title: The P2Y6 receptor mediates Clostridium difficile toxin-induced CXCL8/IL-8 production and intestinal epithelial barrier dysfunction.
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.
authors.affiliation: Department of Physiology & Pharmacology, University of Calgary, Calgary, Alberta, Canada ; Department of Microbiology, Immunology & Infectious Disease, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Ashleigh
authors.initials: A
authors.lastname: Hansen
authors.forename: Laurie
authors.initials: L
authors.lastname: Alston
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Tulk
authors.forename: L Patrick
authors.initials: LP
authors.lastname: Schenck
authors.forename: Michael E
authors.initials: ME
authors.lastname: Grassie
authors.forename: Basmah F
authors.initials: BF
authors.lastname: Alhassan
authors.forename: Arun Teja
authors.initials: AT
authors.lastname: Veermalla
authors.forename: Samir
authors.initials: S
authors.lastname: Al-Bashir
authors.forename: Fernand-Pierre
authors.initials: FP
authors.lastname: Gendron
authors.forename: Christophe
authors.initials: C
authors.lastname: Altier
authors.forename: Justin A
authors.initials: JA
authors.lastname: MacDonald
authors.forename: Paul L
authors.initials: PL
authors.lastname: Beck
authors.forename: Simon A
authors.initials: SA
authors.lastname: Hirota
mesh_terms: D000818:Animals; D001081:Apyrase / Q000378:metabolism; D018938:Caco-2 Cells; D016360:Clostridioides difficile / Q000378:metabolism*; D004195:Disease Models, Animal; D004761:Enterocolitis, Pseudomembranous / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology*; D004768:Enterotoxins / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000096:biosynthesis*; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000382:microbiology / Q000503:physiopathology*; D008297:Male; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D004768:Enterotoxins; D016209:Interleukin-8; D016328:NF-kappa B; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; C099845:purinoceptor P2Y6; D001081:Apyrase