Deletion of P2X7 attenuates hyperoxia-induced acute lung injury via inflammasome suppression

Abstract

Increasing evidence shows that hyperoxia is a serious complication of oxygen therapy in acutely ill patients that causes excessive production of free radicals leading to hyperoxia-induced acute lung injury (HALI). Our previous studies have shown that P2X7 receptor activation is required for inflammasome activation during HALI. However, the role of P2X7 in HALI is unclear. The main aim of this study was to determine the effect of P2X7 receptor gene deletion on HALI. Wild-type (WT) and P2X7 knockout (P2X7 KO) mice were exposed to 100% O₂ for 72 h. P2X7 KO mice treated with hyperoxia had enhanced survival in 100% O₂ compared with the WT mice. Hyperoxia-induced recruitment of inflammatory cells and elevation of IL-1β, TNF-α, monocyte chemoattractant protein-1, and IL-6 levels were attenuated in P2X7 KO mice. P2X7 deletion decreased lung edema and alveolar protein content, which are associated with enhanced alveolar fluid clearance. In addition, activation of the inflammasome was suppressed in P2X7-deficient alveolar macrophages and was associated with suppression of IL-1β release. Furthermore, P2X7-deficient alveolar macrophage in type II alveolar epithelial cells (AECs) coculture model abolished protein permeability across mouse type II AEC monolayers. Deletion of P2X7 does not lead to a decrease in epithelial sodium channel expression in cocultures of alveolar macrophages and type II AECs. Taken together, these findings show that deletion of P2X7 is a protective factor and therapeutic target for the amelioration of hyperoxia-induced lung injury.

acute lung injury (ali) is a major clinical problem in the United States, accounting for one of the primary causes of in-hospital hypoxemic respiratory failure leading to morbidity and mortality (48). Prolonged exposure to hyperoxia has been conclusively demonstrated to cause ALI (22, 58, 62). Furthermore, numerous cardiovascular and pulmonary diseases require oxygen therapy (22, 28–30, 58). Therefore, it is pertinent to understand the mechanism of damage in ALI, wherein the pathology manifests as alveolar damage from immune cell infiltration and pulmonary edema (31, 63).

Prolonged exposure to oxygen concentrations of Fi_O2 > 0.8 has previously been shown to cause mortality in small animal and higher-order primate models (26). Hyperoxia-induced lung injury contributes to increasing the production of reactive oxygen species (ROS), inflammatory cytokines, as well as exudative pulmonary edema, collagen deposition, and damage to the alveolar epithelium (26, 32). Thus, using hyperoxia as a form of oxygen toxicity is clinically relevant to study the pathophysiology of ALI. In ALI, the disruption of the fine balance between proinflammatory and anti-inflammatory agents is the basic pathology with a concomitant activation of apoptotic signals (33, 59, 62). IL-1β was discovered to be one of the early inflammatory cytokines to appear in ALI patients and cause the release of additional cytokines (18). The caspase-1-mediated activation of IL-1β was, in turn, identified to be generated by the NLRP3 inflammasome (34). NLRP3, a member of the NOD-like receptors (NLRs) family, is responsible for the inflammasome-mediated immune responses in association with caspases and apoptosis-associated speck-like protein containing a CARD (ASC) domain (20, 51). ROS are implicated in the stimulation of the NLRP3 inflammasome (11). Recent study from our laboratory implicated hyperoxia in inducing inflammasome complex formation and caspase-1 activation (27, 28). The precursors of IL-1β and IL-18 (i.e., pro-IL-1β and pro-IL-18) were, in turn, activated by caspase-1 (49). We further showed that hyperoxia-induced inflammasome complex formation was inhibited by P2X7 suppression (28).

The membrane receptor P2X7 becomes activated by extracellular ATP, which then stimulates the NLRP3 inflammasome to mediate the production of IL-1β (14, 36). Previous studies have demonstrated the augmented NLRP3 inflammasome action and IL-1β levels in the presence of LPS in ALI mice (65). Furthermore, LPS increased P2X7 levels in lung parenchyma, while P2X7 knockout mice demonstrated decreased amount of immune response cells and collagen deposition (36). Our previous research on P2X7 with antagonist oxATP exhibited inhibitory action on hyperoxia-induced inflammasome activation (28). However, it is unclear whether deletion of P2X7 in vivo protects against hyperoxia acute lung injury (HALI). Therefore, in this study, we investigate whether P2X7 deletion would lead to the attenuation of HALI.

MATERIALS AND METHODS

Mice.

