Protective Role of P2Y2 Receptor against Lung Infection Induced by Pneumonia Virus of Mice

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y2 receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y2 receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y2 −/− mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y2 −/− mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4+ T cells and CD8+ T cells in P2Y2 −/− compared to P2Y2 +/+ infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y2 −/− infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y2 −/− compared to P2Y2 +/+ bronchoalveolar fluids. The increased morbidity and mortality of P2Y2 −/− mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y2 receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.


Introduction
Acute viral bronchiolitis represents a major challenge in both developing and industrialized countries. Indeed, amongst many viruses who can induce bronchiolitis, studies have shown that respiratory syncytial virus is the cause of 70% of all cases of viral bronchiolitis [1]. Human respiratory syncytial virus (hRSV) is a negative-sense, single-strand RNA virus of the family Paramyxoviridae. hRSV is the most common cause of airway morbidity among children under 1 year of age and can cause subsequent infections throughout life [2].
Infection of mice with pneumonia virus of mice (PVM) is used as a natural host experimental model for studying the pathogenesis of infection with the closely related hRSV [3][4][5]. PVM infection induces a disease that begins on day 6 post-infection and inoculation of more than 300 PFUs is generally lethal by day 9 post-infection [6]. The primary targets of PVM in vivo are respiratory epithelial cells [7]. In infected mice, virus replication is accompanied by a profound inflammatory response with recruitment of granulocytes, marked edema, mucus production, and airway obstruction, leading to significant morbidity and mortality [7][8][9][10]. This is associated with marked respiratory dysfunction and by local production of inflammatory mediators including MIP-1a (CCL3), MIP-2 (CXCL2), MCP-1 (CCL2) and IFN-c [7]. Subsequently, a predominant Th1 adaptive response occurs from day 8 post-infection, with a pronounced influx of CD8 + cytotoxic T cells [11,12]. This cytotoxic response is enhanced by type I interferon production (IFN-a and IFN-b) and plays a crucial role in anti-PVM immunity, as it contributes to control PVM replication and is correlated to the severity of the disease in a viral dose-dependent fashion.
Metabotropic P2Y receptors have been recognised as important regulators of cell functions [13][14][15]. Amongst the P2Y receptors family, P2Y 2 is an ubiquitous receptor that is fully activated by ATP and UTP [16]. Metabotropic receptors are coupled to intracellular signalling pathways through heterotrimeric G proteins [15]. Several studies have demonstrated that extracellular nucleotides regulate lung inflammation: P2Y 1 and P2Y 2 receptors exert a protective role against infection of the lungs by P. aeruginosa [17] and P2Y 2 was described as a target for cystic fibrosis therapy [18]. Moreover, the role of ATP in eosinophil recruitment and dendritic cell activation during asthma has been previously shown [19]. We have previously shown that P2Y 2 receptor acts also as a regulator of membrane and soluble forms of VCAM-1 mediating the adhesion and migration of eosinophils in an asthma model [20].
In this study, we investigated the consequences of P2Y 2 loss in lung inflammation initiated after PVM infection.

Ethics statement
This study was carried out using mice in strict accordance with the national, european (EU Directives 86/609/EEC) and international guidelines in use at the Université Libre de Bruxelles. All procedures were reviewed and approved by the ethics committee (Commission d'Ethique du Bien-Etre Animal, CEBEA) of the Université Libre de Bruxelles (Permit Number: 338N, 146N and 341N). All efforts were made to minimize suffering: mice were placed in a ventilated room with all appropriate hygiene and feeding conditions throughout the experiments. Housing, inoculation, data collection, and euthanasia procedures complied with National Institutes of Health guidelines, and the experimental protocol was approved by the Bioethics Committee of the University of Liège. Mice were monitored twice daily and in the case of a weight loss exceeding 30% of the body weight or clear signs of animal suffering, mice were euthanized by cervical dislocation and integrated in the mortality curve data.

