Eosinophil extracellular traps in asthma: implications for pathogenesis and therapy.

Asthma is a common, chronic inflammatory disease of the airways that affects millions of people worldwide and is associated with significant healthcare costs. Eosinophils, a type of immune cell, play a critical role in the development and progression of asthma. Eosinophil extracellular traps (EETs) are reticular structures composed of DNA, histones, and granulins that eosinophils form and release into the extracellular space as part of the innate immune response. EETs have a protective effect by limiting the migration of pathogens and antimicrobial activity to a controlled range. However, chronic inflammation can lead to the overproduction of EETs, which can trigger and exacerbate allergic asthma. In this review, we examine the role of EETs in asthma.

Calcium ionophore-activated platelets induce eosinophil extracellular trap formation.

BACKGROUND: Platelets play a modulatory role in inflammatory response by secreting a vast array of granules and disintegrating into membrane-bound microparticles upon activation. The interplay between eosinophils and platelets is postulated to be implicated in the pathology of allergic airway inflammation. In this study, we investigated whether activated platelets can induce eosinophil extracellular trap (EET) formation, a cellular process by which activated eosinophils release net-like DNA fibers. METHODS: Platelets were stimulated with the calcium ionophore, A23187, and the platelet agonists, thrombin and adenosine diphosphate (ADP). Platelet cultures were fractionated into conditioned medium (CM) and pellet, which were then overlaid on eosinophils to examine EET formation. RESULTS: The CM and pellet from A23187-activated platelets stimulated eosinophils to generate EET, whereas those from thrombin- or ADP-activated platelets failed to induce such generation. The EET-inducing activity of the A23187-activated platelet culture was linearly proportional to the number of activated platelets. Interestingly, while EET formation induced by the direct stimulation of eosinophils with A23187 was NADPH oxidase (NOX)-dependent, EET formation induced by A23187-activated platelets was NOX-independent and significantly inhibited by necroptosis pathway inhibitors. CONCLUSIONS: Activated platelets and their products may induce EET formation, thereby potentiating their role in eosinophilic airway inflammation.

Cysteinyl leukotriene induces eosinophil extracellular trap formation via cysteinyl leukotriene 1 receptor in a murine model of asthma.

PURPOSE: Eosinophils are one of the main cells responsible to the inflammatory response in asthma by the release of inflammatory molecules such as cytokines, reactive oxygen species (ROS), cytotoxic granule, eosinophil extracellular trap (EET), and lipid mediators as cysteinyl leukotriene (cysLT). The interconnections between these molecules are not fully understood. Here, we attempted to investigate the cysLT participation in the mechanisms of EET formation in an asthma model of OVA challenge. MATERIALS AND METHODS: Before intranasal challenge with OVA, BALB/cJ mice were treated with a 5-lipoxygenase-activating protein (FLAP) inhibitor (MK-886), or with a cysLT1 receptor antagonist (MK-571) and the lung and bronchoalveolar lavage fluid (BALF) were analyzed. RESULTS: We showed that OVA-challenged mice treated with MK-886 or MK-571 had a decrease in inflammatory cells, goblet cells hyperplasia, and eosinophil peroxidase (EPO) activity in the airway. However, only OVA-challenged mice treated with MK-571 had an improvement in lung function. Also, treatments with MK-886 or MK-571 decreased Th2 cytokines levels in the airway. Moreover, we observed that OVA-challenged mice treated with MK-886 or MK-571 had a decrease in EET formation in BALF. We also verified that EET release was not due to cell death because the cell viability remained the same among the groups. CONCLUSION: We revealed that the decrease in cysLT production or cysLT1 receptor inhibition by MK-886 or/and MK-571 treatments, respectively reduced EET formation in BALF, showing that cysLT regulates the activation process of EET release in asthma.

The Enigma of Eosinophil Degranulation.

Eosinophils are specialized white blood cells, which are involved in the pathology of diverse allergic and nonallergic inflammatory diseases. Eosinophils are traditionally known as cytotoxic effector cells but have been suggested to additionally play a role in immunomodulation and maintenance of homeostasis. The exact role of these granule-containing leukocytes in health and diseases is still a matter of debate. Degranulation is one of the key effector functions of eosinophils in response to diverse stimuli. The different degranulation patterns occurring in eosinophils (piecemeal degranulation, exocytosis and cytolysis) have been extensively studied in the last few years. However, the exact mechanism of the diverse degranulation types remains unknown and is still under investigation. In this review, we focus on recent findings and highlight the diversity of stimulation and methods used to evaluate eosinophil degranulation.

Lysophosphatidylserine induces eosinophil extracellular trap formation and degranulation: Implications in severe asthma.