The C57BL/6 (wild type) and P2X7 knockout (KO) mice (6 wk old, 50% female and 50% male, n = 20) were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free animal facility at the University of South Florida. All experiments were in accordance with the Institutional Animal Care and Use Committee regulations of the University of South Florida. A total of five mice were used per experiment. The experiments were repeated three or four times.

Reagents.

Materials required for cell culture, including growth media, FBS, and buffers were obtained from Life Technologies, (Grand Island, NY). All of the other reagents were purchased from Sigma (St. Louis, MO). Protein concentration of cell lysates was quantified using a BSA assay kit (Thermo Scientific, Rockford, IL). Microscope slides, ethanol, and all other solvents were bought from Fisher Scientific (Houston, TX).

Survival study.

The wild-type (WT; n = 20) and P2X7 knockout (P2X7 KO; n = 20) mice were subjected to continuous 100% O₂ and observed at 24-h intervals to assess survival. Observation was continued until the last mouse survived, as previously described (15, 17).

Hyperoxia exposure.

Six-week-old mice were placed in a closed chamber of size (75 ×50 ×50 cm) and exposed to 100% O₂ for 24, 48, and 72 h, respectively. The controls were exposed to room air. A proOx P100 sensor (BioSpherix) was used to monitor the oxygen concentration, as previously described (30, 62).

Bronchoalveolar lavage fluid collection.

Mice were anesthetized with ketamine/xylazine mixture via intraperitoneal injection, as previously described. Surgically, the trachea was exposed and incised after cervical dislocation, and a 0.6-mm catheter was inserted (15, 58, 62). The lungs were perfused with sterile PBS and bronchoalveolar lavage fluid (BALF) was collected, as previously described (6, 15, 17). For each mouse, BALF perfusion was performed three times, and the cell-free BALF was stored at −80°C until required for further experiments.

ELISA.

Commercial ELISA kits were used to measure the levels of IL-1β (eBioscience, San Diego, CA), IL-6 (BD Bioscience, San Diego, CA), TNF-α (RayBiotech, Norcross, GA), and monocyte chemoattractant protein 1 (MCP-1) (eBioscience) in BALF or in cell supernatants, as per the manufacturer's instructions (6, 15, 17).

Caspase-1 activity assay.

Caspase-1 activity was measured in cell lysates of WT or P2X7 KO mice alveolar macrophages and determined by a fluorogenic substrate Ac-WEHD-AMC and caspase-1 fluorometric assay kit from Abcam (Cambridge, MA), according to manufacturer's instructions (12).

Alveolar fluid clearance.

The alveolar Evans Blue-labeled albumin concentrations were used to estimate the alveolar fluid clearance (AFC) rate, as previously described (41). Evans Blue-dyed 5% BSA (0.15 mg/ml) in 1 ml of sterile warm saline (5 ml/kg mice body wt) was injected into the lung by intratracheal administration, with 2 ml O₂ to facilitate distribution. At an airway pressure of 7 cmH₂O, the lungs were ventilated with 100% O₂ in a humidified incubator at 37°C for 1 h. The alveolar fluid was then aspirated and measured for labeled albumin by a spectrophotometer at 620 nm (41). AFC was calculated using the formula: AFC = [(V_i − V_f/V_i)] × 100% V_f = (V_i × E_i)/E_f, where V_i stands for the volume of injected albumin solution, V_f stands for the volume of the final alveolar fluid, E_i stands for the injected and (E_f) final concentrations of the Evans Blue-labeled 5% albumin solution.

Analysis of BALF.

BALF was centrifuged for 10 min at 200 g at 4°C. The supernatants obtained were stored at −80°C for further analysis, while the cell pellets were resuspended with ice-cold sterile PBS. A glass hemocytometer was used to measure the total number of cells in the cell suspension. Each cell suspension was then aliquoted into 100–300 μl and centrifuged at 800 g for 3 min in a cytocentrifuge (Shandon Cytospin 2, Pittsburg, PA) and transferred onto glass slides. These cells were then stained with Diff-Quik stain (Andwin Scientific, Schaumburg, IL) and were analyzed under a microscope for differential white blood cell count (minimum of 200 cells) at ×200 magnification (15, 17). The concentration of protein in BALF, a key indicator of alveolar leak, was assessed by BCA protein estimation assay (Pierce, Rockford, IL) following the manufacturer's protocol (6).

Lung perfusion and tissue collection.

Following BALF collection, the lungs were perfused with 10% formalin in PBS at pH 7.40 through the right ventricle via an abdominal incision. The left lobe of the lung was fixed in 0.5 ml of 10% neutral buffered formalin and was removed, histologically processed, and paraffin-embedded (15, 17, 58, 62). The rest of the tissue was stored at −80°C until required for further experiments.

