Despite active research for many decades the aetiology of Crohn's disease (CD) is still enigmatic. Most of the research has focused on a potential dysregulation of specific mucosal immunology. These investigations have elegantly described the mucosal cellular populations and cytokine profiles associated with inflammatory bowel disease (IBD) but have not succeeded in finding the aetiological culprit. The alternative hypothesis of a primary defect in the mucosal barrier also has never been substantiated at the molecular level. In this review we will outline a novel concept of how epidemiological, pathophysiological, genetic, molecular, clinical and pharmacological sets of data may be synthesized into a unifying hypothesis compatible with many features of this disease.
Several years ago we became interested in the mucosal system of antibiotic peptides contributing to the defensive array of substances and structures opposed to the invasion of luminal bacteria and other potential invaders. Most of the work in the field of defensins had focused on the skin as another border of the body exposed to a multitude of bacteria and has resulted in the isolation of various peptides exhibiting potent antibiotic activity towards both Gram-positive and Gram-negative bacteria as well as enveloped viruses and fungi. A very similar system of antibiotic peptides is apparently synthesized and secreted by the intestinal mucosa as part of innate immunity but has been given little attention. The topic of defensins has recently been covered by a concise overview in this journal and is now presented in some detail with respect to IBDs .
Epidemiology: the role of hygiene
There is a clear-cut north to south gradient of IBD incidences worldwide as well as in Europe . In developing countries infectious intestinal diseases are common, whereas idiopathic IBDs, especially CD, are the rare exception. Similarly, the incidence rates in Scandinavia are several fold higher compared to southern Europe but the reasons for this difference are not clear . Since migration in many instances is associated with adaptation to the incidence rates in the immigrant country there is little doubt that environmental factors are involved , although a genetic background cannot be excluded. The increase in incidence rates in these countries appears to be associated with the adaptation to ‘Western lifestyle'. The dominant role of hygiene in this cultural–medical evolution is likely but unproven.
It is supported by the finding that good domestic hygiene in infancy has been shown to be a risk factor for CD but not for ulcerative colitis (UC), even within a country . Thus, the risk of developing CD is increased 3-fold if a separate toilet is available and 5-fold if there is hot tap water in the household . Similarly, Helicobacter pylori seroprevalence was substantially reduced in CD (odds ratio 0.18) but not in UC. CD was also associated with childhood eczema and frequent use of a swimming pool . In addition, CD occurs more often in members of small families as opposed to those with many children. Since intrafamilial transmission of common pathogens is frequent, the single child is particularly prone to be raised under more hygienic conditions with lower risk of acquiring gut infections [6,7]. Most likely, these various factors associated with the incidence of CD serve as indicators of a rather clean environment, leading to a diminished confrontation with pathogenic or non-pathogenic microorganisms. As a result the intestinal innate immune system is probably not ‘trained’ to confront minor infections without recruiting the full array of specific immune functions which act only at the expense of a relevant inflammation.
Another important aspect in this regard is the apparently frequent association of a recent intestinal infection with the first appearance of CD and the prevalence of superinfection in pre-established IBD . Although these relationships have not been fully understood the interpretation has been made that an infection in some way triggers a relapse of the idiopathic bowel disease by breaking mucosal tolerance. Despite their self-limited character, these infections may initiate a cascade of inflammatory events leading to chronic relapsing disease in genetically susceptible hosts (the ‘hit and run’ hypothesis). Alternatively, the host with IBD may be more likely to contract an intestinal infection because of a defective innate defence system (see below).
