Knotting the NETs: Analyzing histone modifications in neutrophil extracellular traps

than a decade since their discovery, neutrophil extracellular traps (NETs) remain in the headlines. Initially, interest focused on the structure of extracellular NET chromatin and its capacity to capture and damage bacteria. Soon, however, researchers began to see the impli cations of extracellular chromatin for the development of autoimmune diseases. One quintessential auto-immune disease, systemic lupus erythematosus (SLE), is known to arise together with autoantibodies to DNA and chromatin, although the immediate trigger for the pro-duc tion of these autoantibodies is unclear. A connection between NETs and autoimmunity was made by discovering that histones, a set of proteins that act as a structural harness for DNA in chromatin, are modifi ed by peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginines to citrullines. Researchers had long suspected that autoantigen modifi cations could provide the initial stimuli in autoimmunity because subtle alterations in a protein's primary sequence can break tolerance. PAD4 is implicated in the development of rheumatoid arthritis (RA) because the most reliable clinical test for RA uses the detection of anti-citrulline antibodies in the sera of patients. In a sophisticated set of experiments reported in the previous issue of Arthritis Research & Th erapy, Liu and colleagues [1] accomplished an extensive inventory of post-translational modifi cations in NET histones. Th e researchers induced NETs from human neutrophils, as well as two cell lines that assume neutrophil-like characteristics , and used a panel of 40 commercially available antisera to identify histone modifi cations that arise in parallel with NETs. Stimuli that were used to elicit NET release also induced histone H3 and H4 citrullination in human neutrophils and the EPRO cell line. However, other modifi cations such as histone H4 lysine 20 methy-la tion and H4 lysine 16 acetylation showed inconsistent results in neutrophils versus the EPRO cells. To survey histone modifi cations, Liu and colleagues [1] confronted technical diffi culties in that histone amino terminal tails contain the highest concentration of histone modifi ca-tions yet are also highly susceptible to proteases secreted by activated neutrophils [2,3]. Th e histone tails act as fl exible tethers that organize chromatin into higher-order structures. Interestingly, purifi ed NETs failed to induce an immune response in mice, although a subset of SLE sera reacted strongly with citrullinated histone H3 [1]. Th erefore, mechanisms that regulate histone modifi ca-tion deserve further attention. Neeli and colleagues [4] were the fi rst to identify citrullinated histone …