MATERIALS & METHODS

The species used in this study were described as in Chapter I. All animal experiments conducted in this study were approved by the Institutional Animal Care and Use Committee of Ajou University (IACUC 2015-11-09).

B. Experimental procedure for a secondary challenge, eosinophilic asthma mouse model of

BALB/c mice were intraperitoneally sensitized with OVA/Alum solution at 10 ug/1mg on days 0 and 14. On days 28, 29, and 30, mice underwent the primary allergen challenges by nebulization with 0.2% OVA for 20 minutes, using an ultrasonic nebulizer (NE-SM1, KTMED Inc, Seoul, South Korea). On days 44-46, mice underwent the secondary challenge using 1% OVA aerosols.

C. Protocols of drug administration

Mice were given either Clo (10 mg/kg) orally, Mon (10 mg/kg) orally, dexamethasone (1 mg/kg) intraperitoneally 30 minutes prior to OVA challenge.

Forty-eight hours after the last challenge, mice were assayed for further experiments. For the negative control group, mice received PBS containing DMSO as the solvent control.

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Figure 8. The schema of a secondary challenge, OVA-induced eosinophilic asthma mouse model. After two times of intraperitoneal injection with OVA, mice underwent the primary allergen challenges by exposure to OVA aerosols (0.2%) for 30 minutes on days 28, 29 and 30 in OVA. On days 44, 45 and 46, mice were subjected to the secondary allergen challenge with OVA aerosols 1% along with drug administration.

D. Airway resistance measurement and sample collection

As in Chapter I, the values of airway resistance to inhaled MCh were recorded using the flexiVent system (SCIREQ), except that mice were challenged to a series of MCh dilution from 1.56 to 25 mg/mL

The BAL fluid, lung tissues were harvested and processed for differential cell count and histological analysis as described in Chapter I.

E. Measurement of cytokines level

The BAL fluid levels of IL-4, IL-5, IL-13 (obtained from eBioscience, San Diego, CA), PF4 (from Abcam, Cambridge, UK) and eosinophil peroxidase (EPX)

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(MyBiosource, Inc, San Diego, USA) were measured by ELISA, according to the manufacturer’s instructions.

F. Identification of PEA in BAL fluid and whole blood of mice

Flow cytometric determination of PEA in whole blood was conducted as described elsewhere (Evangelista et al., 2005; Harding et al., 2006). Mouse whole blood was harvested by cardiac puncture into tubes containing 3.8% sodium citrate to prevent the coagulation. Mouse whole blood was incubated with PE-conjugated anti-mouse Siglec-F and FITC-conjugated anti-mouse CD61 for 30 minutes, RT, avoid from light. Next, red blood cells were lysed with RBC Lysis/Fixation solution (Biolegend, San Diego, CA, USA) for 10 minutes and washed once with PBS 1X. Cells were analyzed immediately by using flow cytometry. PEA was identified as Siglec-F⁺/CD61⁺ cells, at least 5,000 events were recorded for each sample.

In some experiments, PEA was visualized using the immunocytochemistry.

By using the methods of double immunofluorescence staining as described in Chapter II, we labeled BAL cells with anti-P-selectin and anti-EPX antibody as markers for activated platelet and eosinophils, and counterstained with mounting medium for fluorescence containing DAPI (Vector). Signals of fluorescence were visualized by Zeiss Zen Microscope software.

G. Isolation of mouse eosinophil and platelets

Isolation of mouse eosinophil was modified from the previous protocol (Carlens et al., 2009). A single cell suspensions were incubated with peridinin chlorophyll protein complex (PerCP) conjugated anti-Ly6G and allophycocyanin (APC) conjugated anti CD11c antibodies for 30 minutes. Mouse eosinophils were sorted using fluorescence-activated cell sorting. The leukocyte population was

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identified as the FCS⁺/SSC⁺ cells, the Ly6G^-/CD11c^- eosinophils were sorted out and suspended in the RPMI-1640 medium supplemented with 10% foetal bovine serum and 100 U/mL penicillin G sodium and 100 µg/mL streptomycin sulfate (all from Gibco, Grand Island, NY, USA) until further experiment. (Fig. 9)

