Eosinophil extracellular traps in asthma: implications for pathogenesis and therapy

Asthma is a chronic inflammatory airway disease in which neutrophils and eosinophils play an important role [1, 2]. Activated neutrophils can release granules through degranulation, internalize and degrade pathogens through phagocytosis, and release neutrophil extracellular traps (NETs) to defend against external pathogens [3]. Studies have shown that excessive levels of NETs can damage the airway epithelium and trigger an inflammatory response, increasing the severity of asthma [4].

Eosinophils are known for their role in asthma as end-effector cells in allergic diseases [5]. Upon receiving stimulatory signals, they undergo tissue migration and perform immunomodulatory and pro-inflammatory functions by releasing immunomodulatory factors (cytokines, chemokines, growth factors), and cytotoxic proteins that are pre-formed and stored intracellularly [1, 6].

In addition, recent studies have observed that eosinophils can form extracellular traps similarly to neutrophils. By releasing nuclear DNA into an extracellular backbone and embedding granule proteins in this backbone, a new cell death pathway distinct from necrosis and apoptosis is formed, called eosinophil extracellular trap cell death (EETosis; Fig. 1) [7]. A variety of microorganisms and their products, as well as non-infectious stimuli, can activate this pathway, which is regulated by multiple activation mechanisms, including toll-like receptors, chemokines, cytokines, and adhesion receptors. These mechanisms can initiate transmembrane signaling leading to the formation of eosinophil extracellular traps (EETs) [6, 8].

Like NETs, EETs are important for host defense against extracellular pathogens by providing scaffolding structures that offer attachment points for agents such as cytotoxic proteins, limiting the immune response to an effective and safe area [1]. However, unlike NETs, the chromatin structure on EETs is more stable and concentrated, and EETs are less susceptible to proteolytic degradation, allowing them to remain in the body for a longer period of time [9]. In chronic and uncontrolled asthma, overproduction of EETs can interact with other immune cells and exacerbate the inflammatory response [6, 9]. Studies have shown that EETs are elevated in asthmatic patients and correlate with the severity of the disease [10]. In ovalbumin (OVA)-challenged mice, increased EETs secretion has been associated with greater cellular infiltration, airway inflammation, and mucus secretion [11]. Additionally, it has been shown that EETs may be associated with a failure to respond to steroid hormone therapy for asthma [12].

Although the exact mechanisms by which EETs contribute to chronic inflammatory airway disease remain unknown, the importance of studying EETs should not be underestimated. A deeper understanding of EETs may provide new insights for the development of biomarkers to aid in the diagnosis of asthma type and severity, as well as potential new targets for treatment. This review provides a summary of the general characteristics of EETs, their relevance to asthma, and potential mechanisms of action and therapeutic targets in the development of asthma.

Degranulation is a critical function of eosinophils associated with many allergic and inflammatory diseases [13]. It refers to the process by which living or dead cells release intact or ruptured specific granules [6]. These granules, also known as secondary or secretory granules, are membrane-surrounded structures that contain a dense crystalline core and a matrix containing various mediators that can induce inflammation and/or tissue damage. These mediators include basic proteins, cytokines, chemokines, growth factors, and enzymes, with proteins being the most important [14, 15].

Eosinophils contain both enzymatic and non-enzymatic cationic proteins in specific granules that are selectively secreted in response to stimulation [16]. These eosinophil-derived granule proteins (EDGPs) include eosinophil peroxidase (EPO), major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) [17]. These proteins have potent cytotoxic effects and may act by disrupting lipid bilayer integrity [18], exhibiting neurotoxicity and RNase activity, and/or participating in the generation of reactive oxidants and free radicals [19]. In addition to their cytotoxicity, EDGPs also play a critical role in eosinophil development [20]. Studies have shown that the simultaneous absence of MBP-1 and EPO can result in a loss of eosinophil precursors in the bone marrow as well as eosinophils in the peripheral blood [21].

In addition, activated eosinophils are capable of releasing galectin-10 (Gal10) and undergoing a phase transition to form Charcot-Leyden crystals (CLCs). These crystals persist in the tissues for several months and contribute further to the progression of asthma [22]. A study conducted by Persson et al. discovered that biomimetic crystals of Gal10 elicited an innate immune response rich in neutrophils and monocytes upon injection into the airways of young rats. Furthermore, when combined with OVA, it not only induced dendritic cell uptake and initiated type 2 helper T cell response but also resulted in an increase in airway eosinophils and an immunoglobulins (Ig) G1 response [23].