BACKGROUND: Recent evidence demonstrates that activated eosinophils undergo a distinct form of lytic cell death, accompanied by formation of DNA-based eosinophil extracellular trap (EET) and degranulation, enhancing inflammatory immune responses in asthmatic airways. We previously showed that human blood eosinophils undergo degranulation in response to lysophosphatidylserine (LysoPS), an inflammatory lipid mediator, and strongly express P2Y10, a LysoPS receptor. METHODS: We evaluated EET, degranulation, and cell death of eosinophils in response to various concentrations of LysoPS. We also compared responsiveness to LysoPS between eosinophils from severe and nonsevere asthmatics. RESULTS: Extensive EET formation was elicited from a substantial fraction of stimulated eosinophils in response to 50 µmol/L LysoPS. Analyses for LDH and eosinophil-derived neurotoxin release showed that both lytic cell death and degranulation accompanied EET formation in response to LysoPS. Cytological analyses demonstrated that citrullinated histone 3 was present in the extracellular, filamentous DNA structure embedded with eosinophil granules. The LysoPS-induced EET was independent of ROS production and irrelevant to several signaling pathways examined, but dependent on protein arginine deiminase 4. A low concentration of LysoPS (5 µmol/L) did not induce EET or degranulation, but significantly increased platelet-activating factor-induced degranulation. Eosinophils from severe asthmatics exhibited greater degranulation, but not EET formation, in response to LysoPS (50 µmol/L), than those from nonsevere asthmatics, along with great expression of surface P2Y10. CONCLUSIONS: We identified a novel function of LysoPS, namely induction of EET in association with cytolysis and degranulation. LysoPS-dependent EET or degranulation plays a potential role in eosinophilic inflammation of severe asthma.

Presence of eosinophil extracellular trap cell death in the affected skin of eosinophilic granulomatosis with polyangiitis.

Eosinophil activation in tissue might be associated with disease severity. Eosinophil cytolysis, a process of active cell death, has been referred to as eosinophil extracellular trap cell death (EETosis). In the present study, the authors investigated EETosis in the affected skin of four patients with eosinophilic granulomatosis with polyangiitis (EGPA) using an immunofluorescence staining method. Immunofluorescence staining for myelin basic protein, galectin-10, and DNA revealed various degrees of EETosis and Charcot-Leyden crystals in skin tissue, suggesting the different degree of eosinophil activation status. The histopathological characteristic features may help physicians establish an earlier diagnosis of intractable eosinophilic-related disease including EGPA. Furthermore, ETotic eosinophil infiltration in perineurium of skin tissue might play a primary role in peripheral neuropathy of this disorder.

Eosinophil-derived galectin-10 upregulates matrix metalloproteinase expression in bullous pemphigoid blisters.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune bullous disease in which abundant eosinophils accumulate in the blisters. Galectin-10 abounds in the cytoplasm of eosinophils and is released as a result of eosinophil extracellular trap cell death (EETosis). OBJECTIVE: To identify EETosis and the pathological roles of galectin-10 in BP. METHODS: EETosis and galectin-10 in BP blisters were confirmed by immunofluorescence and transmission electron microscopy. The concentrations of galectin-10 in serum and blister fluid from BP patients were studied by ELISA. The matrix metalloproteinase (MMP) expression in BP blisters was immunohistochemically compared to that in healthy controls. As an in vitro assay, normal human epidermal keratinocytes (NHEKs) and normal human dermal fibroblasts (NHDFs) were stimulated with galectin-10, followed by MMP expression measurement by real-time PCR and ELISA. The signaling pathways activated by galectin-10 were studied using Western blotting and confirmed by inhibition assays. RESULTS: Galectin-10-containing eosinophil infiltration and the extracellular deposition of major basic protein were observed in BP blisters. The ultrastructural characteristics of tissue eosinophils indicated piecemeal degranulation and EETosis. In the BP patients, the concentration of galectin-10 was higher in the blister fluid than in the serum. Several types of MMPs were upregulated in BP blisters. Galectin-10 upregulated the production of MMPs through the pathways of p38 MAPK, ERK and JNK in NHEKs and NHDFs. CONCLUSION: In the BP blisters, the eosinophils underwent EETosis and released galectin-10. Galectin-10 might contribute to BP blister formation through the production of MMPs by keratinocytes and fibroblasts.

Mono-n-butyl phthalate regulates nuclear factor erythroid 2-related factor 2 and nuclear factor kappa B pathway in an ovalbumin-induced asthma mouse model.

Recent studies have emphasized the role of endocrine-disrupting chemicals in asthma development, especially in eosinophilic asthma. However, the exact mechanism was unknown. Among all the endocrine-disrupting chemicals, mono-n-butyl phthalate (MnBP) was a chemical that was most frequently detected in human urine. Our study was performed with the aim of investigating the harmful effects of MnBP on airway epithelial cells (AECs), T cells, and eosinophils by using eosinophilic asthma mouse models. Mice that received OVA with MnBP had higher levels of airway hyperresponsiveness, total and eosinophil cell counts, as well as T cell proliferation and T helper 2 cytokine release than those which only received OVA. Moreover, MnBP contributed to directly enhancing the eosinophilic activation which was shown in. Long-term exposure MnBP activated AECs through the nuclear factor kappa B (NF-kB) pathway, decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression, and increased interleukin-33 expression. Additionally, MnBP can induce human eosinophil activation to release eosinophil extracellular traps (EETs). Taken together, our study suggested the roles of MnBP exposure increase the risk of asthma development and severity. Furthermore, vitamin E treatment (anti-inflammatory and antioxidant effects) can reduce MnBP-induced harmful effects through inhibiting EETs, restoring Nrf2, and suppressing the NF-kB pathway.