In parallel, mouse platelets were isolated as described in the previous reports (Evangelista et al., 2005). The whole blood was centrifuged for 3000 rpm, 15 minutes, RT. The platelet-rich plasma (PRP) were collected and centrifuged for 400g, 10 minutes, RT to isolate the platelets. Cells were primed with CaCl2 (Sigma Aldrich) (final concentration = 5 mM) for 10 minutes prior to experiment. (Fig. 9)

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Figure 9. The schema of platelet and eosinophil enrichment. (A) Eosinophils were isolated from the homogenized mouse lung tissues. Cell suspension was labeled with PerCP conjugated Ly6G antibody and APC conjugated anti-CD11c antibody. Cells were sorted out by flow cytometry. A gate of leukocytes was set based on the FSC/SSC characteristics. The eosinophils were defined as Ly-6G -/CD11c^- leukocytes. (B) Representative images of flow cytometric data and HE stained samples were shown. (C) Platelets were isolated from mouse whole blood by centrifugation. The PRP was centrifuged one more time to isolate platelet.

H. In vitro assay of PEA

On the basis of possible ligands between platelets and eosinophils (Mitsui et al., 2016) (Fig.10), mouse eosinophils and platelets were co-cultured, stimulated with ADP, LTE4, LTC4 and treated with Mon (1µM), Clo (1 µM), anti-CD40 antibody (100 ng/mL), and tirofiban hydrogen sulfate (1 µM) for 3 hours. Cells were incubated with PE-anti mouse Siglec-F and FITC-anti mouse CD41, PE/Cy7-anti mouse CD62P for 20 minutes. Fluorescence signals were analyzed immediately by flow cytometry and at least 1000 events were recorded.

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Figure 10. Platelet-adherent eosinophils (Mitsui et al., 2016).

I. Antibodies, reagents.

Antibodies used against mouse target proteins were, anti-P-selectin antibody (sc-8419, Santa Cruz Biotechnology), anti-EPX antibody (sc-19147, Santa Cruz Biotechnology), Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 594-conjugated rabbit anti-goat IgG were from ThermoFisher Scientific (Waltham, MA, USA). PerCP-conjugated anti-Ly6G (127654, Biolegend), APC-conjugated CD11c (117309, Biolegend), PE-conjugated anti-Siglec-F (552126, BD Pharmingen), FITC-conjugated anti-CD41 (133904, Biolegend), PE/Cy7-conjugated anti-CD62P (148310, Biolegend) antibodies were used for flow cytometry and cell sorting. All drugs used for treatment were obtained from the Sigma Aldrich (USA), except otherwise indicated.

J. Study design to validate the clinical effects of Clo in patients with asthma

33 and/or AR

Based on the electronic medical records from 1998 to 2015 in Ajou Medical Center, Suwon, South Korea, a retrospective, cross-sectional study was undertaken.

We recruited patients who were diagnosed as asthma and/or ARby physicians.

Subsequently, the patients who received Clo for more than 7 days after the diagnosis of asthma were selected. The exclusion criteria were patients without the complete blood count (CBC) or the CBC was conducted outside the exposure period to Clo. Totally, 596 subjects were included and the eosinophil count, including the percentage and the absolute count, within 60 days were recorded (Fig.

11).

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Figure 11. Study design to validate the clinical effectiveness of Clo. AR, allergic rhinitis; CBC, complete blood count.

K. Statistical analysis

Data are presented as the mean ± SEM. The differences between groups were analyzed by the Mann-Whitney U test, except otherwise indicated. The statistical analysis were performed by SPSS version 23.0 (SPSS Inc, Chicago, IL, USA), and a P value of less than 0.05 was considered statistically significant.

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III- RESULTS A. Effects of Clo/Mon on AHR

Mice received Clo/Mon showed a further decrease in AHR compared to the single treatment at the highest MCh dose (P=0.003), and the effect was as similar to those received dexamethasone treatment (Fig. 12A). In addition, Clo/Mon suppressed the increased total cell count (P=0.005) and eosinophil count significantly (P<0.001) (Fig. 12B).

Figure 12. Additional effects of clopidogrel to montelukast in reducing airway inflammation. (A) Changes in the airway hyperresponsivess were recorded by FlexiVent at 48 hours after the last challenge; (B) Total and differential cell counts were counted using haemocytometer . P values were analyzed by one way ANOVA with the Tukey post hoc test; *, **, ***, P<0.05, 0.01, 0.001 comparing between the indicated groups; #, P<0.05 comparing between the drug treated groups; NS, not significant.

