P2Y12 antagonist attenuates eosinophilic inflammation and airway hyperresponsiveness in a mouse model of asthma

Abstract Leukotriene E4 (LTE4) that plays a key role in airway inflammation is expressed on platelets and eosinophils. We investigated whether blocking of the P2Y12 receptor can suppress eosinophilic inflammation in a mouse model of asthma because platelets and eosinophils share this receptor to be activated. BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA), followed by OVA nebulization. On each challenge day, clopidogrel, a P2Y12 antagonist was administered 30 min. before each challenge. Forty‐eight hours after the last OVA challenge, mice were assessed for airway hyperresponsiveness (AHR), cell composition and cytokine levels, including chemokine ligand 5 (CCL5), in bronchoalveolar lavage (BAL) fluid. EOL cells were treated with LTE4, with or without clopidogrel treatment, and intracellular and extracellular eosinophil cationic protein (ECP) expressions were measured to find the inhibiting function of P2Y12 antagonist on eosinophilic activation. The levels of P2Y12 expression were increased markedly in the lung homogenates of OVA‐sensitized and ‐challenged mice after platelet depletion. Administration of clopidogrel decreased AHR and the number of airway inflammatory cells, including eosinophils, in BAL fluid following OVA challenge. These results were associated with decreased levels of Th2 cytokines and CCL5. Histological examination showed that inflammatory cells as well as mucus‐containing goblet cells were reduced in clopidogrel‐administered mice compared to vehicle‐treated mice. Clopidogrel inhibited extracellular ECP secretion after LTE4 stimulation in EOL‐1 cells. Clopidogrel could prevent development of AHR and airway inflammation in a mouse model of asthma. P2Y12 can be a novel therapeutic target to the suppression of eosinophils in asthma.


Introduction
Eosinophils are key effector cells inducing airway inflammation and airway hyperresponsiveness (AHR) in allergic asthma. They contain abundant preformed and de novo inflammatory mediators, such as eosinophil cationic protein (ECP) and CysLTs [1][2][3]. Human eosinophils not only produce and secrete CysLTs but also express both CysLT receptor 1 (CysLTR1) and CysLT receptor 2 (CysLTR2) on the cell surface as well as the outer membranes of their granules [4].
CysLTs, including LTC4, D4 and E4, are well-known proinflammatory lipid mediators that are synthesized from membrane-derived arachidonic acid via the 5-lipoxygenase pathway and contribute to the pathogenesis of asthma by affecting leukocyte recruitment, enhancing activities of eosinophils [5]. Among the 3 CysLTs, leukotriene E4 (LTE4) is the most stable and abundant form [6], and known to have great inflammatory effects on inducing bronchial constriction and enhancing inflammatory cell infiltration compared to LTC4 and LTD4 [7,8]; however, LTE4 binds to CysLTR1 and CysLTR2 with low affinity [9][10][11][12] compared to LTC4 and LTD4.
Using an in silico modeling, it is predicted that LTE4 could bind to purinergic receptors, such as P2Y12, and elicit calcium flux and phosphorylation of extracellular signal-regulated kinase in the P2Y12 receptor overexpressing transfected cells [13]. However, a more recent study suggested that LTE4 cannot activate signalling through P2Y12R [14], and G-protein coupled receptor (GPR) 99 has been identified as a direct LTE4-specific receptor because mice lacking CysLT1, CysLT2 and GPR 99 receptors lose the ability to respond to the CysLTs, including LTE4 [15].
P2Y12 is an adenosine diphosphate chemoreceptor expressed on platelet membrane, and eosinophil granule membranes also express the P2Y12 receptor [4]. A recent study reported an increased number of platelet-adherent eosinophils in the upper and lower airways of patients with asthma [16]. In addition, genetic polymorphisms of P2RY12 can affect platelet and eosinophil activation in patients with asthma [17].
Clopidogrel, known as an antagonist to P2Y12, is a thienopyridine class antiplatelet agent that has been used to inhibit blood clots in the treatment of various cardiovascular diseases [16]. In addition, the drug has been proposed to be a potential therapeutic for various inflammatory diseases due to its anti-inflammatory and immunomodulatory effects observed in cardiovascular diseases [18]. However, their anti-inflammatory therapeutic effects in allergic asthma have not yet been completely understood.
In this study, we evaluated the blocking effect of the P2Y12 receptor antagonist on eosinophilic asthmatic inflammation because platelets and eosinophils share this receptor to be activated.

