Klaus Fellermann

A Chromosome 8 Gene-Cluster Polymorphism with Low Human Beta-Defensin 2 Gene Copy Number Predisposes to Crohn Disease of the Colon

Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin-gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2-10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with < or = 3 copies have a significantly higher risk of developing colonic CD than did individuals with > or = 4 copies (odds ratio 3.06; 95% confidence interval 1.46-6.45). An HBD-2 gene copy number of < 4 was associated with diminished mucosal HBD-2 mRNA expression (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression.

NF-κB- and AP-1-Mediated Induction of Human Beta Defensin-2 in Intestinal Epithelial Cells by Escherichia coli Nissle 1917: a Novel Effect of a Probiotic Bacterium

ABSTRACT 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-κB and AP-1 in the hBD-2 promoter are required for induction of hBD-2 through E. coli Nissle 1917. Treatment with the NF-κB 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.

Inducible and constitutive β‐defensins are differentially expressed in Crohn's disease and ulcerative colitis

Antimicrobial peptides such as defensins provide nonspecific mucosal defense against a multitude of microorganisms. Recently, it has been shown that luminal bacteria may invade the mucosa in inflammatory bowel diseases, suggesting a defect in innate mucosal immunity. The aim of this study was to investigate the expression of human &bgr;-defensins (HBD) in controls, Crohns disease (CD), ulcerative colitis (UC), and unspecific inflammation. Up to 4 biopsies were taken from 103 patients (33 controls, 24 with Crohns disease, 36 with ulcerative colitis, 10 with unspecific colitis). Mucosal mRNA was measured using real-time fluorescence temperature cycler reverse-transcription polymerase chain reaction with primers for HBD-1, HBD-2, HBD-3, tumor necrosis factor &agr;, and interleukin 8. Mucosal HBD-1 expression was marginally decreased in both CD and UC. HBD-2 was increased exclusively in UC but not in CD. The expression of the novel defensin HBD-3 was strongly correlated with HBD-2 and also raised predominantly in UC. The expression of both inducible &bgr;-defensins was enhanced in the state of inflammation. Expression of HBD-2 showed a weak correlation with interleukin 8 only in inflamed CD biopsies but not with tumor necrosis factor &agr;. The missing induction of both inducible &bgr;-defensins in CD as compared with UC may cause a defect in barrier function that predisposes to bacterial invasion.

Probiotic lactobacilli and VSL#3 induce enterocyte β-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)‐κB 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‐κB and AP‐1 as well as MAPKs.

The Paneth Cell α-Defensin Deficiency of Ileal Crohn’s Disease Is Linked to Wnt/Tcf-4

Ileal Crohn′s disease (CD), a chronic mucosal inflammation, is characterized by two pertinent features: a specific decrease of Paneth cell-produced antimicrobial α-defensins and the presence of mucosal-adherent bacteria. A mutation in NOD2, the muramyl dipeptide recognition receptor, is found in some patients, which leads to an even more pronounced α-defensin decrease. However, the underlying mechanism remains unclear for the majority of patients. In this study, we report a reduced expression in ileal CD of the Wnt-signaling pathway transcription factor Tcf-4, a known regulator of Paneth cell differentiation and α-defensin expression. Within specimens, the levels of Tcf-4 mRNA showed a high degree of correlation with both HD5 and HD6 mRNA. The levels of Tcf-4 mRNA were decreased in patients with ileal disease irrespective of degree of inflammation, but were not decreased in colonic CD or ulcerative colitis. As a functional indicator of Tcf-4 protein, quantitative binding analysis with nuclear extracts from small intestine biopsies to a Tcf-4 high-affinity binding site in the HD-5 and HD-6 promoters showed significantly reduced activity in ileal CD. Furthermore, a causal link was shown in a murine Tcf-4 knockout model, where the comparably reduced expression of Tcf-4 in heterozygous (+/−) mice was sufficient to cause a significant decrease of both Paneth cell α-defensin levels and bacterial killing activity. Finally, the association between Paneth cell α-defensins and Tcf-4 was found to be independent of the NOD2 genotype. This new link established between a human inflammatory bowel disease and the Wnt pathway/Tcf-4 provides a novel mechanism for pathogenesis in patients with ileal CD.

