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Am J Respir Crit Care Med
Am J Respir Crit Care Med
ajrccm
American Journal of Respiratory and Critical Care Medicine
1073-449X
1535-4970
American Thoracic Society

38598776
202403-0599ED
10.1164/rccm.202403-0599ED
Editorials
NO Casting of NETs in Allergic Asthma
https://orcid.org/0000-0003-4419-7258
Mincham Kyle T.
Sanghavi Krish
Snelgrove Robert J.
National Heart and Lung Institute
Imperial College London
London, United Kingdom
10 4 2024
1 9 2024
10 4 2024
210 5 533535
Copyright © 2024 by the American Thoracic Society
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0. For commercial usage and reprints, please e-mail Diane Gern (dgern@thoracic.org).
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pmcAllergic asthma has prototypically been considered a T-helper cell type 2 (Th2)-driven disease characterized by elevated airway eosinophilia, augmented systemic IgE, mucus hypersecretion, and airway hyperreactivity, whereas neutrophils conversely have been associated with a nonallergic, Th2-low phenotype of asthma. However, neutrophils are readily recruited into the airways of patients with allergic asthma and mice upon exposure to allergen, with numbers further elevated in virus-driven exacerbations and nocturnal crisis (1). Moreover, allergic asthma–associated neutrophils have been purported to exhibit marked phenotypic and functional differences (2–4). However, the precise role of neutrophils in the context of allergic asthma remains incompletely understood, although it is anticipated they mediate generally deleterious effects in part by potentiating Th2 inflammation and ensuing disease pathology (1). Neutrophil extracellular traps (NETs) are weblike scaffolds released from neutrophils that contain host extracellular double-stranded DNA in conjunction with histones and neutrophil proteases, which can operate as important antimicrobial agents but also harness the capacity to impart substantial tissue pathology. Of note, in the context of allergic asthma and associated viral exacerbations, elevated NET release from neutrophils has been demonstrated to promote antigen presentation by dendritic cell populations, which has in turn resulted in augmented ensuing Th2-driven pathology (3, 5). Accordingly, abrogation of NETs reduced airway eosinophilia, serum IgE, mucus hypersecretion, and airway hyperreactivity.

In this issue of the Journal, Chacón and colleagues (pp. 593–606) present an elegant study identifying a novel mechanism of IgE-dependent, inducible nitric oxide synthase (iNOS)-independent nitric oxide (NO) production by neutrophils, which regulates NET formation by neutrophils derived from patients with intermittent bronchial allergic asthma (6). It is well recognized that NO is elevated in patients with allergic asthma and associated with adverse clinical outcomes (7, 8); however, the routine failure of iNOS-targeting therapies in alleviating clinical disease and airway inflammation (9, 10) infers alternate mechanisms of NO production. Within the study presented here, peripheral blood and sputum neutrophils from patients with allergic asthma demonstrated a robust capacity to produce NO after allergen/IgE stimulation, attributed to cross-linking via galectin-3. IgE-dependent NO production was independent of iNOS and instead was driven by a reaction between reactive oxygen species (ROS; generated by NADPH oxidase [NOX-2]) and L-arginine. Both ROS and NO generation required signaling via phosphoinositide 3-kinase and mitogen-activated protein kinases. The authors went on to highlight the physiological importance of this pathway, whereby IgE/NOX-2/ROS-dependent NO accumulation within neutrophils potentiates H3 citrullination and NET formation, a process governed by NO-dependent myeloperoxidase (MPO) activation and mediated in part by a positive feedback loop promoting ROS generation. However, given the interdependency of ROS, NO, and MPO in this complex pathway, untangling the relative contribution of each component during NET formation remains difficult and would benefit from further scrutiny. One question that remains, however, is to what extent IgE-dependent NO production contributes to the abundance of NETs in the airways of patients with allergic asthma and the relative importance of NETs generated by this pathway in governing ensuing pathology. Addressing such complex pathways in a clinical setting is challenging, and although mouse models present their own limitations, their use in dissecting galectin-3/NOX-2/ROS/NO/NET signaling would be of great benefit.

The findings of this study further reinforce the clear interdependency of neutrophils and Th2 responses in the context of allergic airway inflammation. Previous studies have demonstrated the capacity of allergen-induced cross-linking of IgE receptors on the surface of neutrophils derived from patients with asthma to augment the release of an array of products encompassing ROS, IL-8, matrix metalloproteinase 9, neutrophil elastase, and MPO (1), a list that can now be expanded to include NETs. Thus, the function of neutrophils readily recruited to the airways of patients with allergic asthma will be potentiated by the prototypical type 2 antibody IgE, and, through ensuing release of NETs, the neutrophils themselves may then function to reciprocally promote Th2 inflammation, reinforcing Th2-driven pathology. However, it is noteworthy that IL-4 and IL-13 signaling through neutrophil-expressed IL-4R has been demonstrated to suppress neutrophil recruitment and effector functions (11), and thus type 2 inflammation can also mediate inhibitory effects on neutrophils. Furthermore, although neutrophils have often been purported to promote Th2 inflammation and pathology in the context of allergic asthma, other studies have demonstrated important regulatory roles for neutrophils whereby they may operate to restrain type 2 responses (12–14), thus highlighting the complexity and likely context dependence of this relationship.

