Role of basal cells in nasal polyp epithelium in the pathophysiology of eosinophilic chronic rhinosinusitis (eCRS).

BACKGROUND: Basal cell hyperplasia is commonly observed in nasal polyp epithelium of eosinophilic chronic rhinosinusitis (eCRS). We examined the function and mechanisms of basal cell hyperplasia in the pathophysiology of eCRS. METHODS: We found that normal human bronchial epithelial (NHBE) cells obtained basal cell characteristics when cultured with PneumaCult™-Ex Plus Medium. Most of the cells passaged three times expressed basal cell surface markers CD49f and CD271 by flow cytometry, and basal cell nuclear marker p63 by immunohistochemical staining. We named these NHBE cells with basal cell characteristics cultured Basal-like cells (cBC), and NHBE cells cultured with BEGM™ cultured Epithelial cells (cEC). The characteristics of cBC and cEC were examined and compared by RNA sequencing, RT-PCR, ELISA, and cell proliferation studies. RESULTS: RNA sequencing revealed that cBC showed higher gene expression of thymic stromal lymphopoietin (TSLP), IL-8, TLR3, and TLR4, and lower expression of PAR-2 compared with cEC. The mRNA expression of TSLP, IL-8, TLR3, and TLR4 was significantly increased in cBC, and that of PAR-2 was significantly increased in cEC by RT-PCR. Poly(I:C)-induced TSLP production and LPS-induced IL-8 production were significantly increased in cBC. IL-4 and IL-13 stimulated the proliferation of cBC. Finally, the frequency of p63-positive basal cells was increased in nasal polyp epithelium of eCRS, and Ki67-positive proliferating cells were increased in p63-positive basal cells. CONCLUSIONS: Type 2 cytokines IL-4 and IL-13 induce basal cell hyperplasia, and basal cells exacerbate type 2 inflammation by producing TSLP in nasal polyp of eCRS.

Immunological effects of sublingual immunotherapy with Japanese cedar pollen extract in patients with combined Japanese cedar and Japanese cypress pollinosis.

Sublingual immunotherapy (SLIT) with Japanese cedar (JCe) pollinosis was expected to be effective for Japanese cypress (JCy) pollinosis. However, only a half of JCy pollinosis patients clinically improved. Therefore, we examined the immunological effect of SLIT for JCy pollinosis. Peripheral blood mononuclear cells (PBMCs) from patients with JCe and JCy pollinosis who did and did not receive SLIT were incubated with Cry j 1, Cha o 1 and Cha o 3 antigens. Basophil activation test (BAT) were performed. Production of IL-5 and IL-17 induced by antigens was inhibited in the SLIT group. Cry j 1-specific production of IL-10 was increased, and serum Cry j 1-specific IgE and -IgG4 were elevated. However, Cha o 1- or Cha o 3-specific production of IL-10 and specific IgG4 was not increased. Antigens-specific BAT did not decrease after SLIT. New SLIT with JCe and JCy is needed for patients with combined JCe and JCy pollinosis.

Evidence for the induction of Th2 inflammation by group 2 innate lymphoid cells in response to prostaglandin D2 and cysteinyl leukotrienes in allergic rhinitis.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play important roles in allergic inflammation. However, their roles in the pathophysiology of allergic rhinitis (AR) are poorly understood. OBJECTIVE: Prevalence of ILC2s in the inferior nasal turbinate (INT) tissues and the activating mechanisms of ILC2s were examined in patients with house dust mite (HDM)-induced AR. METHODS: Eighteen patients with HDM-induced AR and 13 control subjects were recruited. Fresh INT tissues and peripheral blood mononuclear cells (PBMCs) were analysed using flow cytometry. Nasal lavage fluids (NLF) were collected at 10 minutes after the nasal provocation test (NPT) with HDM disc, and released mediators were measured by ELISA. Sorted ILC2s were cultured and stimulated with mediators associated with AR. RESULTS: The prevalence of ILC2s was significantly increased in nasal mucosa of patients with HDM-induced AR, and it was positively correlated with the number of infiltrating eosinophils. ILC2s in the INT tissues expressed a prostaglandin D2 (PGD2 ) receptor, chemoattractant receptor-homologous molecule-expressed TH2 cells (CRTH2) and a cysteinyl leukotriene (cysLTs) receptor, CysLT1. After NPT, the number of eosinophils and concentrations of PGD2 and cysLTs were significantly increased in the NLF from AR patients. PGD2 and cysLTs significantly induced IL-5 production from cultured PBMC-derived ILC2s dose-dependently. PGD2 -induced and cysLTs-induced productions of IL-5 and IL-13 from ILC2s were completely inhibited by ramatroban, a dual CRTH2 and thromboxane receptor antagonist, and montelukast, a CysLT1 antagonist, respectively. CONCLUSIONS: PGD2 -CRTH2 and cysLTs-CysLT1 axes may activate tissue-resident ILC2s to produce Th2 cytokines, IL-5 and IL-13, leading to the development of allergic inflammation in AR.