Pathophysiology: the role of luminal bacteria
It has always been an intriguing hypothesis that IBDs are caused by a specific, hitherto unrecognized, infection. For example, Mycobacterium paratuberculosis has been considered by various groups to cause not only Johne's disease in cattle but also CD in humans [9,10]. The debate has been ongoing for many years and is beyond the scope of the present considerations. It should be noted, however, that even very recently, using novel techniques like granuloma isolation with laser capture microdissection, many more Crohn's samples were shown to be positive for mycobacteria than were controls . Thus, although mycobacteria are far from proven to be causative agents, it is apparent that the mucosa in CD frequently harbours unusual and potentially pathogenic bacteria. In some instances listeria have been isolated  or specific mucosal adherent Escherichia coli . Interestingly, there is a tremendous increase in the mucosal associated bacterial counts in the neoterminal ileum after ileocaecal resection for CD and this colonization may be related to postoperative relapse . Measles infection in CD is also a very controversial issue but it adds up to the list of transmissible agents recovered from Crohn's mucosa . Taken together, these findings indicate that Crohn's mucosa is often the target of various infections but positive proof that the disease is caused by these agents is missing. Most importantly, the immune response in the gut mucosa is not specific to any of these suspicious agents but rather unspecific to a multitude of organisms.
Since 1939, a series of clinical reports and laboratory investigations have suggested that the intestinal faecal stream may play a significant part in the pathogenesis of CD . This has been corroborated by experiments where small bowel contents have been infused into the distal loop of an ileostomy which triggered an inflammatory response . The human large intestine is known to contain hundreds of culturable bacterial species and morphological and molecular analysis suggests at least an equal number of unculturable species . Also, distal small intestinal contents are contaminated by upwardly mobile colonic flora. Thus, CD is obviously localized mostly in sites with a heavy bacterial load, i.e. in the ileocaecum or large bowel and only rarely in the proximal gut or stomach. Possibly, the colonization of ileal mucosa with an unusual luminal bacterial content after formation of an ileoanal pouch may pose a problem in pouchitis.
However, it has only recently been appreciated that the mucosal immune response in IBD is directed towards a multitude of common luminal bacteria. The most convincing evidence for a break in mucosal tolerance in intestinal inflammation stems from the observation that knockout mice lacking several relevant genes, including interleukins 2 or 10, develop experimental colitis only when raised in contaminated but not in sterile conditions . This fits well with the consistent finding of a break in mucosal tolerance towards various luminal bacteria in IBDs [20,21]. It may be concluded that these diseases are not autoimmune diseases in the strict sense, i.e. reactivity against autologous tissues, but only in a more general sense, i.e. immune response towards commensal bacteria. The permeable mucosal barrier may also explain the development of anti-Saccharomyces cerevisiae antibodies especially in familial CD  as well as antibodies to various other microbes, including E. coli.
The most surprising finding in this regard is the demonstration by Swidsinski et al. that the mucosa in IBDs is heavily contaminated by adherent and sometimes invading bacteria entering from the lumen . In contrast, normal mucosa is virtually sterile when washed a few times in saline. These findings are difficult to reconcile with an immunological dysregulation as the sole basis of intestinal inflammation in these diseases. Rather, there may be a primary defect in the chemical barrier of intestinal defensins which protects the normal mucosa extremely efficiently against adherent or entering microbes. Thus, a thorough understanding of these functionally relevant peptides is paramount to understanding the true pathogenesis of IBD. Indirectly, a change in the expression or function of this chemical defence may indeed explain the changes in bacterial flora in IBDs reviewed by Linskens et al. .
Defensins: genetics, expression and regulation
The question why frogs have an undisturbed wound healing gave rise to the intensive search for antimicrobials in vertebrates and invertebrates. Several potential peptides and proteins have been identified so far (for review of antimicrobial peptides see ) including defensins, lysozyme, bactericidal permeability increasing protein, chemokines and many others (an overview of known human antimcrobial peptides is listed in Table 1). Probably the most important peptide family of endogenous antibiotics is the still growing number of defensins [1,26,27]. They comprise a class of cationic antimicrobial peptides with a molecular weight of 3–5 kDa conserved throughout phylogeny. All defensins have been mapped to chromosome 8 in humans [28–30] as well as in mice and the genomic organization suggests one ancestral gene which has been duplicated during evolution [29,31]. The common key feature is three intramolecular disulphide bonds between cysteine residues. Their position allows differentiation between α- and β-defensins.