Lung injury evaluation.

To assess lung edema, we calculated wet/dry ratio by using six lungs per group, as previously described (53). To calculate wet weight, we dry blotted the lungs and weighed them. These lungs were then desiccated by incubation overnight at 130°C in a vacuum oven and reweighed to estimate the dry weight. Wet/dry ratio was calculated by dividing wet and dry weights (15, 17, 53). The rest of the tissue was stored at −80°C in liquid nitrogen for further analysis.

Lipid peroxidation.

Indicators of oxidative stress like F2-isoprostanes and isofurans were estimated using Cayman 8-isoprostane ELISA kit (Ann Arbor, MI) using lung homogenates as per the manufacturer's instructions. Other protein adducts, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were analyzed by ELISA in lung tissue homogenates, as previously described (16).

Measurement of albumin flux.

A coculture model using type II alveolar epithelial cells (AECs) and alveolar macrophages (AM) from WT or P2X7 KO mice was used to evaluate transepithelial albumin flux in normoxic and hyperoxic conditions. Type II AECs were isolated, as previously described (2) and grown in 24-well transwell plates until cells reached adequate confluence. Alveolar macrophages (1.5 × 10⁵) were then added to the type II AECs, cocultured for 3 h, and exposed to hyperoxia for 1 h. Transepithelial albumin flux across the monolayers was evaluated by the addition of 0.05 μCi [¹²⁵I]-labeled albumin to each upper compartment and incubated for 1 h. Lower-compartment contents were then collected and counted in a Wizard γ-counter (Perkin-Elmer, Waltham, MA), as previously described (28).

Determination of epithelial monolayer bioelectric properties.

Type II AEC monolayers isolated from mice lungs were cultured in an air-liquid interface for 4 days and were exposed to supernatants of WT and P2X7 KO AM, which were treated with hyperoxia for 24 h. The potential difference (PD; millivolts) and transepithelial resistance (TER; kΩ/cm²) was measured using a Millicell-ERS Voltohmmeter (Millipore, Bedford, MA) with Ag/AgCl electrodes, as described previously (54). Transepithelial current (TEC; microamps/cm²) was calculated by using Ohm's Law equation: TEC = PD/R_t. Data are represented as a percentage of control.

Measurement of epithelial sodium transport channel expression.

Type II AECs were cultured in an air-liquid interface for 4 days and were exposed to the supernatants of WT and P2X7 KO alveolar macrophages that were treated with hyperoxia for 24 h. Total RNA was extracted from cell lysates using an RNeasy mini kit (Qiagen, Valencia, CA) and analyzed for epithelial sodium channel, α-subunit (αENaC) expression by quantitative real-time reverse transcriptase PCR (qRT-PCR), as described previously (54). Data are presented as a percentage of control.

Statistics.

We used WT and P2X7 KO mice (n = 20 per group) for survival studies. Survival curve was generated using Kaplan-Meier analysis, while survival differences between groups were calculated using Sigma2 analysis. Values are given as means ± SE. For statistical analysis, GraphPad Prism (GraphPad Software, San Diego, CA) version 10.0 was used. Differences between the groups were analyzed using one-way ANOVA with post hoc Tukey test or Student's unpaired t-test. P < 0.05 was considered statistically significant.

RESULTS

Increased survival rates in P2X7 KO mice.

Hyperoxia causes deleterious effects through oxidative stress at the cellular level leading to molecular dysfunction (28, 30, 32). In lung epithelial cells, the resultant inflammation is mediated by the P2X7/NLRP3 inflammasome pathway (57). Our previous research provided the first documented evidence that hyperoxia-induced cell permeabilization was inhibited by blocking the P2X7 receptor with oxATP, demonstrating the critical role of the P2X7 receptor in hyperoxia-induced pore formation (28). To further test this pathway, we investigated whether deletion of P2X7 could provide protection from hyperoxia-induced injury. As the first step, we sought to determine the survival potential of P2X7 knockout (P2X7 KO) mice subjected to hyperoxia. We compared the survival in 100% O₂ of P2X7 KO mice with their WT littermate controls. In keeping with observations from our laboratory and others (15, 17), all WT mice died within 6 days of 100% O₂ exposure. In contrast, the P2X7 KO mice were more resistant to hyperoxia, surviving up to 8 days under hyperoxic exposure (Fig. 1). From these results, we conclude that the knockdown of P2X7 increases net survival of mice subjected to 100% O₂.

Fig. 1.

