Alimentary Tract
Molecular evidence for Mycobacterium avium subspecies paratuberculosis (MAP) in Crohn's disease correlates with enhanced TNF-α secretion

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Abstract

Background

Support for a role of Mycobacterium avium subspecies paratuberculosis in Crohn's disease is largely based on epidemiological evidence, as no data on mechanisms linking the presence of M. avium subspecies paratuberculosis with gut damage is available.

Aims

To determine whether the presence of M. avium subspecies paratuberculosis contributes to the pathogenesis of Crohn's disease by promoting cytokine secretion within gut mucosa.

Patients and methods

A total of 235 subjects were recruited: 63 with Crohn's disease, 53 with ulcerative colitis, 45 with irritable bowel syndrome and 74 normal controls. M. avium subspecies paratuberculosis status was defined by nested PCR using IS900 sequence. Gut mucosal organ cultures were established to detect cytokine secretion patterns.

Results

Significantly higher tumour necrosis factor-α concentrations were found in culture supernatants for Crohn's disease compared to ulcerative colitis (p < 0.05), irritable bowel syndrome (p < 0.01) and controls (p < 0.0001). When tumour necrosis factor-α levels were correlated with the presence of M. avium subspecies paratuberculosis, significantly greater concentrations were only found in M. avium subspecies paratuberculosis-positive Crohn's disease patients (p < 0.05). Tumour necrosis factor-α levels in M. avium subspecies paratuberculosis-positive Crohn's disease were significantly higher than in M. avium subspecies paratuberculosis-positive ulcerative colitis (p < 0.01), M. avium subspecies paratuberculosis-positive irritable bowel syndrome (p < 0.05) and M. avium subspecies paratuberculosis-positive controls (p < 0.01) and all M. avium subspecies paratuberculosis-negative specimens.

Conclusions

The data link M. avium subspecies paratuberculosis with a pathogenic mechanism in Crohn's disease and is consistent with abnormal macrophage handling of M. avium subspecies paratuberculosis.

Introduction

The cause of chronic inflammation in the gut of subjects with Crohn's disease (CD) is unclear, though two hypotheses are widely held. The first is that genetically influenced defects in mucosal immune regulation and/or mucosal barrier protection, underpin a heightened inflammatory response to enteric bacteria that have accessed the gut mucosal compartment [19]. The second is that a specific infection drives mucosal inflammation, with the most popular candidate being Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in CD would appear to vary, although much of this variation may be a reflection of the different methodologies used [13], [10], [18]. Any relationship between MAP infection and disease is complicated by the observations that MAP may not be necessary for CD; that MAP is found in gut disease other than CD, as well as in normal subjects [10], [18]; and that the clinical benefits claimed for rifabutin/macrolide antibiotic combinations developed to eradicate MAP have not yet been correlated with the presence of MAP infection [4]. The argument for a role of MAP in the pathogenesis of CD would be strengthened by identifying a mechanism whereby MAP could induce tissue damage. The aim of this study was to determine whether the presence of MAP in CD correlated with a particular pattern of cytokine secretion by the gut mucosa. We report data that link the presence of MAP in CD with a selective enhancement of tumour necrosis factor-α (TNF-α) secretion from the gut mucosa, a known contributor to the inflammatory response that mediates tissue damage characteristic of this disease.

Section snippets

Subjects

The study was approved by the Centre for Digestive Diseases Human Research Ethics Committee, Sydney and Human Research Ethics Committee, University of Newcastle, Australia, and was performed in accordance with the principles of the 2000 Declaration of Helsinki. Informed consent was obtained from all subjects. Diagnosis was based on clinical criteria and the results of the pathological examination [15]. Patients presenting both for review and initial diagnosis were included. During colonoscopic

Results

Subjects were assessed in four groups: Crohn's disease (CD) (n = 63), ulcerative colitis (UC) (n = 53), irritable bowel syndrome (IBS) (n = 45) and normal controls (n = 74). The diagnosis was based on classical clinical, endoscopic and histological criteria. Of the 63 subjects with CD, 52 subjects had active inflammation (defined at colonoscopy as inflammation and/or ulceration and confirmed in biopsies) (11 for MAP-positive and 41 for MAP-negative) observed at colonoscopy and 11 had no inflammation (4

Discussion

The demonstration of enhanced TNF-α secretion in the gut of subjects with CD who are positive for MAP is the first potential evidence of a mechanism that could link the presence of MAP with gut disease. The selective effect of MAP infection on TNF-α secretion in CD compared to an absence of such an effect in normal and disease controls, points to a specific defect in the cellular handling of MAP in CD.

The high concentration of TNF-α in the supernatants of gut mucosal cultures in subjects with

Conflict of interest statement

None declared.

Acknowledgements

The study was funded by the Broad Medical Research Program of the Eli and Edythe L. Broad Foundation, USA. Authors are grateful to Evelyn Vigano for her skillful technical assistance. We thank Professor Tom Borody for providing clinical data and comments.

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