Six α-defensins and four β-defensins have been identified in humans so far. The α-defensins comprise human neutrophil peptide 1–4, abundant in granulocytes, and human defensins 5 and 6 synthesized in Paneth cells. The β-defensins are of epithelial origin and abundant in skin, intestine and lung (representative stains for HD-5, HBD-1 and HBD-2 in inflamed colonic tissue are given in Fig. 1). The concept of a certain defensin exclusively formed by specialized tissues or cells has to be revised as inflammation induces epithelial expression of human neutrophil peptides  and β-defensins in monocytes and lymphocytes .
Defensins can be divided into constitutive forms, e.g. HBD-1 with its widespread stable distribution  and inducible peptides such as HBD-2 . The mechanisms of activation are currently under investigation. A cytokine driven induction e.g. by IL-1β and TNF-α has been shown in addition to a direct response to bacterial components such as lipopolysaccharides (LPSs) and lipoproteins. Possible signalling pathways involve Toll-like receptors, especially TLR2 and 4 eventually leading to nuclear factor κB (NFκB) mediated activation of transcription [36,37]. Promoter analysis revealed that transactivation depends on the proper function of NFκB response elements . In the signalling pathway triggered by Salmonella enteritidis both calcium and inositol triphosphate appear to play a role  whereas the Src dependent Raf-MEK1/2-ERK system is involved in mediating the IL-1 induction . NOD2/CARD15, as an intracellular LPS receptor, induces NFκB  which, in turn, is known to trigger HBD-2 transcription. Interestingly, this NFκB response is impaired in the NOD2 insertion mutation associated with CD [42,43] suggesting a diminished innate response to bacterial components.
Human defensin 5 is released as a propeptide from Paneth cells and activated by trypsinogen in the lumen of the intestinal crypts . This differs from mice, where the active peptide is cleaved by the matrix metalloproteinase matrilysin . Post-transcriptional processing and the activation of other defensins remain obscure.
Physiological role and dysfunction of defensins
The antimicrobial spectrum varies from one defensin to another. Maximum activity is achieved in the micromolar range and in a low-salt environment. Antibacterial activity of HD-5 has been characterized extensively with activity against E. coli, Listeria monocytogenes, Salmonella typhimurium and Candida albicans . In comparison, HBD-2 is most active against Gram-negative strains  whereas HBD-3 has a more Gram-positive spectrum [47,48]. Current proposals for the mode of action include formation of micropores by defensin multimeres within the phospholipid moiety of bacterial membranes resulting in disruption of the membrane [49,50]. Of special interest is the link to adaptive immunity as defensins act as chemokines attracting effector cells [51–53] and their ability to amplify acquired immune responses .
The expression of defensins has already been linked to several diseases. For example, necrotizing colitis is associated with an induction of α-defensins . HBD-2 is bactericidal against Helicobacter pylori in vitro and is induced in H. pylori gastritis resolving after eradication treatment [56–58]. Bacterial pneumonia is accompanied by increased systemic and local HBD-2 peptide levels [59,60]. The functional significance in bacterial infection has recently been shown in HD-5 transgenic mice which are protected from lethal salmonella infection . On the other hand, matrilysin deficient mice fail to process defensins efficiently and exhibit higher bacterial counts .
If defensin induction is a physiological process in infectious diseases one might postulate that malfunction of this innate defence gives rise to an increase in frequency and/or severity of infections. Improper function of defensins may occur in two ways, lack of function (inactivation) or lack of induction. A model disease for inactivation may be cystic fibrosis. It was postulated that defensins, expressed in respiratory epithelia and present in airway surface fluid of cystic fibrosis patients, are inactivated in the high-salt environment and may account for recurrent pulmonary infections [62,63]. A lack of induction, especially regarding HBD-2, has been encountered in CD (see below) and atopic dermatitis . Bacterial invasion and superinfection is almost absent in psoriasis. In contrast, neurodermatic skin is often colonized by Gram-positive bacteria and superinfections do often occur. This observation has now been attributed to HBD-2, which is largely induced in psoriatic but not in atopic dermatitis skin lesions. Obviously, current efforts to better understand this complex defence system will help clarify many inflammatory as well as immunological features of various infectious as well as ‘idiopathic’ diseases.
Defensins and inflammatory bowel diseases
Although a decrease or missing induction of antimicrobial peptides may, in principle, lead to a gradual bacterial invasion which could trigger inflammation and loss of mucosal tolerance, only little is known about this aspect of innate immunity in IBD.