P2X7 KO mice display extended survival rates during hyperoxia. Kaplan-Meier survival curve of wild-type (WT; circles) and P2X7 knockout (KO) mice (squares) (n = 20) subjected to 100% O₂ for 72 h. Values represent percent survival, with baseline at 100%. The numbers of surviving mice were counted at 24-h intervals.

P2X7 KO confers protection against alveolar leakage and lung edema.

Alveolar protein extravasation and pulmonary edema are pathognomonic of ALI (17). Quantification of the protein level in BALF would be proportional to the extent of immune response in ALI. Protein levels in P2X7 KO mice were found to be reduced to approximately half of the level relative to WT mice under 100% O₂ (Fig. 2A). However, the levels were unchanged in both groups under normoxia. Additionally, we assessed lung edema by measuring wet/dry ratio in room air (normoxia) and hyperoxia. There was a twofold increase in lung edema in the WT mice compared with P2X7 KO mice under hyperoxia (Fig. 2B). However, the edema volume was the same for both the groups under room air exposure. Collectively, these results suggest that the knockdown of P2X7 decreases alveolar leakage and lung edema under hyperoxic conditions.

Fig. 2.

P2X7 deficiency attenuates hyperoxia-mediated alveolar leak and lung edema, while increasing alveolar fluid clearance. WT and P2X7 KO mice were exposed to room air (normoxia) or 100% O₂ (hyperoxia) for 72 h. A: total protein content was assessed using BCA assay. BALF, bronchioalveolar lavage fluid. B: lung edema was measured by calculating wet/dry ratio of mouse lungs. C: alveolar fluid clearance (AFC) was measured using spectrophotometric assay with Evans Blue-labeled 5% albumin. Alveolar Evans Blue-labeled albumin concentrations were used to estimate the AFC rate, as described in materials and methods. Data are expressed as means ± SE are representative of at least three independent experiments, with five mice used per experiment. *P < 0.05 and †P < 0.05, comparing PBS, WT, and P2X7 KO hyperoxic groups.

Alveolar fluid clearance is improved in P2X7 KO mice.

As the data suggest that P2X7 KO ameliorated protein leakage and pulmonary edema, we further addressed the role of P2X7 in alveolar fluid transport. Alveolar fluid clearance was determined as previously described (41). The results obtained varied with the level of O₂ exposure in the two groups of mice. Under normoxia, WT mice showed an alveolar fluid clearance of 43.3%. P2X7 KO mice exposed to hyperoxia showed a significant increase in the alveolar fluid clearance, relative to WT and PBS-treated mice exposed to hyperoxia (Fig. 2C). These results suggest that alveolar fluid clearance is accelerated in P2X7 KO mice and further validate our earlier observation that lung edema is drastically attenuated in the absence of P2X7.

P2X7 deletion attenuates hyperoxia-triggered immune cell infiltration.

ALI-mediated P2X7/NLRP3 pathway-mediated inflammatory sequelae lead to the accumulation of immune cells (15, 57). To assess the alveolitis generated as a consequence of P2X7 activation, we analyzed accumulated immune cells. BALF was collected from the WT and P2X7 KO mice under normoxia and hyperoxic conditions. BALF from hyperoxic WT mice displayed nearly twice the amount of total cells relative to the KO mice (Fig. 3A). Infiltration of various immune cells was analyzed using differential cell count (15). Macrophage count was drastically reduced in P2X7 KO mice exposed to hyperoxia compared with WT control (Fig. 3B). Similar results were obtained when neutrophil counts were compared between the two groups (Fig. 3C). Under hyperoxia, the WT showed approximately twice the number of neutrophils relative to the P2X7 KO mice, while mice exposed to normoxia showed no significant changes in the number of inflammatory cells. Moreover, MCP-1, a potent chemoattractant recruiting largely monocytes/macrophages (25), was assessed by ascertaining MCP-1 levels under the same conditions. Interestingly, WT mice displayed a threefold increase in MCP-1 concentration that correlated well with an increase in the number of macrophages observed (Fig. 3, D and B, respectively). Taken together, these data support the anti-inflammatory effect of P2X7 KO under hyperoxia.

Fig. 3.

Hyperoxia-induced recruitment of immune cells and elevation of monocyte chemoattractant protein 1 (MCP-1) were attenuated in P2X7 KO mice. WT and P2X7 KO mice were exposed to room air or 100% O₂ for 72 h. BALF was collected and analyzed for total cell counts (A), total number of macrophages (B), total number of neutrophils (C), and levels of MCP-1 (D). Data are expressed as means ± SE are representative of at least three independent experiments, with five mice used per experiment. *P < 0.05, **P < 0.01.

Cytokine levels are suppressed by P2X7 deletion.

