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INTESTINAL EPITHELIAL CELLS MODULATE DENDRITIC CELL ACTIVATION THROUGH A TLR9-MEDIATED MECHANISM
JL Backer, N Hotte, K Madsen
University of Alberta, Edmonton, Alberta
BACKGROUND: Epithelial-microbial interactions involving Toll-like receptors are critical to the maintenance of intestinal tract homeostasis. Factors derived from epithelial cells (IECs) alter gut dendritic cell phenotypes and ability to activate Th1 cells. We have shown that bacterial DNA will alter IECs immune activity in a strain-dependent fashion through interactions with TLR9.
AIM: The aim of this study was to determine if probiotic and pathogenic bacterial DNA stimulation of IEC would modulate dendritic cell phenotype in a differential manner, and if this response was altered under inflammatory conditions.
METHODS: Bone marrow cells were extracted from the long bones of 129/SvEv mice and cultured for 10 days in mrGM-CSF to derive dendritic cell populations (BM-DCs). BM-DCs were treated with either Salmonella dublin or Bifidobacterium breve DNA (50ug/ml) for 24 hrs. Treatment with LPS was used as a positive control. In co-culture experiments, BM-DCs were placed in the basolateral compartment under cultured mouse colonic cells (ModeK) and the apical surface of ModeK cells treated with bacterial DNA. In separate experiments, BM-DCs were exposed to conditioned media (CM) from ModeK cells treated with DNA. As a model of stress and inflammation, ModeK cells were pre-treated with either 2,4, dinitrophenol (DNP) or IFNgamma and TNF-alpha prior to DNA stimulation. BM-DC activation and cytokine secretion was assessed by flow cytometry and ELISA. To identify differences between proteins secreted in the presence of probiotic and pathogenic DNA, supernatant from IEC was analyzed using the EttanTM DIGE (Difference Gel Electrophoresis) system
RESULTS: BM-DCs responded to both S. dublin and B. breve DNA with enhanced secretion of IL-10 and IL-12. In contrast, when BM-DCs were cultured with ModeK cells or with CM from ModeK cells treated with bacterial DNA, both IL-10 and IL-12 secretion from BM-DCs was reduced. Treatment of IEC with B. breve DNA completely suppressed IL-12 secretion from BM-DC. BM-DCs incubated with ModeK cells metabolically stressed with DNP responded with an altered ratio of IL-10/IL-12 secretion, and also responded to B. breve DNA in a similar fashion to S. dublin DNA. DIGE analysis showed a statistical difference in >100 discrete proteins secreted by ModeK cells in response to probiotic compared with pathogenic bacterial DNA.
CONCLUSION: These results suggest that in response to TLR9 agonists, IECs release soluble mediators that dampen dendritic cell activation and cytokine release. Further, the IEC differential response to probiotic and pathogenic DNA is lost in a metabolically stressed epithelial cell, and this is reflected in dendritic cell responses. Finally, in contrast to IECs, dendritic cells do not differentiate between probiotic and pathogenic DNA.