This complexity may in part align with our rapidly evolving view of neutrophils, which are now recognized to exhibit significant heterogeneity, a pronounced awareness of proximal environmental cues and pleiotropic functionality (15). It has previously been demonstrated in mice that common risk factors for allergic asthma, such as low environmental endotoxin, pollution, and respiratory viral infection, operate to elicit a pulmonary neutrophilic population defined by elevated expression of CXCR4, Lamp-1, and CD49d and with a heightened proclivity to release NETs and drive Th2-mediated pathology (3). These findings are reinforced by seminal studies demonstrating elevated CXCR4 expression on blood neutrophils derived from allergy-susceptible Hutterite children raised in low-endotoxin environments relative to Amish children who exhibit a reduced susceptibility to allergy (2). Similarly, nasal lavage and blood-derived neutrophils from atopic patients exhibited greater CD49d expression, which was further enhanced upon allergen challenge (4). These studies support the notion that neutrophils are heterogeneous and plastic cells that are adaptable to context-specific cues to modulate their phenotype and function. In the context of the present study, it would be of interest to ascertain whether distinct neutrophil populations, such as those defined by enhanced CXCR4 and a proclivity to release NETs, display greater responsiveness to allergen/IgE, and indeed whether responsiveness is modulated by common risk factors associated with allergic asthma or with increasing severity of disease.

The present study has clear implications for therapeutic interventions in the context of allergic asthma (Figure 1). Although NETs represent an attractive therapeutic target in this setting, the present study identifies the opportunity for upstream intervention through application of antioxidants or scavengers of NO. One would envision that such approaches may ameliorate not only pathology directly attributable to NETs but also potentially the magnitude of the ensuing Th2 response and adverse sequelae. The present findings also have potential consequences for biologics currently used for the treatment of severe T2-high asthma. Treatments such as Omalizumab that target the IgE pathway may inadvertently also modulate neutrophil functionality, resulting in reduced NET-mediated pathology and associated induction of Th2 inflammation. Other biologics such as Dupilumab and Tezepelumab may also operate to ameliorate IgE-driven neutrophil NETosis, but, conversely, given the reported potential of IL-4/IL-13 to curtail neutrophil responses, such treatments could operate to potentiate facets of neutrophilic inflammation. Overall, these exciting studies identify the neutrophil–ROS (NOX2-dependent)–NO (NOS-independent)–NET axis as a druggable target in allergic asthma. Moreover, they highlight the clear interplay between neutrophils and type 2 inflammation and the necessity to consider the impact of type 2 biologics on neutrophil phenotype and function.

Figure 1. Opportunities for and implications of therapeutic intervention in the context of the proposed IgE–ROS–NO–NET axis. A simplified schematic of the IgE-dependent–ROS (NOX2-dependent)–NO (NOS-independent)–NET axis proposed by Chacón and colleagues. Cross-linking of the IgE receptor galectin-3 on human neutrophils with allergen triggers a signaling cascade whereby PI3K and MAPKs drive activation of NOX-2 and ROS formation, leading to NO production, subsequent activation of MPO and PAD-4, and culminating in H3 citrullination and NET formation. It is noteworthy that previous studies have described the capacity of NETs to promote Th2-mediated pathology in the context of allergic asthma via the potentiation of antigen presentation by dendritic cells. Thus, a prototypical type 2 IgE can feasibly exacerbate Th2 inflammation indirectly through induction of NET release from neutrophils. Current strategies that seek to inhibit NET formation or augment NET degradation (green) hold therapeutic potential in the context of allergic asthma. The present study highlights upstream opportunities for therapeutic intervention by reducing bioavailability of ROS or NO (blue) to reduce NET formation. It is feasible that biologics currently used in patients with asthma that target the IgE axis may impede this IgE-mediated NET release and ameliorate ensuing pathology (red). DC = dendritic cell; Eos = eosinophils; H3 cit = H3 citrullination; IgE = immunoglobulin E; MAPK = mitogen-associated protein kinase; MPO = myeloperoxidase; MPOi = myeloperoxidase inhibitor; NETs = neutrophil extracellular traps; NO = nitric oxide; NOX-2 = NADPH oxidase; NOX-2i = NADPH oxidase inhibitor; PAD-4 = peptidyl arginine deaminase-4; PAD-4i = peptidyl arginine deaminase-4 inhibitors; PI3K = phosphorinositide 3-kinase; ROS = reactive oxygen species; TH2 = T-helper type 2 cell.

R.J.S. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences (209458/Z/17/Z). Supported by Imperial College London through an Imperial College Research Fellowship grant awarded to K.T.M.

Originally Published in Press as DOI: 10.1164/rccm.202403-0599ED on April 10, 2024

Author disclosures are available with the text of this article at www.atsjournals.org.
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