PVM inoculation
PVM strain J3666 (generously supplied by A. Easton, University of Warwick, Coventry, UK) was first passed in 10-week-old BALB/c mice and then grown once onto BS-C-1 cells to produce the stock solution. The stock solution was then diluted to 10 25 in MEM, divided into aliquots, and stored at280uC to serve as inoculum. Randomly selected aliquots yielded highly reproducible titers on BS-C-1 cells, amounting to <5610 5 PFU/mL. The inoculation procedure consisted of slowly instilling 50 mL of the viral suspension into the nostrils of the anesthetized mouse maintained in a vertical position (35 mg/kg pentobarbital sodium intraperitoneally). Mice were inoculated under brief anaesthesia (ketamine, Pfizer, 50 mg/kg, and xylazine, Bayer, 10 mg/kg, i.p.) by intranasal instillation of 50 ml of a viral suspension containing 1000 PFU and 1% BSA in PBS [10].
At selected time intervals (8, 9, 10 and 12 day post-infection), groups of minimum 6 mice were sacrificed with sodium thiopental (5 mg/animal, i.p.) and exsanguination. Broncho-alveolar lavage fluids (BALFs) were obtained by flushing the lungs with sterile 0.9% NaCl, and cell counts were performed on cytospin preparations after Diff-Quick staining (Dade Behring, Deerfield, IL) and by flow cytometry.
Quantification of ATP level in the BALF of PVM-infected mice P2Y 2 +/+ and P2Y 2 2/2 mice were infected with PVM and their BALF was collected at day 8, 9 and 10 post-infection. ATP level was quantified in the BALF using the luminescence ATP detection assay system ATPlite (PerkinElmer, Zaventem, Belgium) as previously described [20].

Quantification of leukocyte infiltration by flow cytometry analysis
Flow-cytometry data acquisition was performed on a dual-laser FACSCalibur flow cytometer running CELLQuest software (BD Biosciences, Erembodegem, Belgium). WinMDI software was used for data analysis. Cells were stained with mAbs directed against Quantification of cytokine levels in BALFs of P2Y 2 +/+ and P2Y 2 2/2 mice Cytokines such as KC, MIP-2, MIP-1a, MCP-1, IL-12p40, IFN-c, TNF-a, IL-6, IFN-b, IL-17, MIP-3a, IP-10 were measured in P2Y 2 +/+ and P2Y 2 2/2 BALFs using ELISA kits from BD Biosciences and R&D Systems (Abingdon, U.K.), following the manufacturer's instructions. BRAK was measured by RT-qPCR using the following primers set: 59-GAT GAA GCG TTT GGT GCT CT-39 and 59-AGT ACC CAC ACT GCG AGG AG-39, with Power SYBR Green PCR Master Mix (Applied Biosystem). Reactions were run on a 7500 Fast Real-Time PCR System (Applied Biosystems). The cycling conditions were 10 min for polymerase activation at 95uC and 40 cycles at 95uC for 15 s and 60uC for 60 s. Mean 6 SD values were obtained for each gene using qBase software. Each assay was performed in duplicate.

Viral Titration
At day 8 and day 10 post-infection, mice were euthanized to quantify lung virus titers by quantitative polymerase chain reaction (qPCR) as previously described [21]. The lungs were homogenized in ice-cold BSA 1% in PBS, and clarified (1000 g for 10 min). Viral RNA was extracted using Nucleospin RNA Virus columns according to the user manual (Macherey Nagel). Homogenates were treated with Fermentas DNase I and an aliquot of each RNA extract (100 ng RNA) was then reverse-transcribed using commercial high capacity cDNA reverse transcription kit (Invitrogen), and PCR was conducted using the following PVM SH gene primers set: 59-GCC GTC ATC AAC ACA GTG TGT-39 and 59-GCC TGA TGT GGC AGT GCT-39, with SYBR green PCR Master Mix (Applied Biosystem). SDHA was selected as control gene after analysis for its stability in our system. Reactions were run on a 7500 Fast Real-Time PCR System (Applied Biosystems). The cycling conditions were 10 min for polymerase activation at 95uC and 40 cycles at 95uC for 15 s and 60uC for 60 s. Mean 6 SD values were obtained using qBase software. Each assay was performed in duplicate.
Histological analysis of inflamed lungs of P2Y 2 +/+ and P2Y 2 2/2 mice Left lungs were insufflated with 700 ml of 4% paraformaldehyde, and embedded in paraffin. Sections (7 mm) were stained with haematoxylin and eosin and assessed by light microscopy. Histological analysis has been performed according to the score related to PVM infection defined by Anh et al [10]. Lung slides have been examined independently and blindly by two individuals.

