Abstract
C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI) is driven by toxin A (TcdA) and toxin B (TcdB), secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s) that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y6 receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y6 receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y6 inhibitor (MRS2578). This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y6 receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y6 receptor antagonists (MSR2578) attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y6 receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.
Highlights
Clostridium difficile (C. difficile), a Gram-positive sporeforming anaerobic bacterium, is a leading cause of nosocomial diarrhea worldwide
The release of CXCL8/IL-8 from TcdA/B treated intestinal epithelial cells (IECs) has been well documented, the mechanism(s) through which this occurs have yet to be fully elucidated [8,44,45,46]. We hypothesized that this process involves the release of UDP and autocrine/paracrine activation of the P2Y6 receptor and downstream induction of NFκB-dependent CXCL8/IL-8 transcription
To examine whether P2Y6 receptor activation could trigger the production of CXCL8/IL-8, an observation published previously [31,32], we stimulated Caco-2 cells with 5-OMe-UDP for 16 hr and measured CXCL8/IL-8 release by ELISA. 5-OMe-UDP triggered a dose-dependent increase in CXCL8/IL-8 release, which was completely blocked by pretreating the cells with MRS2578 (10 μM; Figure 1C), a selective inhibitor of the P2Y6 receptor [47]
Summary
Clostridium difficile (C. difficile), a Gram-positive sporeforming anaerobic bacterium, is a leading cause of nosocomial diarrhea worldwide. Through the release of two large toxins, toxin A (TcdA) and toxin B (TcdB), C. difficile triggers intestinal tissue damage and a robust inflammatory response resulting in relapsing diarrhea, pseudomembranous colitis, toxic megacolon and, in severe cases, death [4]. In addition to damaging the intestinal epithelial layer, TcdA and TcdB trigger the release of inflammatory mediators from intestinal epithelial cells (IECs) and monocytes/macrophages [6,7,8]. Toxin-induced CXCL8/IL-8 release from IECs is well documented and thought to play a key role in the recruitment of inflammatory cells into intestinal tissue [9]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
