P2X receptor inhibition protects THP‐1 monocytes against cytolysis by α‐hemolysin from Escherichia Coli and leukotoxin from Aggregatibacter actinomycetemcomitans

Abstract

The Repeats in Toxins (RTX) α‐hemolysin (HlyA) from E. coli and leukotoxin A (LtxA) from A. actinomycetemcomitans are important virulence factors for ascending urinary tract infections and aggressive forms of periodontitis respectively. Upon insertion into the plasma membrane ATP is released through the toxin pore and is as signaling molecule essential for the HlyA‐ and LtxA‐induced erythrocyte damage. The immediate effects after ATP release are a massive erythrocyte shrinkage and phosphatidylserine exposure in the outer leaflet of the plasma membrane. These events are eminent for the erythrocytes to be recognized and engulfed by the monocyte cell line THP‐1. Here, we investigate how these toxins and ATP, affect the survival and function of THP‐1 monocytes.By measurements of [Ca2+]i, we demonstrate that the THP‐1 express functional P2X (P2X1, P2X4 and P2X7) and P2Y2 receptor. We show that desensitization of either P2X1 by either low (0.001 μM) or more pronounced of P2X4/P2X7 by high concentrations of ATP (0.1 to 1 mM) increases the survival rate of THP‐1 cells attacked by either HlyA or LtxA in a calcein release assay. This protection is also found by specific blockage of the ATP sensitive receptors P2X1 (100 μM NF449) or P2X7 (100 μM oxATP or A804589). By erythrocyte phagocytosis assay, we determined that low dose HlyA (EC25) preconditioning is increasing the function of erythrocyte clearance at the place of injury.These findings suggest a new target for protecting monocytes during pore‐former toxemia – both pharmacologically through P2X blockers or receptor desenzitisation. Both mechanisms may be relevant for improving monocyte survival and function in patients suffering from infection with RTX producing bacteria.Support or Funding InformationFunded by the Danish Council for Independent Research, MEMRANES and Lundbeck Foundation