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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 3

ATP levels and P2X4R expression increase during sepsis.

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ATP levels and P2X4R expression increase during sepsis.
(A and B) CLP in...
(A and B) CLP increases ATP levels in the peritoneum. (A) pmeLUC HEK293 cells (1 × 107) were injected retro-orbitally to recipient C57Bl/6J mice before sham operation or CLP. (B) Alternatively, pmeLUC mice were subjected to sham operation or CLP surgery. In both A and B, 3 hours after the CLP, 75 mg/kg luciferin was injected i.p. to animals and whole body luminometry was performed using IVIS 200 preclinical in vivo imaging system. Representative images are shown from sham/CLP-subjected mice from (A) pmeLUC HEK293-injected C57BL/6J mice or (B) pmeLUC transgenic mice. All results are representatives of 3 experiments. (C and D) mRNA expression of P2XRs in the liver and lung of sham/CLP-subjected (SH/C-subjected) C57Bl/6J mice. Mice were subjected to CLP or sham operation and, 16 hours later, liver and lung specimens were harvested. RNA was extracted from the tissues, transcribed, and analyzed by qPCR. *P < 0.05 vs. sham-operated mice; n = 3 (sham) and 6 (clp). ***P < 0.001. (E–J) tdTomato-P2X4R expression on blood and peritoneal macrophages. Peritoneal lavage fluid and blood were harvested from sham- or CLP-operated tdTomato-P2X4R transgenic reporter mice. Recovered cells were stained with anti-CD45, anti-F4/80, and anti-Ly6G antibodies, and tdTomato-P2X4R expression was monitored using flow cytometry. **P < 0.01 and ***P < 0.001 vs. neutrophils; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham group. n = 4. Data are expressed as mean ± SEM. All results are representatives of 2 experiments. Data obtained by two-tailed Student’s t test.

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