In general, α-defensins appear to be induced in both CD and UC (Table 2). Human neutrophil peptides 1–3 as well as lysozyme are expressed in surface enterocytes of mucosa with active IBD but surprisingly not in controls . HD-5 is stored in a precursor form in normal Paneth cells and is expressed by metaplastic colonic Paneth cells . Notably, both α-defensins HD-5 and HD-6 are induced in the colonic mucosa of IBD patients [65–67]. The induction of HD-6 but not of HD-5 is specific for idiopathic IBD and was not observed in infectious colitis.
The alterations in β-defensins are more intriguing because there is a conspicuous difference between CD and UC. It has been suggested that HBD-1 is constitutively expressed in the intestinal epithelium  and qualitative investigations indeed showed constitutive expression in normal tissue and IBD mucosa . Using qualitative reverse transcriptase–polymerase chain reaction (RT-PCR), constitutive expression was observed in 50–60% of normal subjects and IBD patients. Furthermore, we described HBD-1 on the protein level by immunohistochemistry in the colonic epithelium. To extend our previous qualitative RT-PCR findings we quantified β-defensin expression using real-time PCR in colonic mucosa in a recent study . With the quantitative approach a decrease of HBD-1 was found in inflamed mucosa of both CD and UC, respectively. However, it remains to be shown that such a decrease actually translates into a diminished mucosal antibacterial activity.
The inducible HBD-2, which was described originally in skin , is also expressed in the colon during inflammation , particularly in UC . Based on the qualitative RT-PCR in our recent study HBD-2 was detected more often in biopsies from UC than in controls or CD. On the protein level we confirmed these findings by immunohistochemistry with a preferential expression in UC. The real-time PCR and the possibility to measure mRNA expression levels further explored the difference between CD and UC. Using light cycler technique the inducible β-defensin 2 was found almost exclusively in inflamed and much less in non-inflamed UC . In CD the inflamed mucosa exhibited a low level of HBD-2 expression comparable to unspecific colitis. In non-inflamed CD the level of HBD-2 was comparable to non-inflamed controls. Most likely, there is a lack of β-defensin induction in CD contributing to a defective antimicrobial barrier or, alternatively, there is an excessive induction in UC.
The third defensin studied was HBD-3 which was reported by Harder et al. as a novel inducible β-defensin in skin . Another group described HBD-3 based on genomic analysis . In a recent study expression of HBD-3 was a rare event, almost limited to inflamed specimens (Wehkamp et al., submitted). Interestingly, HBD-3 levels in the different patient groups closely correlated with that of HBD-2, although defensins are known to be regulated independently. Although HBD-3 was also slightly induced in inflamed Crohn's mucosa, its expression was preferentially enhanced in inflamed and non-inflamed UC. This was unexpected because cell culture experiments indicated a divergent regulation of both defensins [47,48]. A deficiency in the antimicrobial defence systems of defensins may be a reasonable and plausible explanation for the break of the antibacterial barrier function in IBDs.
In conclusion, the decrease of HBD-1 in both IBDs and the lack of induction of both inducible β-defensins HBD-2 and HBD-3 in CD suggest a deficient mucosal barrier function. This may in part be compensated by the induction of the α-defensins. A lack in the innate defence system of antimicrobial peptides may lead to a permanent but slow bacterial invasion triggering the inflammatory process but further direct studies on antimicrobial peptide activity in IBD mucosa are required to validate this hypothesis.
Therapy: the role of antibiotics and probiotics
If a deficiency of these endogenous antibiotics was triggering relapse one would expect exogenous antibiotics to be an efficacious treatment option. Therefore the different trials using antibiotics for various indications are discussed.
Remission induction in Crohn's disease
In active CD metronidazole has been shown to be equivalent to sulfasalazine  and significantly better than placebo . Similarly, ciprofloxacin was equivalent to mesalazine in the treatment of mild to moderate flare-ups . The combination of both antibiotics may even be as effective as systemic steroids . The best study to date tested the combination of both antibiotics in addition to budesonide in a placebo controlled fashion. Although the authors were unable to show significant differences between the two groups there was a trend towards efficacy in colonic disease . Thus, although not completely convincing antibiotics may offer a therapeutic option in acute flares of CD at least in cases with mild to moderate disease activity.
