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Macrophage P2X4 receptors augment bacterial killing and protect against sepsis
Balázs Csóka, … , Pál Pacher, György Haskó
Balázs Csóka, … , Pál Pacher, György Haskó
Published June 7, 2018
Citation Information: JCI Insight. 2018;3(11):e99431. https://doi.org/10.1172/jci.insight.99431.
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Research Article Cell biology

Macrophage P2X4 receptors augment bacterial killing and protect against sepsis

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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 2

ATP augments bacterial killing by increasing ROS generation.

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ATP augments bacterial killing by increasing ROS generation.
(A and B) A...
(A and B) ATP augments bacterial killing in a manner that is independent of phagolysosome fusion. Bacterial killing assay was performed in the presence of (A) 1-butanol or (B) bafilomycin A. Macrophages were infected with E. coli for 90 minutes and then pretreated with 1-butanol or bafilomycin A 30 minutes before ATP treatment for 5 minutes. The cells were then incubated with gentamicin for 2 hours, and intracellular bacterial CFUs were determined at the end of the gentamicin incubation period. *P < 0.05, **P < 0.01 vs. E. coli; n = 4–6. (C–H) ATP increases bacterial killing through enhancing ROS generation. (C–F) Peritoneal macrophages were infected with E. coli for 90 minutes and then treated with (C) N-acetyl-L-cysteine (NAC), (D) superoxide dismutase-polyethylene glycol (SOD-PEG), (E) 1,3-PB-ITU (nitric oxide synthase 2 inhibitor), or (F) rotenone for 30 minutes, followed by a 5-minute ATP pulse. The cells were then incubated with gentamicin for 2 hours, and intracellular CFUs were determined at the end of the incubation period. *P < 0.05, **P < 0.01 vs. E. coli treatment; #P < 0.05, ##P < 0.01 vs. E. coli + ATP treatment; n = 6. (G and H) Measurement of (G) cellular or (H) mitochondrial ROS production. Peritoneal macrophages were incubated with (G) 25 μM 2′,7′-dichlorofluorescin diacetate (DCFDA) (cellular ROS dye) for 45 minutes or with (H) 5 μM MytoSOX dye (mitochondrial ROS dye) for 10 minutes. Thereafter, cells were incubated with E. coli for 90 minutes and then pulsed with ATP for 5 minutes. Following a 1-hour incubation with 400 ng/ml gentamicin, DCFDA- and mytosox-related fluorescence were detected using Victor2 (Perkin Elmer) luminometer. *P < 0.05, **P < 0.01 vs. vehicle treatment; n = 6. Data are expressed as mean ± SEM. All results are representatives of 3 experiments. Data obtained by one-way ANOVA followed by Mann Whitney test.

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