Histone H4 potentiates neutrophil inflammatory responses to influenza A virus: Down-modulation by H4 binding to C-reactive protein and Surfactant protein D.

Neutrophils participate in the early phase of the innate response to uncomplicated influenza A virus (IAV) infection but also are a major component in later stages of severe IAV or COVID 19 infection where neutrophil extracellular traps (NETs) and associated cell free histones are highly pro-inflammatory. It is likely that IAV interacts with histones during infection. We show that histone H4 binds to IAV and aggregates viral particles. In addition, histone H4 markedly potentiates IAV induced neutrophil respiratory burst responses. Prior studies have shown reactive oxidants to be detrimental during severe IAV infection. C reactive protein (CRP) and surfactant protein D (SP-D) rise during IAV infection. We now show that both of these innate immune proteins bind to histone H4 and significantly down regulate respiratory burst and other responses to histone H4. Isolated constructs composed only of the neck and carbohydrate recognition domain of SP-D also bind to histone H4 and partially limit neutrophil responses to it. These studies indicate that complexes formed of histones and IAV are a potent neutrophil activating stimulus. This finding could account for excess inflammation during IAV or other severe viral infections. The ability of CRP and SP-D to bind to histone H4 may be part of a protective response against excessive inflammation in vivo.

Histone H4 directly stimulates neutrophil activation through membrane permeabilization.

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. Here, it is shown that histone H4 acts as neutrophil activator by inducing hydrogen peroxide production, degranulation, cell adhesion, and IL-8 generation. Histone H4 caused permeabilization of the neutrophil membrane (a phenomenon described in other cell types) leading to accelerated cell death. H4 caused sustained rise in neutrophil intracellular calcium that is necessary for respiratory burst activation and degranulation. Convincing evidence was not found for TLRs or ATP receptors in H4 mediated activation. However, pertussis toxin and wortmannin (inhibitors of G protein and PI3K) inhibited H4-induced hydrogen peroxide production and degranulation. These studies suggest that release of histone H4 in sites of infection or inflammation may potentiate neutrophil activation and promote additional inflammatory responses. These studies may provide a better basis for developing novel therapeutic strategies to block neutrophil extracellular trap (NET) and H4-related pathology in sepsis and various forms of lung injury including that induced by viruses like influenza or SAR-CoV2.