Background: Plasma levels of the polymorphonuclear neutrophil (PMN)-derived enzyme myeloperoxidase (MPO) correlate with prognosis and severity of myocardial infarction (MI). Apart from its capacity to catalyse the generation of highly reactive oxygen species (ROS), MPO attracts and activates PMN. However, MPO´s transport and its effect on circulating monocytes and infiltrating macrophages after MI remains to be elucidated.

Purpose: Herein we evaluate the role of MPO on monocyte and macrophage activation in vitro and ex vivo, and extrapolate this using a murine model of MI. We further investigate the role of extracellular vesicles (EV) as a novel transport mechanism in MPO-induced monocyte and macrophage activation in MI.

Methods and Results: Peritoneal macrophages (PM) were harvested after thioglycolate stimulation in wild-type (WT) and MPO-deficient (Mpo^-/-) mice. mRNA analyses by quantitative RT-PCR revealed enhanced macrophage activation as shown by increased expression of TNF-α in cells from WT compared to Mpo^-/- animals. Similarly, WT bone marrow derived macrophages (BMDM) showed increased mRNA expression of proinflammatory cytokines (TNF-α, IL-6 and IL-23) 24h after MPO treatment. In vivo, intraperitoneal injection of MPO (15 µg) in Mpo^-/- mice resulted in a marked increase in peritoneal leukocyte recruitment compared to mice injected with saline. These leukocytes were subsequently identified as macrophages (F4/80⁺) by flow cytometry. Using a murine MI model of permanent ligation of the left descending coronary artery (LAD) we showed a reduced macrophage infiltration in the infarct- and peri-infarct area in Mpo^-/- mice as compared to WT animals after three days. Furthermore, splenic monocyte mobilisation was significantly decreased in Mpo^-/- mice one day after LAD ligation suggesting a role for MPO in monocyte/macrophage recruitment in MI. Given that EV mediate splenic monocyte recruitment after MI, we analysed plasma-derived EV of WT mice for mechanistic insight. ELISA analyses revealed enhanced MPO levels in EV isolated 1 day after LAD ligation compared to sham operated mice. Immunoblotting showed that plasma-derived EV isolated one day after LAD ligation are of neutrophil and endothelial origin. Treatment of RAW264.7 macrophages with MPO or MPO-competent EV increased chemokine receptor CCR2 expression as compared to unstimulated cells or treatment with MPO-deficient EV, suggesting CCR2 as a potential mechanistic link in MPO-induced macrophage recruitment after MI.  

Conclusions: Herein we identify MPO as a pro-inflammatory mediator and propose the release of MPO-containing neutrophil-derived EV as a novel mechanism of monocyte and macrophage recruitment in myocardial infarction.