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. 2018 Jun 7;3(11):e99431.
doi: 10.1172/jci.insight.99431.

Macrophage P2X4 receptors augment bacterial killing and protect against sepsis

Balázs Csóka  1 , Zoltán H Németh  1   2   3 , Ildikó Szabó  4 , Daryl L Davies  5 , Zoltán V Varga  6 , János Pálóczi  6 , Simonetta Falzoni  7 , Francesco Di Virgilio  7 , Rieko Muramatsu  8 , Toshihide Yamashita  8 , Pál Pacher  6 , György Haskó  1
Affiliations

Macrophage P2X4 receptors augment bacterial killing and protect against sepsis

Balázs Csóka et al. JCI Insight. .

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.

Keywords: Cell Biology; Innate immunity.

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Conflict of interest statement

Conflict of interest: GH owns stock in Purine Pharmaceuticals, Inc., which has a patent license (62/532,619 and 62/562,770) for the use of P2X4 agonists in infections and sepsis. FDV is a member of the Scientific Advisory Board and receives compensation from Biosceptre Ltd., which is developing anti-P2X7 Abs for therapeutic purposes.

Figures

Figure 1
Figure 1. ATP augments bacterial killing in macrophages in a P2X4R-dependent fashion.
(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.
Figure 2
Figure 2. ATP augments bacterial killing by increasing ROS generation.
(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.
Figure 3
Figure 3. ATP levels and P2X4R expression increase during sepsis.
(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.
Figure 4
Figure 4. P2X4Rs decrease mortality, bacterial load, inflammatory cytokines and chemokines, and organ injury in sepsis.
(A) WT mice have improved survival compared with P2X4R–/– mice. Male WT and P2X4R–/– mice were subjected to CLP, and survival was monitored for 7 days. *P = 0.019 (WT and P2X4R–/–; n = 24 and 25, respectively). (B and C) 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 hour after CLP. *P < 0.05; **P < 0.01 vs. WT (WT and P2X4R–/–; n = 10 and 14, respectively, for blood; n = 10 and 14, respectively, for lavage). (D–N) WT and P2X4R–/– mice were subjected to sham or CLP operation and (D and I) IL-1β, (E and J) IL-6, (F and K) IL-10, (G and L) TNF-α, and (H and M) MIP-2 levels were determined with ELISA from blood and peritoneal lavage fluid collected at 16 hours after the operation. (N) Blood urea nitrogen (BUN) was measured in plasma of sham- or CLP-subjected WT and P2X4R–/– mice 16 hours after CLP. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT sham- and CLP-operated WT and P2X4R–/–; n = 3, 4 for sham WT and P2X4R–/–; n = 6 and 10 for CLP WT. Data are expressed as mean ± SEM. All results are representatives of 3 experiments. Mortality curves were analyzed using Kaplan-Meier curve and log rank test and in the rest of the experiments one-way ANOVA followed by Mann Whitney test or two-tailed Student’s t test was used
Figure 5
Figure 5. P2X4Rs protect against sepsis-induced apoptosis in the spleen.
Apoptosis in spleen and thymus was examined by fluorescent TUNEL labeling. (A and C) Representative images of TUNEL and DAPI-stained (A) spleen and (C) thymus samples from sham- or CLP-operated WT and P2X4R–/– animals 16 hours after sham/CLP. The slides were scanned with a Zeiss LSM 710 confocal laser-scanning microscope. Arrows indicate apoptotic cells. (B and D) Quantitative analysis of TUNEL-stained slides of (B) spleen and (D) thymus specimens from sham- or CLP-subjected WT and P2X4R–/– mice. Quantification of TUNEL-positive nuclei was done on 8 high-power fields per section using the ImageJ software. **P < 0.01, ***P < 0.001 vs. WT (WT and P2X4R–/–; n = 24 and 24, respectively). Data are expressed as mean ± SEM. Results are representatives of 3 experiments. Data obtained by one-way ANOVA followed by Mann Whitney test was used.
Figure 6
Figure 6. P2X4Rs on macrophages decrease bacterial burden, inflammation, and organ injury during sepsis.
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.
Figure 7
Figure 7. Ivermectin, an allosteric activator of P2X4Rs improves survival, decreases bacterial burden and organ injury in mice after sepsis, and augments bacterial killing by macrophages.
(A) Ivermectin-treated WT mice showed improved survival compared with vehicle-treated (Veh-treated) WT mice. The survival of ivermectin-treated P2X4R–/– and Veh-treated P2X4R–/– mice was comparable. WT and P2X4R–/– mice were injected with 10 mg/kg ivermectin or its vehicle and subjected to CLP. The survival of mice was monitored for 7 days. *P < 0.05, P2X4R–/– injected with vehicle (n = 21) vs. WT injected with vehicle (n = 25); ***P < 0.001, WT injected with ivermectin (n = 20) vs. WT injected with vehicle (n = 25). (B and C) 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. *P < 0.05 vs. Veh (Veh and 10 mg/kg ivermectin; n = 10 and 9, respectively). (D) BUN was determined from plasma of ivermectin- or Veh-treated mice 16 hours after CLP. *P < 0.05 vs. Veh; n = 10 and 9, respectively. (E and F) Ivermectin increases intracellular killing of E. coli in cultured macrophages. Peritoneal macrophages were infected with E. coli for 90 minutes and then pulsed with ATP for 5 minutes. ATP was then removed, and the macrophages were incubated with ivermectin for 30 minutes. Thereafter, the ivermectin was removed and the cells were incubated in medium containing gentamicin for another 2 hours. The cells were then lysed, and serial dilutions of intracellular content were spread onto LB agar plates. *P < 0.05 (n = 5–6); **P < 0.01 (n = 5–6). Data are expressed as mean ± SEM. All results are representatives of 3 experiments. Mortality curves were analyzed using Kaplan-Meier curve and log rank test and in the rest of the experiments one-way ANOVA followed by Mann Whitney test or two-tailed Student’s t test was used.
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