Induction of Human β-Defensin 2 by the Probiotic Escherichia coli Nissle 1917 Is Mediated through Flagellin

ABSTRACT Human β-defensin 2 (hBD-2) is an inducible antimicrobial peptide synthesized by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certain bacterial strains, have been identified as potent endogenous inducers. Recently, we have found that hBD-2 induction by probiotic Escherichia coli Nissle 1917 was mediated through NF-κB- and AP-1-dependent pathways. The aim of the present study was to identify the responsible bacterial factor. E. coli Nissle 1917 culture supernatant was found to be more potent than the pellet, indicating a soluble or shed factor. Chemical analysis demonstrated the factor to be heat resistant and proteinase digestible. Several E. coli Nissle 1917 deletion mutants were constructed and tested for their ability to induce hBD-2 expression in Caco-2 cells. Deletion mutants for flagellin specifically exhibited an impaired immunostimulatory capacity. Reinsertion of the flagellin gene restored the induction capacity to normal levels. Isolated flagellin from E. coli Nissle 1917 and from Salmonella enterica serovar Enteritidis induced hBD-2 mRNA significantly in contrast to the flagellin of the apathogenic E. coli strain ATCC 25922. H1 flagellin antiserum abrogated hBD-2 expression induced by flagellin as well as E. coli Nissle 1917 supernatant, confirming that flagellin is the major stimulatory factor of E. coli Nissle 1917.

Steroid-unresponsive acute attacks of inflammatory bowel disease: immunomodulation by tacrolimus (FK506)

Objective:Steroid treatment failure in acute Crohns disease and ulcerative colitis frequently necessitates surgical intervention. Several alternative therapeutic strategies have been raised. The most promising so far has been intravenous cyclosporine, but the results in the long term have been discouraging. We assessed the efficacy and safety of the new macrolide immunomodulator tacrolimus as an alternative to cyclosporine A.Methods:Eleven patients with steroid-refractory disease (six ulcerative colitis, two indeterminate colitis, two Crohns disease, one pouchitis) and severe activity according to the Truelove and Witts criteria or Crohns disease activity index > 150, respectively, were eligible for the study. All patients were treated with intravenous tacrolimus for 7–10 days followed by oral treatment over a median period of 7 months (range 0.25–16). Azathioprine and mesalamine were given concomitantly. Steroids were tapered according to clinical activity.Results:Seven of 11 patients achieved remission rapidly, whereas a modest improvement was noted in two. Only two patients required an early and one a delayed colectomy. Moreover, a rectovaginal fistula closure in a case of Crohns disease and an improvement of pouchitis was observed. A tapering to low dose steroids was possible during oral tacrolimus therapy in all nine responders and remission was maintained in five of them (mean follow-up 9.2 months). The drug was well tolerated and side effects were managed conservatively.Conclusion:Tacrolimus induced rapid remission in steroid resistant inflammatory bowel disease in the majority of cases. It appears to be an effective treatment modality that may be superior to cyclosporine with respect to maintenance of remission.

Validation of treatment strategies for enterohaemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic syndrome: case-control study

Objective To evaluate the effect of different treatment strategies on enterohaemorrhagic Escherichia coli O104:H4 induced haemolytic uraemic syndrome. Design Multicentre retrospective case-control study. Setting 23 hospitals in northern Germany. Participants 298 adults with enterohaemorrhagic E coli induced haemolytic uraemic syndrome. Main outcome measures Dialysis, seizures, mechanical ventilation, abdominal surgery owing to perforation of the bowel or bowel necrosis, and death. Results 160 of the 298 patients (54%) temporarily required dialysis, with only three needing treatment long term. 37 patients (12%) had seizures, 54 (18%) required mechanical ventilation, and 12 (4%) died. No clear benefit was found from use of plasmapheresis or plasmapheresis with glucocorticoids. 67 of the patients were treated with eculizumab, a monoclonal antibody directed against the complement cascade. No short term benefit was detected that could be attributed to this treatment. 52 patients in one centre that used a strategy of aggressive treatment with combined antibiotics had fewer seizures (2% v 15%, P=0.03), fewer deaths (0% v 5%, p=0.029), required no abdominal surgery, and excreted E coli for a shorter duration. Conclusions Enterohaemorrhagic E coli induced haemolytic uraemic syndrome is a severe self limiting acute condition. Our findings question the benefit of eculizumab and of plasmapheresis with or without glucocorticoids. Patients with established haemolytic uraemic syndrome seemed to benefit from antibiotic treatment and this should be investigated in a controlled trial.