B. Effects of Clo/Mon on histological score

At the low dose, Clo could not suppress as effectively the inflammatory cells.

Clo/Mon attenuated the number of inflammatory cells into the lung tissues (P<0.05) (Fig. 13A). In the same manner, the single treatment with Mon could not deplete

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the mucus containing cells in the asthma mouse significantly. Thus, the administration of Clo/Mon improved the mucus secretion more effectively than the single treatment in the asthma mice (P<0.05) (Fig. 13B).

Figure 13. The inflammatory cell counts from (A) H&E stained lung tissues and mucus containing cells from (B) PAS-stained lung tissues, respectively, were calculated. P values were analyzed by one way ANOVA with the Tukey post hoc test.*, **, ***, P<0.05, 0.01, 0.001 comparing between groups; NS, not significant.

C. Additional effects of P2Y12 antagonists on cytokine levels

Compared to the OVA/OVA, Clo/Mon suppressed the increased levels of IL-4, IL-13 more effectively than the single treatment and as similar to dexamethasone (P<0.001; P=0.001) (Fig. 14A). Clo/Mon reduced IL-5 level significantly (P<0.001)

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but the effect was not prominent than the single therapy. Interestingly, Clo/Mon suppressed significantly the activation status of platelet and eosinophil, demonstrating by the decreased PF4 and EPX, respectively (P=0.016, P=0.001) (Fig. 14B, C).

Figure 14. Effects of Clo/Mon on Th2 cytokines and the activation state of platelet and eosinophil. BAL fluid was harvested and stored at -70⁰C until further analysis. The BAL fluid levels of (A) IL-4, IL-5, IL-13, (B) PF4 and (C) EPX were measured by ELISA. Data are presented as means ± SD. N=10 mice per group. P values were analyzed by one way ANOVA with the Tukey post hoc test; except for the IL-4 and PF4 levels were analyzed by Mann-Whitney U test *, **, ***, P<0.05, 0.01, 0.001 comparing between the indicated groups.

D. Clo/Mon depleted the PEA formation

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In Fig.15, PEA in mouse whole blood was visualized based on the cells containing the P-selectin and EPX. On the basis of flow cytometric determination, the increased level of PEA in asthma was abrogated by Clo/Mon in mouse whole blood (P=0.014) and BAL fluid. Dex did not show much effect on the PEA formation (Fig.15B). The suppressive effect of Clo/Mon was stronger than that of Dex (P=0.023).

Figure 15. Upregulation of PEA in asthma. (A) The peripheral blood eosinophil in whole blood was labeled with EPX. The activated platelets (P-selectin) bound to eosinophil surfaces were identified by P-selectin and EPX. (B) The percentage of PEA in mouse whole blood. PEA was defined as Siglec-F⁺/CD41⁺ cells based on the flow cytometric determination. Data are presented as means ± SD. P values were analyzed by Mann-Whitney U test. N=5 for each group. (C) The circulating PEA in mouse BAL fluid. Representative images from at least 3 independent experiments were shown.

E. Ligands of PEA

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In in vitro assay, ADP induced significantly the formation of PEA (P=0.004) and the fold induction of P-selectin (P=0.031). Although LTC₄, LTE₄ tended to induce PEA but we could not achieve any statistical significance. Clo/Mon suppressed significantly the ADinduced PEA aggregation (P=0.008) and P-selectin overexpression (P=0.003) (Fig. 16).

Figure 16. Effects of Clo/Mon on the ligands of PEA. Mouse platelets and eosinophils were isolated from whole blood and tissues as described in the Materials & Methods section. After being primed with CaCl₂, mouse platelet and eosinophils were co-incubated within 1 hour, with the addition of ADP, LTC4, LTE4. In some experiments, cells were primed with Mon (1 µM), Clo (1 µM), anti CD40

40

antibody (100 ng/mL), Tirofiban (1 µM). PEA was incubated with antibodies to Siglec-F, to CD41 and to P-selectin and then subsequently analyzed by flow cytometry. (A) The percentage of Siglec-F⁺/CD41⁺ was calculated. (B) Clo/Mon reduced significantly the PEA formation. (C) Clo/Mon reduced significantly the P-selectin expression on platelets. (D) Representative flow cytometric images were shown. Data are presented as means ± SD, from 3 independent experiments with duplicate results. P values were analyzed by Wilcoxon-signed Rank test. *, **, P<0.05, 0.01 compared between the indicated groups.

