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PATIENTS WITH INFLAMMATORY BOWEL DISEASE EXHIBIT DYSREGULATED RESPONSES TO TLR9 AGONISTS IN COLONIC BIOPSIES
P Bach, H Diaz, K St. Arnaud, N Hotte, R Fedorak, KL Madsen
University of Alberta, Edmonton, Alberta
The intestinal epithelium is constantly exposed to microflora at high levels and extensive cross-talk between microbes, intestinal epithelial cells (IECs), and immune cells is required to maintain homeostasis. Cells interact with bacterial DNA through TLR9. Patients with Crohn’s disease (CD) exhibit deficiencies in innate immune function, and associations between specific TLR9 polymorphisms and CD have been demonstrated. We have previously shown that IECs respond to probiotic and pathogenic in a differential manner, and probiotic bacterial DNA has been shown to reduce pro-inflammatory cytokine secretion in a mouse model of colitis.
AIM: The aim of this study was to examine the response of biopsies from patients with Crohn’s disease (CD) and ulcerative colitis (UC) to bacterial DNA.
METHODS: Biopsy samples were collected from non-inflamed areas in transverse colon of CD and UC patients and in controls. Two biopsies were snap-frozen for RNA extraction. Six biopsy samples underwent epithelial cell isolation using an EDTA/EGTA method. Equal amounts of cells were incubated at 37°C in DMED/antibiotic media ± Bifidobacterium breve or Salmonella dublin DNA (50 µg/ml). After 24 hrs, supernatants were collected and TNFalpha and IFNgamma measured in the supernatants by ELISA. Tissues were assessed for mRNA levels of TLR9 by real-time PCR.
RESULTS: In UC patients (n=8), S. typhimurium DNA induced a 2.5 fold increase in TNFalpha secretion over basal values, while B. breve DNA reduced TNFalpha secretion. In contrast, in CD patients (n=13) TNFalpha secretion did not change in response to either S. typhimurium or B. breve DNA. Biopsies from both UC and CD patients responded to B. breve, but not S. typhimurium DNA with enhanced IFNgamma secretion. Biopsies from control patients (n=3) exhibited increased IFNgamma secretion to both bacterial DNAs. TLR9 mRNA levels were reduced in CD biopsies compared with UC and controls.
CONCLUSIONS: In comparison with controls, CD and UC patients demonstrate different and distinct patterns of cytokine secretion in response to probiotic and pathogenic DNA. In particular, CD patients appear to exhibit reduced levels of TLR9 and a decreased innate response to pathogenic bacterial DNA. This failure to respond to bacterial DNA in CD patients supports the concept that CD results from impaired innate immunity.