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Infectious agents and Crohn's disease

1999; Elsevier BV; Volume: 5; Issue: 10 Linguagem: Inglês

10.1111/j.1469-0691.1999.tb00415.x

1469-0691

P. Bulois, Pierre Desreumaux, Christel Neut, Arlette Darfeuille-Michaud, Antoine Cortot, Jean‐Frédéric Colombel,

Mycobacterium research and diagnosis

Since its definition in 1932, it has been recognized that Crohn's disease contains elements that might be the result of microbial processes in the gut [1Sartor RB Microbial factors in the pathogenesis of Crohn's disease, ulcerative colitis and experimental intestinal inflammation.in: Kirsner JB Shorter RG Inflammatory bowel disease. 4th edn. Williams & Wilkins, Baltimore1995: 96-124Google Scholar, 2Sartor RB Enteric microflora in IBD: pathogens or commensals?.Inflamm Bowel Dis. 1997; 3: 230-235Crossref PubMed Scopus (107) Google Scholar, 3Van Kruiningen HJ On the use of antibiotics in Crohn's disease.J Clin Gastroenterol. 1995; 20: 310-316Crossref PubMed Scopus (31) Google Scholar, 4Colombel JF Louvet B Müller-Allouf H Desreumaux P Recent advances in the etiology of Crohn's disease. Environment and genetics.in: Galmiche JP Gournay J Recent advances in the pathophysiology of gastro-intestinal and liver diseases. John Libbey Eurotext, Paris1997: 109-131Google Scholar]. These include evidence of onset in the Peyer's patches and in lymphoid aggregates of the colon, ulcerations, microabscesses, fissures, fistulas, granulomas and lymphangitis. Aphthous ulcers in the intestine represent the earliest lesions, and we know that these occur in viral and bacterial infections. Crohn's lesions occur in segments that suggest the distribution of Peyer's patches. Ulcerations in colonic Crohn's disease originate in the follicle-associated epithelium of colonic lymphoid nodules. Recent studies in animal models have led to the widespread belief that microbial constituents provide the antigenic stimulus in Crohn's disease, and the most common hypothesis is that chronic inflammation results from an abnormal host immune response to normal luminal flora. Although present data do not convincingly incriminate a single, persistent pathogen as a universal cause of Crohn's disease, this hypothesis should not be abandoned. Our current understanding of the complexity of the intestinal flora is very limited, and it is quite possible that one or more etiologic agents for Crohn's disease exist but have not been detected. We will review in this note the infectious agents which have been implicated in the etiology of Crohn's disease within the last few years. The etiologic role of mycobacteria in Crohn's disease has been a controversial issue for some time. In 1984, Chiodini et al recovered slow-growing Mycobacterium paratuberculosis from tissues of three patients with Crohn's disease [5Chiodini RJ Van Kruiningen HJ Thayer Jr, WR Merkal RS Coutu JA Possible role of mycobacteria in inflammatory bowel disease. I. An unclassified Mycobacterium species isolated from patients with Crohn's disease.Dig Dis Sci. 1984; 29: 1073-1079Crossref PubMed Scopus (272) Google Scholar], and it was encouraging that other laboratories also recovered this organism, albeit rarely. However, 15 years later, data subsequently obtained do not strongly support this hypothesis. Six separate immunocytochemistry studies on varied groups of patients have failed to find M. paratuberculosis in diseased tissues, and numerous serologic studies have failed to document elevated titers or changes in titer in relation to disease activity. With the exception of a paper by Prantera et al [6Prantera C Kohn A Mangiarotti R Andreoli A Luzi C Antimycobacterial therapy in Crohn's disease: results of a controlled, double-blind trial with multiple antibiotic regimen.Am J Gastroenterol. 1994; 89: 513-518PubMed Google Scholar], controlled trials with antibiotics directed against mycobacteria have failed to make patients well, and the clinical improvement of Prantera's patients may reflect a broad-spectrum antibiotic effect on secondary invaders. A recent 5-year follow-up study of patients receiving rifampicin, isoniazid, and ethambutol for up to 2 years failed to show any benefit in treated patients [7Thomas GAO Swift GL Green JT et al.Controlled trial of antituberculous chemotherapy in Crohn's disease: a five year follow up study.Gut. 1998; 42: 497-500Crossref PubMed Scopus (80) Google Scholar]. Dairy cattle with Johne's disease shed great numbers of M. paratuberculosis into the environment, and it has been reported that M. paratuberculosis strains isolated from patients with Crohn's disease had a common clonal origin with strains isolated from cows with Johne's disease. However, there has never been any recorded case of Crohn's disease occurring in farm staff or farmer's families associated with these herds. Studies involving the use of PCR techniques to amplify the IS900 insertion sequence, specific for M. paratuberculosis, in intestinal biopsies have given controversial results. At the present time, the remote possibility remains that only a specifically susceptible subset of Crohn's disease patients might have a mycobacterial etiology. A more recent thrust at discovery has been the work of Wakefield et al, first put forward 8 years ago, when it was proposed that microvascular occlusion constitutes the earliest lesion and basis of Crohn's disease [8Wakefield AJ Ekbom A Dhillon AP Pittilo RM Pounder RE Crohn's disease: pathogenesis and persistent measles virus infection.Gastroenterology. 