Abstract
Neutrophils respond to various stimuli by decondensing and releasing nuclear chromatin characterized by citrullinated histones as neutrophil extracellular traps (NETs). This achieves pathogen immobilization or initiation of thrombosis, yet the molecular mechanisms of NET formation remain elusive. Peptidyl arginine deiminase-4 (PAD4) achieves protein citrullination and has been intricately linked to NET formation. Here we show that citrullination represents a major regulator of proteolysis in the course of NET formation. Elevated cytosolic calcium levels trigger both peptidylarginine deiminase-4 (PAD4) and calpain activity in neutrophils resulting in nuclear decondensation typical of NETs. Interestingly, PAD4 relies on proteolysis by calpain to achieve efficient nuclear lamina breakdown and chromatin decondensation. Pharmacological or genetic inhibition of PAD4 and calpain strongly inhibit chromatin decondensation of human and murine neutrophils in response to calcium ionophores as well as the proteolysis of nuclear proteins like lamin B1 and high mobility group box protein 1 (HMGB1). Taken together, the concerted action of PAD4 and calpain induces nuclear decondensation in the course of calcium-mediated NET formation.
Highlights
In recent years, a functional role of neutrophil extracellular traps (NETs) has been implicated in the setting of multiple diseases as diverse as autoimmunity, cancer, thrombosis, crystalopathies, and the response to large pathogens [1, 2]
NET formation has been described in response to calcium ionophores such as ionomycin [9, 12, 22, 32]
Calcium ionophore-induced NET formation is largely independent of NADPH oxidase, whereas mitochondrial ROS may play an important role [39, 40]
Summary
A functional role of neutrophil extracellular traps (NETs) has been implicated in the setting of multiple diseases as diverse as autoimmunity, cancer, thrombosis, crystalopathies, and the response to large pathogens [1, 2]. NETs serve an important role in the containment and remodeling of inflammatory foci. Chromatin decondensation is a crucial event in the formation of NETs [3]. Nuclear chromatin condensation and caspasedependent DNase activity result in characteristic DNA fragmentation at the internucleosomal level [4]. NETs capable of pathogen immobilization and thrombus stabilization depend on large undegraded tangles of chromatin, which functionally depend on the framework of extracellular DNA [5, 6]. The mechanism of chromatin decondensation in the setting of NET formation is incompletely understood. Proteolytic cleavage events by neutrophil serine proteases such as neutrophil elastase have been implicated in neutrophils treated with phorbol
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