Compromised Anti-inflammatory Action of Neutrophil Extracellular Traps in PAD4-Deficient Mice Contributes to Aggravated Acute Inflammation After Myocardial Infarction

Kaveh Eghbalzadeh,Ugur Keser,Haizhi Zhao,Carolyn Weber,Leena Georgi,Theresa Louis,Andreas Conforti,Martin Mollenhauer,Adnana Paunel-Görgülü,Thorsten Wahlers

doi:10.3389/fimmu.2019.02313

Kaveh Eghbalzadeh, Ugur Keser + Show 8 more

Open Access

https://doi.org/10.3389/fimmu.2019.02313

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Abstract

Innate immune responses and rapid recruitment of leukocytes, which regulate inflammation and subsequent healing, play a key role in acute myocardial infarction (MI). Peptidylarginine deiminase 4 (PAD4) is critically involved in chromatin decondensation during the release of Neutrophil Extracellular Traps (NETs) by activated neutrophils. Alternatively, activated macrophages (M2) and accurate collagen deposition determine the repair of the infarcted heart. In this study, we investigated the impact of NETs on macrophage polarization and their role for acute cardiac inflammation and subsequent cardiac healing in a mouse model of acute MI. NETs were found to promote in vitro macrophage polarization toward a reparative phenotype. NETs suppressed pro-inflammatory macrophages (M1) under hypoxia and diminished IL-6 and TNF-α expression. Further on, NETs strongly supported M2b polarization and IL-10 expression. In cardiac fibroblasts, NETs increased TGF-ß expression under hypoxic culture conditions. PAD4−/− mice subjected to permanent ligation of the left anterior descending artery suffered from overwhelming inflammation in the acute phase of MI. Noteworthy, PAD4−/− neutrophils were unable to release NETs upon ex vivo stimulation with ionomycin or PMA, but produced significantly higher amounts of reactive oxygen species (ROS). Increased levels of circulating cell-free DNA, mitochondrial DNA and cardiac troponin were found in PAD4−/− mice in the acute phase of MI when compared to WT mice. Reduced cardiac expression of IL-6, IL-10, and M2 marker genes, as well as increased TNF-α expression, suggested a pro-inflammatory state. PAD4−/− mice displayed significantly increased cardiac MMP-2 expression under baseline conditions. At day 1, post-MI, PAD4−/− mice showed increased end-diastolic volume and increased thinning of the left ventricular wall. Interestingly, improved cardiac function, as demonstrated by significantly increased ejection fraction, was found at day 21. Altogether, our results indicate that NETs support macrophage polarization toward an M2 phenotype, thus displaying anti-inflammatory properties. PAD4 deficiency aggravates acute inflammation and increases tissue damage post-MI, partially due to the lack of NETs.

Highlights

Myocardial infarction (MI) leads to the death of up to one billion cardiac cells in response to occurred ischemia and, in consequence, the affected myocardium undergoes a series of remodeling processes accompanied by inflammatory responses, fibrosis and scar formation [1]
To imitate ischemia in the infarcted heart, cells were cultured under hypoxic condition (2% O2) and cells cultured under normoxia (21% O2) served as a control
Acute MI leads to the death of cardiomyocytes, which induces the release of DAMPs, including DNA [39], and a strong proinflammatory response, in order to remove the damaged tissue

Summary

Introduction

Myocardial infarction (MI) leads to the death of up to one billion cardiac cells in response to occurred ischemia and, in consequence, the affected myocardium undergoes a series of remodeling processes accompanied by inflammatory responses, fibrosis and scar formation [1]. Activated neutrophils release decondensated chromatin covered by elastase, myeloperoxidase (MPO) and cytoplasmic proteins, known as neutrophil extracellular traps (NETs). This process of NETs formation is called NETosis and largely differs from apoptosis and necrosis. Impaired resolution of inflammation and prolonged M1 macrophage activation may aid adverse cardiac remodeling and impair MI outcomes. In this regard, silencing of interferon regulatory factor 5 (IRF5), which regulates polarization toward the M1 phenotype, shifted macrophage phenotype from M1 to M2 in the heart and attenuated heart failure post-MI [14]

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