Of the numerous inflammatory cytokines involved in ALI, IL-1β is the most prominent molecule associated with early ALI (18, 64). The mice were subjected to 72 h of hyperoxia followed by ELISA to determine IL-1β levels in BALF. Hyperoxia caused elevated levels of IL-1β in the WT mice (Fig. 4A). On the other hand, IL-1β levels were suppressed by 90% in the BALF of P2X7 KO mice relative to the WT mice under hyperoxia (Fig. 4A). In addition, other cytokines, including IL-6 and TNF-α, were analyzed by using a similar approach. After hyperoxia challenge, IL-6 release was reduced in P2X7 KO mice compared with continued increase in WT mice, although still greater than room air (Fig. 4B). Similarly, TNF-α levels were increased three-fold in WT mice in relation to the P2X7 KO group (Fig. 4C). Under normoxic conditions, no significant variation in the levels of the various cytokines was observed between the two groups. These results show that deletion of P2X7 decreases cytokine infiltration and release under hyperoxic conditions.

Fig. 4.

Hyperoxia-induced elevation of pro-inflammatory cytokines was suppressed in P2X7 KO mice. WT and P2X7 KO mice were exposed to room air or 100% O₂ for 72 h. After euthanization, BALF was collected and analyzed for the concentration of proinflammatory cytokines: IL-1β (A), IL-6 (B), and TNF-α (C). Cytokines were quantified by using ELISA, according to manufacturer's instructions. Data are presented as means ± SE and are representative of at least three independent experiments, with five mice used per experiment. **P < 0.01.

Hyperoxia-induced lipid peroxidation products are decreased in P2X7 KO mice.

F₂-isoprostane, a lipid peroxidation by-product is a biomarker for oxidative stress (24, 56). More recently, another marker isofuran was identified, whose concentration increases under elevated O₂ levels, making combined detection of these molecules more efficient for evaluation of hyperoxia-induced oxidative stress (60). Previously, a connection has been made between P2X7 and its involvement in mediating lipid peroxidation (21, 39). P2X7 was shown to increase the production of superoxide, which is known to be formed by hyperoxia-induced oxidative stress and contribute to the formation of lipid peroxidization by-products. Thus, we hypothesized that knockout of P2X7 will reduce the production of these peroxidized molecules. Data analysis of lipid peroxidation products using Cayman-8-isoprostane ELISA kit showed decreased levels of F₂-isoprostane in P2X7 KO mice, compared with the WT under hyperoxia (Fig. 5A). However, this result was not statistically significant when evaluated by Students t-test (hyperoxic WT vs. P2X7 KO mice). On the other hand, F₂-isofurans were significantly increased by twofold in WT mice relative to P2X7 KO mice under hyperoxia (Fig. 5B). Room air samples showed a marginal decrease in F₂-isofurans levels in P2X7 KO mice that was not statistically significant (Fig. 5B). MDA and 4-HNE levels were also analyzed in mice lung homogenates. WT mice exposed to hyperoxia showed a significant twofold increase in MDA levels, compared with P2X7 KO mice (Fig. 5C). Similarly, WT mice displayed a significant increase in 4-HNE levels relative to P2X7 KO mice under hyperoxia (Fig. 5D). These results further validate the protective effect of P2X7 deletion under oxidative stress.

Fig. 5.

Markers of oxidative stress were decreased by the knockdown of P2X7. The WT and P2X7 KO mice were subjected to room air and hyperoxia for 72 h. The lungs were isolated and tested for F2-isoprostane (A) and F2-isofurans using Cayman-8-isoprostane ELISA kit (B). MDA (C) and 4-HNE (D) levels were measured in mice lung homogenates by ELISA. Data are expressed as means ± SE are representative of at least four independent experiments, with five mice used per experiment. *P < 0.05.

Caspase-1 cleavage and generation of IL-1β was inhibited in P2X7 KO mice.

