Blocking P2Y2 purinergic receptor prevents the development of lipopolysaccharide-induced acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality resulting from a direct or indirect injury of the lung. It is characterized by a rapid alveolar injury, lung inflammation with neutrophil accumulation, elevated permeability of the microvascular-barrier leading to an aggregation of protein-rich fluid in the lungs, followed by impaired oxygenation in the arteries and eventual respiratory failure. Very recently, we have shown an involvement of the Gq-coupled P2Y2 purinergic receptor (P2RY2) in allergic airway inflammation (AAI). In the current study, we aimed to elucidate the contribution of the P2RY2 in lipopolysaccharide (LPS)-induced ARDS mouse model. We found that the expression of P2ry2 in neutrophils, macrophages and lung tissue from animals with LPS-induced ARDS was strongly upregulated at mRNA level. In addition, ATP-neutralization by apyrase in vivo markedly attenuated inflammation and blocking of P2RY2 by non-selective antagonist suramin partially decreased inflammation. This was indicated by a reduction in the number of neutrophils, concentration of proinflammatory cytokines in the BALF, microvascular plasma leakage and reduced features of inflammation in histological analysis of the lung. P2RY2 blocking has also attenuated polymorphonuclear neutrophil (PMN) migration into the interstitium of the lungs in ARDS mouse model. Consistently, treatment of P2ry2 deficient mice with LPS lead to an amelioration of the inflammatory response showed by reduced number of neutrophils and concentrations of proinflammatory cytokines. In attempts to identify the cell type specific role of P2RY2, a series of experiments with conditional P2ry2 knockout animals were performed. We observed that P2ry2 expression in neutrophils, but not in the airway epithelial cells or CD4+ cells, was associated with the inflammatory features caused by ARDS. Altogether, our findings imply for the first time that increased endogenous ATP concentration via activation of P2RY2 is related to the pathogenesis of LPS-induced lung inflammation and may represent a potential therapeutic target for the treatment of ARDS and predictably assess new treatments in ARDS.


Introduction
Acute respiratory distress syndrome (ARDS) is a life-threating disease often linked to lung infection and systemic inflammation, and characterized by rapid disease progression (1,2).An acute onset of respiratory distress, edema formation and severe hypoxemia associated with high mortality are the most characteristic features of ARDS (3,4).Despite advances in ventilatory strategies, general care of the critically ill patient (5,6) and imaging techniques (7) in the past two decades, efficient pharmacotherapy for ARDS has not yet been developed (8).The excessively high rate of failure in drug efficacy in human models underscores the significant need for investigating its underlying mechanisms and subsequently introducing candidate components with the potential to reduce ARDS symptoms (2).Many researchers believe that ARDS is disclosed by a demonstrative inflammatory response (9).Bacteria-derived pneumonia and sepsis are the most common causes of ARDS (10-13).Neutrophilic granulocytes (neutrophils) are the main drivers of the inflammation process, which often proceeds and leads to breakdown of the endothelial barrier and immune-mediated injury of pulmonary tissue (14,15).The rapid recruitment of neutrophils to the lung vasculature peaks as early as after four hours (16).Accumulation and clustering of neutrophils at sites of tissue injury are classical hallmarks of acute inflammation (17,18).
Previously, it has been shown that the extracellular purines such as ATP exert an important role during the induction and maintenance of asthmatic inflammatory reactions (31).Furthermore, cell types implicated in the pathophysiology of bacterial infectious diseases of the lung or ARDS development are affected by purinergic signaling in various ways, e.g.chemotaxis and extended survival of neutrophils (32).Hence, upon activation, neutrophils and macrophages express functional P2Y and P2X receptors, which are sensitive to increasing extracellular ATP concentrations and are known to increase the release of proinflammatory cytokines (33).Therefore, the role of extracellular nucleotides and their corresponding receptors in ARDS as well as the pathophysiology of infectious respiratory diseases has been suggested.
In this study, we provide for the first time direct in vivo evidence of the contribution of P2RY2 in acute LPS-induced ARDS.We observed that P2ry2 expression was upregulated both in lung tissue and immune cells in an ARDS mouse model.P2ry2 expression is critical for the amelioration of ARDS inflammatory effects.Furthermore, neutrophils expressing P2ry2 are mainly involved in LPS-induced inflammation.Altogether, these data indicated a pivotal role of P2RY2 signaling in hematopoietic cells for the development of ARDS.

