The NET Effect: Platelet Factor 4 and DNA-Histone Interactions in Sepsis

In response to infection and inflammation, neutrophils release neutrophil extracellular traps (NETs), web like structures composed of nuclear DNA associated with histones that may have both beneficial and deleterious effects.  The formation of NETs alters the course of late-stage sepsis and the associated release of histones has been shown to contribute to many of the observed pathologic complications of sepsis.  Histones are octamers comprised of two copies of H2A, H2B, H3 and H4, each of which is highly positively charged. NET formation is dependent on chromatin decondensation mediated by the enzyme peptidylarginine deiminase 4 (PAD4). PAD4 potentiates chromatin decondensation by decreasing the overall positive charge of histones through citrullinating many of their lysines residues, forming Cit-histones, which have a decreased affinity for negatively charged DNA. Platelets also contribute to events in late sepsis by undergoing significant activation and degranulation. We propose that one way platelets may affect outcomes in late sepsis is through the release of large amounts of the highly positively charged chemokine platelet factor 4 (PF4, CXCL4).  After its release, we believe that PF4 can displace histones and cit-histones from cell free DNA, altering the composition of NETs. We chose to investigate whether PF4 might liberate cit-histones from NET fibers more effectively than non-citrullinated histones. We initially sought to examine the effect of PF4 on histone attachment to DNA. In a competitive binding assay, we found that PF4 binds to DNA with greater affinity than histones. Of note, cit-histones were approximately 5 times more easily displaced from DNA than non-cit-histones consistent with a model of decreased DNA affinity of cit-histones. Furthermore, using immunofluorescence studies and confocal microscopy, we showed that when NETs are generated in the presence of platelets, endogenous PF4 adheres readily to NET DNA.  We have also demonstrated that exogenous PF4 avidly binds to NETs generated from neutrophils isolated with minimal platelet contamination. Based on the results of these experiments, we decided to investigate the interaction between PF4, NETs and histones in a novel microfluidic system that is designed to mimic intravascular flow conditions. We isolated neutrophils from fresh whole blood samples obtained from healthy human donors and stimulated them with TNFα to promote adherence to fibronectin coated microfluidic channels. After the neutrophils had firmly bound to the channels, we exposed them to NET stimuli, including lipopolysaccharide (LPS) and calcium ionophore and visualized NET formation. Extracellular DNA was detected using the cell membrane impermeable dye, SYTOX Green. After NET formation occurred, PF4 was flowed through the channels at 25-100 μg/mL, concentrations similar to those observed in terminal sepsis.  Exposure to PF4 at these concentrations lead to the dissolution of NET fibers.  Interestingly, although the residual NET fibers continued to stain positive for non-cit-histones, they no longer stained positive for cit-histones.  In conclusion, cit-histones are present in NETs and may contribute to the pathobiology of late sepsis. We propose that cit-histones are competitively displaced from NETs by PF4. This may be due to their decreased relative affinity for DNA binding. These studies provide new insights into how histones are released from NET fibers into the circulation during sepsis. This information sheds new light on the interaction of chemokines and NETs and may lead to the identification of new therapeutic strategies in the treatment of sepsis.