Eosinophil Extracellular Traps Pave the Way for the Identification of Novel Therapeutics in Severe Asthma

The release of chromatin and granule proteins against microorganisms by forming extracellular fibers was first reported in neutrophils, named neutrophil extracellular traps (NETs).¹ Afterward, the release of extracellular traps (ETs) was observed in other immune cells, including mast cells, monocytes, tissue macrophages, and lymphocytes.² In contrast to the original concept of ETs as a defense mechanism, accumulated data implicate ETs in the development of numerous medical conditions, especially regarding NETs in infections, autoimmunity, and malignancies.³,⁴

Over the past decade, researches also shed light on the biological role of eosinophil extracellular traps (EETs) in type 2 inflammation. In addition to the presence of EETs during bacterial and fungal infections, they have been found in tissues obtained from patients with allergic and eosinophilic diseases, such as asthma, atopic dermatitis, eosinophilic esophagitis, and chronic rhinosinusitis.² With the availability of biologic agents targeting type 2 inflammation in the aforementioned diseases, understanding the contribution of eosinophils in the development, severity, and prognosis of pathologic conditions has become an important research field. In this regard, the biological role of EETs in eosinophilic inflammation needs to be clarified.

In this issue of Allergy, Asthma & Immunology Research, Lee and colleagues⁵ demonstrated a vicious cycle in severe asthma composed of cytokeratin (CK)-18, its immunoglobulin G (IgG) antibody, and autoantibody-induced EETs, which further enhances the production of CK-18 in airway epithelial cells. The authors noted the elevated level of CK-18 and CK-18-specific IgG in severe asthma using samples from patients and murine models. They also conducted in vitro experiments which showed that CK-18-specific IgG enhanced the production of extracellular DNA and eosinophil-derived neurotoxin from isolated eosinophils. In addition, intranasal administration of EETs in mice led to upregulated CK-18 and CK-18-specific IgG in the lungs in a dose-dependent manner, which was not diminished by treatment with dexamethasone. These results collectively indicated the presence of a mutually enhancing autoimmune-EET pathway in steroid-unresponsive severe asthma.

In 2011, the infiltration of EETs colocalized with the major basic protein (MBP) in human asthmatic airways was first reported in all 20 participants of the study.⁶ EETs colocalized with the eosinophil cationic protein (ECP) in the bronchoalveolar lavage fluid (BALF) were observed in patients with asthma, but not in healthy controls. Moreover, the level of EETs in BALF was significantly correlated with lung function and severity of asthma.⁷ Among asthmatic patients, the circulating EET levels were significantly higher in patients with poorly controlled asthma than those with well-controlled asthma. Meanwhile, circulating EETs did not differ according to the severity of asthma.⁸ However, the proportion of EET-forming eosinophils from patients with severe asthma was higher than those with non-severe asthma during stimulation with interleukin (IL)-5 and lipopolysaccharide (LPS). Further, it was negatively correlated with lung function.⁹ These findings suggested the potential of EET as a biomarker in asthma.

Mechanistically, various triggers including IL-5, LPS, reactive oxygen species, autophagy, and viral infections have been reported to stimulate eosinophils to release EETs.¹⁰ Since EETs are web-like structures made up of DNA fibers and granule proteins, namely MBP and ECP, they exert tissue toxicity and recruit or activate inflammatory cells.¹¹ In asthma, the authors previously identified an association between EETs and type 2 innate lymphoid cells, which stimulate the airway epithelium to release alarmins and consequently exacerbate type 2 inflammation.¹² Most recently, the contribution of EETs in aggravating allergic inflammation by activating pulmonary neuroendocrine cells has been reported.⁷ The present study adds a novel pathologic role of EET in asthma progression linked with autoimmunity against the airway epithelium.

The involvement of autoimmunity in asthma has been suggested for decades. In addition to antigens derived from bronchial epithelial cells, including CK-18, endothelial or platelet proteins, extracellular matrix protein, and cellular junction have been reported as self-antigens in asthma.¹³ Particularly, immunoprecipitated sputum immunoglobulins from patients with severe eosinophilic asthma reporting elevated anti-eosinophil peroxidase IgG and antinuclear antibodies promoted the production of EET, which was not suppressed by dexamethasone.¹⁴ In the present study, CK-18 served as a key inducer as well as a mediator of persistent eosinophilic inflammation. Given that CK-18 is derived from bronchial epithelium, the study by Lee et al.⁵ elucidated the impact of EETs on airway structure cells and suggested a possible association with progressive airway remodeling observed in severe eosinophilic asthma, despite treatment with corticosteroids.

EETs have gained attention as a potential therapeutic target in severe eosinophilic asthma due to their involvement in the progress of type 2 inflammation. However, there are many unresolved questions in the pathophysiology of EETs. First, regarding autoimmunity, the initiator of autoreactivity is still unclear. Secondly, under the same stimulation, some eosinophils release EETs, while others do not. This implicates the presence of distinct subtypes of eosinophils. Thirdly, the interaction between DNA fibers and granule proteins in the traps requires further investigation. Fourthly, a methodological way of preventing or dissolving EETs is needed. In addition, the outcomes of depleting EETs should be assessed in terms of efficacy and safety. Finally, working EETs are difficult to determine readily from the patients. Without stimulation, the circulating levels of EETs and the proportion of EET-forming eosinophils were similar between patients with severe and non-severe asthma in vivo.⁸ Therefore, identifying patients whose asthma is affected by EETs is still challenging in clinical practice.

Although biologics have broadened the therapeutic options for managing severe eosinophilic asthma, there is a subpopulation of patients who show limited response to either corticosteroid or biologics, and a gradual decline of lung function despite anti-inflammatory treatment.¹⁵ For these patients, novel biomarkers and therapeutic agents are necessary. Beyond the numerical measurement of eosinophils and suppressing type 2 cytokines, more elaborate strategies are required to modulate specific components of eosinophils. At present, EETs are regarded as one of the potential therapeutic targets in severe eosinophilic asthma, which became more cogent with the research by Lee and colleagues.⁵ Even though we still have a long way to go before fully understanding the biology of disease-related eosinophils, the accumulated data can enable the search for novel therapeutics in severe asthma.