Figure 17. Schema of the platelet-eosinophil interactions and the effects of Clo/Mon in asthma.

F. Clinical effectiveness of Clo on eosinophil count in patients with asthma Generally, the trend of eosinophil counts (% and absolute count) fluctuated

41

during the follow up (Fig. 18). The eosinophil counts (% and absolute count) were declined at day 60 comparing to day 1 (Table 2). No significant differences were found at day 60 compared to day 1.

Figure 18. Therapeutic effects of Clo on eosinophil count. (A) Percentage of eosinophil count; (B) absolute eosinophil count in CBC were recorded. Means of eosinophil count (% and absolute) were linearized.

Table 2. Eosinophil counts during the follow-up period

Day Eosinophil count (%) Absolute eosinophil count (x10⁴)

P values

1 2.32 ± 2.55

179.74 + 194.35 0.381

60 1.2 ± 0.61 94.38 ± 44.46 0.381

Data are presented as means ± SD.

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IV- DISCUSSION

Recent data supported the function of platelets as the key effector cells as well as antiplatelet therapies in treatment of inflammatory disease (von Hundelshausen and Weber, 2007; Semple et al., 2011; Idzko et al., 2015; Takeda et al., 2017).

Clopidogrel was documented to inhibit the platelet-leukocyte aggregation and the platelet-dependent leukocyte activation in the diabetes mellitus, atherosclerosis and coronary syndromes (Evangelista et al., 2005; Harding et al., 2006). We are the first group to report the synergistic effect of Clo to Mon in reducing the airway inflammation. Clo/Mon impaired not only the PEA formation and platelet activation mediated by ADP, but also the platelet-dependent eosinophil degranulation.

In the current study, the combination of Clo/Mon was more effective than the single therapy with either Mon or Clo. Comparing to the efficacy of Dex, Clo/Mon showed the similar or stronger effect on attenuating the AHR, IL-4, IL-5 and IL-13, although Dex was able to suppress the eosinophil count and changes of histological structure. These results are relevant because of (1) the lower concentrations of drugs, and (2) the secondary challenge, eosinophilic asthma mouse model. As compared to the previous experimental studies, we used the considerably lower doses of Mon and Clo (10mg/kg for each drug) (Henderson et al., 2006; Shin et al., 2013; Suh et al., 2016). The secondary, eosinophilic asthma mouse model manifested higher eosinophilic infiltration than the model that we have used previously, in order to see clearly the therapeutic effects of Clo/Mon. Besides, Clo/Mon treatment suppressed lymphocyte count in BAL fluid, which could explain for the decreased levels of Th2 cytokines.

In agreement with the hypothesis, Clo/Mon synergistically inhibited not only PEA formation but also the activation of platelets (PF4) and eosinophils (EPX).

The symbiotic association between platelets and eosinophils was reinforced in our study. By direct contact via the surface ligands (e.g., P-selectin) or soluble

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mediators, platelets and eosinophils can interact, leading to cellular activation.

Platelets expressed both high-and low- affinity IgE receptors, which could be functional in asthma (Idzko et al., 2015; Shah et al., 2017; Takeda et al., 2017). A large number of studies demonstrates the increased PEA after allergen challenge in asthma (Pitchford et al., 2003; Benton et al., 2010). Platelet and eosinophil activity correlated positively in asthmatic patients and negatively with asthma-related quality of life (Benton et al., 2010). In the current study, the increased PEA formation was impaired by Clo/Mon, possibly resulting in reduced eosinophil recruitment to lung tissues. Moreover, the adherent platelets from the PEA were more activated, leading to the release of PF4, which further trigger eosinophil activation (Owen et al., 1987; Laidlaw et al., 2012). Indeed, we found the increased level of PF4 and EPX, PF4, also known as CXCL4, released from the α-granules of the activated platelets, is known as a potent eosinophil chemoattractant and an augmentative agent for eosinophil adhesion (Hayashi et al., 1994; Idzko et al., 2015). In our study, the increased PF4 in asthma mice could also enhance the eosinophilia which amplifies the EPX production. It is also noteworthy that PF4 and EPX can contribute directly to inducing the airway hyperactivity in the animal models (Gundel et al., 1991; Coyle et al., 1994). PEA is markedly increased in patients with aspirin-exacerbated respiratory disease, which is characterized by LT overproduction. Although we could not find the significant increase of PEA induced by LTE₄ in in vivo assay, PEA is known to correlate with LT synthesis (Laidlaw et al., 2012). Taken together, we speculated that the inhibitory effects of Clo/Mon may rely on the interactions of platelet-eosinophils.