Mice
C57BL/6 mice (6-8-week-old) wild type, CCT-Cre × P2ry2 fl/fl , LysM-Cre × P2ry2 fl/fl , CD4-Cre × P2ry2 fl/fl were bred at the animal facilities of the University Hospital Freiburg and Medical University of Vienna.The animal experiments described within this study were approved by the Animal Ethics Committee of the University of Freiburg (Germany) and the Medical University of Vienna (Austria).All mice were maintained in specific pathogen-free (SPF) conditions.Genetically manipulated mice used in this study were described previously (34).To stimulate an LPS-induced acute lung inflammation, anesthetized mice received an intratracheal (i.t.) injection of LPS (Sigma Aldrich, Germany) diluted to a final dose of 300 µg/kg in PBS (Sigma Aldrich, Germany) to a total volume of 50 µL.The applied dosage was based on previous studies (34, 35).Mice were sacrificed 24 hours or 48 hours after LPS exposure depending on the experiment setting.Bone marrow (BM) chimera experiments was performed as described previously (9).Briefly, irradiated wild type or P2ry2 -/-recipients (900 cGy) were reconstituted with 5×10 6 P2ry2 +/+ or P2ry2 −/− BM suspensions intravenously.The following donor/recipient pairs were combined and treated with LPS i.t.: P2ry2 +/+ BM in P2ry2 +/+ recipient, P2ry2 +/+ BM in P2ry2 -/- recipient, P2ry2 -/-BM in P2ry2 +/+ recipient, P2ry2 -/-BM in P2ry2 -/-recipient.

Collection of lung tissue, bronchoalveolar lavage fluid and BALF cells
The animals were killed by i.p. injection of thiopental (200 mg/ kg) (WDT veterinary company, Germany) and exsanguinated.BALFs were collected by flushing the lung with one mL of sterile PBS supplemented with 0.1 mM sodium EDTA, three times.The first mL was collected in a separate tube for cytokine measurement using enzyme-linked immunosorbent assay (ELISA).Bronchoalveolar lavage fluid (BALF) was kept on ice until processing.BALF neutrophils and macrophages were isolated using fluorescence activated cell sorting (FACS).Therefore, BALF cells were incubated with anti-Ly-6G (Gr-1) FITC-and anti-F4/80 PE-labeled antibodies in PBS containing 0.5% BSA and 0.01% sodium azide for 20 min after blocking unspecific binding with an unlabeled anti-CD16/32 antibody.The lungs were resected for qPCR analysis.For qPCR, lung pieces were homogenized and stored in Qiazol (Qiagen, Hilden, Germany).

ATP measurements in BALF of mice
The ATP levels were measured in BALF of mice, immediately after collection using ATPlite assay (Perkin Elmer, Waltham, MA) according to the instructions of the manufacturer.The cell lysis step was omitted to avoid any contamination of intracellular ATP (36).

Histology
As previously described (34) for H&E (Haematoxylin and Eosin) staining, we embedded lung lobes in Tissue-Tek O.C.T. compound (Sakura Fintek, USA) and then froze them in liquid nitrogen.The molds were sectioned into 5 mm slices followed by staining with filtered 0.1% Mayers Hematoxylin (Sigma; MHS-16) and rinsing in cool running ddH 2 O. Next, they were immersed in 0.1% HCl water and then in undiluted Eosin.Subsequently the slides were rinsed in cool running ddH 2 O and dehydrated in 70% EtOH and then in 95% EtOH.Finally, they were placed in xylene and this was repeated in fresh xylene mount and coversliped with Entellan new (Sigma-Aldrich, Germany).

