HlyA cause platelet activation and neutrophil/ monocyte interaction during urosepsis

Abstract

The urinary tract is frequently a primary infection site in patients with severe sepsis or septic shock 1. Urinary tract infections are most commonly caused by E.coli sub‐strains that produce several virulence factors 4– 8, of which α‐haemolysin (HlyA) is the most abundant 9. Our previous data demonstrate that the biological effects of HlyA are intimately associated with extracellular ATP signalling secondarily to ATP release from cells through the HlyA‐pore. Our previous data support that HlyA is largely responsible for the septic symptoms observed in response to bacteraemia with uropathogenic E.coli. ATP and its degradation products are aggressive activators of immune cells 10 and platelets 11– 13, and prime candidates for promoting the intravascular coagulation observed during septic shock. We tested the effect of antagonising platelet P2Y receptors (P2Y12 and P2Y1) during sepsis with uropathogenic E.coli(ARD6, O6:K13:H1) in anaesthetised mice. In aggreement with the literature, infusion of the P2Y12 antagonist cangrelor (8.6 or 86mg/hour) did not change survival or any tested sepsis parameters. However, the P2Y1 inhibitor MRS2500 reduced platelet activation by pore‐forming bacterial toxins (70–80%) in vitro, measured as surface exposure of fibronectin, DC63 and P‐selectin. In vivo, infusion of MRS2500 (624μg/hour) increased the survival in mice exposed to uropathogenic E. coli. Moreover, infusion of MRS2500 partially prevented the 60% fall in platelet count otherwise seen in response to sepsis in control mice. Interestingly, targeting of P2Y2 receptors, which is highly expressed on neutrophils and monocytes 14 but not in platelets, showed that lack of P2Y2 receptors entirely prevented the fall in circulating platelets in response to sepsis. Moreover, P2Y2 receptor‐deficient mice die much earlier in response to intravenous application of uropathogenic E. coli compared to controls. By flow cytometry, we are able to detect increased complex formation between thrombocytes (CD42) and neutrophils (Ly6‐G) /monocyte (Ly6‐G/CD11B). These data could potentially suggest that complex formation between thrombocytes and neutrophils and/or monocytes are important for the observed reduction in free thrombocyte numbers during sepsis and that the complexes could dampen the septic reaction to the circulating bacteria and improve survival. Support or Funding Information This project is funded byThe Independent Research Foundation ‐ DenmarkThe Augustinus Foundation NicolleCritCareClin29699715KennedyChest15625568GottsMatthayBMJ353i1585JohnsonClinMicrobiolRev480128ConnellPNAS93982732SvanborgAdvExpMedBiol76420510WulltMolMicrobiol3845664JohnsonStellJInfectDis18126172CavalieriMicrobiolRev4832643LandAmJTransplant16332237JinJBiolChem273203034DanielJBiolChem273202429JarvisBrJPharmacol12927582CliffordAmJPhysiol273C97387