Modulation of pathogen-induced CCL20 secretion from HT-29 human intestinal epithelial cells by commensal bacteria

BACKGROUND: Human intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-kappaB activation were measured using enzyme-linked immunosorbent assays. RESULTS: Compared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-kappaB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-kappaB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis-induced CCL20 secretion. CONCLUSION: This study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens

Mycobacterium avium subsp. Paratuberculosis (MAP) as a modifying factor in Crohn's disease

BACKGROUND: Crohn's disease (CD) is a multifactorial syndrome with genetic and environmental contributions. Mycobacterium avium subspecies paratuberculosis (MAP) has been frequently isolated from mucosal tissues of patients with CD but the cellular immune response to this bacterium has been poorly described. Our aim was to examine the influence of MAP on T-cell proliferation and cytokine responses in patients with inflammatory bowel disease (IBD). METHODS: Peripheral blood mononuclear cells (PBMCs) and mesenteric lymph node cells (MLNCs) were obtained from IBD patients and non-IBD controls. PBMC T-cell proliferation in response to MAP was determined using CFSE labeling and flow cytometry. The specificity of cytokine responses to MAP was controlled by parallel exposure to Listeria monocytogenes (LM) or Salmonella typhimurium (ST). RESULTS: Coincubation of PBMCs with MAP induced significantly more T-cell proliferation (P < 0.0001) in PBMCs isolated from CD patients compared to PBMCs obtained from ulcerative colitis (UC) patients or healthy volunteers. In addition, PBMCs from CD patients secreted significantly higher (P < 0.05) levels of tumor necrosis factor-alpha (TNF-alpha; 2302 +/- 230 pg/mL) and interleukin (IL)-10 (299 +/- 48 pg/mL) in response to MAP compared to UC patients (TNF-alpha: 1219 +/- 411 pg/mL; IL-10: 125 +/- 19 pg/mL) and controls (TNF-alpha: 1447 +/- 173 pg/mL; IL-10: 127 +/- 12 pg/mL). No difference in cytokine responses was observed in response to LM or ST. MLNCs from both CD and UC patients secreted significantly more TNF-alpha and IL-8 in response to MAP compared to MLNCs from non-IBD control patients. CONCLUSIONS: Increased proliferation of T cells and an altered cytokine response suggest that prior exposure to MAP and engagement of the immune system is common in patients with CD. This does not imply causation but does support further examination of this bacterium as an environmental modifying factor