Histone H4 activates neutrophils alone or with influenza A virus, unless countered by C-reactive protein and surfactant protein-D

Extracellular histones have been implicated as a cause of tissue inflammatory injury in a variety of disorders including sepsis, lung and liver diseases. However, little is known about their interactions with neutrophils and how this might contribute to injury. We used human neutrophils isolated from healthy donors in this study, and we conducted functional and mechanistic studies of the effects of histone H4 on neutrophils in vitro. We found that histone H4 acts as a strong neutrophil activator, inducing cytokine release, hydrogen peroxide production, adhesion and degranulation. H4 had a distinctive profile as compared to other neutrophil agonists in that it caused a marked and sustained elevation of intracellular calcium, which resulted from permeabilization of the neutrophil plasma membrane. Prior studies showed that neutrophils predominate in the early response to influenza A virus (IAV) infection in the lung, that IAV induces formation of neutrophil extracellular traps (NETs), and IAV infection in vivo is worsened by release of free histones in the lung. Neutrophils predominate in the early response in the lung to IAV infection. We found that IAV-induced NETs contain histones. Of interest, we found that histone H4 binds to and aggregates IAV particles, inhibits IAV infectivity in vitro, and increases the uptake of IAV by neutrophils. On the other hand, histone H4 potentiated IAV-induced neutrophil responses, including hydrogen peroxide production and calcium flux. These findings may in part explain the injurious effect of histones during IAV infection in vivo. We found that C-reactive protein (CRP) and surfactant protein-D (SP-D) or its carbohydrate binding domain bound to histone H4. CRP markedly reduced all of the effects of histone H4 on IAV and/or neutrophils. We also found that SP-D or its isolated binding domain reduced neutrophil activation induced by histone H4 which may in part account for its ability to reduce lung inflammation. We hope these findings provide a better understanding of the pro-inflammatory effects of histones (in IAV or other types of injury) and how the body protects itself against histone-induced injury. We also hope our results will aid development of novel strategies to ameliorate tissue damage caused by histones