A278
CLOSTRIDIUM DIFFICILE TOXIN-INDUCED UDP RELEASE TRIGGERS THE PRODUCTION OF IL-8 FROM INTESTINAL EPITHELIAL CELLS - A NOVEL ROLE FOR THE P2Y6 RECEPTOR IN THE INDUCTION OF CLOSTRIDIUM DIFFICILE-ASSOCIATED DISEASE
S Hirota, V Lam, M Grassie, J MacDonald, P Beck
University of Calgary, Calgary, AB
Aims: C. difficile is a gram-positive spore-forming bacterium that is the leading cause of nosocomial diarrhea in the developed world. Recent outbreaks have been caused by strains with enhanced virulence due to their increased production of toxin A (TcdA) and toxin B (TcdB), the major secreted factors that contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis triggered by TcdA and TcdB. Previous reports have described that intestinal epithelial cell (IEC) intoxication triggers the release of IL-8, a potent neutrophil chemokine; however, little is known about the surface receptors mediating this effect. In the current study we sought to assess whether toxin-induced IL-8 release is driven by the activation of the P2Y6 receptor following the release of UDP, a danger signal, from intoxicated IECs.
Methods: Caco-2 cells were grown to confluence on standard culture plates and differentiated for seven days. Cells were treated with a mixture of TcdA and TcdB, purified from C. difficile cultures, and the supernatant and cell lysates isolated. UDP and IL-8 were detected in the supernatant with HPLC and ELISA, respectively. To probe the signaling pathways involved we utilized the following: MRS 2578-P2Y6 receptor antagonist; BAY 11-7085-NFκB inhibitor; PD98059-MEK1 inhibitor; AG1478-EGFR inhibitor.
Results: Treatment of Caco-2 cells with TcdA/TcdB (10 µg/mL; 16 h) led to UDP release into the culture supernatant which was also associated with the release of IL-8. TcdA/TcdB treatment also led to a significant increase in ERK activation. Inhibition of the P2Y6 receptor completely abolished toxin-induced IL-8 release, suggesting a role for UDP in this response; however, no inhibitory effect on ERK was observed. To probe the pathways downstream of the P2Y6 receptor we inhibited the ERK pathway and blocked EGFR activation. Both inhibition of ERK and the EGFR led to a significant reduction (~50% reduction) in TcdA/TcdB-induced IL-8 release. Interestingly, inhibition of NFκB signaling completely abolished TcdA/TcdB-induced IL-8 release.
Conclusions: Exposure of IECs to TcdA/TcdB triggers the release of UDP that acts in an autocrine fashion to activate the P2Y6 receptor and induce NFκB-dependent IL-8 release. These data suggest that strategies aimed at inhibiting the inflammatory effects of endogenous danger signals, such as UDP, may prevent the production of mediators that drive neutrophil migration to site of cellular intoxication, thus reducing immune-mediated tissue damage and disease progression.