CD69 Signaling in Eosinophils Induces IL-10 Production and Apoptosis via the Erk1/2 and JNK Pathways, Respectively.

INTRODUCTION: Eosinophils contribute to the pathogenesis of allergic diseases, including asthma, allergic rhinitis, and atopic dermatitis. We previously reported that human tissue eosinophils have high CD69 expression compared to blood eosinophils, and its expression is correlated with disease severity and the number of infiltrated eosinophils. However, biological CD69 signaling activity in eosinophils remains unclear. METHODS: CD69 expression on lung tissue eosinophils obtained from mice with ovalbumin-induced asthma was measured using flow cytometry. CD69 crosslinking was performed on eosinophils purified from the spleen of IL-5 transgenic mice to investigate CD69 signaling and its function in eosinophils. Then, qPCR, Western blot, enzyme-linked immunosorbent assay, and survival assay results were analyzed. RESULTS: Surface CD69 expression on lung tissue eosinophils in the asthma mice model was 2.91% ± 0.76%, whereas no expression was detected in the healthy group. CD69-expressed eosinophils intrinsically have an upregulation of IL-10 mRNA expression. Moreover, CD69 crosslinking induced further pronounced IL-10 production and apoptosis; these responses were mediated via the Erk1/2 and JNK pathways, respectively. CONCLUSIONS: Our results suggested that CD69+ eosinophils play an immunoregulator role in type 2 inflammation, whereas activated tissue eosinophils contribute to the pathogenesis of asthma.

The Neutralization of the Eosinophil Peroxidase Antibody Accelerates Eosinophilic Mucin Decomposition.

Eosinophilic airway inflammation, complicated by bronchial asthma and eosinophilic chronic rhinosinusitis (ECRS), is difficult to treat. The disease may become refractory when eosinophilic mucin associated with eosinophil peroxidase (EPX) and autoantibodies fills in the paranasal sinus and small airway. This study investigated the functional role of an anti-EPX antibody in eosinophilic mucin of ECRS in eosinophilic airway inflammation. Eosinophilic mucin was obtained from patients with ECRS. The effects of the anti-EPX antibody on dsDNA release from eosinophils and eosinophilic mucin decomposition were evaluated. Immunofluorescence or enzyme-linked immunosorbent assays were performed to detect the anti-EPX antibody and its supernatant and serum levels in eosinophilic mucin, respectively. The serum levels of the anti-EPX antibody were positively correlated with sinus computed tomography score and fractionated exhaled nitrogen oxide. Patients with refractory ECRS had higher serum levels of the anti-EPX antibody than those without. However, dupilumab treatment decreased the serum levels of the anti-EPX antibody. Immunoglobulins (Igs) in the immunoprecipitate of mucin supernatants enhanced dsDNA release from eosinophils, whereas the neutralization of Igs against EPX stopped dsDNA release. Furthermore, EPX antibody neutralization accelerated mucin decomposition and restored corticosteroid sensitivity. Taken together, the anti-EPX antibody may be involved in the formulation of eosinophilic mucin and be used as a clinical marker and therapeutic target for intractable eosinophilic airway inflammation.

CCL4 Regulates Eosinophil Activation in Eosinophilic Airway Inflammation.

Eosinophilic chronic rhinosinusitis (ECRS) is a refractory airway disease accompanied by eosinophilic inflammation, the mechanisms of which are unknown. We recently found that CCL4/MIP-1ß-a specific ligand for CCR5 receptors-was implicated in eosinophil recruitment into the inflammatory site and was substantially released from activated eosinophils. Moreover, it was found in nasal polyps from patients with ECRS, primarily in epithelial cells. In the present study, the role of epithelial cell-derived CCL4 in eosinophil activation was investigated. First, CCL4 expression in nasal polyps from patients with ECRS as well as its role of CCL4 in eosinophilic airway inflammation were investigated in an in vivo model. Furthermore, the role of CCL4 in CD69 expression-a marker of activated eosinophils-as well as the signaling pathways involved in CCL4-mediated eosinophil activation were investigated. Notably, CCL4 expression, but not CCL5, CCL11, or CCL26, was found to be significantly increased in nasal polyps from patients with ECRS associated with eosinophil infiltration as well as in BEAS-2B cells co-incubated with eosinophils. In an OVA-induced allergic mouse model, CCL4 increased eosinophil accumulation in the nasal mucosa and the bronchoalveolar lavage (BALF). Moreover, we found that CD69 expression was upregulated in CCL4-stimulated eosinophils; similarly, phosphorylation of several kinases, including platelet-derived growth factor receptor (PDGFR)ß, SRC kinase family (Lck, Src, and Yes), and extracellular signal-regulated kinase (ERK), was upregulated. Further, CCR5, PDGFRß, and/or Src kinase inhibition partially restored CCL4-induced CD69 upregulation. Thus, CCL4, which is derived from airway epithelial cells, plays a role in the accumulation and activation of eosinophils at inflammatory sites. These findings may provide a novel therapeutic target for eosinophilic airway inflammation, such as ECRS.

