Paneth cell α-defensins HD-5 and HD-6 display differential degradation into active antimicrobial fragments.

Antimicrobial peptides, in particular α-defensins expressed by Paneth cells, control microbiota composition and play a key role in intestinal barrier function and homeostasis. Dynamic conditions in the local microenvironment, such as pH and redox potential, significantly affect the antimicrobial spectrum. In contrast to oxidized peptides, some reduced defensins exhibit increased vulnerability to proteolytic degradation. In this report, we investigated the susceptibility of Paneth-cell-specific human α-defensin 5 (HD-5) and -6 (HD-6) to intestinal proteases using natural human duodenal fluid. We systematically assessed proteolytic degradation using liquid chromatography-mass spectrometry and identified several active defensin fragments capable of impacting bacterial growth of both commensal and pathogenic origins. Of note, incubation of mucus with HD-5 resulted in 255-8,000 new antimicrobial combinations. In contrast, HD-6 remained stable with consistent preserved nanonet formation. In vivo studies demonstrated proof of concept that a HD-5 fragment shifted microbiota composition (e.g., increases of Akkermansia sp.) without decreasing diversity. Our data support the concept that secretion of host peptides results in an environmentally dependent increase of antimicrobial defense by clustering in active peptide fragments. This complex clustering mechanism dramatically increases the host's ability to control pathogens and commensals. These findings broaden our understanding of host modulation of the microbiome as well as the complexity of human mucosal defense mechanisms, thus providing promising avenues to explore for drug development.

Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial ß-Defensin 1.

The antimicrobial peptide human ß-defensin 1 (hBD1) is continuously produced by epithelial cells in many tissues. Compared to other defensins, hBD1 has only minor antibiotic activity in its native state. After reduction of its disulfide bridges, however, it becomes a potent antimicrobial agent against bacteria, while the oxidized native form (hBD1ox) shows specific activity against Gram-negative bacteria. We show that the killing mechanism of hBD1ox depends on aerobic growth conditions and bacterial enzymes. We analyzed the different activities of hBD1 using mutants of Escherichia coli lacking one or more specific proteins of their outer membrane, cytosol, or redox systems. We discovered that DsbA and DsbB are essential for the antimicrobial activity of hBD1ox but not for that of reduced hBD1 (hBD1red). Furthermore, our results strongly suggest that hBD1ox uses outer membrane protein FepA to penetrate the bacterial periplasm space. In contrast, other bacterial proteins in the outer membrane and cytosol did not modify the antimicrobial activity. Using immunogold labeling, we identified the localization of hBD1ox in the periplasmic space and partly in the outer membrane of E. coli However, in resistant mutants lacking DsbA and DsbB, hBD1ox was detected mainly in the bacterial cytosol. In summary, we discovered that hBD1ox could use FepA to enter the periplasmic space, where its activity depends on presence of DsbA and DsbB. HBD1ox concentrates in the periplasm in Gram-negative bacteria, which finally leads to bleb formation and death of the bacteria. Thus, the bacterial redox system plays an essential role in mechanisms of resistance against host-derived peptides such as hBD1.

Chronic Heavy Alcohol Use is Associated with Upregulated Paneth Cell Antimicrobials in Gastric Mucosa.

OBJECTIVES: How alcohol consumption affects the integrity and the defense mechanisms of the mucosa in the upper gastrointestinal tract is largely unknown. We examined the effect of heavy alcohol use on gastric and duodenal Paneth-cell-derived and epithelial antimicrobial peptides (AMPs), cytokines, and the Wnt pathway, an important regulator of epithelial regeneration. METHODS: In 22 patients with heavy alcohol use and 17 control subjects, biopsies from gastric corpus, antrum, and duodenum were examined for messenger RNA (mRNA) of AMPs, cytokines, and Wnt pathway factors using real-time PCR. The expression of the α-defensin HD5 was analyzed immunohistochemically. The effect of alcohol exposure on Wnt signaling and AMP production was also studied in a gastric cell line using mRNA and reporter gene assays. RESULTS: Heavy alcohol use was associated with increased expression of Paneth cell HD5 and HD6 mRNA in the antrum, where these products are normally absent (HD5 mRNA in controls vs. PATIENTS: 2100±900 and 365 500±161 600, HD6 mRNA: 320±130 and 58 300±32 600 copies per 10 ng total RNA, means±s.e.m., P value: 0.022 and 0.011). Upregulated HD5 was independent of intestinal metaplasia that was observed in a minority of patients. No significant differences were found for ß-defensins and cytokines (interleukins IL1ß, IL6, IL8, IL10). In patients, Wnt pathway factors showed a trend toward higher levels. In vitro, ethanol exposure induced the production of HD5 and HD6 and activation of the Wnt pathway. CONCLUSIONS: Alcohol exposure can induce gastric Paneth cell AMP expression. This may be linked to Wnt pathway activation, which has an important role in the epithelial regenerative homeostasis.

