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.

Neutrophil extracellular traps (NETs) are web-like structures, which are released upon neutrophil activation. It has previously been demonstrated that NETs are present in atherosclerotic lesions of both humans and animal models thus playing a decisive role in atherosclerosis. Besides, macrophages have a crucial role in disease progression, whereby classically activated M1 macrophages sustain inflammation and alternatively activated M2 macrophages display anti-inflammatory effects. Although NETs and macrophages were found to colocalize in atherosclerotic lesions, the impact of NETs on macrophage function is not fully understood. In the present study, we aimed to investigate the effect of NETs on human and murine macrophages in respect to the expression of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and uptake of oxidized LDL (oxLDL) in vitro. Human THP-1 and murine bone marrow-derived macrophages were cultured under M1 (LPS + IFN-γ)- and M2a (IL-4)-polarizing culture conditions and treated with NETs. To mimic intraplaque regions, cells were additionally cultured under hypoxic conditions. NETs significantly increased the expression of IL-1β, TNF-α and IL-6 in THP-M1 macrophages under normoxia but suppressed their expression in murine M1 macrophages under hypoxic conditions. Notably, NETs increased the number of oxLDL-positive M1 and M2 human and murine macrophages under normoxia, but did not influence formation of murine foam cells under hypoxia. However, oxLDL uptake did not strongly correlate with the expression of the LDL receptor CD36. Besides, upregulated MMP-9 expression and secretion by macrophages was detected in the presence of NETs. Again, hypoxic culture conditions dampened NETs effects. These results suggest that NETs may favor foam cell formation and plaque vulnerability, but exert opposite effects in respect to the inflammatory response of human and murine M1 macrophages. Moreover, effects of NETs on macrophages’ phenotype are altered under hypoxia.

IntroductionDespite multiple studies in the past, the role of peptidylarginine deiminase 4 (PAD4) in atherosclerosis is currently insufficiently understood. In this regard, PAD4 deletion or inhibition of enzymatic activity was previously reported to ameliorate disease progression and inflammation. Besides, strong influence of neutrophil extracellular traps (NETs) on atherosclerosis burden has been proposed. Here, we studied the role of PAD4 for atherogenesis and plaque progression in a mouse model of atherosclerosis.Methods and resultsLethally irradiated ApoE–/– mice were reconstituted with ApoE–/–/Pad4–/– bone marrow cells and fed a high-fat diet (HFD) for 4 and 10 weeks, respectively. PAD4 deficiency did not prevent the development of atherosclerotic lesions after 4 weeks of HFD. However, after 10 weeks of HFD, mice with bone marrow cells-restricted PAD4 deficiency displayed significantly reduced lesion size, impaired lipid incorporation, decreased necrotic core area and less collagen when compared to ApoE–/– bone marrow-transplanted mice as demonstrated by histological staining. Moreover, flow cytometric analysis and quantitative real-time PCR revealed different macrophage subsets in atherosclerotic lesions and higher inflammatory response in these mice, as reflected by increased content of M1-like macrophages and upregulated aortic expression of the pro-inflammatory genes CCL2 and iNOS. Notably, diminished oxLDL uptake by in vitro-polarized M1-like macrophages was evidenced when compared to M2-like cells.ConclusionThese results suggest that pharmacological inhibition of PAD4 may impede lipid accumulation and lesion progression despite no beneficial effects on vascular inflammation.