DISCUSSION

There have been studies implying the role of P2Y12R in allergic conditions and eosinophil-associated diseases (Paruchuri et al., 2009; Lussana et al., 2015;

Muniz et al., 2015). In the present study, we further elucidated the localization in the airway and the interactions between P2Y12R and other CysLTRs. Given that P2Y12R is an important receptor for platelets and modulate platelet functions during inflammation, we aimed to investigate if P2Y12R were expressed on other inflammatory cells rather than platelets. We detected the signals of P2Y12R on the bronchial epithelial cells and eosinophils, based on the colocalization between P2Y12R and eosinophil markers (MBP, EPX). Furthermore, the expression of P2Y12R was elevated on these cells as well as T cells in asthmatic mice. We demonstrated that the levels of CysLTR1, CysLTR2, and P2Y12R were up-regulated in lung tissue of OVA/OVA mice compared to NC mice, and that the increased levels were statistically significant in P2Y12R and CysLT1R, but not in CysLT2R. These findings highlight the importance of P2Y12R and CysLT1R, rather than CysLT2R, in allergic inflammation (Figueroa et al., 2003; Corrigan et al., 2005). There are two plausible hypotheses for the increased expression level of receptors. One hypothesis is that the Th2-rich environment, as displayed in our asthma mouse model, may trigger the expression of receptors. Indeed, IL-13 was shown to up-regulate CysLTR1 on human fibroblasts, human monocytes, and monocytes-derived macrophages; similar patterns was seen on human monocytes for IL-4 (Thivierge et al., 2001; Chibana et al., 2003). The other hypothesis is that cells with higher receptor expression on the surface may infiltrate more readily into lung tissue (Figueroa et al., 2003). Notably, after OVA sensitization and challenge, P2Y12R was dominant in mouse lungs compared to CysLTR1 and CysLTR2, which suggests the relevant role of the antagonists to P2Y12R in asthma.

In addition, we investigated the inhibitory effects of the antagonists to CysLTRs in lung tissues and BAL cells. We used a mouse model of acute asthma

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without platelet depletion because platelets can contribute to initiating airway hyperresponsiveness (Idzko et al., 2015); furthermore, we would like to evaluate the effects of drugs in the context of interactions between platelets and other inflammatory cells. Overall, the expression levels of these CysLTRs were abolished by either Mon, HAMI 3379, or Clo in a receptor-specific fashion. In lung tissues and BAL cells, Mon, a CysLTR1 antagonist, was shown to suppress the expressions CysLTR1 and CysLTR2, while, HAMI 3379 was only able to down-regulate the expression of CysLTR2. The 2 receptors share 38% sequence homology, thus the antagonists to CysLTRs may cross-react to regulate the other receptor’s expression to some extent (Heise et al., 2000). Initially, it was thought that the primary CysLT activation pathway was mainly controlled by CysLTR1.

CysLTR2 might only be activated depending on the CysLTR1 receptor in heterodimer formation (Figueroa et al., 2003). Nevertheless, recent studies elucidated more important functions of CysLTR2. Knockdown of CysLTR2 on a human mast cell line increases CysLTR1 surface expression (Jiang et al., 2007).

CysLTR1 was up-regulated by priming monocytes or monocytes-derived macrophages with IL-4 (Thivierge et al., 2001). In study, administration of HAMI 3379 significantly reduced IL-4 in BALF, for which CysLTR1 expression may be subsequently dampened.

Interestingly, we found that the expression of P2Y12R in lung tissues was significantly suppressed by the administration of Clo, Mon, and HAMI 3379. There was insufficient evidence on the interactions of CysLTR1 or CysLTR2 with P2Y12R. Mamedova et al.(Mamedova et al., 2005) found that montelukast and pranlukast can inhibit nucleotide-induced calcium mobilization through P2Y receptors. Presumptively, CysLTR1 antagonists may intervene the expression of P2Y12R. Regarding the interactions between CysLTR2 and P2Y12R, both receptors were found to be concurrently expressed on various cell types such as T cells and CD34+ progenitor cells. Thus, HAMI 3379 may interfere with the

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development of P2Y12R+ leukocytes. However, further studies are warranted to reveal underlying mechanisms.

In the aspect of clinical implication, it is intriguing that Mon and Clo preferentially attenuated AHR, the infiltration of inflammatory cells and enhanced Th2 cytokines. These findings advocated the combination of antagonists to CysLTRs to achieve better anti-asthmatic effects, as in the Chapter-II. Practically, in this study, the ability of Clo in cytokine reduction may be supportive to montelukast’s effect in eosinophil suppression to attenuate airway inflammation.

Up to now reports on the combination of CysLTR antagonists are still limited in in vivo studies. Sekioka et al. [35] reported the additional effects of a combination therapy of Mon and BayCysLT2RA (a CysLTR2 receptor antagonist) or ONO-6950 (a dual CysLT1/2 receptor antagonist) in reducing the mite-induced bronchoconstriction (Sekioka et al., 2015). In addition, through its inhibitory effects on CysLTRs, the combination therapy may render inflammatory mediators, such as CysLT, more effectively.