Paneth cell α-defensin 6 (HD-6) is an antimicrobial peptide.

Defensins protect human barriers from commensal and pathogenic microorganisms. Human α-defensin 6 (HD-6) is produced exclusively by small intestinal Paneth cells but, in contrast to other antimicrobial peptides (AMPs) for HD-6, no direct antibacterial killing activity has been detected so far. Herein, we systematically tested how environmental factors, like pH and reducing conditions, affect antimicrobial activity of different defensins against anaerobic bacteria of the human intestinal microbiota. Remarkably, by mimicking the intestinal milieu we detected for the first time antibacterial activity of HD-6. Activity was observed against anaerobic gut commensals but not against some pathogenic strains. Antibiotic activity was attributable to the reduced peptide and independent of free cysteines or a conserved histidine residue. Furthermore, the oxidoreductase thioredoxin, which is also expressed in Paneth cells, is able to reduce a truncated physiological variant of HD-6. Ultrastructural analyses revealed that reduced HD-6 causes disintegration of cytoplasmic structures and alterations in the bacterial cell envelope, while maintaining extracellular net-like structures. We conclude that HD-6 is an antimicrobial peptide. Our data suggest two distinct antimicrobial mechanisms by one peptide: HD-6 kills specific microbes depending on the local environmental conditions, whereas known microbial trapping by extracellular net structures is independent of the reducing milieu.

Cell-mediated reduction of human ß-defensin 1: a major role for mucosal thioredoxin.

Human ß-defensin 1 (hBD-1) is an antimicrobial peptide expressed by epithelia and hematopoietic cells. We demonstrated recently that hBD-1 shows activity against enteric commensals and Candida species only after its disulfide bonds have been reduced by thioredoxin (TRX) or a reducing environment. Here we show that besides TRX, glutaredoxin (GRX) is also able to reduce hBD-1, although with far less efficacy. Moreover, living intestinal and lymphoid cells can effectively catalyze reduction of extracellular hBD-1. By chemical inhibition of the TRX system or specific knockdown of TRX, we demonstrate that cell-mediated reduction is largely dependent on TRX. Quantitative PCR in intestinal tissues of healthy controls and inflammatory bowel disease patients revealed altered expression of some, although not all, redox enzymes, especially in ulcerative colitis. Reduced hBD-1 and TRX localize to extracellular colonic mucus, suggesting that secreted or membrane-bound TRX converts hBD-1 to a potent antimicrobial peptide in vivo.

[Human beta-defensin 1: from defence to offence]. / Humanes Beta-Defensin 1: Aus der Defensive gelockt.

The human gut is colonised by about one kilogram of commensal bacteria. These microorganisms are a potential threat, thus an efficient defence system is crucial in preventing bacterial translocation and infection. Besides other mechanisms of protection humans produce antimicrobial peptides (AMPs) that are able to kill a broad range of microorganisms. The human beta-defensin 1 (hBD-1) plays a major role because it is produced constitutively by all human epithelia and some immune cells. In contrast to other AMPs, however, the biological function of hBD-1 has remained unclear since the antibiotic activity of hBD-1 in vitro was only marginal. But still, several diseases have been associated with genetic polymorphisms in the hBD-1 encoding gene. Herein we discuss why the biological role of hBD-1 has been overlooked and how hBD-1 can be activated by chemical reduction. We elaborate on the biological significance of this activation and its importance for inflammatory bowel disease.