Remission maintenance in Crohn's disease
All reported trials have been performed with active disease. Only one placebo controlled study tested the efficacy of ciprofloxacin as adjunct to standard therapy in moderately active CD with a study duration of 6 months. Ciprofloxacin was significantly superior to placebo in remission maintenance . In the postoperative condition metronidazole was effective in preventing endoscopic recurrence after 3 months . In contrast, a prophylactic probiotic treatment did not prevent recurrence after surgery .
Perianal and fistulizing Crohn's disease
No controlled trials for antibiotics exist for this condition, but uncontrolled trials have shown efficacy for metronidazole [79,80] and the combination therapy of metronidazole and ciprofloxacin  with respect to pain relief, reduction of draining fistulas and fistula closure.
Remission induction in ulcerative colitis
Less promising results have been obtained in the treatment of UC. There is no trial testing the efficacy of antibiotics alone. Only an early study on short-term oral tobramycin added to steroids showed a significant benefit in the treatment of acute colitis . In contrast, in addition to steroids neither short-term oral ciprofloxacin in mild to moderate disease  nor the intravenous application in severe disease resulted in better outcome . Similarly, metronidazole  as well as the combination of metronidazole and tobramycin  as an adjunct to steroids failed to improve remission rate in severe UC.
Remission maintenance in ulcerative colitis
Only one controlled trial using ciprofloxacin as a long-term ‘add on’ to steroids and mesalazine has been conducted in UC. Though it proved clinical efficacy after 3 months of continued application the advantage disappeared in the steroid-free trial period at 6 months . The trial was questioned by the lack of good design. A promising therapeutic concept is the application of the probiotic strain E. coli Nissle. In two controlled trials it was equivalent to mesalazine in successful remission maintenance in recurrent disease [88,89].
Pouchitis after ileal pouch–anal anastomosis
In contrast to the disappointing results in UC, antibiotics have a defined role in the special condition of pouchitis. In a controlled trial ciprofloxacin was superior to metronidazole with regard to efficacy and side effects . Chronic pouchitis may be effectively treated with metronidazole alone  or with antibiotic combination therapy of metronidazole and ciprofloxacin  or rifaximin and ciprofloxacin . Interestingly, the probiotic cocktail VSL#3 was able to maintain remission in chronic pouchitis significantly better than placebo .
In conclusion, antibiotics appear to have a limited effect in CD and probiotics in UC. In CD the exogenous antibiotics may compensate for the deficient endogenous antibiotic response to infection or commensal bacterial invasion. In UC the pattern is different with low basal activity but normal induction during inflammation. Therefore antibiotics may not work and the benefit of probiotics may be due to the induction of β-defensins as demonstrated recently in vitro . In contrast to the majority of tested E. coli the Nissle strain potently upregulated HBD-2 expression in colonic cell culture. In pouchitis both approaches work for induction and maintenance, respectively.
Seventy years after Crohn's description of the disease named after him it becomes apparent that CD is not a disease but a syndrome. It is not surprising that the diverse facets of genetic predisposition, where only a minority of patients display a defective NOD2 gene, modified by environmental factors such as childhood hygiene and others, may lead to very different forms of disease with respect to localization, natural course and therapeutic response. Although in no way perfect, the present hypothesis appears to be plausible, for the reasons presented above, but particularly since the multitude of defensins, other antibiotic peptides and related transcription factors or transporters leaves enough room for clinical diversity. UC may indeed not be due to a defensin problem but the fact that anti-neutrophil cytoplasmic antibody is directed against the endogenous antibiotic bactericidal permeability increasing protein [96–98] or that the disease is related to certain MDR-1 polymorphisms  which may alter defensin export leaves enough room for speculation and, more importantly, hypothesis driven future work.
Helpful discussions and joint investigations with J.M. Schröder and J. Harder are gratefully acknowledged. The work was generously supported by the Robert Bosch Foundation, Stuttgart, Germany.
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