Quantitative PCR analysis of purinergic receptor
The cells were lysed in qiazol to extract total RNA, followed by cDNA synthesis using random-hexamer primers and the First Strand cDNA Synthesis Kit (Thermo-Fisher Scientific GmbH, Germany).Specific primers for various murine purinergic receptors were employed to assess mRNA expression levels.Quantitative PCR utilized Taqman Universal PCR Mastermix (Applied Biosystems, USA), along with pre-formulated primers and probe mixes (Applied Biosystems, USA).PCR was conducted under the following conditions: 2 minutes at 50°C, 10 minutes at 95°C, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute using a thermal cycler (iCycler,Bio-Rad, USA).Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) served as the reference gene.Primer sequences are shown in Supplementary Table 1.

Plasma leakage assay
Plasma vascular leakage was examined as previously described (38).Briefly, Evans blue dye conjugated to albumin (EBA, Sigma Aldrich, Germany) (20 mg/kg) was injected into the tail vein of mice.30 minutes later the mice were sacrificed and the lungs were perfused with PBS supplemented with 5 mM EDTA.The perfused lungs were excised en bloc, dried, weighed and snap frozen in liquid nitrogen.The whole lung was homogenized in PBS (1 mL/100 µg tissue) prior to incubation in formamide at 60°C for 18 hours.The optical density of the supernatant was determined spectrophotometrically at 620 nm after centrifugation at 5,000 x g for 30 minutes.The concentration of the extravasated Evans blue in lung homogenate was calculated against the standard curve and the results expressed as µg of Evans blue dye per gram of lung tissue (38).

Polymorphonuclear neutrophil migration into the interstitium and BALF
At different time points after LPS exposure and treatment with suramin or PPADS, the mice were injected intravenously with Alexa 633-labeled rat anti-mouse GR-1 (10 µg) antibody and allowed to circulate for 5 minutes to bind intravascular PMN.After 5 minutes, the mice were killed.After performing BAL, total lung tissue was harvested.The lungs were minced and digested with 125 U/mL collagenase type XI, 60 U/mL hyaluronidase type I-s, and 60 U/mL DNase (all Sigma Aldrich, Germany) at 37°C for 30 minutes.The digested lungs were obtained by being passed through a cell strainer and the cell suspension was centrifuged for 10 minutes at 300 g.The pellet was lysed with red blood-lysis buffer (ammonium chloride lysis buffer, 150 mM NH 4 Cl, 10 mM KHCO 3 , 100 nM EDTA, dH 2 O, pH=7.4) to remove erythrocytes and centrifuged again.The pellet was resuspended in buffer and the cells were counted with a hemocytometer (39).

Statistical analysis
Data are expressed as mean ± SEM and were tested for differences using one-way ANOVA, followed by Bonferroni posttest to correct for multiple comparison with the LPS/vehicle group as reference.Where appropriate, data were analyzed using unpaired t-test for comparisons of data collected from two different treatment groups.For statistical analysis we used GraphPad Prism version 8 (GraphPad Software, USA).Differences were considered significant at P < 0.05 and highly significant at P < 0.005.

ATP-neutralization or blocking of P2RY2 attenuates LPS-induced lung inflammation
To investigate the effect of ATP neutralization on the LPSinduced inflammation, we treated the C57BL/6 wild type mice with 4U/mL apyrase i.t.either one hour prior to or 24 hours after LPS exposure -prophylactic and therapeutic regimes, respectively.The diminished ATP BALF levels due to apyrase treatment (Supplementary Figure 1) were accompanied by reduced numbers of neutrophils in both regimes and macrophages only in the therapeutic regime (Figures 1A, B).We further observed a reduction of inflammatory cytokines in BALF including IL-6, TNFa and KC in both regimes (Figures 1C-F) as well as decreased levels of Evans blue in microvascular plasma leakage to the lung in both regimes compared to vehicle receiving control animals (Figures 1G,  H).Notably, both approaches of apyrase treatment showed only a partial reducing effect on IL-1b and MIP-2 secretion in the BALFs.To explore whether the observed effect on reducing inflammation was due to reduced P2RY2 activation and to assess the causative link between P2RY2 function and LPS-induced inflammation, suramin (an inhibitor of P2Y and, with a lower affinity, P2X receptors) was administrated i.t. in either a prophylactic or a therapeutic treatment regime.As a positive control and proof of principle, the nonspecific antagonists PPADS (P2R non-selective antagonist) was applied i.t. in both regimes.As expected, PPADS showed reduced concentration of proinflammatory cytokines, IL-6 and IFN-g (Figures 2C-F).We observed a reduced number of neutrophils not only in a therapeutic but also in a prophylactic treatment (Figures 2A, B).Moreover, in therapeutic regime we observed reduced concentrations of IL-1b, IL-6, IFN-g, and MIP-2 in suramin-treated animals (Figures 2D, F).Remarkably, in prophylactic regime we observed decreased concentration of IFN-g in suramin-treated mice (Figure 2E).Microvascular plasma leakage within the lung was also reduced in therapeutic regimes with both antagonist treatments (Figures 2G, H).We observed that the effects of both antagonists (suramin or PPADS) on cytokines and Evans blue measurements appeared similar and did not demonstrate clear superiority over each other.In line with our data, H&E staining of the lung indicated reduced inflammatory features of LPS-induced ARDS in both regimes (Supplementary Figure 2).Altogether, our results show that ATP-neutralization or blocking of P2RY2 attenuates LPSinduced lung inflammation.