Materials and methods
Animals Female 6-week-old BALB/c mice weighing 20 AE 2 g were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed under specific pathogen-and ovalbumin (OVA)-free conditions and maintained on a 12-hr light-dark cycle with food and water ad libitum. All animal experiments performed in this study were approved by the Institutional Animal Care and Use Committee of Ajou University (IACUC 2013-0068).

OVA sensitization, challenge and clopidogrel treatment
The experimental protocol for allergen sensitization and challenge was modified from previously described procedures [19]. Briefly, BALB/c mice were sensitized intraperitoneally with 10 lg of OVA (Fisher Scientific, Pittsburgh, PA, USA) emulsified in 1 mg of alum (Imject Alum; Pierce, Rockford, IL, USA) on days 0 and 14. For the allergen challenge, mice were subjected to airway allergen challenges by exposure to 1% OVA aerosols for 20 min. on days 28, 29 and 30 with an ultrasonic nebulizer (NE-U22; Omron, Kyoto, Japan). To treat P2Y12 antagonist, mice were administered by oral gavage with 30 mg/kg of clopidogrel (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 30 min. before each OVA challenge. PBS was given as a vehicle. Each experiment in our study was repeated 3 times with 5 mice per group.
Airway resistance measurement, bronchoalveolar lavage and lung histology Airway resistance to inhaled methacholine (MCh; Sigma-Aldrich) was measured using the flexiVent System (SCIREQ, Montreal, QC, Canada) 48 hrs after the last OVA challenge, as previously described [20]. Bronchoalveolar lavage (BAL) was performed immediately after assessing AHR via the tracheal cannula with 1 ml of Hank's balanced salt solution. Leukocytes were counted with a haemocytometer, and cell differentiation was performed on cytospin slides prepared with Wright-Giemsa stain as previously described [21]. Tissue sections were evaluated using ImageJ (National Institutes of Health, Bethesda, MD, USA). To detect inflammatory cells, sections were stained with haematoxylin and eosin, and mucus-containing cells were stained with periodic acid-Schiff (PAS). The number of inflammatory cells per lm 2 of perivascular and peribronchial areas and the number of mucus-containing cells per lm 2 of basement membrane were determined.

Enzyme-linked immunesorbent assay
The levels of interleukin (IL)-4, IL-5, IL-13, and interferon (IFN)-c in BAL fluid were measured using a sandwich ELISA (eBioscience, San Diego, CA, USA), and ECP levels in human eosinophil cells (EOL-1) supernatants and CCL5 levels in BAL fluid were analysed by a quantitative ECP ELISA kit (Medical & Biological Labs, Nakaku Nagoya, Japan and R&D systems, Minneapolis, MN, USA, respectively).

Platelet depletion
Platelets that express P2Y12 receptors were removed before measuring the P2Y12 level in lung homogenates. To remove platelets, sensitized mice intraperitoneally received 40 lg of rat monoclonal antibody against mouse CD42b (Emfret Analytics, Eibelstadt, Germany) 48 hrs before experiments.
In vitro cell culture and Western blot EOL-1 cells were purchased from Sigma-Aldrich. Cells (1 9 10 6 /ml) were seeded into each well of 6-well plates, grown in RPMI-1640 with 10% FBS and 1% penicillin/streptomycin under a 5% CO 2 humidified atmosphere at 37°C. After reaching the appropriate cell confluence, the cells were pretreated with clopidogrel (10 lg/ml) or montelukast (10 lM) in serum-free media for 30 min. Then, EOL-1 cells were stimulated with 300 nM LTE4 for 30 min.
After stimulation, the cells were lysed in RIPA buffer containing protease inhibitor cocktail (Fisher Scientific). Total 30 lg of protein from each cell lysate were loaded onto a 15% SDS-PAGE gel, subjected to electrophoresis, and transferred to Polyvinylidene fluoride (PVDF) membranes. The membranes were blocked by 5% skim milk in PBS containing 0.05% Tween-20 and incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase. Antibodies used against human target proteins were ECP (Santa Cruz Biotechnology, Dallas, TX, USA) and b-actin (Santa Cruz Biotechnology).

