NET-associated citrullinated histones promote LDL aggregation and foam cell formation in vitro

Abstract

Neutrophils have been recently identified in the atherosclerotic lesion and they can release neutrophil extracellular trap (NET) under the pro-inflammatory conditions prevailing in the lesion. Citrullinated histones (Cit-histones) are the major type of citrullinated proteins associated with NET release. Since elevated levels of citrullinated proteins have been detected in inflammatory diseases including atherosclerosis, this study analysed the role played by NET and Cit-histones in different atherogenic events in vitro.First, neutrophil recruitment and NET release in the presence of low-density lipoprotein (LDL) and oxidised LDL (Ox-LDL) were analysed by Boyden's chamber method and microscopy respectively. Then, LDL oxidation and LDL aggregation in the presence of NET and Cit-histones were analysed spectroscopically. Foam cell formation in the presence of NET or Cit-histone was studied by both microscopic and spectroscopic methods.While neutrophil recruitment was facilitated by Ox-LDL and not by LDL, the extent of NET release was significantly increased in the presence of both LDL and Ox-LDL. In the presence of NET, LDL oxidation, aggregation and foam cell formation were found to be increased. Cit-histones were found to accelerate LDL aggregation and foam cell formation at higher citrulline levels. Altogether, the results suggest that both NET and NET-associated Cit-histone released at the lesion can play major roles as pro-atherogenic mediators. Inhibiting the action of NET or Cit-histone would, therefore, be beneficial in slowing down atherosclerotic progression.