Neutrophil Extracellular Traps in Asthma: Friends or Foes?

Abstract

Asthma is a chronic inflammatory disease characterized by variable airflow limitation and airway hyperresponsiveness. A plethora of immune and structural cells are involved in asthma pathogenesis. The roles of neutrophils and their mediators in different asthma phenotypes are largely unknown. Neutrophil extracellular traps (NETs) are net-like structures composed of DNA scaffolds, histones and granular proteins released by activated neutrophils. NETs were originally described as a process to entrap and kill a variety of microorganisms. NET formation can be achieved through a cell-death process, termed NETosis, or in association with the release of DNA from viable neutrophils. NETs can also promote the resolution of inflammation by degrading cytokines and chemokines. NETs have been implicated in the pathogenesis of various non-infectious conditions, including autoimmunity, cancer and even allergic disorders. Putative surrogate NET biomarkers (e.g., double-strand DNA (dsDNA), myeloperoxidase-DNA (MPO-DNA), and citrullinated histone H3 (CitH3)) have been found in different sites/fluids of patients with asthma. Targeting NETs has been proposed as a therapeutic strategy in several diseases. However, different NETs and NET components may have alternate, even opposite, consequences on inflammation. Here we review recent findings emphasizing the pathogenic and therapeutic potential of NETs in asthma.

Keywords: NETs; PMN; TSLP; VEGF; asthma; inflammation; neutrophil extracellular traps; neutrophils.

Figure 1

Schematic representation of biochemical and cellular events driving lytic NET formation. A multitude of extracellular pathogens, including viruses and bacteria, can induce lytic NET formation, also called NETosis [18,75]. Activation of neutrophils induces actin filament disassembly, an increase in intracellular Ca²⁺ and ROS production. Neutrophil elastase (NE) [68] and myeloperoxidase (MPO) [76] contribute to nuclear membrane permeabilization and unfolding of chromatin. Peptidyl arginine deiminase 4 (PAD4), mainly expressed in the nucleus of granulocytes, mediates citrullination of the nucleosome histones leading to chromatin decondensation [77]. These events favor nuclear rounding and nuclear envelope permeabilization. The following event is the chromatin swelling into the cytoplasm and the activation of gasdermin D [78]. A final step is the plasma membrane rupture and NET release [79].

Figure 2

The most potent inducer of NETosis in vitro is phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC) in human neutrophils. [15]. Several other stimuli such as agonists of FcγRIIIB [86], crystals [86,87], and the Ca²⁺ ionophore A23187 or ionomycin [88] can induce suicidal NETosis. NET formation induced by PMA requires NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activity, but the requirement for this enzymatic activity differs depending on the stimulus [89]. Neutrophil elastase (NE) translocates from the cytoplasmic granules to the nucleus and activates its proteolytic activity in a myeloperoxidase (MPO)-dependent manner [90]. In the nucleus, NE degrades specific histones promoting chromatin decondensation [68]. Peptidyl arginine deiminase 4 (PAD4), which converts arginine residues into citrulline [91], catalyzes histone citrullination [69]. This results in a loss of positive charges on arginine residues in histones, which loosen the forces between DNA and histones and contributes to chromatin decondensation [92]. After nuclear envelope disruption, the chromatin comes into contact with several cytoplasmic granules (e.g., NE, MPO, PR3 (proteinase 3), PTX3 (pentraxin 3)) [12]. The rupture of the plasma membrane and the consequent NET release are mediated by gasdermin D [78,93,94]. In vital NET formation, the cell remains intact and normal cellular functions of neutrophils, such as chemotaxis and phagocytosis, can still be carried out [18,72,73,80,95]. This process is biochemically distinct from suicidal NETosis. A variety of stimuli such as bacterial products [73,95], GM-CSF and C5a [72,96], immune complexes [81,96,97], lipopolysaccharide (LPS) [80,98,99], and conditioned media of cancer cells [18] can rapidly (within 30 min) induce vital NET formation. Vital NET formation occurs without nuclear membrane disruption and has been reported with DNA leaving the cytoplasm in vesicles [73,80,95]. DNA becomes decorated with granule proteins (e.g., NE, MPO, PR3) [72,96]. Mitochondria can mediate mitochondrial ROS (mROS) formation [18,97]. (DAMP (damage-associated molecular pattern); E. coli, Escherichia coli; S. aureus, Staphylococcus aureus).

Figure 3

Schematic representation of the pleiotropic and potentially conflicting roles of NETs in human and experimental models of asthma. NETs and their components can exert a pathogenic role in asthma. In particular, they can contribute to epithelial and endothelial damage mediated by ROS, MMP-9 or NE [174]. ROS and NE can also favor airway smooth muscle cell proliferation [175]. MMP-9 contributes to airway edema and remodeling [112] and, together with ROS, stimulates mucus production [176]. High-mobility group box 1 protein (HMBG1) induces the release of TSLP, TNF-α, MMP-9 and VEGF-A from bronchial epithelial cells [150]. Histones exert a cytotoxic effect [177], and MMP-9 and NE participate in airway remodeling of the extracellular matrix (ECM) [178]. In an experimental model, NETs promoted rhinovirus-induced type 2 asthma exacerbations [139]. Neutrophil cytoplasts induced T_H17 differentiation in a non-type 2 model of asthma [179]. NETs amplify neutrophil recruitment in a model of neutrophilic asthma [180]. NETs promoted neutrophil-associated asthma through the activation of macrophages [181]. In experimental models of asthma, NETs promoted the expression of Th2-like cytokines, airway eosinophil infiltration and airway hyperresponsiveness (AHR) [152]. NETs may not only have pathogenic effects but may also exert beneficial effects in human and experimental models of asthma [9]. NETs can capture and/or kill bacteria [15,182], viruses [161,167,183], and fungi [162,172,184]. NETs can also promote the resolution of inflammation by degrading cytokines and chemokines [168,169]. NETs inhibit GM-CSF/IL-4-induced dendritic cell (DC) differentiation [173]. In experimental models of asthma, MMP-9 reduced ROS accumulation and DNA damage [185].