Immunohistochemistry
Immunohistochemistry (IHC) was performed using either the avidinbiotin-complex method or the immunofluorescence technique on 5-lmthick paraffin sections. After deparaffinization, tissue sections were sequentially incubated with blocking buffer (0.05% PBS-Tween 20 containing 5% bovine serum albumin and 10% normal horse serum) for 1 hr at room temperature (RT), and then incubated with rabbit anti-ECP antibody (Santa Cruz Biotechnology), overnight at 4°C. The sections were incubated for 1 hr with goat biotinylated rabbit antibody (Vector, Burlingame, CA, USA) at RT, and then with the avidin-biotinylated horseradish peroxidase complex for 1 hr at RT. Cell nuclei were counterstained with haematoxylin. For immunofluorescence labelling, tissue sections were blocked as previously described and incubated with rabbit anti-P2Y12R antibody (Abcam, Cambridge, UK) overnight at 4°C in a humidified chamber. The sections were incubated with Alexa Fluor 488-labeled donkey anti-rabbit antibody (Life Technologies, Carlsbad, CA, USA) for 50 min. at 37°C. Sections were counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI; 0.5 lg/ml) for 2 min. at RT and mounted using Biomeda Mounting Media (Biomeda, Foster city, CA, USA).

Immunocytochemistry
Bronchoalveolar lavage fluid cells (29 to 5 9 10 4 cells/ml) were seeded onto Poly-L-lysine-coated coverslips and allowed to attach to coverslips by incubation for 1 hr at 37°C. Bronchoalveolar lavage fluid cells were fixed with 4% paraformaldehyde for 10 min. at RT and consecutively incubated with blocking buffer for 1 hr. Primary antibodies against the P2Y12 receptor (Santa Cruz Biotechnology) and ECP (Santa Cruz Biotechnology) were added to the sections, followed by incubation in a humidified chamber for 1 hr at 37°C. Thereafter, the sections were incubated with appropriate secondary antibodies. The Alexa Fluor 594 donkey anti-goat antibody (Life Technologies) and Alexa Fluor 488 donkey anti-rabbit antibody (Life Technologies) were applied for 50 min. at 37°C. Finally, after nuclear stain with DAPI (0.5 lg/ml) for 5 min., Biomeda Mounting solution (Biomeda) was dropped on the glass slide, and the coverslips were inverted and placed onto glass microscope slides.

Statistical analysis
The results are presented as the mean AE SEM. Comparisons among the study groups were performed by one-way ANOVA, followed by Tukey's post hoc test. All data were analysed using SPSS version 19.

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(SPSS Inc, Chicago, IL, USA), and a P-value of less than 0.05 was considered statistically significant.

Results
The expression of P2Y12 is increased after OVA sensitization and challenge To determine the expression of P2Y12, the signals of P2Y12 were detected through western blot after depleting platelets with antiplatelet antibody. The expression of P2Y12 was increased markedly following OVA challenge in OVA-sensitized mice compared to OVAsensitized and saline-challenged mice, and this expression was not attenuated by clopidogrel administration in OVA-sensitized and -challenged mice (Fig. 1).

Clopidogrel treatment prevents the development of AHR and airway inflammation
To find out the blocking effects of clopidogrel on allergen-induced AHR and airway inflammation, mice were treated with 30 mg/kg of clopidogrel during the OVA-challenge phase. Following OVA sensitisation and challenge, vehicle-treated mice had significantly a higher lung resistance responding to MCh as well as a larger numbers of inflammatory cells, including eosinophils in BAL fluid compared to OVA-sensitized and saline-challenged mice (P < 0.01). Clopidogrel treated mice developed significantly a lower airway responsiveness to inhaled MCh and a smaller number of eosinophils in BAL fluid compared to vehicle-treated mice (P < 0.01) ( Fig. 2A and B). As shown in Figure 2C, clopidogrel treatment reduced the levels of IL-4, IL-5 and IL-13 in BAL fluid, but we did not find a significant difference in the IFN-c level compared to vehicle-treated mice. Histopathological examination of lung tissue sections revealed that the numbers of inflammatory cells, including eosinophils, in peribronchial and perivascular areas were increased in OVA-sensitized and -challenged mice. Similarly, the numbers of PAS-positive mucuscontaining goblet cells were also increased in the same mice. However, administration of clopidogrel significantly decreased the numbers of inflammatory cells and PAS-positive mucus-containing goblet cells in lung tissue (Fig. 3A and B). As shown in Figure 3C, the expression of ECP in peribronchial and perivascular areas was increased in OVA-sensitized and -challenged mice compared to OVA -sensitized and saline-challenged mice. However, clopidogrel treatment significantly decreased the expression of ECP in lung tissue.
Eosinophils present P2Y12 receptors, and clopidogrel treatment decreases the secretion of chemokine CC ligand 5 in lung tissue Next, the presence of P2Y12 receptors on eosinophils was investigated. Double immunocytochemistry with anti-ECP and anti-P2Y12 antibodies revealed that P2Y12 was co-localized to eosinophils in BAL fluid (Fig. 4).
To investigate how clopidogrel could attenuate airway eosinophilic inflammation, CCL5, an eosinophilic chemoattractant, in BAL fluid, the expressions of adherent molecules in lung homogenates were measured in clopidogrel-treated and vehicle-treated mice. The level of CCL5 in BAL fluid was significantly decreased in clopidogrel-treated mice compared to vehicle-treated mice (Fig. 5). However, clopidogrel treatment did not affect the expression of VCAM-1 or ICAM-1 in lung homogenates (data not shown).

