Peptidylarginine deiminase 4 (PAD4) citrullinates proteins. In neutrophils, it causes chromatin decondensation and release of NETs, which are injurious. Martinod et al. show in this study that NETs promote fibrosis in a cardiac model and that PAD4-deficient mice have reduced age-related organ fibrosis.
Background Neutrophil serine proteases have been implicated in coagulation and neutrophil extracellular trap (NET) formation. In human neutrophils, neutrophil elastase (NE) translocates to the nucleus during NETosis and cleaves histones, thus aiding in chromatin decondensation. NE−/− mice were shown not to release NETs in response to microbes. However, mouse studies evaluating the role of NE in NET formation in sterile inflammation and thrombosis are lacking. Objective We wished to establish if neutrophils from NE−/− mice have a defect in NETosis, similar to peptidylarginine deiminase 4 (PAD4−/−) mice, and how this might impact venous thrombosis, a model where NETs are produced and are crucial to thrombus development. Methods We performed in vitro NET assays using neutrophils from wild-type (WT), NE−/−, SerpinB1 (SB1−/−), and NE−/−SB1−/− mice. We compared WT and NE−/− animals in the inferior vena cava stenosis model of deep vein thrombosis (DVT). Results NE-deficiency resulted in a small reduction in ionomycin-induced NET formation in vitro without affecting histone citrullination. However, NET production in response to PMA or PAF was normal in neutrophils from two independent NE-deficient mouse lines, or in NE−/−SB1−/− as compared to SB1−/− neutrophils. NE-deficiency or inhibition did not prevent NETosis in vivo and DVT outcome. Conclusions NE is not required for NET formation in mice. NE−/− mice, which form pathological venous thrombi containing NETs, do not phenocopy PAD4−/− mice in in vitro NETosis assays or experimental venous thrombosis. Our study suggests that NET-targeted therapies need to be highly effective to have an impact on DVT.
BACKGROUND: Platelets store large amounts of serotonin that they release during thrombus formation or acute inflammation. This facilitates hemostasis and modulates the inflammatory response. METHODS:Infarct size, heart function, and inflammatory cell composition were analyzed in mouse models of myocardial reperfusion injury with genetic and pharmacological depletion of platelet serotonin. These studies were complemented by in vitro serotonin stimulation assays of platelets and leukocytes in mice and men, and by measuring plasma serotonin levels and leukocyte activation in patients with acute coronary syndrome. RESULTS:Platelet-derived serotonin induced neutrophil degranulation with release of myeloperoxidase and hydrogen peroxide (H 2 O 2 ) and increased expression of membrane-bound leukocyte adhesion molecule CD11b, leading to enhanced inflammation in the infarct area and reduced myocardial salvage. In patients hospitalized with acute coronary syndrome, plasmatic serotonin levels correlated with CD11b expression on neutrophils and myeloperoxidase plasma levels. Longterm serotonin reuptake inhibition-reported to protect patients with depression from cardiovascular events-resulted in the depletion of platelet serotonin stores in mice. These mice displayed a reduction in neutrophil degranulation and preserved cardiac function. In line, patients with depression using serotonin reuptake inhibition, presented with suppressed levels of CD11b surface expression on neutrophils and lower myeloperoxidase levels in blood. CONCLUSIONS:Taken together, we identify serotonin as a potent therapeutic target in neutrophil-dependent thromboinflammation during myocardial reperfusion injury.
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