Galectin-10 as a Potential Biomarker for Eosinophilic Diseases.

Galectin-10 is a member of the lectin family and one of the most abundant cytoplasmic proteins in human eosinophils. Except for some myeloid leukemia cells, basophils, and minor T cell populations, galectin-10 is exclusively present in eosinophils in the human body. Galectin-10 forms Charcot-Leyden crystals, which are observed in various eosinophilic diseases. Accumulating studies have indicated that galectin-10 acts as a new biomarker for disease activity, diagnosis, and treatment effectiveness in asthma, eosinophilic esophagitis, rhinitis, sinusitis, atopic dermatitis, and eosinophilic granulomatosis with polyangiitis. The extracellular release of galectin-10 is not mediated through conventional secretory processes (piecemeal degranulation or exocytosis), but rather by extracellular trap cell death (ETosis), which is an active cell death program. Eosinophils undergoing ETosis rapidly disintegrate their plasma membranes to release the majority of galectin-10. Therefore, elevated galectin-10 levels in serum and tissue suggest a high degree of eosinophil ETosis. To date, several studies have shown that galectin-10/Charcot-Leyden crystals are more than just markers for eosinophilic inflammation, but play functional roles in immunity. In this review, we focus on the close relationship between eosinophils and galectin-10, highlighting this protein as a potential new biomarker in eosinophilic diseases.

Successful management of recurrent allergic bronchopulmonary aspergillosis after changing from mepolizumab to dupilumab: A case report.

An 81-year-old woman presented to our hospital due to an abnormal shadow on a chest X-ray and a 4-week-old persistent cough. Laboratory examination revealed increased serum eosinophils and immunoglobulin E. The Asthma Control Test (ACT) score and forced expiratory volume in 1 sec indicated airway obstruction. Chest computed tomography (CT) revealed mucoid impaction in the dilated left-lingular lobar bronchus. She was diagnosed with bronchial asthma and treated with a high-dose inhaled corticosteroid/long-acting ß2 agonist. Two months later, her mucoid impaction in the CT image worsened; moreover, bronchoscopy revealed the white mucus plug with Charcot-Leyden crystals and filamentous fungi. The patient was diagnosed with Allergic bronchopulmonary aspergillosis (ABPA) and treatment with 30 mg/day prednisolone was started. Both the blood eosinophil count and the chest image improved almost substantially, and the steroid was discontinued after a year. Sixteen months after cessation of prednisolone treatment, peripheral eosinophilia and mucoid impaction in the left B3b recurred. For the treatment of bronchial asthma and recurrent ABPA, administration of mepolizumab was initiated. Subsequently, although her peripheral eosinophils count decreased, chest CT showed expansion of the mucoid impaction and IgE increased despite mepolizumab treatment. Alternative subcutaneous injection therapy with dupilumab improved chest image, serum IgE level, and her ACT score. After changing from mepolizumab to dupilumab, her ABPA, asthma, and pulmonary function improved remarkably. This case illustrates the potential utility of dupilumab for ABPA without re-administration of oral prednisolone. Additional research is needed to identify an effective therapy for ABPA with asthma.

An unusual case of eosinophilic lung disease with multiple cyst formation.

We report here an unusual case of eosinophilic necrotizing inflammation of the lung that mimicked chronic eosinophilic pneumonia. A 71-year-old man who lived in an unsanitary environment and was referred to our hospital with suspected pneumonia. Peripheral blood eosinophilia was observed, and computed tomography indicated extensive consolidation with multiple cystic lesions, mainly in the left lung. A histological analysis using video-assisted thoracic surgery revealed diffuse infiltration of inflammatory cells into the alveolar wall and massive accumulation of macrophages and eosinophils in the airspace. Many tiny eosinophilic abscesses were scattered through the tissue. These findings were more severe than those associated with chronic eosinophilic pneumonia. Immunostaining revealed the deposition of eosinophil granular protein and the presence of extracellular traps and Charcot-Leyden crystals, which suggested excessive eosinophil activation. Interestingly, the patient's symptoms and clinical findings gradually improved without treatment after admission. He was discharged to a clean residence and did not have a recurrence for 19 months. The observations suggest a hypersensitivity reaction to an environmental allergen and consequent multiple cyst formation in association with eosinophilic necrotizing inflammation, although further studies are warranted.