell-specific product, which localizes to secretory granules, and is expressed as a multimeric protein in the small intestine. In surgical specimens of ileal CD, ITLN2 mRNA levels were reduced approximately five-fold compared to control specimens. The ileal expression of ITLN2 was unaffected by previously reported disease-associated variants in ITLN2 and CD-associated variants in neighboring ITLN1 as well as NOD2 and ATG16L1. ITLN2 mRNA expression was undetectable in control colon tissue; however, in both ulcerative colitis (UC) and colonic CD, metaplastic Paneth cells were found to express ITLN2. Together, the data reported establish the groundwork for understanding ITLN2 function(s) in the intestine, including its possible role in CD.
Project description:BACKGROUND:Paneth cell dysfunction has been implicated in a subset of Crohn's disease (CD) patients. We previously stratified clinical outcomes of CD patients by using Paneth cell phenotypes, which we defined by the intracellular distribution of antimicrobial proteins. Animal studies suggest that Paneth cells shape the intestinal microbiome. However, it is unclear whether Paneth cell phenotypes alter the microbiome complexity in CD subjects. Therefore, we analyzed the correlation of Paneth cell phenotypes with mucosal microbiome composition and ileal RNA expression in pediatric CD and noninflammatory bowel disease (non-IBD) patients. METHODS:Pediatric CD (n = 44) and non-IBD (n = 62) patients aged 4 to 18 were recruited prior to routine endoscopic biopsy. Ileal mucosal samples were analyzed for Paneth cell phenotypes, mucosal microbiome composition, and RNA transcriptome. RESULTS:The prevalence of abnormal Paneth cells was higher in pediatric versus adult CD cohorts. For pediatric CD patients, those with abnormal Paneth cells showed significant changes in their ileal mucosal microbiome, highlighted by reduced protective microbes and enriched proinflammatory microbes. Ileal transcriptome profiles showed reduced transcripts for genes that control oxidative phosphorylation in CD patients with abnormal Paneth cells. These transcriptional changes in turn were correlated with specific microbiome alterations. In non-IBD patients, a subset contained abnormal Paneth cells. However, this subset was not associated with alterations in the microbiome or host transcriptome. CONCLUSION:Paneth cell abnormalities in human subjects are associated with mucosal dysbiosis in the context of CD, and these changes are associated with alterations in oxidative phosphorylation, potentially in a feedback loop. FUNDING:The research was funded by Helmsley Charitable Trust (to T.S. Stappenbeck, R.J. Xavier, and D.P.B. McGovern), Crohn's and Colitis Foundation of America (to N.H. Salzman, T.S. Stappenbeck, R.J. Xavier, and C. Huttenhower), and Doris Duke Charitable Foundation grant 2014103 (to T.C. Liu).
Project description:Enteric ?-defensins, termed cryptdins (Crps) in mice, and lysozymes secreted by Paneth cells contribute to innate host defense in the ileum. Antimicrobial factors including lysozymes and ?-defensins are often embedded in luminal glycosylated colonic Muc2 mucin secreted by goblet cells that form the protective mucus layer critical in gut homeostasis and pathogen invasion. In this study we investigated ileal innate immunity against Entamoeba histolytica (Eh), the causative agent of intestinal amebiasis, by inoculating parasites in closed ileal loops in Muc2+/+ and Muc2-/- littermates and quantifying Paneth cell localization (lysozyme expression) and function (Crps secretion). Relative to Muc2+/+ littermates, Muc2-/- showed disorganized mislocalization of Paneth cells that was diffusely distributed with elevated lysozyme secretion in the crypts and on villi in response to Eh Inhibiting Eh Gal-lectin binding with exogenous galactose and EhCP5- Eh had no effect on parasite-induced erratic Paneth cell lysozyme synthesis. Although basal ileal expression of Crp genes was unaffected in Muc2-/- mice in response to Eh there was robust release of pro-inflammatory cytokines and Crp peptide secretions in luminal exudates that was also present in the colon. Interestingly, Eh secreted cysteine proteinases cleaved the pro-region of Crp 4 but not the active form. These findings define Muc2 mucin as an essential component of ileal barrier function that regulates localization and function of Paneth cells critical in host defense against microbes.