Abstract
Neutrophils release extracellular traps (NETs) during inflammation as an important immune defence mechanism to clear infection. However, NETs are also implicated in the pathology of many diseases, particularly atherosclerosis and sepsis. NETs are composed of a DNA network containing histones and antimicrobial proteins like myeloperoxidase (MPO). MPO remains enzymatically active, generating hypochlorous acid (HOCl), a highly reactive oxidant that can modify proteins, including histones in NETs. Whether these oxidative histone modifications are generated in vivo during infection and affect the ability of histones to kill bacteria is currently unknown. To assess the extent of NET release induced by bacteria, primary neutrophils from healthy donors were stimulated with different types of bacteria. S. aureus and E. coli induced substantial NET release as indicated by Sytox Green staining of the DNA. We then examined the ability of histones to kill these bacteria and whether this is impacted by HOCl modification. It was shown that 2.5 μg/mL of an unfractionated histone preparation completely inhibits growth of S. aureus, while 325 μg/mL was necessary to inhibit E. coli growth. Modification of histones with HOCl (7.5-fold excess, 24 h) significantly decreased this toxicity to both types of bacteria. A quantitative multiple reaction monitoring (MRM) mass spectrometry method was established to detect specific histone H4 peptides containing methionine sulfoxide and/or 3-chlorotyrosine residues, HOCl-induced modifications observed on NETs in vitro. The quantification was linear over a large range (0.0625 ng/mL to 100 ng/mL) with a lower limit of detection of 0.125 ng/mL. Using this MRM method, we were able to detect oxidative histone modifications in samples from patients with infectious syndromes. This study is the first to show that histones are modified by HOCl in vivo during infection, which influences their ability to kill bacteria. This provides new insight into pathways that impact on inflammation in vivo.