Innate immune dysfunction in inflammatory bowel disease.

The pathogenetic mechanisms that cause the two types of inflammatory bowel disease (IBD), Crohn's disease (CD) and ulcerative colitis (UC), are still under investigation. Nevertheless, there is broad agreement that luminal microbes are of particular relevance in the development of these conditions. In recent years, increasing evidence has shown that defects in the innate immunity are at the centre of both types of IBD. The innate intestinal barrier is provided by the epithelium which secretes antimicrobial peptides (so-called defensins) that are retained in the mucus layer. In ileal CD, the alpha-defensins are lacking owing to several Paneth cell defects. In colonic CD, the expression of beta-defensins is inadequate. This may be related to downregulation of the transcription factor peroxisome proliferator-activated receptor-gamma and in some cohorts is associated with a reduced HBD2 gene copy number. In UC, the mucus layer, which protects the host from the enormous amounts of luminal microbes, is defective. This is accompanied by an insufficient differentiation from intestinal stem cells towards goblet cells. All these disturbances in the gut barrier shift the balance from epithelial defence towards bacterial offence. The current treatment for CD and UC is based on suppression of this secondary inflammatory process. In future, patients may benefit from new therapeutic approaches stimulating the protective innate immune system.

Infliximab as rescue medication for patients with severe ulcerative/indeterminate colitis refractory to tacrolimus.

BACKGROUND: The calcineurin inhibitor tacrolimus and the anti-TNF-antibody infliximab are established options in steroid-refractory ulcerative colitis (UC). AIM: To evaluate the efficacy of infliximab-salvage therapy in patients with refractory UC failing to respond to tacrolimus. METHODS: Twenty-four patients were enrolled in this evaluation. Reasons for tacrolimus therapy were steroid-refractory disease in 19 patients and steroid-dependency in five patients. All patients receiving infliximab had tacrolimus-refractory active disease (Lichtiger score >10) and were treated with 5 mg/kg at weeks 0, 2 and 6 and every 8 weeks thereafter, if tolerated. RESULTS: Six of 24 patients (25%) achieved remission following infliximab infusion and four of 24 (17%) had an initial response only, but underwent proctocolectomy later because of loss of response (3) or development of a delayed hypersensitivity reaction (1). Fourteen patients (58%) completely failed to respond with 10 undergoing colectomy. Eight patients experienced side effects under infliximab, including two infectious complications (herpes zoster and herpes pneumonia). CONCLUSIONS: Infliximab offers a therapeutic option as rescue therapy in about a quarter of patients with active UC after failing to respond to tacrolimus. This benefit has to be weighed against the risks of infectious complications.

A novel role for constitutively expressed epithelial-derived chemokines as antibacterial peptides in the intestinal mucosa.

Intestinal-derived chemokines have a central role in orchestrating immune cell influx into the normal and inflamed intestine. Here, we identify the chemokine CCL6 as one of the most abundant chemokines constitutively expressed by both murine small intestinal and colonic epithelial cells. CCL6 protein localized to crypt epithelial cells, was detected in the gut lumen and reached high concentrations at the mucosal surface. Its expression was further enhanced in the small intestine following in vivo administration of LPS or after stimulation of the small intestinal epithelial cell line, mIC(c12), with IFNgamma, IL-4 or TNFalpha. Recombinant- and intestinal-derived CCL6 bound to a subset of the intestinal microflora and displayed antibacterial activity. Finally, the human homologs to CCL6, CCL14 and CCL15 were also constitutively expressed at high levels in human intestinal epithelium, were further enhanced in inflammatory bowel disease and displayed similar antibacterial activity. These findings identify a novel role for constitutively expressed, epithelial-derived chemokines as antimicrobial peptides in the intestinal mucosa.

Influence of standard treatment on ileal and colonic antimicrobial defensin expression in active Crohn's disease.

