Macrophage P2X4 receptors augment bacterial killing and protect against sepsis
Balázs Csóka, Zoltán H. Németh, Ildikó Szabó, Daryl L. Davies, Zoltán V. Varga, János Pálóczi, Simonetta Falzoni, Francesco Di Virgilio, Rieko Muramatsu, Toshihide Yamashita, Pál Pacher, György Haskó
Balázs Csóka, Zoltán H. Németh, Ildikó Szabó, Daryl L. Davies, Zoltán V. Varga, János Pálóczi, Simonetta Falzoni, Francesco Di Virgilio, Rieko Muramatsu, Toshihide Yamashita, Pál Pacher, György Haskó
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Research Article Cell biology

Macrophage P2X4 receptors augment bacterial killing and protect against sepsis

Abstract

The macrophage is a major phagocytic cell type, and its impaired function is a primary cause of immune paralysis, organ injury, and death in sepsis. An incomplete understanding of the endogenous molecules that regulate macrophage bactericidal activity is a major barrier for developing effective therapies for sepsis. Using an in vitro killing assay, we report here that the endogenous purine ATP augments the killing of sepsis-causing bacteria by macrophages through P2X4 receptors (P2X4Rs). Using newly developed transgenic mice expressing a bioluminescent ATP probe on the cell surface, we found that extracellular ATP levels increase during sepsis, indicating that ATP may contribute to bacterial killing in vivo. Studies with P2X4R-deficient mice subjected to sepsis confirm the role of extracellular ATP acting on P2X4Rs in killing bacteria and protecting against organ injury and death. Results with adoptive transfer of macrophages, myeloid-specific P2X4R-deficient mice, and P2rx4 tdTomato reporter mice indicate that macrophages are essential for the antibacterial, antiinflammatory, and organ protective effects of P2X4Rs in sepsis. Pharmacological targeting of P2X4Rs with the allosteric activator ivermectin protects against bacterial dissemination and mortality in sepsis. We propose that P2X4Rs represent a promising target for drug development to control bacterial growth in sepsis and other infections.

Authors

Balázs Csóka, Zoltán H. Németh, Ildikó Szabó, Daryl L. Davies, Zoltán V. Varga, János Pálóczi, Simonetta Falzoni, Francesco Di Virgilio, Rieko Muramatsu, Toshihide Yamashita, Pál Pacher, György Haskó

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Figure 6

P2X4Rs on macrophages decrease bacterial burden, inflammation, and organ injury during sepsis.

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P2X4Rs on macrophages decrease bacterial burden, inflammation, and organ...
CLP-induced sepsis increased bacterial counts, inflammation, and BUN levels in WT recipients of adoptively transferred P2X4R–/– macrophages (P2X4R–/–→WT) more than in WT recipients of WT macrophages (WT→WT). (A and B) Bacterial burden was determined by counting the number of CFUs on blood agar plates after serial dilution of blood and peritoneal lavage samples. Blood and peritoneal lavage fluid were collected at 16 hours after CLP. (C–H) Cytokine and chemokine levels in WT recipient mice that were adoptively transferred with WT or P2X4R–/– peritoneal macrophages. Cytokines were measured 16 hours after CLP. (I) BUN was measured from plasma of CLP-subjected WT→WT and P2X4R–/–→WT mice 16 hours after CLP. *P < 0.05, **P < 0.01, *** P < 0.001 vs. WT→WT transfer (n = 8–10 recipient mice in each group). (J–Q) Bacterial load and inflammation are higher in myeloid-specific P2X4R–/– mice (P2X4Rfl/fl-LysM-Cre+) when compared with control (LysM-Cre+) mice following sepsis. (J and K) Bacterial burden was determined by counting the number of CFUs on blood agar plates after serial dilution of blood and peritoneal lavage samples. Blood and lavage fluid were collected at 16 hours after CLP. (L–Q) Cytokine and chemokine levels in P2X4Rfl/fl-LysM-Cre+ vs. LysM-Cre+ mice. Cytokines were measured 16 hours after CLP. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. LysM-Cre+ mice (n = 10 mice in each group, except n = 11 in the P2X4Rfl/fl-LysM-Cre+ group of Q). Data are expressed as mean ± SEM. All results are representatives of 2 experiments. Data obtained by two-tailed Student’s t test.