Yoshiki Kobayashi

In obstructive sleep apnea (OSA), repeated airway obstruction alters mucosal inflammation, which increases exhaled nitric oxide (NO) production in the nasal cavity. However, the underlying mechanism remains unclear. Accordingly, we aimed to examine the mechanism underlying NO production in patients with OSA. We included eight patients with moderate‐to‐severe OSA who underwent continuous positive airway pressure (CPAP) therapy. Nasal cavity NO levels were measured before and after CPAP; additionally, the levels of inflammatory cytokines in the supernatants of nasal mucosal samples and mRNA expression of inflammation‐related molecules in epithelial cells were analysed. Additionally, we examined changes in inflammation‐related molecules following stimulation of airway epithelial cells with the aforementioned supernatants and after CPAP treatment. Consistent with previous reports, nasal cavity NO levels improved after the introduction of CPAP therapy. There was a significant post‐treatment decrease in the levels of macrophage migration inhibitory factors (MIF) in the nasal mucosal cavity. Additionally, there was a post‐treatment increase in mRNA expression of SIRT1, as well as decreased mRNA expression of HIF‐1α and inducible nitric oxide synthase (iNOS), in nasal mucosal epithelial cells. Similar results were obtained in airway epithelial cells stimulated with supernatants from nasal mucosal samples. Furthermore, airway epithelial cells stimulated with recombinant MIF showed decreased SIRT1 expression, as well as increased HIF‐1α and iNOS expression. This study describes local inflammatory changes in the nasal mucosa of patients with OSA, suggesting that MIF is involved in NO production. Appropriate therapeutic intervention with CPAP can effectively control this inflammation.

We report a case of eosinophilic granulomatosis with polyangiitis (EGPA), manifesting as orbital cellulitis and scleritis following anti-interleukin (IL)-4 receptor α antibody dupilumab treatment, which was successfully treated with anti-IL-5 antibody mepolizumab. Slit-lamp examination, diagnostic imaging, and biopsy were performed. A 53-year-old male patient presented with sudden swelling of the left eyelid, severe conjunctival edema, and hyperemia. He had received dupilumab for 6 months for refractory nasal polyps. On initial examination, slit-lamp examination and computed tomography of the orbit revealed the orbital cellulitis and scleritis in his left eye. Although orbital swelling resolved by the administration of systemic steroids, scleritis persisted. Six months after initial presentation, the patient developed abdominal pain, vomiting, diarrhea, and skin rashes. Biopsy of upper gastrointestinal tract and skin revealed eosinophilic infiltration surrounding the blood vessel with an increase in peripheral blood eosinophil count. The patient was diagnosed with EGPA and subsequently started on subcutaneous anti-IL-5 antibody mepolizumab with systemic steroid. Following initiation of mepolizumab, his eosinophil counts normalized, gastroenteritis symptoms resolved, and refractory scleritis disappeared within a month. Topical and systemic steroids were discontinued after mepolizumab administration without recurrence. This case highlights that physicians should be aware of the potential ocular side effects associated with biologics administration, such as those associated with EGPA.

Eosinophilic chronic rhinosinusitis (ECRS), a CRS with nasal polyps (CRSwNP), is characterized by eosinophilic infiltration with type 2 inflammation and is highly associated with bronchial asthma. Intractable ECRS with poorly controlled asthma is recognized as a difficult-to-treat eosinophilic airway inflammation. Although eosinophils are activated and coincubation with airway epithelial cells prolongs their survival, the interaction mechanism between eosinophils and epithelial cells is unclear. This study examined the effect of eosinophils on mucin glycoprotein 1 (MUC1), a member of membrane-bound mucin, in the airway epithelial cells, to elucidate the mechanisms of the eosinophil–airway epithelial cell interaction. Nasal polyp samples from patients with CRSwNP and BEAS-2B airway epithelial cells, coincubated with purified eosinophils, were stained with two MUC1 antibodies. To confirm the involvement of CCL4, an anti-CCL4 neutralizing antibody or recombinant CCL4 was used as needed. The immunofluorescence results revealed a negative correlation between the expression of full-length MUC1 and eosinophil count in nasal polyps. In BEAS-2B coincubated with eosinophils, full-length MUC1, but not the C-terminal domain, was reduced, and eosinophil survival was prolonged, which was concomitant with CCL4 increase, whereas the anti-CCL4 neutralizing antibody decreased these reactions. The survival of eosinophils that contacted recombinant MUC1 without the N-terminal domain was prolonged, and recombinant CCL4 increased the expression of metalloproteases. Increased CCL4 induces the contact between eosinophils and airway epithelial cells by shedding the MUC1 N-terminal domain and enhances eosinophil survival in eosinophilic airway inflammation. This novel mechanism may be a therapeutic target for difficult-to-treat eosinophilic airway inflammation.

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.

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.

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-1β—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)β, SRC kinase family (Lck, Src, and Yes), and extracellular signal-regulated kinase (ERK), was upregulated. Further, CCR5, PDGFRβ, 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.

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.

Eosinophilic airway inflammatory disease is associated with bronchial asthma, with eosinophilic chronic rhinosinusitis (ECRS) typical of refractory type 2 airway inflammation. CCL4 produced at local inflammatory sites is involved in them via the accumulation and activation of type 2 inflammatory cells, including eosinophils. The detailed mechanism of CCL4 production remains unclear, and also the possibility it could function as a biomarker of type 2 airway inflammation remains unresolved. In this study, we evaluated CCL4 mRNA expression and production via the TSLP receptor (TSLPR) and toll-like receptors (TLRs) or proteinase-activated receptor-2 (PAR2) in BEAS-2B bronchial epithelial cells co-incubated with purified eosinophils or eosinophil peroxidase (EPX). We examined serum chemokine (CCL4, CCL11, CCL26, and CCL17) and total IgE serum levels, fractionated exhaled nitrogen oxide (FENO), and CCL4 expression in nasal polyps in patients with severe ECRS and asthma. CCL4 was induced by TSLP under eosinophilic inflammation. Furthermore, CCL4 was released via TLR3 signaling, which was enhanced by TSLP. CCL4 was mainly located in nasal polyp epithelial cells, while serum CCL4 levels were reduced after dupilumab treatment. Serum CCL4 levels were positively correlated with FENO, serum IgE, and CCL17 levels. Thus, CCL4 released from epithelial cells via the innate immune system during type 2 airway inflammation may function as a useful biomarker for the condition.