Statistical analysis
For all experiments, data are presented as mean 6 S.E.M. and the statistical significance between samples was calculated using the Student's t test or one-way analysis of variance, using the Prism 5 software (GraphPad). The normal distribution of the data was checked using Kolmogorov-Smirnov, D'Agostino-Pearson, and Shapiro-Wilk tests. Kaplan-Meier survival curves were compared using the Log-rank (Mantel-Cox) Test and the Gehan-Breslow-Wilcoxon Test.
We then analysed the cellular infiltrates in the lung by flow cytometry analysis of BALF samples obtained at day 8 and day 10 post-infection. The total number of neutrophils and monocytes recovered in BALF was comparable in P2Y 2 +/+ and P2Y 2 2/2 mice (Fig. 3E). We observed a weak but not significant increase of neutrophils and monocytes in P2Y 2 2/2 BALF at day 10 (Fig. 3E). Additionally, cytospin preparations of BALF were performed to identify leukocyte subpopulations at day 8. The neutrophil and macrophage populations observed in the BALFs of P2Y 2 +/+ and P2Y 2 2/2 mice (Fig. 3F) were counted and these results confirmed the flow cytometry analysis (data not shown).

Discussion
The present study investigated the role of P2Y 2 receptor in a mouse model of viral pneumonia induced by PVM, the mouse counterpart of human RSV. The two viruses are closely related and provoke similar immune responses. The natural mouse pathogen PVM replicates efficiently following a minimal virus inoculum, and recapitulates many of the clinical and pathologic features of the most severe forms of RSV infection in human infants. By contrast, mice are relatively resistant to infection by human RSV [5]. Besides their phylogenic proximity and genomic similarities, another shared characteristic of the PVM and the RSV pneumoviruses is their capacity to enhance airway hyperreactivity and Th2 response induced after an allergic sensitization and challenge by ovalbumin in mouse [22]. Indeed, PVM infection acts similarly to early-life hRSV infection in human, which is known to increase the risk of subsequent development of childhood asthma.
We demonstrated that P2Y 2 2/2 mice display a severe increase in morbidity and mortality rate in response to PVM. The difference in mortality appeared before a difference in body weight was observed between P2Y 2 +/+ and P2Y 2 2/2 mice, possibly because the rate of weight loss was very high between days 6 and 10 and already maximal in P2Y 2 +/+ mice. We investigated first if the lower survival of P2Y 2 2/2 mice could be correlated with an excessive inflammation. We observed that several inflammatory chemokines were increased in the BALFs of P2Y 2 2/2 mice. More precisely, P2Y 2 2/2 mice presented increased levels of KC/CXCL-1 and MIP-2/CXCL-2 chemokines in their BALFs, but no significant higher infiltration of neutrophils or macrophages was observed in the inflamed lungs of P2Y 2 2/2 mice until day 10 post-infection. ATP, released upon tissue damage and concomitant early inflammatory process, constitutes a danger signal, which initiates several pro-inflammatory responses upon binding to purinergic receptors. In particular it has been shown that ATP, released into the airways during asthmatic airway inflammation, can modulate the function of myeloid dendritic cells thereby triggering and maintaining asthmatic airways inflammation [19]. Indeed, DCs are crucial for asthmatic inflammation because they recruit Th2 lymphocytes to the airway wall and trigger local Th2 effector cytokine production. It was also reported that P2Y 2 receptor exerts a protective role during lung infection such as in the Pseudomonas aeruginosa infection model [17].
