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).
34
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.
35
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
36
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)
37
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
38
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
39
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.
42
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
43
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.
46
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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