BACKGROUND: Crohn's Disease (CD), a chronic intestinal inflammation, is currently treated primarily by therapeutics which are directed against inflammatory responses. Recent findings though suggest a central role of the innate immune barrier in the pathophysiology. Important factors providing this barrier are antimicrobial peptides like the alpha- and beta-defensins. Little is known about in vivo effects of common drugs on their expression. AIM: To analyse the influence of corticosteroids, azathioprine and aminosalicylate treatment on ileal and colonic antimicrobial peptides in active CD and also assess the role of inflammation. METHODS: We measured the expression of antimicrobial peptides and pro-inflammatory cytokines in 75 patients with active CD. RESULTS: Ileal and colonic alpha- and beta-defensins as well as LL37 remained unaffected by corticosteroids, azathioprine or aminosalicylate treatment. Additionally, we did not observe a negative coherency between Paneth cell alpha-defensins and any measured cytokines. HBD2 and LL37 unlike HBD1 levels were linked to inflammatory cytokines and increased in highly inflamed samples. CONCLUSIONS: Current oral drug treatment seems to have no major effect on the expression of antimicrobial peptides. In contrast to HBD2 and LL37, ileal levels of HD5 and HD6 and colonic HBD1 level are independent of current inflammation. Innovative drugs should aim to strengthen protective innate immunity.

Probiotic E. coli treatment mediates antimicrobial human beta-defensin synthesis and fecal excretion in humans.

Inducible epithelial human beta-defensins (hBD) play an important role in intestinal barrier function. In vitro studies showed that clinically effective probiotics induce antimicrobial hBD-2. Here, we aimed to assess the in vivo effect in healthy volunteers and also addressed how defensins affect probiotic survival. Symbioflor 2 containing one strain of several viable genotypes of Escherichia coli was administered to 23 healthy individuals. After 3 weeks, fecal hBD-2 peptide was increased in 78% (mean 3.7-fold; P<0.0001). Interestingly, the fecal hBD-2 peptide was still elevated 9 weeks after treatment (P=0.008). In vitro studies revealed that this effect was mediated by only one out of three tested E. coli genotypes and comparable to probiotic E. coli Nissle 1917 (10- to 15-fold). Functional assays showed that all tested bacteria were similarly killed by defensins allowing to speculate about a suicidal character of this effect. Defensin induction seems to be a common and important mechanism of probiotic treatment.

Defensin-immunology in inflammatory bowel disease.

Defensins are endogenous antibiotics with microbicidal activity against Gram-negative and Gram-positive bacteria, fungi, enveloped viruses and protozoa. A disturbed antimicrobial defense, as provided by Paneth- and other epithelial cell defensins, seems to be a critical factor in the pathogenesis of inflammatory bowel diseases. Conspicuously, there is a relative lack of Paneth cell beta-defensins HD-5 and HD-6 in ileal Crohn's disease, both in the absence of a pattern recognition receptor NOD2 mutation and, even more pronounced, in its presence. This deficit is independent of concurrent active inflammation and results in a diminished antibacterial killing by the mucosa. The Crohn's disease mucosa has not only a significant lack in killing different Escherichia coli but also an impaired ability in clearing Staphylococcus aureus as well as anaerobic micro-organisms. Thus, this dysfunction in antibacterial barrier seems to be broad and is not restricted to a single bacterial strain. In addition to directly controlling barrier function, Paneth cell defensins also regulate the composition of the bacterial stool flora. In the majority of patients, the Paneth cell deficiency is mediated by WNT signalling which suggests a disturbed Paneth cell differentiation in ileal Crohn's disease. In contrast, colonic Crohn's disease is characterised by an impaired induction of mucosal beta-defensins, partly due to a low copy number of the beta-defensin gene cluster. Therefore it seems plausible that bacteria take advantage of a niche formed by defensin deficiency. This would represent a paradigm shift in understanding Crohn's disease and provides a target for future therapeutic strategies.

Barrier dysfunction due to distinct defensin deficiencies in small intestinal and colonic Crohn's disease.