Next, we investigated the mechanism by which Clo/Mon exert the effects. To date, there are several stimulators for platelet activation, including ADP, LTC4

(Dorsam and Kunapuli, 2004; Cummings et al., 2013). ADP was the most potent agonist to induce the PEA formation and P-selectin overexpression. We could not demonstrate the effects of LTC4 on platelet activation as demonstrated elsewhere,

44

(Cummings et al., 2013) possibly due to the prolonged activation of platelets during the induction period in our asthma mouse model. Remarkably, Clo/Mon suppressed the most effectively ADP-induced PEA formation or P-selectin expression. ADP is known to stimulate platelets by phosphoinositide 3-kinase (PI3K)/Akt dependent pathway (Sun et al., 2005; O’Brien et al., 2012; Jiang et al., 2013). Notably, both P2Y12 and CysLTR1 are involved in the PI3K/Akt pathway (Dorsam and Kunapuli, 2004; Burke et al., 2016), which can advocate for the synergistic effects of two respective antagonists, Clo/Mon. In regard to eosinophil, P2Y12R antagonist can inhibit the eosinophil degranulation (Muniz et al., 2015).

Therefore, addition of P2Y12R can support the effect of Mon in suppressing eosinophil protease activity and infiltration (Langlois et al., 2006). Additionally, the antagonists against other surface ligands such as CD40L and glycoprotein IIb/IIIa, anti-CD40L antibody and tirofiban, respectively, were applied. Tirofiban has been documented to influence the interaction of platelets and leukocytes (Xiao et al., 1999). The effectiveness of tirofiban in suppressing platelet activation and platelet-eosinophil interaction could be exploited conceivably in asthma treatment.

In this study, we revealed the slight effects of Clo on the peripheral eosinophil count although none of these differences were statistically significant.

Despite of that, our findings appear to be well substantiated by the in vivo data showing that Clo could inhibit the airway inflammation. As reported in PRINA study, prasugrel was found to decrease AHR in patients with asthma without any significant changes, which was in line to our study, demonstrating that the anti-inflammatory effects are not significant to make statistical significance (Lussana et al., 2015). We are aware that our research may have two limitations. Firstly, the control group was not able to be included in the study. Secondly, since we conducted a retrospective study, we could not evaluate other events which might bias the eosinophil count. In general, there are multiple ligands involving in eosinophil migration except for P-selectin/PSGL-1, such as CD40/CD40L and

45

GpIIb/IIIa/Mac-1(Mitsui et al., 2016), therefore, blockage of the remaining ligands should be cautiously considered for the application of antiplatelet drugs in asthma treatment.

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V. CONCLUSION

This thesis investigated the association of Cys-LT related pathways and platelet-eosinophil interactions in asthma. In allergic inflammation, the expression of CysLT-related receptors, including CysLTR1, CysLTR2 and P2Y12R were up-regulated at the ratio of 1:0.65:1.34, suggesting the 1:1 ratio for Clo:Mon treatment in the subsequent in vivo studies. In a secondary challenge asthma mouse model, we detected increased PEA formation, platelet and eosinophil activation status along with increased airway inflammation, which were suppressed by Clo/Mon treatment. Clo/Mon may function by inhibiting the ADP-induced PEA formation and platelet activation. In conclusion, we demonstrated the synergistic effects of Clo and Mon in suppressing platelet-eosinophil interaction in airway inflammation.

The combination of two antagonists may be potential in asthma treatment, especially in more severe eosinophilic asthma.

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