A mechanism of interleukin-25 production from airway epithelial cells induced by Japanese cedar pollen.

IL-25 likely has vital roles in initiating and activating type-2 immune responses in AR. We hypothesized that the molecules produced IL-25 by allergen-producing organisms such as JC is involved in the pathogenesis of AR. Participants included 13 patients with Japanese cedar pollinosis and 10 HCs. We measured the IL-25 protein concentration in nasal secretions and in culture supernatants of PNECs. NHBE cells were stimulated with pharmacological and immunological agents and JC. The IL-25 concentration in nasal secretions was significantly higher in patients with Japanese cedar pollinosis than in HCs. JC stimulated IL-25 production from PNECs. TNF-α, IL-4, and IL-13 significantly enhanced JC-induced IL-25 production; their activation by serine proteases was sufficient to enhance IL-25 production. Furthermore, the NADPH oxidase activity, including JC enhanced IL-25 production. A better understanding of JC-induced IL-25 production by epithelial cells may allow the development of novel therapeutic and preventive strategies for Japanese cedar pollinosis.

The epidermal growth factor receptor inhibitor AG1478 inhibits eosinophilic inflammation in upper airways.

Mucus hypersecretion and eosinophil infiltration are important characteristics of eosinophilic inflammation in upper airways, such as allergic rhinitis and chronic rhinosinusitis with nasal polyp. EGFR transactivation induces mucus and inflammatory cytokine secretion from airway epithelial cells. However, the roles of EGFR in eosinophilic inflammation in upper airways are still unknown. The purpose of the study is to elucidate the effects of the EGFR inhibitor AG1478 on eosinophilic airway inflammation. AG1478 significantly inhibited thrombin-induced GM-CSF secretion from nasal epithelial cells and thrombin-induced secretion of eotaxin-1 and RANTES from nasal fibroblasts. Intranasal instillation of AG1478 inhibited OVA-induced goblet cell metaplasia, mucus production and eosinophil/neutrophil infiltration in rat nasal epithelium, as did intraperitoneal injection of AG1478. These results indicate that EGFR transactivation plays an important role in eosinophilic airway inflammation. Intranasal instillation of an EGFR inhibitor may be a new therapeutic approach for the treatment of intractable eosinophilic inflammation in upper airways.

Endogenous Protease Inhibitors in Airway Epithelial Cells Contribute to Eosinophilic Chronic Rhinosinusitis.

RATIONALE: Cystatin A and SPINK5 are endogenous protease inhibitors (EPIs) that may play key roles in epithelial barrier function. OBJECTIVES: To investigate the roles of EPIs in the pathogenesis of chronic rhinosinusitis (CRS). METHODS: We examined the expression of cystatin A and SPINK5 in the nasal epithelial cells of patients with CRS. Additionally, the in vitro effects of recombinant EPIs on the secretion of the epithelial-derived cytokines IL-25, IL-33, and thymic stromal lymphopoietin in airway epithelial cells, and the in vivo effects of recombinant EPIs in the nasal epithelium of mice exposed to multiple airborne allergens (MAA) were examined. MEASUREMENTS AND MAIN RESULTS: Compared with control subjects and patients with noneosinophilic CRS, patients with eosinophilic CRS showed significantly lower protein and mRNA expression of cystatin A and SPINK5 in the nasal epithelium. Allergen-induced production of IL-25, IL-33, and thymic stromal lymphopoietin in normal human bronchial epithelial cells was inhibited by treatment with recombinant cystatin A or SPINK5. Conversely, the production of these cytokines was increased when cystatin A or SPINK5 were knocked down with small interfering RNA. Chronic MAA exposure induced goblet cell metaplasia and epithelial disruption in mouse nasal epithelium and decreased the tissue expression and nasal lavage levels of cystatin A and SPINK5. Intranasal instillations of recombinant EPIs attenuated this MAA-induced pathology. CONCLUSIONS: Cystatin A and SPINK5 play an important role in protecting the airway epithelium from exogenous proteases. The preservation of EPIs may have a therapeutic benefit in intractable airway inflammation, such as eosinophilic CRS.

