Purinergic P2Y12 Receptor Activation in Eosinophils and the Schistosomal Host Response

Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5’-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection

P2Y12R blockade reduces the area of the hepatic inflammatory infiltrate in a murine model of <i>S</i>. <i>mansoni</i> infection.

<p>Representative granuloma images from infected mice (Ai) untreated or (Aii) treated with clopidogrel. Slide sections were stained with hematoxylin-eosin and examined under a light microscope. (B) The individual granuloma area was estimated from 10–20 granulomas acquired for each individual mouse. Data represent the mean ± SE and were analyzed by Student’s <i>t</i> test. (*p<0.05). <i>n</i> = 5–8 mice/group and represents animals from at least 2 independent infection studies.</p

P2Y12R blockade promotes an increase in blood eosinophilia but decreases the eosinophil count in the bone marrow.

<p>(A) The number of eosinophils was estimated in the blood of infected, clopidogrel-untreated and clopidogrel-treated mice (Panoptic kit-stained smears). (B) The number of eosinophils in the individual bone marrow was evaluated in clopidogrel-untreated and -treated mice (Panoptic kit-stained cytospins). Data represent the mean ± SE and were analyzed by Student’s <i>t</i> test. (*p<0.05). <i>n</i> = 5–8 mice/group and represent animals from at least 2 independent infection studies.</p

ADP did not induce/prevent eosinophil apoptosis.

<p>Human eosinophils were cultured for 24 h in the presence of IL-5 (30 ng/mL) or ADP (100 and 10 nM) or MRS2395 (10 μM), followed by labeling with annexin V and flow cytometry analysis. Data are representative histograms of 3 independent experiments (n = 3). Vehicle = MRS2395 diluent (DMSO).</p

Th2 response to <i>S</i>. <i>manoni</i> infection is not altered by P2Y12R blockade.

<p><i>S</i>. <i>mansoni-</i>infected, clopidogrel-treated mice displayed a slight difference in plasma concentrations of (A) IL-13, but no difference in (B) IL-4 levels was detected. Data represent the mean ± SE and were analyzed by Student’s <i>t</i> test. (*p<0.05). <i>n</i> = 5–8 mice/group and represents animals from at least 2 independent infection studies.</p

ADP is not chemotactic for human eosinophils.

<p>Human eosinophil-enriched suspensions containing 3x10⁵ cells/100 μL were left to migrate in transwell plates against eotaxin (20 ng/mL) (positive control) or ADP (100 and 10 nM) in (A) the absence or (B) presence of IL-5 (30 ng/mL) for 3 h at 37°C. (C) MRS2395 (10 μM), a P2Y12R antagonist, failed to inhibit the eotaxin-induced eosinophil recruitment. Data are representative dot plots (SSC-A/FSC-A) of 3 independent experiments (n = 3). Vehicle = MRS2395 diluent (DMSO).</p

P2Y12R blockade impairs hepatic granulomatous eosinophilia.

<p>Representative granuloma images from infected mice (Ai) untreated or (Aii) treated with clopidogrel. Slide sections were stained with Syrius red (modified method) and examined under a light microscope. (B) The number of eosinophils per granuloma area were calculated from images of 10–20 granulomas acquired for each individual mouse. Data represent the mean ± SE and were analyzed by Student’s <i>t</i> test. (*p<0.05). <i>n</i> = 10 mice/group and represents animals from at least 2 independent infection studies. (C) Immunofluorescence showing Siglec-F and P2Y12R staining of cytospin smears of murine eosinophil-enriched suspensions. P2Y12R, green; Siglec-F, red; Ab = antibody. Data are from one experiment, representative of 3 experiments. (D) Flow cytometry dot plots showing isotype control (left panel) and Siglec-F versus P2Y12R staining (right panel) of mice bone marrow derived eosinophils. Data are from one experiment, representative of 3 experiments.</p

P2Y12R blockade does not alter the course of <i>S</i>. <i>mansoni</i> parasite oviposition.

<p>Mice were infected with 60 <i>S</i>. <i>mansoni</i> cercariae through a percutaneous route, and the livers and intestines were collected 55 days post-infection. Graphs show the number of eggs per gram tissue within (A) the liver and (B) intestines. Data represents the mean ± SE; 5–8 infected mice were used as individual tissue donors and represents animals from at least 2 independent infection studies.</p

Resveratrol Reverses Functional Chagas Heart Disease in Mice

<div><p>Chronic chagasic cardiomyopathy (CCC) develops years after acute infection by <i>Trypanosoma cruzi</i> and does not improve after trypanocidal therapy, despite reduction of parasite burden. During disease, the heart undergoes oxidative stress, a potential causative factor for arrhythmias and contractile dysfunction. Here we tested whether antioxidants/ cardioprotective drugs could improve cardiac function in established Chagas heart disease. We chose a model that resembles B1-B2 stage of human CCC, treated mice with resveratrol and performed electrocardiography and echocardiography studies. Resveratrol reduced the prolonged PR and QTc intervals, increased heart rates and reversed sinus arrhythmia, atrial and atrioventricular conduction disorders; restored a normal left ventricular ejection fraction, improved stroke volume and cardiac output. Resveratrol activated the AMPK-pathway and reduced both ROS production and heart parasite burden, without interfering with vascularization or myocarditis intensity. Resveratrol was even capable of improving heart function of infected mice when treatment was started late after infection, while trypanocidal drug benznidazole failed. We attempted to mimic resveratrol’s actions using metformin (AMPK-activator) or tempol (SOD-mimetic). Metformin and tempol mimicked the beneficial effects of resveratrol on heart function and decreased lipid peroxidation, but did not alter parasite burden. These results indicate that AMPK activation and ROS neutralization are key strategies to induce tolerance to Chagas heart disease. Despite all tissue damage observed in established Chagas heart disease, we found that a physiological dysfunction can still be reversed by treatment with resveratrol, metformin and tempol, resulting in improved heart function and representing a starting point to develop innovative therapies in CCC.</p></div

P2Y12R is expressed in human eosinophils and mediates ADP-induced eosinophil peroxidase (EPO) secretion.

<p>Analysis of the expression of P2Y12R in human eosinophils by (A) flow cytometry and (B) immunofluorescence. P2Y12R, green; nuclei, blue (after staining with DAPI). Shaded histogram represents staining with control antibody (Ab). Dashed and solid lines represent staining with polyclonal antibodies (pAbs) against P2Y12R and anti-P2Y12R pAbs neutralized by absorption with their immunogen peptide, respectively. Data are from one experiment, representative of 3 experiments. (C) ADP-induced EPO secretion after 1 h of stimulation is mediated by (D) P2Y12R. Graphs represent the mean ± SE (<i>n</i> = 3). Results were analyzed by one-way ANOVA, followed by the Newman-Keuls test (+ and *p<0.05 for EPO released compared with non-stimulated and ADP-stimulated eosinophils, respectively). NS = not stimulated, OD = optical density.</p