Clopidogrel increases intracellular expression of ECP in eosinophils and decreases its extracellular expression after LTE4 stimulation
Finally, intracellular and extracellular expressions of ECP were measured using EOL-1 cells that were stimulated with LTE4, with or without clopidogrel. The intracellular level of ECP was decreased by LTE4 stimulation without clopidogrel; however, clopidogrel treatment significantly inhibited the decrease in the intracellular ECP level induced by LTE4 stimulation. These results were more significantly observed in the clopidogrel-treated group than in the montelukasttreated group (P < 0.01, Fig. 6A). In contrast, as shown in Figure 6B, the levels of ECP in the supernatant were significantly increased by LTE4 stimulation without clopidogrel (P < 0.01); both clopidogrel and montelukast treatments inhibited the increase in the ECP level induced by LTE4 stimulation (P < 0.01 for each). Notably, the inhibiting effect in the supernatant was significantly greater in the clopidogrel group than in the montelukast group (P < 0.01). Taken together, clopidogrel inhibited extracellular ECP secretion induced by LTE4 stimulation.

Discussion
Asthma is characterized by increased infiltration of inflammatory cells, including eosinophils, AHR, reversible airway obstruction along with mucus hypersecretion, and lung remodeling enhanced by cysLTs, such as LTC4, LTD4 and LTE4 [22,23]. There are at least 3G-protein-coupled receptors for cysLTs, including CysLT1R, CysLT2R [16], and newly investigated CysLT3R [14,24]. LTD4 and LTC4 strongly bind to CysLT1R and CysLT2R, respectively, which are found to be expressed by various cells of the immune system [22]. Montelukast, is a well-known CysLTR1R antagonist that competes with LTD4 to bind its receptor, and thereby attenuate bronchoconstriction and airway inflammation induced by CysLTs [25]. However, the last and most stable metabolite of CysLT is LTE4, which has been shown to be highly increased in the urine of asthma patients compared to healthy individuals [26]. Nasal administration of LTE4 increases infiltration and accumulation of various inflammatory cells in the murine lung tissue [24]. LTE4 has been found to bind weakly to CysLT1R as well as CysLT2R; therefore, many investigators have attempted to identify high-affinity receptors for LTE4. Recently, several novel candidate receptors for LTE4, including GPR 99 and Fig. 4 The presence of the P2Y12 receptor in eosinophils in BAL fluid. Detection of P2Y12 in BAL cells from a representative OVA-sensitized and -challenged mouse. Cells were incubated with anti-P2Y12 receptor or anti-ECP antibodies. Upper and lower panels are two representative photographs in each group. Analysis was performed by confocal microscopy. P2Y12R, have been suggested [7,14,15]. First, P2Y12R is predicted to be the third CysLTR through in silico analysis and in vitro investigation which showed that LTE4 elicits calcium flux and that phosphorylation of extracellular signals regulats kinase levels in P2Y12Roverexpressing transfected cells in a dependent manner [13]. In addition, Paruchuri et al. [24] have shown that P2Y12R is required in LTE4-mediated pulmonary eosinophilic inflammation. However, a more recent study has demonstrated that LTE4 does not activate signalling through P2Y12R because no calcium mobilization is elicited in P2Y12-expressing cells [14]. Instead, GPR 99 has been identified as a new candidate receptor for LTE4 [15,27]. Therefore, P2Y12R is simply regarded as a co-receptor for LTE4 [28].
Several studies have reported the inhibitory effect of P2Y12 receptor antagonists on inflammatory responses [24,29]. We found that the expression of P2Y12 was increased after allergen challenge in allergen-sensitized mice. Next, we evaluated the protective role of clopidogrel, a P2Y12 receptor antagonist, in the pathogenesis of asthma by using a mouse model of asthma and in vitro experiments. A previous study has reported that mucus secretion and inflammatory cell infiltration into the airways of asthmatic mice are decreased in P2Y12 À/À and clopidogrel-treated mice [24]. This is consistent with our result that administration of clopidogrel decreased the numbers of airway inflammatory cells, including eosinophils, in BAL fluid as well as lung tissue. In addition, we first provided the evidence to indicate that clopidogrel treatment decreases AHR to Mch and secretion of Th2 cytokines in mouse BAL fluid.