Upregulation of P2RY2 on neutrophils, macrophages and lung tissue in LPSinduced ARDS
To evaluate the effect of LPS-induced inflammation on the ATP BALF levels and P2Y2 receptor regulation, C57BL/6 wild type mice were exposed to LPS (300 µg/kg) via i.t.instillation.The measurement of ATP concentration in BALFs after 24 and 48 hours by ATPlite assay showed that LPS administration resulted in increasing ATP concentrations in the BALFs after 24 hours and 48 hours, respectively (Supplementary Figure 3).As shown in Figure 3, LPS exposure led to an upregulation of P2ry2 mRNA in BALF macrophages (Figure 3A), BALF neutrophils (Figure 3B) and total lung tissue (Figure 3C) conducted after 24 hours.These data highlight the possible role of P2RY2 in regulating ARDS in vivo.
The expression of some other family members of purinergic receptors (P2rx1, P2rx4, P2rx7, P2ry4 and P2ry6) was also screened in BALF macrophages and neutrophils after LPS instillation in WT mice.Among them, higher expression of P2rx4 and P2ry6 in BALF macrophages and neutrophils was observed (Supplementary Figure 4).

P2ry2 expression has a pivotal role in hematopoietic cells in ARDS
Next, to provide a conclusive evidence about the contribution of P2RY2 in the pathogenesis of ARDS, we used P2ry2 knockout mice.P2ry2 -/-mice were treated with LPS (300 µg/kg) for 24 h and then we analyzed the immune cells and cytokines in BALF of LPSchallenged mice.We observed that the number of neutrophils and macrophages was significantly decreased in the BALFs obtained from P2ry2 -/-mice when compared to wild type mice (Figure 4A).We further observed that the concentration of inflammatory cytokines IL-1b, IL-6, MIP-2 and KC was significantly lower in the P2ry2 -/-mice compared to wild type (Figures 4B, C).Consecutively, to investigate the discrimination between the relevance of P2ry2 expression in the hematopoietic system and nonhematopoietic system, we performed bone marrow transplantation experiments.We treated different bone marrow chimeric mice with LPS i.t.: P2ry2 +/+ BM in P2ry2 +/+ recipient, P2ry2 +/+ BM in P2ry2 -/-recipient, P2ry2 -/-BM in P2ry2 +/+ recipient, P2ry2 -/-BM in P2ry2 -/-recipient, and then analyzed them for lung inflammation.As shown in Figure 5, wild type mice with a lack of P2ry2 expression in the hematopoietic system (P2ry2 -/-BM in P2ry2 +/+ ) displayed a significant decrease in lung inflammation, as evidenced by reduced numbers of neutrophils and macrophages in BALF (Figure 5A) and decreased concentration of IL-6, TNF-a and MIP-2 in BALFs (Figures 5B, C).In contrast, P2ry2 -/-animals reconstituted with a wild type hematopoietic system (P2ry2 +/+ BM in P2ry2 -/-) showed no protection against LPS-induced lung inflammation.The data indicate that P2ry2 expression in hematopoietic system has a central role in LPS-induced ARDS inflammatory features.

