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

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 1

ATP augments bacterial killing in macrophages in a P2X4R-dependent fashion.

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ATP augments bacterial killing in macrophages in a P2X4R-dependent fashi...
(A and B) ATP increases intracellular bacterial killing independently of P2X7Rs. Peritoneal macrophages from WT and P2X7R–/– mice were infected with E. coli (A) or with S. aureus (B) for 90 minutes, which was followed by pulsing the cells with ATP for 5 minutes. Subsequently, after a 2-hour incubation with 400 ng/ml gentamicin, the macrophages were lysed and serial dilutions of their intracellular content were spread onto Luria Bertani (LB) agar plates. *P < 0.05, **P < 0.01 vs. E. coli; n=5–6. (C and D) ATP augments bacterial killing independently of adenosine. Macrophages from WT and CD39–/– mice (a gift from Simon Robson, Beth Israel Deaconess Medical, Harvard University, Cambridge, MA; ref. 85) were infected with E. coli, pulsed with ATP (C) or adenosine (D), and then incubated with gentamicin for 2 hours, which was followed by intracellular CFU counting. *P < 0.05, **P < 0.01 vs. E. coli; n = 4–6. (E) Expression of P2Rs in peritoneal macrophages. Peritoneal macrophages were isolated from WT mice, and RNA was extracted from untreated cells. RNA was transcribed and qPCR was conducted. n = 6. (F–K) P2X4Rs are responsible for the ATP-stimulated increase in bacterial killing in macrophages. Peritoneal macrophages were infected with E. coli for 90 minutes, pretreated with (F) P2X4R antagonist (5-BDBD) or (G) P2X7R antagonist (A438079) for 30 minutes before an ATP pulse for 5 minutes, and killing was determined. As above*P < 0.05, ***P < 0.001 vs. E. coli treatment; ##P < 0.01 vs. ATP/E. coli treatment; n = 5–6. (H) Macrophages were transfected with scrambled siRNA or P2X4 siRNA, and the effect of ATP on bacterial killing was determined. **P < 0.01 vs. E. coli treatment; n=6. (I) Peritoneal macrophages from WT and P2X4R–/– mice were infected with E. coli, pulsed with ATP, and then bacterial killing was determined. **P < 0.01 vs. E. coli treatment; n = 6. *P < 0.05. (J and K) PMA-differentiated human monocytic THP-1 cells were infected with E. coli for 90 minutes and exposed to ATP for 5 minutes, and then killing was determined as described above for peritoneal macrophages (J). In other experiments, THP-1 cells were infected with E. coli and pretreated with 5-BDBD or vehicle for 30 minutes before a 5-minute ATP pulse (K); 2 hours later, intracellular CFUs were determined. **P < 0.01 vs. E. coli treatment; ##P < 0.01 vs. ATP/E. coli treatment. n = 6–7. Data are expressed as mean ± SEM. All results are representatives of 3 experiments. Data obtained by one-way ANOVA followed by Mann Whitney test.