Defensins are endogenous antibiotics with broad microbicidal activity. A disturbed antimicrobial defense, as provided by Paneth and other epithelial defensins, seems to be a critical factor in the pathogenesis of inflammatory bowel diseases. Conspicuously, there is a relative lack of Paneth-cell alpha-defensins in ileal Crohn's disease (CD), both in the absence of a pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) frameshift mutation and, even more pronounced, in its presence. This deficit is independent of concurrent active inflammation and cannot be seen in active small intestinal ulcerative colitis (UC; pouchitis) as well as NOD2 wild-type graft vs. host ileitis. After intestinal transplantation, in case of NOD2 mutation, defensins are decreased before the onset of inflammation. In the majority of patients, the Paneth-cell deficiency is mediated by Wnt-TCF4, which suggests a disturbed Paneth-cell differentiation. In contrast, colonic CD is characterized by an impaired induction of mucosal beta-defensins, partly because of a low copy number of the beta-defensin gene cluster. In both ileal and colonic CD, the lack in defensins results in a broadly diminished antibacterial killing by the mucosa, which can also be found independent of inflammation. In summary, the main disease locations can be linked to distinct mechanisms of epithelial barrier dysfunction.

Probiotic lactobacilli and VSL#3 induce enterocyte beta-defensin 2.

Recent evidence suggests that probiotic bacteria may stabilize gut barrier function via induction of anti-microbial peptides such as defensins. This study aimed to elucidate the induction mechanism of the human beta defensin-2 (hBD-2) gene by different probiotic lactobacillus strains. The expression of hBD-2 mRNA peaked at 6 h of incubation upon treatment of Caco-2 cells and increased with higher dosage of various probiotic bacteria. Deletion of nuclear factor (NF)-kappaB and activator protein-1 (AP-1) binding sites on the hBD-2 promoter resulted in a complete abrogation of promoter activation by probiotics. As revealed by the use of specific mitogen-activated protein kinase (MAPK) inhibitors the hBD-2 induction was dependent on the MAPK extracellular regulated kinase (ERK 1/2), p38 and c-Jun N-terminal kinase (JNK), although to varying degrees. Several Lactobacillus strains and VSL#3, a probiotic cocktail of four lactobacilli, three bifidum and one streptococcus species, induced the secretion of the hBD-2 peptide into the culture media as shown by enzyme-linked immunosorbent assay (ELISA). Thus, the present study suggests that lactobacilli and the VSL#3 bacterial mixture strengthen intestinal barrier functions through the up-regulation of hBD-2 via induction of proinflammatory pathways including NF-kappaB and AP-1 as well as MAPKs.

[Chronic inflammatory bowel diseases (IBD): novel pathophysiological concepts and their clinical relevance]. / Chronisch-entzündliche Darm- erkrankungen (CED): Neue pathophysiologische Erkenntnisse--Folgerungen für die Klinik.

Despite many years of intensive research the pathogenesis of inflammatory bowel diseases is still enigmatic. All efforts to identify the cause of Crohn's disease and ulcerative colitis in a dysregulation of specific immune mechanisms have failed. This review presents a novel pathogenetic concept, based on the expression of natural mucosal antibiotic peptides. These so called defensins are part of innate immunity and defend the mucosa as antimicrobial peptides against intraluminal potentially pathogenic and invasive bacteria. In contrast to ulcerative colitis, Crohn's disease is characterised by a diminished defensin expression. This defect may represent the molecular pathogenesis of this disease.

NOD2 (CARD15) mutations in Crohn's disease are associated with diminished mucosal alpha-defensin expression.