Efficacy of Combination Therapy with Lenvatinib and Radioactive Iodine in Thyroid Cancer Preclinical Model.

Patients with differentiated thyroid cancer (DTC) usually have good prognosis, while those with advanced disease have poor clinical outcomes. This study aimed to investigate the antitumor effects of combination therapy with lenvatinib and 131I (CTLI) using three different types of DTC cell lines with different profiling of sodium iodide symporter (NIS) status. The radioiodine accumulation study revealed a significantly increased radioiodine uptake in K1-NIS cells after lenvatinib treatment, while there was almost no uptake in K1 and FTC-133 cells. However, lenvatinib administration before radioiodine treatment decreased radioiodine uptake of K1-NIS xenograft tumor in the in vivo imaging study. CTLI synergistically inhibited colony formation and DTC cell migration, especially in K1-NIS cells. Finally, 131I treatment followed by lenvatinib administration significantly inhibited tumor growth of the NIS-expressing thyroid cancer xenograft model. These results provide important clinical implications for the combined therapy that lenvatinib should be administered after 131I treatment to maximize the treatment efficacy. Our synergistic treatment effects by CTLI suggested its effectiveness for RAI-avid thyroid cancer, which retains NIS function. This potential combination therapy suggests a powerful and tolerable new therapeutic strategy for advanced thyroid cancer.

Omalizumab Restores Response to Corticosteroids in Patients with Eosinophilic Chronic Rhinosinusitis and Severe Asthma.

Eosinophilic chronic rhinosinusitis (ECRS), which is a subgroup of chronic rhinosinusitis with nasal polyps, is characterized by eosinophilic airway inflammation extending across both the upper and lower airways. Some severe cases are refractory even after endoscopic sinus surgery, likely because of local steroid insensitivity. Although real-life studies indicate that treatment with omalizumab for severe allergic asthma improves the outcome of coexistent ECRS, the underlying mechanisms of omalizumab in eosinophilic airway inflammation have not been fully elucidated. Twenty-five patients with ECRS and severe asthma who were refractory to conventional treatments and who received omalizumab were evaluated. Nineteen of twenty-five patients were responsive to omalizumab according to physician-assessed global evaluation of treatment effectiveness. In the responders, the levels of peripheral blood eosinophils and fractionated exhaled nitric oxide (a marker of eosinophilic inflammation) and of CCL4 and soluble CD69 (markers of eosinophil activation) were reduced concomitantly with the restoration of corticosteroid sensitivity. Omalizumab restored the eosinophil-peroxidase-mediated PP2A inactivation and steroid insensitivity in BEAS-2B. In addition, the local inflammation simulant model using BEAS-2B cells incubated with diluted serum from each patient confirmed omalizumab's effects on restoration of corticosteroid sensitivity via PP2A activation; thus, omalizumab could be a promising therapeutic option for refractory eosinophilic airway inflammation with corticosteroid resistance.

The multiple functions and subpopulations of eosinophils in tissues under steady-state and pathological conditions.

