Inhibition of P2X Receptors Protects Human Monocytes against Damage by Leukotoxin from Aggregatibacter actinomycetemcomitans and α-Hemolysin from Escherichia coli

Abstract

␣-Hemolysin (HlyA) from Escherichia coli and leukotoxin A (LtxA) from Aggregatibacter actinomycetemcomitans are important virulence factors in ascending urinary tract infections and aggressive periodontitis, respectively. The extracellular signaling molecule ATP is released immediately after insertion of the toxins into plasma membranes and, via P2X receptors, is essential for the erythrocyte damage inflicted by these toxins. Moreover, ATP signaling is required for the ensuing recognition and phagocytosis of damaged erythrocytes by the monocytic cell line THP-1. Here, we investigate how these toxins affect THP-1 monocyte function. We demonstrate that both toxins trigger early ATP release and a following increase in the intracellular receptors markedly reduces toxin-induced cytolysis. This pattern is paralleled in freshly isolated human monocytes from healthy volunteers. Interestingly, only a minor fraction of the toxin-damaged THP-1 monocytes eventually lyse. P2X 7 receptor inhibition generally prevents cell damage, except from a distinct cell shrinkage that prevails in response to the toxins. Moreover, we find that preexposure to HlyA preserves the capacity of THP-1 monocytes to phagocytose damaged erythrocytes and may induce readiness to discriminate between damaged and healthy erythrocytes. These findings suggest a new pharmacological target for protecting monocytes during exposure to poreforming cytolysins during infection or injury.␣ -Hemolysin (HlyA) is an important virulence factor frequently produced by strains of pathogenic Escherichia coli (1-3). The frequency with which HlyA-producing E. coli strains are isolated from patients increases with severity of the disease (for a review, see reference 2). HlyA is a pore-forming repeat in toxin (RTX) family member which inserts itself receptor independently into cell membranes (1). The cytotoxic effect of HlyA is massively amplified by ATP release, presumably through the HlyA pore (4) and following P2X receptor activation (5, 6). In erythrocytes, P2X 1 and P2X 7 receptors have been implicated in HlyA-induced hemolysis, and blocking of either of these receptors substantially reduces the hemolysis (5, 6). Interestingly, insertion of a HlyA pore does not cause immediate cell swelling and rupture but initially triggers a significant volume reduction that results from an increase in the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) followed by activation of the Ca 2ϩ -sensitive K ϩ and Cl Ϫ channels K Ca 3.1 and TMEM16A (7). During erythrocyte shrinkage, cells expose phosphatidyl serine (PS) in the outer plasma membrane leaflet (7). Recently, we discovered that THP-1 monocytes are more likely to recognize and phagocytose erythrocytes that have been exposed to HlyA (8). This phagocytosis is prevented if HlyA-induced cell damage is diminished by P2X receptor antagonists or if cell shrinkage and PS exposure are blocked (8).Leukotoxin A (LtxA) is a virulence factor often released from Aggregatibacter actinomycetemcomitans in the periodontal connective tissue (9-11). ...

P2X1, P2X4, and P2X7 Receptor Knock Out Mice Expose Differential Outcome of Sepsis Induced by α-Haemolysin Producing Escherichia coli

“…Previous studies from our group demonstrate a sizable reduction of HlyA-induced cell damage by P2X receptor antagonists on human and murine erythrocytes (Skals et al, 2009, 2014) and monocytes (Fagerberg et al, 2016). Therefore, it was exceedingly surprising to find that ${\text{P}2\text{X}}_7^{-/-}$ mice were not relatively protected, but rather distinctively more sensitive ARD6-induced sepsis.…”

α‐Haemolysin production, as a single factor, causes fulminant sepsis in a model ofEscherichia coli‐induced bacteraemia

“…The overall consensus about clearance of bacteria in the bloodstream is that the bacteria are phagocytosed by neutrophils and monocytes similar to what is known for tissue elimination of bacteria (for a review, see Christoffersson & Phillipson, ). Because HlyA certainly has the potential to change the behaviour or even lyse circulating immune active cells like monocytes (Fagerberg, Jakobsen, Skals, & Praetorius, ), the presence of HlyA could potentially interfere with intravascular bacterial clearance. Interestingly, the intravascular clearing of bacteria mechanistically has recently been argued to be markedly different from more solid tissues (for a review, see Minasyan, ).…”

Aggregatibacter actinomycetemcomitans leukotoxin: From mechanism to targeted anti‐toxin therapeutics

“…The authors observed that certain pharmacological P2X antagonists, including pyridoxalphosphate‐6‐azolphenyl‐2′,4′‐disulfonic acid (PPADS), inhibited LtxA‐mediated hemolysis (Munksgaard et al, 2012). A subsequent study identified a similar effect in human monocytes (THP‐1 cells; Fagerberg, Jakobsen, Skals, & Praetorius, 2016). While these reports suggest a role for the P2X receptors in LtxA‐mediated lysis of erythrocytes and monocytes, it is important to note that P2X inhibitors have been demonstrated to interfere directly with oligomerization of the Staphylococcus aureus α‐toxin (Schwiering, Husmann, & Hellmann, 2017) another toxin that had been reported to require P2X receptor activation (Skals, Leipziger, & Praetorius, 2011).…”