Cell-type specific P2ry2 knockout in non-hematopoietic cells resulted no changes in lung inflammation after LPS exposure
To further characterize the cell-specific influence of P2RY2 function in the pathogenesis of ARDS, we conducted the experiments with conditional P2ry2 fl/fl mice crossed with LysM-Cre animals (macrophages/neutrophils), CD4-Cre animals (CD4 + cells) and CCT-Cre animals (airway epithelial cells).All conditional mice were treated i.t. with LPS for 24 hours.Subsequently, the mice were euthanized and the BALF was collected.In treated animals no protective properties with regard to the LPS-induced lung inflammation was observed.However, there was a significant reduction of neutrophils in the BALF in the LysM-Cre × P2ry2 fl/fl animals but not the same effect in CD4-Cre × P2ry2 fl/fl and CCT-Cre × P2ry6 fl/fl (Figures 6A-C).Altogether, these data suggest that the The effect of P2ry2 expression in murine hematopoietic cells.Different bone marrow chimeric mice were treated with LPS i.t.: Wild type BM in P2ry2 +/+ , Wild type BM in P2ry2 -/-, P2ry2 -/-BM in P2ry2 +/+ , P2ry2 -/-BM in P2ry2 -/-, and then analyzed for lung inflammation.P2RY2 in non-hematopoietic system is not involved in LPSinduced airway inflammation.

Blocking P2 receptors in mice reduce PMN migration into the interstitium of the lungs
To describe the impact of P2RY signaling on immune cells during lung inflammation, we evaluated the quantitative movement of polymorphonuclear neutrophils (PMNs) within distinct lung compartments (interstitial and alveolar space) in a spatiotemporal manner.PMN migration was determined in control, PPADS-or suramin-treated mice over 48 hours to detect differences in the time course of PMN trafficking.Following LPS-inhalation, the number of PMNs increased in both lung compartments in a time-dependent manner (Figures 7A, B).In the control C57BL/6 wild type mice, LPS-induced PMN accumulation in the interstitium and BALF was significantly higher than in antagonist-treated mice (Figures 7A, B).However, there was a greater buildup of PMNs in the interstitium compared to the alveolar space.The results suggest that the suppression of P2RY2 reduces the recruitment of circulating PMNs into the interstitium of the lung and alveolar space in the early stage of lung inflammation.Notably, no significant differences on PMN migration were detected between PPADS-and suramintreated mice.

