Post-Translational Modifications in NETosis and NETs-Mediated Diseases

Abstract

: Neutrophils undergo a unique form of cell death that generates neutrophil extracellular traps (NETs) that may help to neutralize invading pathogens and restore homeostasis. However, uncontrolled NET formation (NETosis) can result in numerous diseases that adversely affect health. Recent studies further elucidate the mechanistic details of the different forms of NETosis and their common end structure, as NETs were constantly found to contain DNA, modified histones and cytotoxic enzymes. In fact, emerging evidence reveal that the post translational modifications (PTMs) of histones in neutrophils have a critical role in regulating neutrophil death. Histone citrullination is shown to promote a rapid form of NET formation independent of NADPH oxidase (NOX), which relies on calcium influx. Interestingly, few studies suggest an association between histone citrullination and other types of PTMs to control cell survival and death, such as histone methylation. Even more exciting is the finding that histone acetylation has a biphasic effect upon NETosis, where histone deacetylase (HDAC) inhibitors promote baseline, NOX-dependent and -independent NETosis. However, increasing levels of histone acetylation suppresses NETosis, and to switch neutrophil death to apoptosis. Interestingly, in the presence of NETosis-promoting stimuli, high levels of HDACis limit both NETosis and apoptosis, and promote neutrophil survival. Recent studies also reveal the importance of the PTMs of neutrophils in influencing numerous pathologies. Histone modifications in NETs can act as a double-edged sword, as they are capable of altering multiple types of neutrophil death, and influencing numerous NET-mediated diseases, such as acute lung injury (ALI), thrombosis, sepsis, systemic lupus erythematosus, and cancer progression. A clear understanding of the role of different PTMs in neutrophils would be important for an understanding of the molecular mechanisms of NETosis, and to appropriately treat NETs-mediated diseases.

Keywords: apoptosis; epigenetics; histone acetylation; histone citrullination; histone deacetylase inhibitors; histone methylation; neutrophil extracellular trap formation; neutrophils; post translational modification.

Figure 1

Citrullination (deimination) and acetylation in regulating neutrophil extracellular trap (NET) formation. Neutrophils can be activated during infection and inflammation to undergo NETosis. Calcium ionophores, such as A23187 and ionomycin, activate the NADPH oxidase (NOX)-independent, whereas phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) induce NOX-dependent pathways of NETosis. Both pathways require increased levels of the reactive oxygen species (ROS) from NOX and/or mitochondrial origin, which results in kinase activation. After several intermediate steps, histones start to decondense and ultimately become NETs. During NOX-independent NETosis, increased intracellular Ca²⁺ influx activates peptidyl arginine deiminase, type IV (PADI4), which enables it to translocate into the nuclei. In the resting neutrophil nuclei, the negatively-charged DNA is tightly wrapped around the highly positively-charged histones (e.g., amino acids arginine and lysine) to form condensed chromatin. However, the active form of PADI4 (PADI4: Ca²⁺ complex) deiminate the positively-charged arginine into citrulline, which results in chromatin relaxation at promoters and transcription initiation. Similarly, histone deacetylase (HDAC) inhibitors (e.g., Belinostat and Panobinostat) are able to alter histone acetylation, where adding an acetyl group (H₃C–C=O) to the tip of the side chain (–NH₃) of the N-terminal lysines of histones (e.g., H4 histone with K5 acetylation; H4K5ac, or acetylated histone H4 (AcH4)), results in eliminating the positive charge (middle inset), weakening the overall chromatin structure, and enabling the entry of various proteins to access DNA (e.g., transcription machinery). By suppressing HDACs (18 in neutrophils), histone deacetylase inhibitors (HDACis) ultimately promote histone acetyl transferases (HAT) activity. This modification promotes baseline as well as NOX-dependent and -independent NETosis, without altering ROS production, as HDACis modify histones at a downstream level. However, increasing concentrations of HDACis induces NOX ROS production and activates the cleavage of caspase-3. As a result, neutrophils undergo apoptosis at a baseline condition. When the NETosis is induced with agonists, in the presence of high levels of AcH4, most of the neutrophils neither undergo NETosis nor apoptosis. In summary, histone citrullination and acetylation can alter NETosis and apoptosis, and the overall outcome depends upon the degree of the post-translational modification of histones, and the presence or absence of neutrophil death-inducing stimuli.