Extracellular ATP (eATP) is known to trigger the opening of the P2X7 receptor (45) and mediate inflammasome activation. eATP is implicated in having a role in the generation of IL-1β by facilitating cleavage of caspase-1 (28). Thus, we tested whether ATP would have an effect in alveolar macrophages, a cell that is well known to regulate inflammasome activity. Alveolar macrophages were isolated from WT and P2X7 KO mice and treated with ATP. Caspase-1 cleavage was assessed in the cell lysates exposed to room air (normoxia) or hyperoxia (1 h). Cleavage of caspase-1 is a well-known indicator of inflammasome activation. Caspase-1 activity was measured by fluorometric assay kit (28). Lysates from hyperoxic WT mice showed an approximate three-fold increase in cleaved caspase-1 relative to P2X7 KO mice (Fig. 6A). Lysates collected from the lungs of normoxic mice showed minimal levels of caspase-1 cleavage. These results suggest that deletion of P2X7 induces a reduction in caspase-1 activation. We further measured IL-1β levels in the cell supernatants of isolated alveolar macrophages from WT and P2X7 KO mice exposed to room air and hyperoxia. Previous research demonstrates that hyperoxia induces elevated levels of eATP, leading to increased production of IL-1β (28). We explored whether hyperoxia-induced eATP generation has an effect on IL-1β release by macrophages. Under hyperoxia, cell supernatants from WT mice showed a significant three-fold increase in IL-1β release compared with P2X7 KO mice (Fig. 6B). Under normoxic conditions, IL-1β release was reduced in the supernatants from P2X7 KO mice relative to WT mice, yet was not significant (Fig. 6B). Our results demonstrate that IL-1β production correlated well with the activation or cleavage of caspase-1 (Fig. 6A). Furthermore, these results suggest that P2X7 has a critical role in the release of IL-1β under hyperoxia-induced oxidative stress.

Fig. 6.

Alveolar macrophage caspase-1 activity and IL-1β levels were reduced in knockout of P2X7. Alveolar macrophages were isolated from WT and P2X7 KO mice, treated with ATP, and exposed to room air or hyperoxia for 72 h. A: caspase-1 activity was measured by flourometric assay in cell lysates. B: IL-1β levels were measured by ELISA in the supernatant. Data are expressed as means ± SE are representative of at least three independent experiments, with five mice used per experiment. *P < 0.05, **P < 0.01.

P2X7 deletion inhibits transepithelial albumin flux in alveolar epithelial type II cells and alveolar macrophages cocultures.

To mimic in vivo hyperoxia-induced epithelial cell permeability and dysfunction, we utilized a coculture model of type II AECs and AMs. Type II AECs were isolated and cultured, as previously described (2). Macrophages and alveolar epithelial cells are vulnerable to damage from oxidative stress and provide vital defense mechanisms, together serving as a barrier to inhaled pathogens and mediating inflammatory responses (23). They are among the first responders to signals of oxidative stress and exhibit interaction (5); thus, a coculture method using AM and type II AECs is appropriate for the study of in vivo macrophage-epithelial responses during exposure to hyperoxic lung injury. Type II AECs were isolated from WT mice exposed to room air or hyperoxia (1 h) and grown to reach adequate confluency. AM from WT or P2X7 KO mice were then added to type II AECs. When WT AMs were added to type II AECs, transepithelial albumin flux was substantially increased under hyperoxic conditions (Fig. 7A). In the P2X7 KO cell coculture, albumin flux was significantly decreased, compared with the WT group. We additionally measured IL-1β levels using the coculture model of type II AECs and AM. Knockout of P2X7 considerably inhibited the production of IL-1β, compared with WT (Fig. 7B). These results demonstrate that deletion of P2X7 protected against albumin transport across the epithelial barrier and production of IL-1β in vitro. Thus, P2X7 deletion inhibits transepithelial albumin flux in type II AECs and plays a role in reducing hyperoxia-induced cellular permeability and inflammatory responses.

Fig. 7.

Knockdown of P2X7 reduces protein permeability in a coculture model of type II alveolar epithelial cells (AECs) and alveolar macrophages. Type II AECs and alveolar macrophage (AM) were isolated from WT or P2X7 KO mice as described in materials and methods. Type II AECs were cultured and AM were added to the confluent cells, exposed to hyperoxia for 1 h, and compared with room air controls. A: transepithelial albumin flux was evaluated by the addition of [¹²⁵I]-labeled human serum albumin to each upper compartment, and contents from each lower compartment was counted in a Wizard γ-counter. B: IL-1β levels were measured in extracellular media by ELISA. Data are expressed as means ± SE and are representative of at least three independent experiments, with five mice used per experiment. **P < 0.01, †P < 0.05, comparing hyperoxic WT vs. P2X7 KO groups.

P2X7 deletion protects sodium transport across the epithelium in coculture.