Taken together, our findings implicate the distribution and interactions of the CysLTRs in various inflammatory and non-inflammatory cells. OVA enhanced the expression levels of CysLTR1, CysLTR2, and P2Y12R, especially in whole lung homogenates, T cells, airway epithelium bronchial smooth muscles, and alveolar leukocytes, which were diminished by the blockage of CysLTRs. Understanding of the CysLTRs and their interactions may facilitate the development of novel therapies for asthma treatment.

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CHAPTER-II

Additional Effects of Clopidogrel to Montelukast in

Asthma Treatment

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I- INTRODUCTION

Platelets are anucleated blood cells derived from bone marrow megakaryocytes, which are dispensable for hemostasis and thrombosis (Idzko et al., 2015; Takeda et al., 2017). During the last decades, platelets has gained increasing attention as an effector cells in the allergic inflammation as a bridge between the adaptive and innate immune disease (von Hundelshausen and Weber, 2007; Semple et al., 2011).

Recent data showed that platelets contribute to the allergic response by multiple mechanisms as an immunological barrier. Platelets express various adhesion molecules and surface receptors on the membrane, such as P-selectin (CD62P) which recruits neutrophils, monocytes and lymphocytes via its ligand, P-selectin glycoprotein ligand-1 (PSGL-1) (Diacovo et al., 1996; von Hundelshausen and Weber, 2007). Platelets store the intracellular immune-associated molecules such as α-granules, dense granules and lots of inflammatory cytokines (Takeda et al., 2017).

Through the antimicrobial protein in the α-granules, for instance, platelet factor 4 (PF4), and the pathogenic cytokines, platelets are able to kill the pathogen as well as to induce directly the bronchoconstriction (Yeaman et al., 2007; Takeda et al., 2017). The surface molecules of platelets bind to the surfaces of neutrophils, eosinophil, lymphocytes via the ligands, thereby forming the platelet-adherent leukocytes which subsequently amplify the CysLT production (Laidlaw et al., 2012). Platelets store IL-33 which is crucial for eosinophilic inflammation in the asthma mouse model (Takeda et al., 2016). The released hypertrophic factors and extracellular matrix enzymes from platelets induce the smooth muscle hyperplasia and collagen deposition as features of airway remodeling (Idzko et al., 2015).

There is increasing evidence showing the role of platelets in asthma. In patients with bronchial asthma, platelets are more activated than the healthy controls (Knauer et al., 1981; Sullivan et al., 2000) although patients presented a mild hemostatic defect (Szczeklik et al., 1986; Buyukyilmaz et al., 2014).

Activated platelets release the intracellular mediators and produce the

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microparticles, which are elevated in the patients with asthma (Yamamoto et al., 1993; Duarte et al., 2013). Practically, a randomized, double-blind, placebo-controlled crossover study demonstrated the effects of prasugrel in improving slightly the airway responsiveness to mannitol (Lussana et al., 2015).

The association between platelets and eosinophils is partly elucidated to date.

Activated platelets adhere to eosinophils through P-selectin/PSGL-1, CD40/CD40L, glycoprotein IIb/IIIa/MAC-1 (Takeda et al., 2016), forming the platelet-eosinophil aggregations (PEA). In addition, platelet-leukocyte aggregation was increased after allergen challenge and the percentage of eosinophil bound P-selectin, eosinophil-bound αIIb correlated with the lung function in asthmatic patients (Johansson et al., 2012). Platelets and eosinophils share similar surface receptors, including the CysLTR1 and P2Y12R. The adenosine diphosphate (ADP), a specific platelet activator, is able to trigger eosinophil degranulation through the P2Y12R (Muniz et al., 2015). Our recent study suggested the inhibitory effect of clopidogrel, an antiplatelet drug that targets the P2Y12R, on eosinophilic inflammation and degranulation (Suh et al., 2016). Furthermore, LTE4, an agonist for P2Y12R and CysLTR1, can induce the eosinophilic inflammation following a platelet-dependent mechanism (Paruchuri et al., 2009). Taken together, we hypothesized that LTE₄ could amplify the activation of platelet and eosinophil, thereby initiating the PEA and subsequently airway inflammation. Furthermore, the co-administration of antagonists to CysLTR1 (Mon) and to P2Y12R (Clo) may synergistically beneficial in asthma treatment, especially in severe asthma. Therefore, we established the eosinophilic asthma mouse model and aimed to evaluate the synergistic effect of these two drugs. Based on the ratio the CysLTR1 to P2Y12R in our previous finding, we combined Mon and Clo (Clo/Mon) at the ratio 1:1. Next, we investigated the mechanism by which Clo/Mon showed the synergistic effect.

Subsequently, we aimed to investigate whether addition of Clo could attenuate eosinophilic inflammation in asthmatic patients. Thus, a retrospective study was

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carried out on the patients with asthma and/or allergic rhinitis (AR) to validate the clinical effects of Clo.

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II- MATERIALS & METHODS