NETs: Important players in asthma?

Asthma is a chronic airway disease affecting more than 4.5% of the global population; it contributes to more than half a million deaths annually. Thus far, there is no cure for asthma, although treatments are available to manage its symptoms and improve the quality of life of affected patients. Inhaled corticosteroids (CSs), together with long-acting bronchodilators, are the most effective long-term medications currently used for the treatment of asthma, yet not all patients respond to CSs owing to the fact that, at least partially, asthma is a highly heterogenous disease.1Wenzel S.E. Asthma phenotypes: the evolution from clinical to molecular approaches.Nat Med. 2012; 18: 716-725Crossref PubMed Scopus (1768) Google Scholar On the basis of inflammatory pattern, asthma can be divided into 2 subgroups: type 2 and non–type 2 asthma. In type 2 asthma, TH2 cells, type 2 innate lymphoid cells, eosinophils, mast cells, and basophils release TH2 cytokines, such as IL-4, IL-5, and IL-13 (Fig 1, A). These cytokines mediate increased production of IgE, recruitment of eosinophils to the site of inflammation, and mucus production in the airways (Fig 1, A). Eosinophils have been considered to play a major role in the pathogenesis of type 2 asthma. Recent studies have demonstrated that neutralizing anti–IL-5 or eosinophil death–mediating anti–IL-5 receptor-α chain antibodies were effective in reducing eosinophil numbers and treating asthma in humans. Notably, patients receiving such specific antieosinophil therapies may develop neutrophilic asthma.1Wenzel S.E. Asthma phenotypes: the evolution from clinical to molecular approaches.Nat Med. 2012; 18: 716-725Crossref PubMed Scopus (1768) Google Scholar Neutrophilic airway inflammation is widely recognized as a hallmark of non–type 2 asthma. Several cell types, including TH1 and TH17 cells, as well as cytokines such as IL-6 and IL-17, play a role in the mechanisms underlying non–type 2 asthma. Patients with severe asthma often exhibit neutrophil-predominant inflammation associated with high levels of IL-17, IL-8, and TNF-α in the development of CS-resistant asthma2Poto R. Shamji M. Marone G. Durham S.R. Scadding G.W. Varricchi G. Neutrophil extracellular traps in asthma: friends or foes?.Cells. 2022; 11: 3521Crossref PubMed Scopus (5) Google Scholar (Fig 1, B). Recently, neutrophil extracellular traps (NETs), formed by activated neutrophils,3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. et al.Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6855) Google Scholar have been observed in the airways and mediastinal lymph nodes of asthmatic patients during both type 2 and non–type 2 immune responses.4Dworski R. Simon H.U. Hoskins A. Yousefi S. Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways.J Allergy Clin Immunol. 2011; 127: 1260-1266Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar NETs are extracellular structures composed of double stranded DNA (dsDNA) scaffolds colocalized with released granule proteins.3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. et al.Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6855) Google Scholar This process, characterized by the active release of mitochondrial DNA (mtDNA) by viable neutrophils, does not lead to cell death and is regarded as both a beneficial mechanism of innate immunity against infections and a potential contributor to cell and tissue injury.4Dworski R. Simon H.U. Hoskins A. Yousefi S. Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways.J Allergy Clin Immunol. 2011; 127: 1260-1266Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar Large quantities of extracellular DNA in sputum has been linked with a subset of patients with severe asthma, providing further evidence for the possible presence of NETs in the airways under these conditions.2Poto R. Shamji M. Marone G. Durham S.R. Scadding G.W. Varricchi G. Neutrophil extracellular traps in asthma: friends or foes?.Cells. 2022; 11: 3521Crossref PubMed Scopus (5) Google Scholar NETs have been shown to mediate a direct impact on bronchial epithelial cells, leading to the release of alarmins, such as thymic stromal lymphopoietin (TSLP), IL-33, and IL-25, thereby compromising the integrity of the bronchial epithelium. In addition, the protease components of NETs have the ability to activate IL-1 and IL-36, which are primarily produced by keratinocytes, further exacerbating the inflammatory response in neutrophilic asthma. NETs have also been shown to play a vital role in the early immune response to recruit and activate CD4+ and CD8+ T cells, increase levels of TNF-α and IL-6, and induce airway hyperresponsiveness2Poto R. Shamji M. Marone G. Durham S.R. Scadding G.W. Varricchi G. Neutrophil extracellular traps in asthma: friends or foes?.Cells. 