The ENaC plays an important role in regulating sodium transport across the lung epithelium and ion gradients that facilitate transepithelial reabsorption (54). Thus, the ENaC has a vital role in alveolar fluid clearance and, consequently, pulmonary edema. Earlier studies show that IL-1β decreases sodium transport across rat lung epithelial cell monolayers by reducing the expression of ENaC (47, 54). Further, it has been shown that IL-1β inhibits PD and TEC across type II AEC monolayers (47, 54). Because the generation of IL-1β was inhibited in our P2X7 KO mice, we hypothesized that a relation exists between P2X7 and ENaC expression. Type II AEC monolayers were isolated from mice, cultured, and exposed to supernatants of alveolar macrophages of WT and P2X7 KO mice, which were treated with hyperoxia for 24 h. Measurement of PD in the coculture model showed that deletion of P2X7 significantly reinstated PD that was lost in the WT group (Fig. 8A). However, knockout of P2X7 did not show a substantial alteration in TER, compared with WT (Fig. 8B). TEC was significantly increased when P2X7 was deleted, relative to WT (Fig. 8C). We further evaluated αENaC mRNA expression in type II AEC lysates. Expression of the sodium channel was reduced in WT, yet exhibited a twofold increase compared with the WT group (Fig. 8D). These results suggest the protective effects and reduction of alveolar fluid clearance in P2X7 KO mice is due to rescuing ENaC suppression, which plays an important role on alveolar fluid clearance and resolution of lung edema during ALI.

Fig. 8.

Alveolar epithelial type II cell membrane function was protected by deletion of P2X7. Alveolar macrophages were collected from WT and P2X7 KO mice, treated with hyperoxia for 24 h, and were added to type II AEC monolayers that were cultured in an air-liquid interface for 4 days. Potential difference (PD; A), transepithelial resistance (TER; B), and transepithelial current (TEC; C) were measured with an epithelial Ohm voltmeter containing Ag/AgCl electrodes. D: Type II AEC lysates were collected and analyzed for αENaC expression. Data are expressed as means ± SE and are representative of at least 3 or 4 independent experiments, with five mice used per experiment. *P < 0.05, **P < 0.01.

DISCUSSION

Despite years of research in ALI, the mechanism(s) involved in the disease pathogenesis of ALI has not been completely elucidated. Previous study from our laboratory has shown the role of the NRLP3 inflammasome-P2X7 axis in hyperoxia-induced inflammation in a mouse model of ALI (28). In addition, we provided the first documented evidence that hyperoxia-induced cell permeabilization was inhibited by blocking the P2X7 receptor with oxATP, demonstrating the critical role of the P2X7 receptor in hyperoxia-induced pore formation and NLRP3 inflammasome activation (28). Moreover, several studies have demonstrated that NRLP3 inflammasome activation in various inflammatory conditions is associated with IL-1β production (35, 64). Previous reports have linked P2X7 in the activation of the NLRP3 inflammasome complex (10). This present study has, for the first time, demonstrated that knockdown of P2X7 protects against hyperoxia-induced lung injury in vivo and in vitro.

P2X7, a membrane receptor, is expressed in various types of cells, including endothelial cells (43), mast cells (55), eosinophils (13), lymphocytes (10), dendritic cells (37), macrophages (10), and alveolar type I epithelial cells (4). In human macrophages, the release of IL-1β and IL-18 is stimulated upon activation of P2X7 (40). In the lungs of ALI mice, NLRP3 stimulation has been associated with high levels of P2X7 expression (57). Moreover, inhibition of P2X7 by a selective antagonist suppressed the NLRP3/ASC/caspase-1 signaling cascade and IL-1β release, arresting further lung injury (57). It has been shown that P2X7 also plays a role in the regulation of the immune system, such as in protecting against sepsis (7). Signaling through P2X7 on macrophages was found to reduce sepsis proliferation and provide antibacterial effects (7). A study using P2X7 KO mice demonstrated that the receptor is required for development of the inflammatory response associated with sepsis (50). Therefore, the P2X7 receptor may have additional therapeutic applications. Furthermore, other injury models utilizing LPS have shown association with P2X7 (9, 42). P2X7 can modulate LPS-induced macrophage production of inflammatory mediators and has involvement in the secretion of IL-1β and IL-1α (9, 42).

The increased survival of P2X7 KO mice in the current study can be attributed to inhibition of inflammasome activation through P2X7 suppression, as stated in our previous studies (28). The levels of inflammatory cells in BALF was also decreased in P2X7 KO mice, implicating P2X7 mediation of inflammation under hyperoxic conditions. Lung parenchyma has an abundant macrophage population that is triggered by a variety of cytokines and inflammatory factors (19, 44). MCP-1, a chemoattractant, largely controls the infiltration of macrophages in HALI (38) and is proven to have a concentration-dependent association with the severity of acute respiratory distress syndrome (ARDS) (46). Differential cell count in our study showed drastically diminished macrophages and neutrophils in the P2X7 KO mice with a concomitant decrease in MCP-1 levels, thereby further validating the role of P2X7 as an anti-inflammatory mediator. We have previously demonstrated the effect of hyperoxia on IL-1β cleavage both in vivo and in vitro, which eventually leads to enhanced alveolar epithelial permeability (28). This mechanism is the primary basis for alveolar edema and further lung injury. Moreover, vascular endothelial permeability and alveolar epithelial permeability have been found to be directly caused by IL-1β release (18). It is documented that although pro-IL-1β is produced by peritoneal macrophages, no mature IL-1β is produced or released as a result of ATP challenge in P2X7 deletion (52). Interestingly, the cytokine profile of P2X7 KO mice showed a reduction in IL-1β, lL-6, and TNF-α levels when subjected to hyperoxia, which was not studied earlier. This combined with decreased alveolar edema confirms the hypothesis that P2X7 induces inflammasome activation, IL-1β release, and cytokine activation under hyperoxia. Thus, inhibition of the P2X7-NLRP3 inflammasome axis can lead to the development of novel therapeutic strategies that may prevent pulmonary edema in HALI.