Eosinophils not only play a critical role in the pathogenesis of eosinophil-associated diseases, but they also have multiple important biological functions, including the maintenance of homeostasis, host defense against infections, immune regulation through canonical Th1/Th2 balance modulation, and anti-inflammatory and anti-tumorigenic activities. Recent studies have elucidated some emerging roles of eosinophils in steady-state conditions; for example, eosinophils contribute to adipose tissue metabolism and metabolic health through alternatively activated macrophages and the maintenance of plasma cells in intestinal tissue and bone marrow. Moreover, eosinophils exert tissue damage through eosinophil-derived cytotoxic mediators that are involved in eosinophilic airway inflammation, leading to diseases including asthma and chronic rhinosinusitis with nasal polyps characterized by fibrin deposition through excessive response by eosinophils-induced. Thus, eosinophils possessing these various effects reflect the heterogenous features of these cells, which suggests the existence of distinct different subpopulations of eosinophils between steady-state and pathological conditions. Indeed, a recent study demonstrated that instead of dividing eosinophils by classical morphological changes into normodense and hypodense eosinophils, murine eosinophils from lung tissue can be phenotypically divided into two distinct subtypes: resident eosinophils and inducible eosinophils gated by Siglec-Fint CD62L+ CD101low and Siglec-Fhigh CD62L- CD101high, respectively. However, it is difficult to explain every function of eosinophils by rEos and iEos, and the relationship between the functions and subpopulations of eosinophils remains controversial. Here, we overview the multiple roles of eosinophils in the tissue and their biological behavior in steady-state and pathological conditions. We also discuss eosinophil subpopulations.

A Novel Approach for Investigating Upper Airway Hyperresponsiveness Using Micro-CT in Eosinophilic Upper Airway Inflammation such as Allergic Rhinitis Model.

Airway hyperresponsiveness (AHR) has been proposed as a feature of pathogenesis of eosinophilic upper airway inflammation such as allergic rhinitis (AR). The measurement system for upper AHR (UAHR) in rodents is poorly developed, although measurements of nasal resistance have been reported. Here we assessed UAHR by direct measurement of swelling of the nasal mucosa induced by intranasal methacholine (MCh) using micro-computed tomography (micro-CT). Micro-CT analysis was performed in both naïve and ovalbumin-induced AR mice following intranasal administration of MCh. The nasal cavity was segmented into two-dimensional horizontal and axial planes, and the data for nasal mucosa were acquired for the region of interest threshold. Then, a ratio between the nasal mucosa area and nasal cavity area was calculated as nasal mucosa index. Using our novel method, nasal cavity structure was clearly identified on micro-CT, and dose-dependent increased swelling of the nasal mucosa was observed upon MCh treatment. Moreover, the nasal mucosa index was significantly increased in AR mice compared to controls following MCh treatment, while ovalbumin administration did not affect swelling of the nasal mucosa in either group. This UAHR following MCh treatment was completely reversed by pretreatment with glucocorticoids. This novel approach using micro-CT for investigating UAHR reflects a precise assessment system for swelling of the nasal mucosa following MCh treatment; it not only sheds light on the mechanism of AR but also contributes to the development of new therapeutic drugs in AR patients.

Eosinophilic Upper Airway Inflammation in a Murine Model Using an Adoptive Transfer System Induces Hyposmia and Epithelial Layer Injury with Convex Lesions.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a refractory upper airway disease, accompanied mainly by eosinophilia and/or asthma. In addition, the disease correlates with a high rate of hyposmia, following a marked infiltration of eosinophils into the inflamed site, the paranasal sinus. Although eosinophils are known to contribute to the development of hyposmia and CRSwNP pathology, the underlying mechanisms remain unclear. This study aimed to investigate whether eosinophilic upper airway inflammation induces hyposmia and CRSwNP in a murine model using an adoptive transfer system. METHODS: To induce eosinophilic rhinosinusitis, splenocytes, including a high proportion (over 50%) of activated eosinophils (SPLhEos), were collected from interleukin-5 transgenic mice following double intraperitoneal injections of antigens, such as ovalbumin, house dust mite, or fungus. Activated SPLhEos with corresponding antigens were then transferred into the nasal cavity of recipient mice, which were sensitized and challenged by the corresponding antigen four times per week. Olfactory function, histopathological, and computed tomography (CT) analyses were performed 2 days after the final transfer of eosinophils. RESULTS: Hyposmia was induced significantly in mice that received SPLhEos transfer compared with healthy and allergic mice, but it did not promote morphological alteration of the paranasal sinus. Pathological analysis revealed that epithelial layer injury and metaplasia similar to polyps, with prominent eosinophil infiltration, was induced in recipient tissue. However, there was no nasal polyp development with interstitial edema that was similar to those recognized in human chronic rhinosinusitis. CONCLUSIONS: This study supports the previously unsuspected contribution of eosinophils to CRS development in the murine model and suggests that murine-activated eosinophilic splenocytes contribute to the development of hyposmia due to more mucosal inflammation than physical airway obstruction and epithelial layer injury with convex lesions.