The P2Y6 receptor mediates Clostridium difficile toxin-induced CXCL8/IL-8 production and intestinal epithelial barrier dysfunction.

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

TcdB, but not TcdA triggers CXCL8/IL-8 production and release from Caco-2 cells in a manner dependent on extracellular nucleotides and the P2Y₆ receptor.

<p>(A) CXCL8/IL-8 release from Caco-2 cells treated with purified TcdA or TcdB (16 hr). N = 5; * denotes p<0.05 compared to TcdA. (B) TcdB, but not TcdA, treatment of Caco-2 cells increases cell death as assessed by LDH release. N = 5; * denotes p<0.05 compared to TcdA. TcdB-induced CXCL8/IL-8 release is significantly reduced by (C) MRS2578 (10 μM) and (D) co-treatment with apyrase (20 u/mL). N = 5; * denotes p<0.05 compared to no treatment; # denotes p<0.05 compared to all groups. </p

<i>C. difficile</i> TcdA/B triggers the release of UDP from Caco-2 cells that express a functional P2Y₆ receptor.

<p>(A) Western blot analysis of lysates reveals the expression of the P2Y₆ receptor in differentiated Caco-2 cells and PMA-differentiated THP-1 macrophages (included as positive control). (B) Stimulation of the Caco-2 cells with the selective P2Y₆ receptor agonist 5-OMe-UDP (1 μM) increases intracellular calcium concentrations as assessed by fluorescence imaging. (B-i) Pseudocolour images of Caco-2 cells before and after 5-OMe-UDP treatment. (B-ii) Representative traces of individual cells challenged with 5-OMe-UDP. (B-iii) The mean of the 5-OMe-UDP-induced calcium responses (n=46; grey denotes the standard error of the mean). (C) P2Y₆ receptor agonist 5-OMe-UDP triggers CXCL8/IL-8 release from Caco-2 cells, an effect that blocked by the potent P2Y₆ receptor antagonist MRS2578. N = 6; * denotes p<0.05 compared to control; # denotes p<0.05 compared to vehicle; % denotes p<0.05 compared to vehicle and 1 μM MRS 2578. (D) TcdA/B triggers the release of UDP as assessed by HPLC. i – control treated culture supernatant; ii – UDP-spiked control culture supernatant (100 μM UDP); iii – TcdA/B-spiked control culture supernatant (10 μg/mL); iv – TcdA/B-treated cell culture supernatant (10 μg/mL; 16 hr). (E) Summary data from HPLC measurement of TcdA/B-induced UDP release. N=5; * denotes p<0.05.</p

TcdA/B-induced CXCL8/IL-8 production from Caco-2 IECs involves the NFκB activation, an effect that is inhibited by pharmacological blockade of the P2Y₆ receptor by MRS2578.

<p>(A) TcdA/B-induced CXCL8/IL-8 release is inhibited by pretreatment with the selective NFκB pathway inhibitor BAY 11-7085 (20 μM). N=6; ** denotes p<0.005 compared to vehicle-treated TcdA/B stimulated cells (10 μg/mL). (B) Representative western blot for phosphorylated p65 (P-p65) in lysates from TcdA/B (10 μg/mL) stimulated Caco-2 IECs over the course of 60 min in the presence of the P2Y₆ antagonist MRS2578 (10 μM) or vehicle control (DMSO). (C) The summarized western blot data for P-p65 expressed as a percentage of the total p65. N = 4, *, denotes p<0.05 compared to time 0 min; # denotes p<0.05 compared to respective vehicle control (DMSO). </p

A Sterile 20 Family Kinase and Its Co-factor CCM-3 Regulate Contractile Ring Proteins on Germline Intercellular Bridges

eTOC Blurb Intercellular bridges are proposed to arise from stabilization of a mitotic cytokinetic ring. Rehain-Bell et al. demonstrate that inhibition of non-muscle myosin II is a conserved mechanism of bridge stabilization and find that, in the C. elegans oogenic germline, this is mediated by GCK-1(Ste-20 Kinase), its co-factor CCM-3, and anillin proteins. [Image: see text

TcdA/B-induced intestinal inflammation and permeability are attenuated by inhibiting the P2y₆ receptor <i>in</i><i>vivo</i>.

<p>(A) Intrarectal instillation of TcdA/B (50 μg/100 μL for 4 hrs) triggers a significant increase in colonic tissue myeloperoxidase (MPO), an effect that is significantly reduced by pretreating mice with the P2Y₆ inhibitor MRS 2578 (100 μL of 10 μM; in PBS via intrarectal instillation). N = 6/group; * denotes p<0.05 compared to PBS vehicle groups; # denotes p<0.05 compared to TcdA/B treatment with vehicle (DMSO). (B) Pretreating mice with MRS 2578 (100 μL of 10 μM; in PBS via intrarectal instillation) inhibits TcdA/B-induced increases in permeability as assessed by FITC-flux from the colonic lumen in to the serum. N = 6/group; * denotes p<0.05 compared to PBS vehicle groups; # denotes p<0.05 compared to TcdA/B treatment with vehicle (DMSO). (C) Pretreating mice with MRS 2578 reduces the histological inflammatory score and the (D) percentage of the colonic tissue section exhibiting architectural changes (% architecture change). N = 12 sections/group; * denotes P<0.05 compared to vehicle pretreatment. (E) Representative colonic sections stained with hematoxylin and eosin from mice treated with vehicle, MRS2578 alone, vehicle + TcdA/B and MRS2578 + TcdA/B; N = 6/group.</p

Inhibition of the P2Y₆ receptor attenuates TcdA/B-induced intestinal epithelial barrier dysfunction in Caco-2 IECs.

<p>(A) TcdA/B-induced (10 μg/mL) FITC-flux is significantly reduced by the selective P2Y₆ receptor antagonist MRS 2578 (10 μM). N=4; * denotes p<0.05 compared to vehicle and MRS2578. # denotes p<0.05 compared to TcdA/B. (B) 5-OMe-UDP (100 μM) increases FITC-flux in Caco-2 monolayers, an effect that is significantly attenuated by MRS2578 (10 μM). N=4; * denotes p<0.05 compared to vehicle and MRS2578. # denotes p<0.05 compared to 5-OMe-UDP. (C) Apical administration of TcdA/B (10 μg/mL) or 5-OMe-UDP (100 μM) for 4 hr triggers a redistribution of ZO-1 in Caco-2 monolayers, an effect that is blocked by pretreatment with MRS 2578 (10 μM; N=4).</p