1995; 108: 911-916Abstract Full Text PDF PubMed Scopus (105) Google Scholar]. A further step in the work from this group was the proposal that Crohn's disease may be a persistent measles virus-induced granulomatous vasculitis. Measles virus antigen and mRNA were localized to granulomas and endothelial cells by in situ hybridization, immunofluorescence and immunogold electron microscopy [8Wakefield AJ Ekbom A Dhillon AP Pittilo RM Pounder RE Crohn's disease: pathogenesis and persistent measles virus infection.Gastroenterology. 1995; 108: 911-916Abstract Full Text PDF PubMed Scopus (105) Google Scholar], and antimeasles antibody was reported to be elevated. Epidemiologic arguments supported the hypothesis. Early exposure to measles virus, live-attenuated measles vaccination and prenatal exposure to measles were all associated with an increased risk [9Ekbom A Daszak P Kraaz W Wakefield AJ Crohn's disease after in-utero measles virus exposure.Lancet. 1996; 348: 515-517Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar]. However, the interpretation of studies implicating measles virus has been challenged. Over the past several years it has been shown that mesenteric blood vessels are not primary sites of Crohn's disease. Furthermore, the presence of measles virus in the tissues has not been confirmed by independent investigators [10Liu Y Van Kruiningen HJ West AB Cartun RW Cortot A Colombel JF Immunocytochemical evidence of Listeria, Escherichia coli and Streptococcus antigens in Crohn's disease.Gastroenterology. 1995; 108: 1396-1404Abstract Full Text PDF PubMed Scopus (276) Google Scholar], and measles viral genomic sequences could not be detected in intestinal tissue from Crohn's patients by PCR [11Haga Y Funakoshi O Kuroe K et al.Absence of measles viral genomic sequence in intestinal tissues from Crohn's disease by nested polymerase chain reaction.Gut. 1996; 38: 211-215Crossref PubMed Scopus (73) Google Scholar]. As for serologic studies, we saw no titers that would have suggested measles virus participation in families with a high frequency of Crohn's disease [12Touze Y Dubucquoi S Cortot A Van Kruiningen HJ Colombel JF IgM-specific measles-virus antibody in families with a high frequency of Crohn's disease.Lancet. 1995; 346: 967Abstract PubMed Google Scholar]. Finally, recent epidemiologic studies failed to confirm the original association [13Metcalf J Is measles infection associated with Crohn's disease?.Br Med J. 1998; 316: 166Crossref PubMed Scopus (22) Google Scholar]. Thus the theory of measles as a causative factor in the development of Crohn's disease is difficult to uphold. There has been renewed interest in the transmittable agent hypothesis after the report of Crohn's disease occurring in multiple siblings of two French families within a short time period [14Van Kruiningen HJ Colombel JF Cartun RW et al.An in depth-study of Crohn's disease in two French families.Gastroenterology. 1993; 104: 351-360PubMed Google Scholar]. When a polyclonal antibody against Listeria monocytogenes was applied to the tissues of patients, including those of these families, antigen was demonstrated in 75% and not in controls [10Liu Y Van Kruiningen HJ West AB Cartun RW Cortot A Colombel JF Immunocytochemical evidence of Listeria, Escherichia coli and Streptococcus antigens in Crohn's disease.Gastroenterology. 1995; 108: 1396-1404Abstract Full Text PDF PubMed Scopus (276) Google Scholar]. However, there are several data elements that militate against the hypothesis that Listeria is important in Crohn's disease: Listeria has never been cultivated from Crohn's intestinal tissue, and a recent PCR study has given negative results [15Chiba M Fukushima T Inoue S Horie Y Iizuka M Masamune O Listeria monocytogenes in Crohn's disease.Scand J Gastroenterol. 1998; 33: 430-434Crossref PubMed Scopus (33) Google Scholar]. Finally, there is no association in the literature between the occurrence of Crohn's disease and Listeria meningoencephalitis or abortion. Since the discovery of H. pylori, interest in other Helicobacter species has increased. H. hepaticus is a murine pathogen which can induce colitis and hepatitis in immunodeficient mice and in germ-free monoinfected mice. Scid mice reconstituted with CD4+ T-cells develop severe colitis when maintained in conventional conditions. In this model, the combination of H. hepaticus infection and CD45RBhigh CD4+ T-cell reconstitution resulted in severe disease expression [16Cahill R Foltz CJ Fox JG Dangler CA Powrie F Schauer DB Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus.Infect Immun. 