In the present study, we observed a lower infiltration of DCs, CD4 + and CD8 + T cells in the BALFs of P2Y 2 2/2 mice compared to those of P2Y 2 +/+ mice. This lack of infiltration can be correlated to the data of Müller and colleagues demonstrating that P2Y 2 R is involved in the recruitment of DCs in the lungs [23]. IL-12 level was quantified in the BALFs of P2Y 2 +/+ and P2Y 2 2/2 PVMinfected mice and was significantly lower in P2Y 2 -deficient mice at days 8 and 10 post-infection. DCs are one primary producer of IL-12 which induces the proliferation of NK, T cells, DCs and macrophages, the production of IFN-c and increased cytotoxic activity of these cells. IL-12 also promotes the polarization of CD4 + T cells to the Th1 phenotype involved against viral infection. Higher IL-6 level observed in P2Y 2 2/2 BALFs could also reflect a defective Th1 response in these mice. It was indeed shown that IL-6 production by pulmonary dendritic cells impedes Th1 immune responses [24].
The absence of P2Y 2 receptor and the reduced level of its ligand ATP which are involved in DC recruitment in the lungs [23] could explain lower DC infiltration observed in P2Y 2 2/2 lungs. Lower ATP level in P2Y 2 2/2 lung could be explained by P2Y 2 -mediated ATP release. P2Y 2 activation was shown to open pannexin-1 channels forming non-selective pores permeable to ions and large molecules such as ATP in rat carotid body cells [25]. Lower DC and T lymphocyte infiltration could also have been related to reduced level of DC-recruiting chemokines. A comparative gene profiling analysis of P2Y 2 +/+ and P2Y 2 2/2 PVM-infected lungs focused on inflammatory genes revealed the down-regulation of BRAK (CXCL-14) in P2Y 2 2/2 lungs. The quantification of DC recruiters IP-10 (CXCL10), MIP-3a (CCL20) and BRAK (CXCL-14) by ELISA or qPCR in P2Y 2 +/+ and P2Y 2 2/2 BALFs confirmed lower expression of BRAK at day 10 post-infection in the P2Y 2 2/2 BALFs compared to P2Y 2 +/+ BALFs. Interestingly, BRAK is a potent chemoattractant and activator of dendritic cells [26]. It has also an ability to block endothelial cell chemotaxis resulting in the inhibition of angiogenesis [27]. CXCL14 is constitutively and highly expressed in many normal tissues, where its source is thought to be fibroblasts [28] and epithelial cells [29] which both express P2Y 2 receptors. Reduced DC infiltration in P2Y 2 2/2 PVM-infected lungs could result from a defect in both direct nucleotide-driven and BRAK-mediated DC chemotaxis. Recruitment of T cells was also affected in PVM-infected P2Y 2deficient mice. Both CD4 + and CD8 + T cells contribute to the clearance of PVM from the lung [11]. Genetically T-cell-deficient or T-cell-depleted mice cannot eliminate PVM.
The increased morbidity and mortality of P2Y 2 2/2 mice could be the consequence of a lower viral clearance leading to a more persistent viral load and higher viral titers as observed at day 10 post-infection in the lungs of P2Y 2 2/2 infected mice. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. Additionally, we cannot exclude that P2Y 2 2/2 mice display after 10 days an excessive inflammation with higher neutrophil recruitment compatible with the increase in KC, MIP-2 and IL-6, but this could not be efficiently analysed because of their high and rapid mortality.
In conclusion, our study reveals that the purinergic P2Y 2 receptor, previously described as a mediator of Th2 response in asthma, is also involved in the initiation of Th1 response protecting mice against lung viral infection.