Discussion
In this study, we have established that P2RY2 enhances the typical signs of ARDS disease in an LPS-induced mouse model.Mice treated with apyrase, the ATP-neutralizing enzyme, or PPADS and suramin, non-selective purinergic receptor antagonists, showed a reduction in the proinflammatory cytokine KC concentration, which plays a major role in neutrophil recruitment (40)(41)(42)(43).We also observed that apyrase administration had a partial effect on IL-1b and MIP-2 reduction.It is plausible that the interaction between apyrase and inflammatory cells may function as an additional source of IL-1b and MIP-2, in the initial time of the inflammatory response (44).Our data also displayed diminished production of proinflammatory cytokines IL-6 and IFN-g in LPSexposed ARDS mice models, challenged with suramin or PPADS.This might be explained by the interplay between IL-6 and IFN-g signaling which governs neutrophil trafficking and apoptosis during acute inflammation (45).Further, histological analysis of the lung confirmed reduced inflammatory features in the airway of the lung following administration of suramin or PPADS in both prophylactic and therapeutic regimes.
The excessive infiltration of neutrophils into the pulmonary airspace is the main cause for the acute inflammation and lung injury and a common clinical picture of modern intensive care medicine (41).Exposure to LPS intratracheally triggers a hyperinflammatory ARDS phenotype, leading to a substantial recruitment of neutrophils to the lungs, evident through heightened neutrophil levels in the bronchoalveolar lavage fluid (BALF) and increased PMN-trafficking within lung compartments (interstitial, alveolar space) (39,46).Interestingly, blocking P2R by suramin or PPADS also prevents the development of lung inflammation indicated by reducing PMN influx into the lungs of inflammatory cells of the immune systems.Moreover, we found that i.t.applying of suramin or PPADS, either before or after LPS administration, significantly diminished vascular leakage in the lungs.Given that vascular leakage is a crucial aspect of pulmonary edema in ARDS (47), the significant improvement after antagonist application supports the crucial role of ATP signaling and purinergic receptors in ARDS development.We thereby manifest a functional role for the purinergic receptors in LPS-induced lung inflammation in the mouse lung in vivo.
Our findings showing strong upregulation of P2ry2 at mRNA level in BALF neutrophils, macrophages and lung tissue of the ARDS mouse models suggest that P2RY2 contributes to the accumulation of neutrophils and macrophages in the lung.The mRNA expression does not always directly correspond to protein expression due to various post-transcriptional and translational regulatory mechanisms (48,49).Therefore, future studies to elucidate the protein expression of Frontiers in Immunology frontiersin.orgthe P2RY2 would provide a more comprehensive understanding of its involvement in the observed outcomes.Our observation is in line with the previous findings showing higher expression levels of P2ry2 in chronic obstructive pulmonary disease (COPD) patients (27) and the role of P2Y2 receptor in chemotaxis of dendritic cells and eosinophils in allergic lung inflammation (50).Additionally, our recent research highlights the involvement of the P2Y2 receptor in the HDM-driven allergic airway inflammation model (34).We found that P2Y2 amplifies the production of proinflammatory cytokines in airway epithelial cells, monocytes, and dendritic cells (34).Furthermore, the P2Y2 receptor facilitates the chemotaxis of dendritic cells and eosinophils during allergic lung inflammation (50).
Our sought definitive experimental proof about the role of P2RY2 in ARDS has been derived from mouse model experiments and a study of P2ry2-deficient mice.We demonstrated for the first time that P2ry2 deficient animals have a reduced pulmonary inflammation after LPS i.t.exposure.We found that the number of neutrophils and macrophages combined with inflammatory cytokines dramatically decreased in P2ry2 -/- mice compared to wild type mice.The role of P2RY2 and its specific impact on neutrophils versus macrophages as a driver of inflammation remains an intriguing area for further exploration.In this context, it has been shown that P2RY2 is critical for neutrophil activation (51) and plays a crucial role in controlling inflammatory gene expression on neutrophils (52).On the other hand, signaling through P2RY2 enhances macrophage IL-1b production (53).Moreover, P2RY2 activation in macrophages has been linked to inflammatory cytokine production such as TNF-a and IL-1b, but direct control of inflammation in macrophages is not well understood (54).
In the following series of experiments with chimera BM P2ry2 -/- and wild type animals, it was revealed that expression of P2ry2 on hematopoietic cells might account for an inflammatory effect of ARDS.P2ry2 -/-mice reconstituted with wild type bone marrow did not show a reduction in LPS-induced lung inflammation compared to P2ry2 -/-and wild type mice reconstituted with P2ry2 -/-bone marrow.To verify the chimera results, we used different cell-typespecific conditional P2ry2-deficient mice.Therefore, we specified that neutrophils are mainly involved in LPS-induced inflammation.According to the effective function of CD4 cells in controlling critical aspects of lung immunity (55) and airway epithelial cells as the initial cell type impacted by inhaled components (56) we performed complementary experiments.Conditional knockout mice in non-hematopoietic system including airway epithelial cells and lymphocytes indicated no effect on reducing inflammatory features represented by number of neutrophil and macrophage cells in the BALFs.Our data show for the first time that P2RY2 in the hematopoietic system is important in the pathogenesis of ARDS.
Recent studies have suggested that the peripheral airways play an important role in the pathophysiology of ARDS (57).The mechanism of epithelial injury and denotation are not completely understood but are likely to result from changes in shear stress due to reopening of their collapsed small airways or non-collapsed flooded airways (58,59).To have a better understanding of the disease mechanism, it is therefore suggested to investigate other members of purinergic receptors family for further studies in the future.
Notably, our current study does not exclude the potential contribution of P2X receptors in ARDS.Indeed, we observed similar effects of non-selective P2Y receptors antagonist suramin (60,61) or P2 receptors antagonist PPADS (62,63) on reducing cytokine concentrations and microvascular plasma leakage measurements in the therapeutic regime experiments.In addition, we found that expression of P2x receptors including P2x4 and P2x7 at mRNA level increased in macrophages of LPS-treated mice.These data are in line with previous reports implying the involvement of P2X receptors in the lung inflammatory response (64,65).Moreover, previous study manifested the direct involvement of the P2X7 pathway in triggering the inflammatory reaction responsible for ARDS (9).
In summary, the results of the present study show conclusively for the first time the pivotal role of P2RY2 in the pathogenesis of ARDS.These data showed that enhanced endogenous ATP concentration, via activation of purinergic receptors P2RY2 is related to the pathogenesis of LPS-induced lung inflammation in mouse model, and might be a potential therapeutic target for treatment of ARDS patients and predictably evaluate new treatments on organ function in ARDS.This study shed some light on the underlying mechanism and potential components resulting in the ARDS disease.