Human β-defensin 2 but not β-defensin 1 is expressed preferentially in colonic mucosa of inflammatory bowel disease

Objective Various antimicrobial peptides such as defensins are part of innate immunity and contribute to the intestinal barrier that may be defective in inflammatory bowel disease (IBD). This study investigated β-defensin mRNA and peptide expression in the colon from controls and patients with Crohns disease, ulcerative colitis or unspecific colitis as inflammatory controls. Methods Mucosal mRNA expression was measured by multiplex reverse transcriptase–polymerase chain reaction (RT-PCR) with primers for human β-defensin 1 (HBD-1) and human β-defensin 2 (HBD-2) in CaCo-2 cells and in biopsies from 103 patients (33 controls, 24 Crohns disease patients, 36 ulcerative colitis patients, 10 unspecific colitis patients). Paraffin-embedded tissue from colonic resections was tested for HBD-1 and HBD-2 peptides by immunohistochemistry. Results HBD-1 mRNA was expressed constitutively whereas HBD-2 was induced by pro-inflammatory cytokines in CaCo-2 cells. HBD-1 mRNA was detectable in 61% of control and Crohns disease biopsies and 53% of ulcerative colitis biopsies. HBD-2 transcript was expressed differentially, with 18% of control biopsies positive as opposed to 34% in Crohns disease and 53% in ulcerative colitis. HBD-2 mRNA but not HBD-1 mRNA was expressed preferentially in inflamed areas. Immunohistochemical investigation demonstrated the presence of defensin peptides in colonic epithelium as well as the differential induction in IBD. Conclusions HBD-1 is expressed constitutively in colonic tissue irrespective of inflammation. HBD-2 is barely present in uninflamed colon but it is induced in inflammation. The lower expression of HBD-2 in Crohns disease compared with ulcerative colitis indicates different responses of the mucosal innate defence. Defensins may play a crucial role in controlling pathogen invasion in IBD, although the functional significance remains to be established.

Response of refractory colitis to intravenous or oral tacrolimus (FK506)

Intravenous cyclosporine has proven to be an alternative to emergency colectomy in steroid-refractory ulcerative colitis, whereas the experience with FK506 is limited. In this report we compare intravenous to oral FK506 treatment in 38 patients with refractory ulcerative (n = 33) or indeterminate (n = 5) colitis. FK506 was started intravenously in the first group (n = 18) at a dose of 0.01 to 0.02 mg/kg up to 14 days, followed by 0.1 to 0.2 mg/kg orally, or was started orally at this dose in a second group (n = 20). Additional azathioprine/6-mercaptopurine was given and steroids were tapered in responding patients, followed by a dose reduction of FK506. Clinical disease activity and laboratory parameters were assessed to evaluate efficacy and safety. Primary objectives were the induction of remission (Truelove index of mild) and colectomy-free survival. Treatment lasted for a mean of 7.6 months, and the mean observation period was 16.2 months. Eighteen of 38 patients improved within 14 days, and a complete remission was achieved in 13 patients after 1 month. A colectomy within 1 month was performed in 3 of 38 patients. The overall colectomy rate was 34%. One-half of the patients with a minimum follow-up of 2 years required a colectomy. Intravenous and per oral administration were equally safe and effective. The most frequent adverse events included tremor, hyperglycemia, hypertension, and infection, but none were severe. Renal impairment was rare and subsided upon drug withdrawal. In conclusion, FK506 is effective in the treatment of refractory colitis with per oral dosing being equivalent to intravenous administration.