Figure 1.
C. difficile TcdA/B triggers the release of UDP from Caco-2 cells that express a functional P2Y6 receptor.
(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.
Figure 2.
C. difficile TcdA/B triggers the production and release of CXCL8/IL-8 through the activation of the P2Y6 receptor.
(A) TcdA/B-induced CXCL8/IL-8 release was significantly reduced by the selective P2Y6 receptor antagonist MRS2578. N = 5; * denotes p<0.05 compared to vehicle (BHI culture broth). # denotes p<0.05 compared to vehicle-treated TcdA/B-stimulated cells. (B) TcdA/B-induced CXCL8/IL-8 release is sensitive to the selective P2-receptor antagonist PPADS. N = 5; * denotes p<0.05 compared to vehicle (BHI culture broth). # denotes p<0.05 compared to vehicle-treated TcdA/B stimulated cells. (C) Pharmacological antagonist of the P2Y6 receptor attenuates TcdA/B-induced CXCL8/IL-8 transcription in Caco-2 IECs. N = 5; * denotes p<0.05 compared to time 0. # denotes p<0.05 compared to vehicle-treated TcdA/B stimulated cells (10 μg/mL).
Figure 3.
TcdA/B-induced cell death and Rac1 modification are not affected by MRS2578.
(A) TcdA/B-induced cell death, as assessed by lactate dehydrogenase release (LDH), is not affected by the selective P2Y6 receptor antagonist MRS2578 (10 μM). N = 5; * denotes p<0.05 compared to vehicle control and MRS2578 (10 μM). (B) The detection of unmodified Rac1 is not affected by the selective P2Y6 receptor antagonist MRS2578 (10 μM). TcdA/B-induced Rac1 glucosylation and subsequent loss of a detectable Rac1 band is illustrated in vehicle- or MRS2578-treated Caco-2 cells at various time-points over 16 hrs. The western blot pictured is representative of 4 separate experiments.
Figure 4.
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.
(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).
Figure 5.
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.
(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.
Figure 6.
Inhibition of the P2Y6 receptor attenuates TcdA/B-induced intestinal epithelial barrier dysfunction in Caco-2 IECs.
(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).
Figure 7.
TcdA/B-induced intestinal inflammation and permeability are attenuated by inhibiting the P2y6 receptor in vivo.
(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.