2022; 11: 3521Crossref PubMed Scopus (5) Google Scholar (Fig 1, B). Furthermore, NETs have been discovered to enhance the presentation of antigens by dendritic cells (DCs), thereby enhancing the TH2 inflammatory response in a house dust mite-induced mouse model of allergic asthma5Radermecker C. Sabatel C. Vanwinge C. Ruscitti C. Marechal P. Perin F. et al.Locally instructed CXCR4(hi) neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps.Nat Immunol. 2019; 20: 1444-1455Crossref PubMed Scopus (93) Google Scholar (Fig 1, B). Collectively, these findings indicate that the influx of neutrophils and formation of NETs have a pivotal role in the onset and progression of asthma. In a recent study, Tsai et al6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar put forth findings indicating that the presence of NETs has a detrimental impact on the response to CSs in neutrophilic asthma. Tsai et al6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar integrated human clinical and transcriptomic data with data obtained from a mouse model to explore the underlying mechanisms of the relationship between NETs and inadequate response to inhaled CSs in asthma. Notably, they demonstrated a correlation between expression of the CCL4L2 gene and NET formation in vitro, linked with inhaled nonresponse to CSs in asthma. On the other hand, Tsai et al6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar claim a direct link between "NETosis" and neutrophilic asthma symptoms. However, the data presented do not substantiate this link. For instance, the statistical analysis for the receiver operating characteristic curve of NET release for prediction of response to inhaled CSs shows statistically low values, rendering the prediction unlikely. In addition, why Tsai et al6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar stimulated patients' blood neutrophils with phorbol 12-myristate 13-acetate (PMA), a nonphysiologic stimulus, is unclear. It would be advisable to use lipopolysaccharides (LPS) to mimic their mouse model of neutrophilic asthma. LPS, a major component of the outer membrane in gram-negative bacteria, is a known trigger for NET formation, and it is prevalent in in vivo conditions during bacterial infections. Originally, Brinkmann et al3Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. et al.Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6855) Google Scholar reported that the activation of neutrophils by bacteria or LPS results in NETs in the absence of cell death. In the article by Tsai et al,6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar LPS-induced NETosis (a form of neutrophil death required for NET formation) is not really shown. In fact, the data presented by Tsai et al6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar do not provide evidence for neutrophil death at all, and the colocalization of extracellular DNA with neutrophil granule proteins, which are a hallmark of NETs, was also not demonstrated. It should be noted that whether NETosis is indeed required for the formation of NETs, as suggested earlier, remains unclear.7Yousefi S. Stojkov D. Germic N. Simon D. Wang X. Benarafa C. et al.Untangling "NETosis" from NETs.Eur J Immunol. 2019; 49: 221-227Crossref PubMed Scopus (103) Google Scholar Moreover, Tsai et al6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar suggest that the activation of neutrophils by using ovalbumin and LPS in mice lacking neutrophil elastase (NE–/–) resulted in impaired NETosis compared with that in wild-type mice. dsDNA concentrations and expression of inflammatory cytokines such as IL-1β, IL-6, and IL-17 were shown to be decreased in NE–/– mice, suggesting proof of NETs in neutrophilic airway inflammation. However, it has been shown previously that elastase-deficient mice can still form NETs in an experimental model of deep vein thrombosis.8Martinod K. Witsch T. Farley K. Gallant M. Remold-O'Donnell E. Wagner D.D. Neutrophil elastase-deficient mice form neutrophil extracellular traps in an experimental model of deep vein thrombosis.J Thromb Haemost. 