New insight into the composition of extracellular traps released by macrophages exposed to different types of inducers.

Neutrophil extracellular trap (NET) release plays a key role in many chronic disease settings, including atherosclerosis. They are critical to innate immune defence, but also contribute to disease by promoting thrombosis and inflammation. Macrophages are known to release extracellular traps or "METs", but their composition and role in pathological processes are less well defined. In this study, we examined MET release from human THP-1 macrophages exposed to model inflammatory and pathogenic stimuli, including tumour necrosis factor α (TNFα), hypochlorous acid (HOCl) and nigericin. In each case, there was release of DNA from the macrophages, as visualized by fluorescence microscopy with the cell impermeable DNA binding dye SYTOX green, consistent with MET formation. Proteomic analysis on METs released from macrophages exposed to TNFα and nigericin reveals that they are composed of linker and core histones, together with a range of cytosolic and mitochondrial proteins. These include proteins involved in DNA binding, stress responses, cytoskeletal organisation, metabolism, inflammation, anti-microbial activity, and calcium binding. Quinone oxidoreductase in particular, was highly abundant in all METs but has not been reported previously in NETs. Moreover, there was an absence of proteases in METs in contrast to NETs. Some of the MET histones, contained post-translational modifications, including acetylation and methylation of Lys but not citrullination of Arg. These data provide new insight into the potential implications of MET formation in vivo and their contributions to immune defence and pathology.

Mapping the modification of histones by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl).

Histones are critical for the packaging of nuclear DNA and chromatin assembly, which is facilitated by the high abundance of Lys and Arg residues within these proteins. These residues are also the site of a range of post-translational modifications, which influence the regulatory function of histones. Histones are also present in the extracellular environment, following release by various pathways, particularly neutrophil extracellular traps (NETs). NETs contain myeloperoxidase, which retains its enzymatic activity and produces hypochlorous acid (HOCl). This suggests that histones could be targets for HOCl under conditions where aberrant NET release is prevalent, such as chronic inflammation. In this study, we examine the reactivity of HOCl with a mixture of linker (H1) and core (H2A, H2B, H3 and H4) histones. HOCl modified the histones in a dose- and time-dependent manner, resulting in structural changes to the proteins and the formation of a range of post-translational modification products. N-Chloramines are major products following exposure of the histones to HOCl and decompose over 24 h forming Lys nitriles and carbonyls (aminoadipic semialdehydes). Chlorination and dichlorination of Tyr, but not Trp residues, is also observed. Met sulfoxide and Met sulfones are formed, though these oxidation products are also detected albeit at a lower extent, in the non-treated histones. Evidence for histone fragmentation and aggregation was also obtained. These results could have implications for the development of chronic inflammatory diseases, given the key role of Lys residues in regulating histone function.