Eosinophils have multiple degranulation mechanisms, and the classical modes of degranulation include cytosolic spitting and progressive degranulation (also known as lamellar degranulation or segmental degranulation). Cytosolic degranulation refers to the fusion of the entire granule content with the plasma membrane as a separate granule, resulting in the release of the entire granule content, whereas progressive degranulation refers to the gradual and selective release of the granule content by small vesicles without fusion with other granules or the plasma membrane [13, 24, 25]. In addition, a unique degranulation pattern has been observed in eosinophils that were previously thought to be a form of “necrosis” [9, 26]. However, subsequent research has shown that this degranulation pattern does not exhibit the usual features of necrosis or apoptosis, such as phosphatidylserine expression (as shown by weak Annexin V staining) or necrotic spots, and represents a distinct mode of cell death called extracellular trap cell death (ETosis) [6, 27‐29]. None of the known types of necrosis or extrusion artifacts could explain the mechanism of this degranulation pattern [6, 9].

The discovery of extracellular traps (ETs) dates back to 1975 when Anker et al. demonstrated in vitro that lymphocytes can release DNA into the extracellular space without compromising their activity [30]. In 2004, Brinkmann et al. observed that in addition to phagocytosis and degranulation mechanisms, neutrophils can kill bacteria in the extracellular space by releasing nuclear components that form extracellular fibers with granules, a phenomenon known as NETs [31]. Subsequently, in 2008, Yousefi et al. discovered that eosinophils can also exert their effects by releasing granule proteins and DNA to form extracellular structures later identified as EETs [32].

ETosis is considered a potential type of cell death that promotes inflammation [33]. Eosinophils can be further classified into vital EETosis and suicidal EETosis depending on whether they undergo cell death [34]. It has been observed that eosinophils can release DNA from mitochondria by jetting, and after forming EETs, eosinophils remain viable without evidence of apoptosis or other types of cell death, a process known as vital EETosis [32].

For suicidal EETosis, the process is more complex and involves a series of morphological changes: disassembly of nuclear chromatin followed by rupture of the cytoplasmic membrane, which in turn leads to the release of EDGPs and nuclear material (e.g. histones and DNA) from the ruptured eosinophils into the surrounding environment and the production of EETs [35‐37]. Although the above appears to be a continuous process, it has been observed to occur rapidly in vitro experiments (~ 0.5- 3 h), which may lead to a rapid release of nuclear chromatin and EDGPs after plasma membrane rupture without sufficient mixing, resulting in a detectable extracellular network of DNA fibers with or without intact EDGPs [9, 38]. In the case of suicidal EETosis, it somehow becomes a continuation of the eosinophil function and extends the function of eosinophils[39].

It has been suggested that the decision of whether EETosis is released in a viable or dead form may depend on the source of stimulation in vivo and in vitro [37], but there is still academic debate as to whether mitochondrial-derived DNA alone is sufficient to form such a large number of EETs upon stimulation [39]. The prevailing view is that the DNA for EETosis is nuclear in origin and that the cell must eventually release it through cell death formation [8]. There are three main reasons for this. First, the release of DNA from the mitochondria into the cytoplasm and then into the extracellular compartment requires very high energy; second, a single eosinophil contains a small number of mitochondria with insufficient copies of DNA and is susceptible to structural damage caused by reactive oxygen species (ROS); and finally, mitochondrial DNA is stickier and smaller than nuclear DNA, making it easier to detect, which may partially explain the previous findings [6].

In allergic tissues, cytolysis is the second most common mode of degranulation after lamellar degranulation, accounting for 10–33% of all degranulation patterns [40‐42]. Some studies suggest that this percentage may be as high as 80% in patients with eosinophilic esophagitis [43]. ETosis may also be present at high levels in severe and fatal asthma [26].