Alveolar fluid clearance is another important factor known to have a mortality suppressing potential in ALI (61). According to our data, the increased clearance of Evans Blue-labeled albumin into alveolar fluid is due to deletion of P2X7. Since our data show that IL-1β is significantly increased during hyperoxia-lung injury, in vivo cytokine signaling cascades are impaired in mice exposed to hyperoxic oxidative stress. Furthermore, IL-1β decreases expression of the αENaC in alveolar epithelial cells, which is a key factor in alveolar fluid clearance (47). A reduction in ion transport across the alveolar epithelium can contribute to alveolar edema in ALI (47). In our current study, we validate this by showing increased epithelial monolayer membrane bioelectrics (e.g., PD, TEC, TER) and αENaC expression by deletion of P2X7. Further, these results corroborate the increase in lung edema seen in our in vivo experiments. Thus, enhanced alveolar fluid clearance due to IL-1β suppression suggests protection of hyperoxia-exposed P2X7-deficient mice.

Earlier studies have shown that the ATP-triggered P2X7 receptor (P2X7R) targets NADPH oxidase, which is an important mediator of oxidative stress (39). The augmentation of P2X7 receptor-induced NADPH oxidase activity has been shown in other disease models involving oxidative stressful conditions, such as Alzheimer's disease and nonalcoholic steatohepatitis (8, 39). Moreover, lipid peroxidation by-products F2-isoprostanes and isofurans have been identified as markers of oxidative stress and NADPH activation (60). According to our study, these lipid peroxidation by-products were significantly decreased in the hyperoxic P2X7 KO mice, indicating the protective effect of P2X7 deletion during hyperoxia-induced oxidative stress. 4-HNE and MDA are known to be additional markers of oxidative stress and are produced in response to increased levels of ROS (16). On the basis of our results, knockdown of P2X7 significantly reduced the production of these peroxidized lipid by-products, further signifying the importance of P2X7 deletion-mediated protection in HALI.

Interestingly, early-phase clinical trials are currently under way to test the potential of P2X7 inhibitors in various human diseases. Until recently, inhibitors targeting P2X7 in inflammatory diseases were overlooked. Phase I and Phase II clinical trials have researched two major P2X7 antagonists: AZD9056 and CE 224535 (1). Antagonistic drugs targeting P2X7 in humans have been studied in various disorders, such as arthritis, pain, and multiple sclerosis (3). Two full studies are documented on the efficacy and safety of AZD9056 and CE 224535 in patients with rheumatoid arthritis (3). These inhibitors have not been studied in the context of pulmonary disease or pulmonary inflammation; therefore, further research is warranted for elucidating whether P2X7 antagonists are effective for pulmonary therapeutic interventions.

Taken together, this study delineates a protective role for P2X7 deletion in HALI. In other words, the morbidity and mortality occurring as a consequence of HALI can be targeted by unraveling the methods of P2X7 inhibition. Clinically, this could revolutionize therapeutic strategies, which are presently focused only on oxygen therapy in critically ill patients with activated inflammasomes. However, this study has its limitations with respect to the use of only a single triggering factor, hyperoxia, for activation of the inflammasome. It is plausible that several cytokines and inflammatory mediators may be involved in the P2X7-NLRP3 signaling pathway other than the ones that were studied here. Future studies will explore other key players that may play an important role in hyperoxia-induced lung injury. This study is innovative as it provides mechanistic insights into HALI and may lead to therapeutic drugs targets for HALI and other acute oxidative stress-mediated inflammatory conditions.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.G. performed experiments; L.G. and A.R. prepared figures; A.R. and A.F. drafted manuscript; A.F., R.S., and N.K. edited and revised manuscript; R.F.L. and N.K. approved final version of manuscript; N.K. conception and design of research.