1997; 65: 3126-3131PubMed Google Scholar]. A group of Helicobacter species has also been identified from the lower gastrointestinal tract of humans and, in the case of two species, H. cinaedi and H. fenelliae, human infection has been associated with colitis and proctitis in homosexual men. H. hepaticus does not appear to be involved in chronic intestinal inflammation in mice with functioning T-lymphocytes. Furthermore, no Helicobacter species has been isolated so far from feces or mucosal samples of patients with Crohn's disease. Systemic antibodies to various dietary and bacterial antigens have been reported in sera from patients with Crohn's disease and ulcerative colitis. Using ELISA, which employs whole killed yeast cells as antigens, elevated anti-Saccharomyces cerevisiae antibody (ASCA) levels were reported in the sera from Crohn's patients and not from patients with ulcerative colitis. In 1996, we showed that this serologic response recognizes mannose sequences in the cell wall mannan of S. cerevisiae strain Su1 (formerly S. uvarum 1, a species now classified within S. cerevisiae) [17Sendid B Colombel JF Jacquinot PM et al.Specific antibody response to oligomannosidic epitopes in Crohn's disease.Clin Diagn Lab Immunol. 1996; 3: 219-226PubMed Google Scholar]. ASCAs constitute a specific marker for Crohn's disease and are present in 20% of healthy relatives of patients, suggesting that they could represent a subclinical marker [18Quinton JF Sendid B Reumaux D et al.Anti-Saccharomyces cerevisiae mannan combined with antineutrophil antibodies in inflammatory bowel disease: prevalence and diagnostic role.Gut. 1998; 42: 788-791Crossref PubMed Scopus (534) Google Scholar, 19Sendid B Quinton JF Charrier G et al.Anti-Saccharomyces cerevisiae mannan antibodies in familial Crohn's disease.Am J Gastroenterol. 1998; 93: 1306-1310Crossref PubMed Scopus (147) Google Scholar]. Different hypotheses concerning the origin of ASCAs can be proposed [17Sendid B Colombel JF Jacquinot PM et al.Specific antibody response to oligomannosidic epitopes in Crohn's disease.Clin Diagn Lab Immunol. 1996; 3: 219-226PubMed Google Scholar]. The first one to consider is that they may originate from immunization by yeasts. A second hypothesis worth testing is that yeast mannan oligomannosides could correspond to epitopes shared by other microorganisms. Finally, the ubiquitous character of mannose residue sequences raises a third hypothesis: some S. cerevisiae oligomannosides might share structural homologies with oligomannosides expressed on human glycoconjugates as autoantigens or neo-autoantigens. Abundant experimental and clinical data incriminate normal luminal bacteria or bacterial products in the initiation and perpetuation of chronic intestinal inflammation [1Sartor RB Microbial factors in the pathogenesis of Crohn's disease, ulcerative colitis and experimental intestinal inflammation.in: Kirsner JB Shorter RG Inflammatory bowel disease. 4th edn. Williams & Wilkins, Baltimore1995: 96-124Google Scholar, 2Sartor RB Enteric microflora in IBD: pathogens or commensals?.Inflamm Bowel Dis. 1997; 3: 230-235Crossref PubMed Scopus (107) Google Scholar, 3Van Kruiningen HJ On the use of antibiotics in Crohn's disease.J Clin Gastroenterol. 1995; 20: 310-316Crossref PubMed Scopus (31) Google Scholar, 4Colombel JF Louvet B Müller-Allouf H Desreumaux P Recent advances in the etiology of Crohn's disease. Environment and genetics.in: Galmiche JP Gournay J Recent advances in the pathophysiology of gastro-intestinal and liver diseases. John Libbey Eurotext, Paris1997: 109-131Google Scholar]. The most convincing evidence of the potential role for bacteria comes from genetically engineered rodent models that develop intestinal inflammation. In these models, inflammation is absent or attenuated when animals are kept under germ-free conditions [20Taurog JD Richardson JA Croft JT et al.The germfree state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats.J Exp Med. 1994; 180: 2359-2364Crossref PubMed Scopus (939) Google Scholar, 21Sartor RB The influence of normal microbial flora on the development of chronic intestinal inflammation.Res Immunol. 1997; 148: 567-576Crossref PubMed Scopus (146) Google Scholar]. In humans, there is evidence that bacteria might be involved, at least as secondary invaders, in the pathogenesis of Crohn's disease [3Van Kruiningen HJ On the use of antibiotics in Crohn's disease.J Clin Gastroenterol. 1995; 20: 310-316Crossref PubMed Scopus (31) Google Scholar, 4Colombel JF Louvet B Müller-Allouf H Desreumaux P Recent advances in the etiology of Crohn's disease. Environment and genetics.in: Galmiche JP Gournay J Recent advances in the pathophysiology of gastro-intestinal and liver diseases. John Libbey Eurotext, Paris1997: 109-131Google Scholar]. Bacterial flora might also play a primary role. Endoscopic studies after surgery have suggested that bacteria are important in recurrence: diverting ileostomy prevents postoperative recurrence, which rapidly occurs after reanastomosis [22Sartor RB Postoperative recurrence of Crohn's disease: the enemy is within the fecal stream.Gastroenterology. 