In isolated eosinophils, the induction of stimulated production of EETs occurs in a variety of ways, such that transmembrane signaling may be initiated first by cytokines (e.g., interleukin 5 [IL-5], interferon-γ), chemokines, or adhesion molecules, followed by lipopolysaccharide (LPS), eosinophil chemokine, or complement component 5a (C5a), thymic stromal lymphopoietin (TSLP), or staphylococcus aureus stimulation [8, 32, 44, 45]. In addition, it can also be induced by direct stimulation with sorbitol acetate, calcium ion carriers, immobilized IgA or IgG, phorbol 12-myristate 13-acetate (PMA), respiratory syncytial virus, and the calcium ionophore A23187 [38, 46, 47].

Neutrophil-released NETs have been extensively studied for their role in infectious diseases. Eosinophils are considered to be one of the most important central effector cells in the development of asthma. In contrast to neutrophil phagocytosis, eosinophils promote airway inflammation and remodeling primarily through the selective release of various molecules such as cytokines and granulins [58, 59].

In non-infectious asthma, the production of EETs is significantly increased, contributing to the exacerbation of type 2 immunity. In OVA-challenged mice, both eosinophil and neutrophil extracellular traps were generated and released after allergen stimulation, with significantly higher levels of EETs production than NETs (57.09 ± 10.23% vs. 5.27 ± 3.32%) [10]. Intranasal administration of EETs for five days increased the number of eosinophils and neutrophils in the BALF of wild-type BALB/c mice, along with elevated levels of epithelial-derived cytokines (such as IL-1α, IL-1β, CXCL-1, CCL24, IL-33, and TSLP) and an increased proportion of IL-5 or IL-13-producing ILC2 cells in the lungs [11].

Moreover, several studies have shown that EETs may serve as potential biomarkers of severe asthma and are associated with acute exacerbations of asthma [59, 60]. In humans, levels of EETs in BALF were significantly higher in adult asthmatics than in healthy controls and were positively correlated with levels of type 2 cytokines (i.e., IL-4, IL-5, and IL-13) and asthma severity, while negatively correlated with FEV1% [10]. The study by Choi and colleagues demonstrated that severe asthmatics have a higher release of EETs compared to non-severe asthmatics when peripheral blood eosinophils are stimulated by IL-5 and LPS [51]. The study also showed a negative correlation between EETs and FEV1, and a positive correlation between EETs and serum eosinophil-derived neurotoxin levels. Follow-up studies by Lee et al. and Choi et al. confirmed these findings and further suggested that EETs production is associated with the degree of airway inflammation and obstruction in patients with severe asthma [11, 53]. However, a study by Granger et al. found no association between serum EETs and asthma severity, and EETs expression was not detectable in BALF [61]. Instead, this study showed that only serum NETs levels correlated with asthma severity, control level, age at onset and duration of asthma exacerbation.

Several factors contribute to the variability in the results of studies investigating ETs in asthma. First, the heterogeneity of the populations included in the studies may partially explain the observed differences. In mouse models of asthma following viral infection, an increase in neutrophil generation with NETs has been observed, peaking within 2 days [56, 62]. Respiratory infections are important triggers of asthma exacerbations, which tend to be more severe and have different pathological manifestations compared to non-infected asthma. Secondly, unlike in vitro experiments, the formation of ETs in vivo is influenced by various factors such as the type of stimulus, dose, exposure time, and drug treatment. Moreover, the rapid formation of ETs themselves may pose challenges in their in vivo detection [59, 63]. As noted by Granger et al., the expression of EETs in BALF was not effectively detected, even though 53% of patients with available BALF samples had alveolar eosinophil counts > 1.2% [61]. Therefore, future studies are needed to determine the impact of EETs expression levels on different asthma severities and sample sources.

EETs can be monitored by a variety of means, but like NETs, they serve as screening tools to help clinicians better individualize treatment. This requires readily available test samples and tests that demonstrate good specificity, sensitivity, and reproducibility. Such tests should provide value for the diagnosis, treatment and prognostic monitoring of clinical disease.

As mentioned above, EETs are composed of several components. The DNA and citrullinated histone H3 (citH3), which form the “backbone”, and the granular proteins such as ECP and EPO, which are attached to the “backbone”, can be used as indicators of their levels. However, it should be noted that some markers are not only found in eosinophils with EETosis but can also be released by apoptotic or necrotic eosinophils and other cells, so it is worth considering how to combine and detect these markers. Visualization of EETs production by cellular immunofluorescence or immunohistochemistry is the most accurate method [64‐66], but this is relatively inaccessible in asthmatic patients because it requires obtaining BALF and lung tissue samples in an invasive manner, such as bronchoscopy.

