NETosis is an important component of chronic inflammation in patients with heart failure

Abstract Background and aim We have previously demonstrated that heart failure (HF) is characterized by low-grade myocardial inflammation. However, the role of neutrophils (N), neutrophil extracellular traps (NETs) and neutrophil cell death by NETosis in the myocardium of patients with HF remains largely unknown. The present study investigated the number of neutrophils (N) and their proportion undergoing NETosis and developing NETs in HF. Methods We used quantitative confocal microscopy and NETosis markers in the left ventricular biopsies obtained from 5 control and from patients with HF due to dilated (DCM, n=7), inflammatory (infCMP, n=7) and ischemic cardiomyopathy (ICM, n=7). We used immunolabeling for CD45, CD66b and CD11b for (N) and citrullinated histone3 (citH3), peptidylarginine deiminase-4 (PAD-4), neutrophil elastase (NE) and myeoloperoxidase (MPO) for NETosis. These proteins were also investigated by quantitative fluorescence intensity analysis, Western blot and quantitative polymerase chain reaction (qPCR). Results Compared to control, the number of N was increased 3-4 times in HF. We found that using a single marker for NETosemarkers, 43.2% of N in DCM, 46.7% in ICM and 57.3% in infCMP experienced NETosis. The use of double labeling (NE with CitH3) showed that 55.6% of N developed NETosis in DCM, 57.9% in ICM and 79.4% in infCMP. The difference between the N who underwent NETosis in infCMP and those in DCM was statistically different (p<0.01). The proportion of N who developed NETosis or formed NETs in control tissue was less than 5% and differed significantly from that in HF patients, regardless of etiology (p<0.01). These results were confirmed by quantitative fluorescence analysis, Western blot and qPCR. Conclusions This is the first study to show the occurrence of NETosis in human hearts in situ indicating that NETosis is an important component of low-grade myocardial inflammation in HF. What is new? Low-grade myocardial inflammation is a typical feature of heart failure and neutrophil cell death (NETosis) is an important component of this pathological process. What are the clinical implications? Preventing excessive neutrophil activation and inhibiting the major components involved in the NETosis program (neutrophil elastase, myeloperoxidase and peptidylarginine deiminase-4) are perspective targets for the treatment of HF.