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IntroductionNecrotizing enterocolitis (NEC) is a devastating disease of premature infants. Probiotics decrease the risk of NEC in clinical and experimental studies. Antimicrobial peptides protect the gut against noxious microbes and shape the commensal microbiota, but their role in NEC remains unclear.MethodsTo investigate the expression of antimicrobial peptides in experimental NEC and the impact of probiotics on their expression, premature rats were divided into three groups: dam fed (DF), hand fed with formula (FF), or hand fed with formula containing Bifidobacterium bifidum (FF + BIF). All groups were exposed to asphyxia and cold stress.ResultsLike in human ontogeny, the rat pup has low expression of Paneth cell antimicrobials, which increases rapidly during normal development. The expression of lysozyme, secretory phospholipase A(2) (sPLA(2)), pancreatic-associated proteins 1 and 3 mRNA was elevated in the FF group with a high incidence of NEC, as compared with the DF and FF + BIF groups where the disease was attenuated.DiscussionWe conclude that induction of antimicrobial peptides occurs in experimental NEC similar to that reported in human disease and is attenuated when disease is averted by probiotic B. bifidum. The induction of antimicrobial peptides is likely an adaptive mucosal response that is often not sufficient to prevent disease in the premature gut.

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.

BackgroundThe small intestinal epithelium mediates vital functions of nutrient absorption and host defense. The spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. However, the differences in transcriptional programming and the molecular machinery that governs the migration, adhesion, and differentiation of intestinal epithelial cell lineages in humans remain under-explored. To increase our understanding of these mechanisms, we have evaluated gene expression patterns of ileal epithelial cells isolated by laser capture microdissection from either the villus epithelial or crypt cell regions of healthy human small intestinal mucosa. Expression profiles in villus and crypt epithelium were determined by DNA microarray, quantitative real-time PCR, and immunohistochemistry based methods. The expression levels of selected epithelial biomarkers were also compared between gastrointestinal tissues.ResultsPreviously established biomarkers as well as a novel and distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation were found to be enriched in each of the two corresponding cell populations (GEO accession: GSE10629). Additionally, high baseline expression levels of innate antimicrobials, alpha defensin 5 (HD5) and regenerating islet-derived 3 alpha (Reg3A), were detected exclusively within the small bowel crypt, most notably in the ileum in comparison to other sites along the gastrointestinal tract.ConclusionThe elucidation of differential gene expression patterns between crypt and villus epithelial cell lineages in human ileal tissue provides novel insights into the molecular machinery that mediates their functions and spatial organization. Moreover, our findings establish an important framework of knowledge for future investigations of human gastrointestinal diseases.

Little is known about the defensive mechanisms induced in epithelial cells by pathogenic versus probiotic bacteria. The aim of our study was to compare probiotic bacterial strains such as Escherichia coli Nissle 1917 with nonprobiotic, pathogenic and nonpathogenic bacteria with respect to innate defense mechanisms in the intestinal mucosal cell. Here we report that E. coli strain Nissle 1917 and a variety of other probiotic bacteria, including lactobacilli-in contrast to more than 40 different E. coli strains tested-strongly induce the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) in Caco-2 intestinal epithelial cells in a time- and dose-dependent manner. Induction of hBD-2 through E. coli Nissle 1917 was further confirmed by activation of the hBD-2 promoter and detection of the hBD-2 peptide in the culture supernatants of E. coli Nissle 1917-treated Caco-2 cells. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for induction of hBD-2 through E. coli Nissle 1917. Treatment with the NF-kappaB inhibitor Helenalin, as well as with SP600125, a selective inhibitor of c-Jun N-terminal kinase, blocked hBD-2 induction by E. coli Nissle 1917 in Caco-2 cells. SB 202190, a specific p38 mitogen-activated protein kinase inhibitor, and PD 98059, a selective inhibitor of extracellular signal-regulated kinase 1/2, were ineffective. This report demonstrates that probiotic bacteria may stimulate the intestinal innate defense through the upregulation of inducible antimicrobial peptides such as hBD-2. The induction of hBD-2 may contribute to an enhanced mucosal barrier to the luminal bacteria.

Obesity and metabolic comorbidities are associated with gut permeability. While high-fructose and Western-style diet (WSD) disrupt intestinal barrier function, oral administration of human α-defensin 5 (HD5) and β-defensin 2 (hBD2) is believed to improve intestinal integrity and metabolic disorders. Eighty-four male C57BL/6J mice were fed a WSD or a control diet (CD) ± fructose (F) for 18 weeks. In week 13, mice were randomly divided into three intervention groups, receiving defensin fragment HD51–9, full-length hBD2, or bovine serum albumin (BSA)-control for six weeks. Subsequently, parameters of hepatic steatosis, glucose metabolism, and gut barrier function were assessed. WSDF increased body weight and hepatic steatosis (p < 0.01) compared to CD-fed mice, whereas peptide intervention decreased liver fat (p < 0.05) and number of hepatic lipid droplets (p < 0.01) compared to BSA-control. In addition, both peptides attenuated glucose intolerance by reducing blood glucose curves in WSDF-fed mice. Evaluation of gut barrier function revealed that HD51–9 and hBD2 improve intestinal integrity by upregulating tight junction and mucin expression. Moreover, peptide treatment restored ileal host defense peptides (HDP) expression, likely by modulating the Wnt, Myd88, p38, and Jak/STAT pathways. These findings strongly suggest that α- and β-defensin treatment improve hepatic steatosis, glucose metabolism, and gut barrier function. [ABSTRACT FROM AUTHOR]

Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota (1,2). In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon (3). At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of -200 mV to -300 mV in the colon (4). Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces (5-8). Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity (9,10). Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Grampositive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system (11) is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia., We modified the radial diffusion assay12 to analyse antimicrobial activity of synthetic hBD-1 against anaerobic bacteria of the normal flora under anaerobic conditions. Increasing concentrations of the reducing agent dithiothreitol [...]