This led us to look at the more non-invasive and readily available blood samples. The detection of circulating free DNA (cfDNA) is one of the indicators of response to EETs that was first described in a study by Yousefi et al. [32]. In patients with eosinophilia with polyangiitis (EGPA), it was observed that cfDNA levels were significantly higher in EGPA patients than in healthy subjects and correlated significantly with disease activity and blood eosinophil count, and that the presence of EETs increased cfDNA levels in EGPA [67]. Circulating citH3 is another indicator that can be used as a response to EETs and shows better specificity than cfDNA [10], which can be used as a potential assessment indicator for loss of asthma control [68]. However, it is important to note that for in vivo detection of EETs, other innate immune cells besides eosinophils can also release ETs [69], a process that may also involve citrullination of histones, a point that has been well-documented for NETs [3, 70]. Compared to circulating cfDNA and citH3, a modified ELISA targeting the ECP-DNA complexes characteristic of EETs may be more specific [10, 69, 70].

In addition, several novel biomarkers have been proposed that may reflect the levels of EETs. In patients with chronic rhinosinusitis (CRS), calmodulin was found to be expressed in eosinophils and involved in the innate immune response via EETs, which could be used as a biomarker in response to the severity of CRS [71], but it is unclear whether it could be used as one of the indicators of EETs levels in asthma. Gal10 is an eosinophil-specific marker released by EETosis, and elevated levels of Gal10 in serum and tissues may indicate an increase in the number of eosinophils that undergo EETosis in vivo. Further future studies are needed to clarify the scenario and value of these biomarkers in asthma patients.

The advent of biological agents has opened new avenues for the treatment of patients with severe asthma. However, there remains a subset of patients with a limited response to corticosteroids or biologic agents, highlighting the need for the identification of new therapeutic targets and biomarkers. The therapeutic targets for EETs can be approached from two perspectives: a pre-EET formation block, in which eosinophils receive various signaling stimuli in vivo to initiate the EETosis process, and a post-EET release block, which includes targeting the chromatin backbone structure that constitutes EETs and substances attached to chromatin.

These two phases of treatment should be considered complementary rather than independent. It is important to note that just as the formation and release of NETs is a double-edged sword [45], this feature also exists in EETs (Fig. 2). For example, the released granulocytes restrict this action to specific regions by wrapping granulocyte proteases in the DNA network, which may lead to a strong immune response causing organ dysfunction and even organ failure [8].

Therefore, the feasibility of targeting systemic EETs for degradation therapy remains uncertain, and further studies are needed to evaluate the timing, monitoring metrics, and potential side effects of such therapy. Although several studies have explored the therapeutic targets of ETs, the structural characteristics of ETs currently make it difficult to find an ideal therapeutic strategy [72]. In the following section, we provide a brief overview of potential future drugs that could be used to treat EETs based on the available studies in the literature and their relevance to directly targeting EETs (Table 1; Fig. 3).

EETs not only participate in the pathogenesis of asthma but also in many other autoimmune and inflammatory diseases, including chronic obstructive pulmonary disease, sepsis, and vascular disease. Despite numerous studies investigating the formation and function of EETs, the specific mechanisms of EETs regulation remain unknown. EETosis proceeds through a specific sequence of events (Fig. 1). Most of the current literature have investigated the sources of stimulation and effective inhibitors of EETosis. Further investigation of how they affect the cellular events of EETosis may help us understand the underlying mechanisms and identify new therapeutic targets. EETosis, being a ‘double-edged sword’, has primarily been focused on its negative effects in the field of asthma, while the value of its positive effects has been less explored. Furthermore, as one of the physiologically released substances, there is a lack of large-scale studies to define the range of normal levels. Objective reference values would be valuable for clinicians to assess guidelines for therapeutic intervention. Of all the issues raised in this review, several may deserve special attention as they can significantly contribute to our understanding of the cell biology of EETosis, its biophysics, and its role in the field of asthma (Table 3).