Eosinophil is the most important effector cell, and eosinophilia is one of the characteristics of allergic inflammation [30]. Eosinophils infiltrating into the airway undergo primary lysis and release free eosinophil granules containing most of the important mediators, including ECP. Additionally, the mediators released from eosinophil granules are known to play a crucial role in tissue remodeling in patients with asthma [31]. In this study, attenuation of eosinophilia after treatment with clopidogrel showed a striking feature in BAL fluid compared to other studies that used montelukast [32]. This finding suggested that clopidogrel may have a major effect on eosinophil activation and function.
It has been known that LTE4 stimulation increases the infiltration of eosinophils into the lungs in asthma patients and releases ECP from isolated human eosinophils [4,24]. Therefore, we further investigated the influence of clopidogrel treatment on the activation of eosinophils. As expected, we found that LTE4 stimulation decreases intracellular expression of ECP and increases its extracellular secretion from human eosinophils, which indicates that LTE4 could trigger degranulation of eosinophils. In contrast, clopidogrel treatment inhibited the decrease in intracellular ECP expression induced by LTE4 stimulation. Similar results were obtained in the lung tissue from OVA-sensitized and -challenged mice treated with clopidogrel. These However, clopidogrel may have an impact on other inflammatory cells such as T cells and platelets. CCL5 which was downregulated by clopidogrel is mainly secreted from T cell, and the depletion of inhibition of recruitment of eosinophils did not change cytokines in BAL fluid [33][34][35]. Instead, the function of T cells seems to be altered by the treatment of clopidogrel. T cells can reduce the expression of Th2 cytokine, mucus production and eosinophil number in the airway through reduction of CCL5. However, T cells do not express P2Y12. Therefore, the effects of clopidogrel on T cells could be indirect. Other cell types such as platelets may play a role in suppression of allergic airway dysfunction and inflammation following clopidogrel treatment. There is a possibility that platelet-eosinophil complex in lung tissue and BAL cell have a role in the effect of clopidogrel in our asthma model. The binding of activated platelets to eosinophils by P-selectin occur in both non-severe and severe asthma and this complex may affect the activation of eosinophil [16]. Adhesion molecules are known to mediate migration of intravascular immune cells to surrounding tissues during inflammatory responses [36,37]. In this study, we found the increased expression of VCAM and ICAM in lung tissue homogenates obtained from OVA-sensitized and -challenged mice, which were not inhibited by clopidogrel treatment. Based on these results, it is conceivable that the inhibitory effect of clopidogrel could be mediated by production and/or secretion of several chemokines, but not inhibition of inflammatory cell migration.
Recently, Lussana et al. published the first, randomized, doubleblind, placebo-controlled study that pharmacologic inhibition of P2Y12 receptor may be useful in the treatment of asthma using prasugrel, a third generation P2Y12 inhibitor. They showed that significantly improved AHR after taking 10 mg of prasugrel for 15 days in 26 asthma patients [38]. Further studies are needed to confirm the effect of P2Y12 inhibition in asthma.
In conclusion, our study provides clear evidence to indicate that clopidogrel, a P2RY12 antagonist, attenuates allergic inflammation and AHR in asthma. The results of this study suggest that P2Y12 antagonists can be used as novel therapeutics for asthma.