1998; 114: 398-407Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. This suggests that the fecal stream (and most likely bacteria) triggers the onset of early new ileal lesions of Crohn's disease. We have also shown that, in this situation, the neoterminal ileum above the ileocolonic anastomosis is heavily colonized by a colonic-like bacterial flora, with prominent bacteria being Bacteroides, Clostridium and Escherichia coli. All bacterial species may not have equal activity in inducing inflammation. Defined flora studies showed that Bacteroides spp. preferentially induce colitis in HLA B27 transgenic rats [23Rath HC Herfarth HH Ikeda JS et al.Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis and arthritis in HLA-B27/human β2 microglobulin transgenic rats.J Clin Invest. 1996; 98: 945-953Crossref PubMed Scopus (688) Google Scholar]. Among the numerous bacteria of the intestinal flora, E. coli has also been thought to be involved. E. coli antibody titers are higher in Crohn's patients than in controls. When bacteriologic sampling was undertaken at surgery for Crohn's disease, before bowel opening, E. coli was recovered from 33% of mesenteric lymph nodes versus 5% in controls [24Ambrose NS Johnson M Burdon DW Keighley RB Incidence of pathogenic bacteria from mesenteric lymph nodes and ileal serosa during Crohn's disease surgery.Br J Surg. 1984; 71: 623-625Crossref PubMed Scopus (158) Google Scholar]. E. coli antigen was observed by immunostaining in 69% of diseased intestinal resection specimens versus 27% in controls [25Cartun RW Van Kruiningen HJ Pedersen CA Berman MM An immunocytochemical search for infectious agents in Crohn's disease.Modern Pathol. 1993; 6: 212-219PubMed Google Scholar]. A correlation has been shown between high numbers of E. coli and the presence and severity of inflammatory bowel disease in HLA-B27 rats [26Onderdonk AB Richardson JA Hammer RE Taurog JD Correlation of cecal microflora of HLA-B27 transgenic rats with inflammatory bowel disease.Infect Immunol. 1998; 66: 6022-6023PubMed Google Scholar]. We recently characterized several E. coli isolates that were recovered from ileal mucosa of patients with Crohn's disease [27Darfeuille-Michaud A Neut C Barnich N et al.Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn's disease.Gastroenterology. 1998; 115: 1405-1413Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar]. Our findings indicate that E. coli strains associated with ileal mucosa of such patients adhere preferentially to differentiated Caco-2 cells, corresponding to mature intestinal cells. The identification of the adhesins involved was performed by PCR and hybridization assays. Of the 30 patient strains that adhered to differentiated intestinal cells, 73% harbored an adhesin that was not identified by the use of molecular technniques using the different DNA probes or primers specific to the structural genes encoding known adhesins of diarrheagenic E. coli or E. coli involved in human urinary tract infections. None of the E. coli strains isolated from biopsies of patients with Crohn's disease possess any of the virulence genes harbored by pathogenic E. coli strains. A cytotoxic effect on cultured intestinal cells was observed after a 3-h infection. DNA hybridization experiments with the various probes corresponding to known cytotoxin-encoding genes were positive with the α-hemolysin probe for 21.8% of the strains. Such a percentage was much higher than those observed in diarrheal stools (5.3%) or controls (2.2%) [28Jallat C Livrelli V Darfeuille-Michaud A et al.Escherichia coli strains involved in diarrhea in France: high prevalence and heterogeneity of diffusely adhering strains.J Clin Microbiol. 1993; 31: 2031-2037Crossref PubMed Google Scholar]. The E. coli α-hemolysin is a potent stimulus for the release of inflammatory mediators like O2−, β-glucuronidase, leukotriene and histamine. The combination of adhesive ability and synthesis of a cytotoxin might allow the bacteria to colonize intestinal epithelium, damage intestinal cells and participate in the inflammatory disease. The etiopathogenesis of Crohn's disease remains unknown, although an enormous amount of data has appeared in recent years. Environmental and genetic contributions are evident. Microbial agents appear to be intimately involved in the pathogenesis, but no causative microorganism has been found. Chronic intestinal and systemic inflammation requires the interaction of both genetic and bacterial factors, but neither alone is sufficient to induce chronic colitis or enteritis. Finally, resolution of the enigma may be hampered owing to disease heterogeneity, and the possibility exists that Crohn's disease might represent a syndrome with multiple etiologies.