BACKGROUND: Mutations in NOD2, a putative intracellular receptor for bacterial peptidoglycans, are associated with a subset of Crohn's disease but the molecular mechanism linking this protein with the disease pathogenesis remains unclear. Human alpha defensins (HD-5 and HD-6) are antibiotic effector molecules predominantly expressed in Paneth cells of the ileum. Paneth cells also express NOD2. To address the hypothesis that the function of NOD2 may affect expression of Paneth cell defensins, we compared their expression levels with respect to NOD2 mutations in Crohn's disease. METHODS: Forty five Crohn's disease patients (24 with NOD2 mutations, 21 with wild-type NOD2) and 12 controls were studied. Real time reverse transcription-polymerase chain reaction was performed with mucosal mRNA for HD-5, HD-6, lysozyme, secretory phospholipase A2 (sPLA2), tumour necrosis factor alpha, interleukin 8, and human hypoxanthine phosphoribosyltransferase (housekeeping gene). Immunohistochemistry with anti-HD-5 and histological Paneth cell staining were performed in 10 patients with NOD2 mutations or wild-type genotypes. RESULTS: Ileal expression of HD-5 and HD-6, but not sPLA2 or lysozyme, were diminished in affected ileum, and the decrease was significantly more pronounced in patients with NOD2 mutations. In the colon, HD-5, HD-6, and sPLA2 were increased during inflammation in wild-type but not in NOD2 mutated patients. In both the colon and ileum, proinflammatory cytokines and lysozyme were unaffected by NOD2 status. Immunohistochemistry identified Paneth cells as the sole source of HD-5. CONCLUSION: As alpha defensins are important in the mucosal antibacterial barrier, their diminished expression may explain, in part, the bacterial induced mucosal inflammation and ileal involvement of Crohn's disease, especially in the case of NOD2 mutations.

[The role of defensins in the pathogenesis of chronic-inflammatory bowel disease]. / Die Rolle der Defensine in der Pathogenese chronisch-entzündlicher Darmerkrankungen.

Defensins are endogenous antimicrobial peptides with a broad activity spectrum. Even at micromolar concentrations gramnegative and grampositive bacteria, but also mycobacteria, as well as fungi (candida), viruses (herpes) and protozoa (giardia lamblia) are destroyed. As part of the innate immune system defensins are expressed by the intestinal epithelium and contribute to the maintenance of the mucosal barrier. This barrier appears to be defective in inflammatory bowel diseases since on one hand, the immune response is directed against the "normal" luminal bacterial flora and on the other hand, mucosal adherent and invasive bacteria have been observed in these diseases. A defective defensin expression may well explain these phenomena. Indeed, Crohn's disease of the terminal ileum, especially if associated with a NOD2 mutation, is characterised by a diminished alpha-defensin (human defensin 5 and 6) expression, and in inflamed Crohn's colitis, in contrast to ulcerative colitis, the beta-defensin (human beta-defensins 2 and 3) response is reduced. Through a deficient chemical mucosal barrier this defect could lead to increased bacterial invasion into the intestinal mucosa and might well explain an adequate inflammatory response. Although the final proof that this deficient defensin response leads to a reduced antibacterial activity of the intestinal mucosa is still lacking, the most plausible concept of pathogenesis of Crohn's disease is a defensin deficiency syndrome.

Defensin pattern in chronic gastritis: HBD-2 is differentially expressed with respect to Helicobacter pylori status.

BACKGROUND/AIMS: Recent reports have suggested that Helicobacter pylori infection induces the mucosal antibiotic peptide human beta defensin 2 (HBD-2). Therefore, the present study investigated mRNA and peptide expression of four different defensins in the upper gastrointestinal tract in patients with H pylori positive and negative chronic gastritis. MATERIALS/METHODS: Biopsies from the oesophagus to the duodenum were taken during routine gastroscopy in 71 individuals. Total RNA was extracted and the reverse transcription polymerase chain reaction was performed with primers for human defensins 5 and 6 (HD-5/6) or HBD-1 and HBD-2. Paraffin wax embedded tissue from gastric resections was tested for HD-5, HBD-1, and HBD-2 by immunohistochemistry. RESULTS: Helicobacter pylori colonisation was associated with an increased percentage of positive biopsies with respect to HBD-2 in the corpus (p < 0.05). Helicobacter pylori had no impact on the gastric expression of HD-5 and HBD-1, whereas HD-6 was increased in the fundus. The abundant expression of alpha defensins in the duodenum and beta defensins in the oesophagus served as a positive control in each individual. Immunohistochemical analysis confirmed the presence of the HD-5, HBD-1, and HBD-2 peptides in gastric resection specimens. CONCLUSIONS: The recently described induction of HBD-2 upon H pylori infection was confirmed in a clinical setting of chronic gastritis. This phenomenon may be mediated by components of the pathogen itself or may occur secondary to immune events in chronic inflammation.