1
FIGURE 1The effect of ATP neutralization by apyrase on LPS-induced ARDS.Mice were treated with 4U/mL apyrase in both prophylactic (left) and therapeutic (right) regimes.(A, B) The BALF cells differential count was measured by flow cytometry with both treatment methods.(C-F) Concentrations of cytokines IL-1b, IL-6, KC, MIP-2 and TNF-a in BALF were measured by ELISA in both regimes.(G, H) Plasma leakage was evaluated spectrophotometrically 30 minutes after Evans blue dye albumin (20 mg/kg), which was injected into the tail vein.Values are given as mean ± SEM. n = 4 mice in each group.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 obtained by comparing each group versus LPS (vehicle/LPS in prophylactic or LPS/vehicle in therapeutic regimes).ns means not significant.

2
FIGURE 2 The effect of blocking purinergic receptors on LPS-induced inflammation in mice lungs.The mice were treated with suramin or PPADS in both prophylactic (left) and therapeutic (right) regimes.(A, B) The BALF cells differential count was measured by flow cytometry with both treatment methods.(C-F) Concentrations of cytokines IL-1b, IL-6, KC, MIP-2 and IFN-g in BALF were measured by ELISA in both regimes.(G, H) Plasma leakage was evaluated spectrophotometrically 30 minutes after Evans blue dye albumin (20 mg/kg), which was injected into the tail vein.Values are given as mean ± SEM. n = 5 mice in each group.*p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001 obtained by comparing each group versus LPS (vehicle/LPS in prophylactic or LPS/vehicle in therapeutic regimes).

4 3
FIGURE 4 Screening the effect of lacking P2RY2 on LPS-induced ARDS.P2ry2 knockout mice were treated with either PBS or LPS and the BALFs were collected.(A) The number of neutrophils and macrophages in BALFs were measured by flow cytometry.(B, C) Concentration of cytokines IL-1b, IL-6, KC, MIP-2 and TNF-a in BALF were measured by ELISA.Statistical analysis was based on Bonferroni post-test.Values are given as mean ± SEM. n = 5 mice in each group.*p < 0.05, **p < 0.01, ***p < 0.001 versus P2ry2 +/+ LPS.

6
FIGURE 6The effect of P2ry2 conditional knockout mice in different cell types.The experiments were followed by conditional P2ry2 fl/fl mice respectively crossed with LysM-Cre animals (macrophages/neutrophils), CD4-Cre animals (lymphocytes) and CCT-Cre animals (airway epithelial cells).All conditional mice were treated i.t. with either LPS or PBS for 24 hours.(A) Number of neutrophils and macrophages in P2ry2 conditional knockout mice in (A) macrophages/neutrophils, (B) lymphocytes and (C) airway epithelial cells.Statistical analysis was based on Bonferroni post-test.Values are given as mean ± SEM. n = 5 mice in each group.**p < 0.01 obtained by comparing LPS treated groups.ns means not significant.

7 PMN
FIGURE 7 PMN migration into the interstitium and BALF.PMN migration in the various lung compartments (interstitial, alveolar space) was assessed followed by i.t.injection of Alexa 633-labeled rat anti-mouse GR-1 (10 µg) antibody in control C57BL/6 wild type mice and (A) Suramin 100 µM-and (B) PPADS 100 µM-treated mice over 48 h.Statistical analysis was based on Bonferroni post-test.Values are given as mean ± SEM. n = 4 mice in each group.*p < 0.05, **p < 0.01 versus antagonist LPS.