2016; 14: 551-558Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar The reason(s) for these discrepancies remain unclear and have not been discussed by Tsai et al.6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar There are also certain methodologic aspects within the article by Tsai et al6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar that raise doubts regarding its main conclusions. For instance, as detailed in the article's Methods section, the "NETosis index" was calculated by preculturing human neutrophils with recombinant CCL4L2 for 30 minutes and an additional 3 hours of activation with PMA. Surprisingly however, the result shows no visible NETosis in PMA-treated neutrophils in the absence of recombinant CCL4L2.6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar Furthermore, the NETosis index was determined by measuring the intensity of SYTOX fluorescent dye. Notably, the SYTOX fluorescent dye detects various forms of cell death characterized by membrane perforation, but it is not a specific method for quantifying NETs.7Yousefi S. Stojkov D. Germic N. Simon D. Wang X. Benarafa C. et al.Untangling "NETosis" from NETs.Eur J Immunol. 2019; 49: 221-227Crossref PubMed Scopus (103) Google Scholar In addition, Sytox Green staining was shown to be mostly intracellular, whereas NETs are extracellular structures.6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar Lastly, the genetic modification of human blood neutrophils poses technical challenges, and consequently, modulating gene expression becomes an exceedingly formidable task in these fragile cells. Human peripheral blood neutrophils undergo spontaneous apoptosis within a few hours after isolation.9Simon H.U. Neutrophil apoptosis pathways and their modifications in inflammation.Immunol Rev. 2003; 193: 101-110Crossref PubMed Scopus (305) Google Scholar Therefore, the approach used by Tsai et al6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar to effectively reduce the expression of CCL4L2 protein by using small interfering RNA for 24 hours, followed by a 3-hour incubation with PMA to induce NETosis, appears to be challenging and needs to be supported with additional data demonstrating the viability of neutrophils after 24 hours of small interfering RNA treatment before and after additional 3 hours of incubation of the cells with PMA.6Tsai C.H. Lai A.C. Lin Y.C. Chi P.Y. Chen Y.C. Yang Y.H. et al.Neutrophil extracellular trap production and CCL4L2 expression influence corticosteroid response in asthma.Sci Transl Med. 2023; 15eadf3843Crossref Scopus (1) Google Scholar Additionally, activated neutrophils generate and release CCL410Lapinet J.A. Scapini P. Calzetti F. Perez O. Cassatella M.A. Gene expression and production of tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-8, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and gamma interferon-inducible protein 10 by human neutrophils stimulated with group B meningococcal outer membrane vesicles.Infect Immun. 2000; 68: 6917-6923Crossref PubMed Scopus (99) Google Scholar; thus, whether CCL4 arises predominantly as a consequence of NET formation or whether CCL4 controls NET formation is uncertain. Thus, the data indicate that NETs have been observed in asthma, particularly in neutrophilic asthma. However, whether NETosis indeed occurs in asthma remains uncertain. It is important to mention that NETs have previously been described under in vivo conditions in the absence of cell death in asthma.4Dworski R. Simon H.U. Hoskins A. Yousefi S. Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways.J Allergy Clin Immunol. 2011; 127: 1260-1266Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar Therefore, it is advisable to conduct in vitro experiments involving time-dependent and microscopic analysis of live single cells to gain a deeper understanding of the origin of extracellular dsDNA and the presence or absence of cell death. Moreover, the link between NETs and unresponsiveness to CSs in neutrophilic asthma is still not clear. Hence, obtaining biopsy specimens from both individuals who are responsive to CSs and individuals who are unresponsive to CSs and analyzing the specimens for NET formation would be advantageous to achieve a better understanding of the suggested association between NET formation and responsiveness to CSs. Lastly, the potential inhibitory function of CCL4L2 to suppress NET formation and neutrophilic airway inflammation should be investigated further in an experimental model of neutrophilic asthma using Ccl4 or Ccr5 knockout mice. Supported by the Swiss National Science Foundation (grant 310030_166473 [to H.U.S.]). Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.