α-haemolysin (HlyA)-producing Escherichia coli commonly inflict severe urinary tract infections, including pyelonephritis, which comprises substantial risk for sepsis. In vitro, the cytolytic effect of HlyA is mainly mediated by ATP release through the HlyA pore and subsequent P2X1/P2X7 receptor activation. This amplification of the lytic process is not unique to HlyA but is observed by many other pore-forming proteins including complement-induced haemolysis. Since free hemoglobin in the blood is known to be associated with a worse outcome in sepsis one could speculate that inhibition of P2X receptors would ameliorate the course of sepsis. Surprisingly, this study demonstrates that P2X1−/− and P2X4−/− mice are exceedingly sensitive to sepsis with uropathogenic E. coli. These mice have markedly lower survival, higher cytokine levels and activated intravascular coagulation. Quite the reverse is seen in P2X1−/− mice, which had markedly lower cytokine levels and less coagulation activation compared to controls after exposure to uropathogenic E. coli. The high cytokine levels in the P2X7−/− mouse are unexpected, since P2X7 is implicated in caspase-1-dependent IL-1β production. Here, we demonstrate that IL-1β production during sepsis with uropathogenic E. coli is mediated by caspase-8, since caspase-8 and RIPK3 double knock out mice show substantially lower cytokine during sepsis and increased survival after injection of TNFα. These data support that P2X7 and P2X4 receptor activation has a protective effect during severe E. coli infection.

␣-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). ...

The pore-forming exotoxin α-hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+-sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of either of these channels potentiate the HlyA-induced hemolysis. This means that Ca2+-dependent activation of KCa3.1 and TMEM16A protects the cells against early hemolysis. Simultaneous to the HlyA-induced shrinkage, the erythrocytes show increased exposure of phosphatidylserine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis. We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP-1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods, including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP-1 cells. The HlyA-induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS-exposure in human erythrocytes subjected to both ionomycin and HlyA. Moreover, we show that P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA-induced PS-exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA-insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis.

European Journal of Rheumatology (Eur J Rheumatol) is an international, open access peer reviewed journal committed to promoting the highest standards of scientific exchange and education. The journal is published quarterly on January, April, July and October.The aim of the European Journal of Rheumatology is to cover various aspects of rheumatology for its readers, encompassing the spectrum of diseases with arthritis, musculoskeletal conditions, autoinflammatory diseases, connective tissue disorders, osteoporosis, translational research, the latest therapies and treatment programs. European Journal of Rheumatology publishes original articles, invited reviews, case based reviews, letters to the editor and images in rheumatology. The publication language of the journal is English.Accepted manuscripts are copy-edited for grammar, punctuation, and format. Once the publication process of a manuscript is completed, it is published online on the journal's webpage as an aheadof-print publication before it is included in its scheduled issue. A PDF proof of the accepted manuscript is sent to the corresponding author and their publication approval is requested within 2 days of their receipt of the proof.

Background:The virulence factor HlyA elicits [Ca 2ϩ ] i oscillations in renal epithelial cells. Results: These oscillations and following IL-6 release are reduced by inhibition or lack of P2Y 2 receptors. Conclusion: The effects of HlyA in renal epithelial cells are mediated by P2Y 2 receptors. Significance: ATP release and P2Y 2 receptor activation are essential parts of the early interaction between Escherichia coli and the renal epithelium.