Soluble ST2 suppresses IL-5 production by human basophilic KU812 cells, induced by epithelial cell-derived IL-33.

BACKGROUND: Epithelial cell-derived IL-33 has an important role in the initiation and activation of innate allergic inflammation. IL-33 acts as a cytokine through the ST2 receptor (ST2L) and it stimulates the production of Th2 cytokines. Soluble ST2 (sST2) may regulate Th2 responses by neutralizing the activity of IL-33. Basophils express ST2L and produce IL-5 in response to IL-33. However, the role of the epithelial cell-basophil interaction and sST2 in IL-5 production remains unclear. METHODS: Cultured human bronchial epithelial (hBE33) cells, that contained the human IL-33 gene (i.e., hBE33 cells) and a human basophilic cell line, KU812 cells, were used to study the epithelial cell-basophil interaction in the production of IL-5 induced by HDM. RESULTS: At 15 min after incubation, HDM stimulated the rapid release of IL-33 from cultured hBE33 cells. IL-33 and the supernatant of HDM-treated hBE33 cells stimulated IL-5 production from KU812 cells. Anti-IL-33 antibody and anti-ST2 antibody treatment of KU812 cells suppressed IL-5 production, which had been induced by the supernatant of HDM-treated hBE33 cells. The hBE33 cells secreted sST2 in a time-dependent manner. The production of sST2 by KU812 cells co-cultured with hBE33 cells was significantly increased, compared with KU812 cells cultured with the supernatant of hBE33 cells. Soluble ST2 suppressed IL-5 production by KU812 cells, which was induced by the supernatant of HDM-treated hBE33 cells. CONCLUSIONS: Epithelial cell-derived IL-33 promoted IL-5 production by KU812 cells. The subsequently produced sST2 has important roles in regulating Th2 responses.

Group 2 innate lymphoid cells are increased in nasal polyps in patients with eosinophilic chronic rhinosinusitis.

ILC2s represent a critical innate cellular source of type 2 cytokines and may play important roles in various diseases. We examined the role of ILC2s in the pathogenesis of two subgroups of CRSwNP: ECRS and non-ECRS. We analyzed the prevalence of ILC2s in sinonasal tissues and in blood from patients with ECRS, non-ECRS, CRSsNP, and control. The prevalence of ILC2s in nasal tissues was higher in patients with ECRS as compared to those with non-ECRS or CRSsNP. The prevalence of blood ILC2s was not different between patients with ECRS and non-ECRS. The prevalence of blood ILC2s was higher in patients with allergic rhinitis and elevated serum IgE levels. Alternaria-induced IL-33 secretion was increased in nasal epithelial cells derived from patients with ECRS as compared to those from patients with non-ECRS or CRSsNP. ILC2s may be involved in the pathogenesis of CRSwNP, in particular in patients with tissue eosinophilia (i.e., ECRS).

Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa.

Although type 2 immune responses to environmental Ags are thought to play pivotal roles in asthma and allergic airway diseases, the immunological mechanisms that initiate the responses are largely unknown. Many allergens have biologic activities, including enzymatic activities and abilities to engage innate pattern-recognition receptors such as TLR4. In this article, we report that IL-33 and thymic stromal lymphopoietin were produced quickly in the lungs of naive mice exposed to cysteine proteases, such as bromelain and papain, as a model for allergens. IL-33 and thymic stromal lymphopoietin sensitized naive animals to an innocuous airway Ag OVA, which resulted in production of type 2 cytokines and IgE Ab, and eosinophilic airway inflammation when mice were challenged with the same Ag. Importantly, upon exposure to proteases, uric acid (UA) was rapidly released into the airway lumen, and removal of this endogenous UA by uricase prevented type 2 immune responses. UA promoted secretion of IL-33 by airway epithelial cells in vitro, and administration of UA into the airways of naive animals induced extracellular release of IL-33, followed by both innate and adaptive type 2 immune responses in vivo. Finally, a potent UA synthesis inhibitor, febuxostat, mitigated asthma phenotypes that were caused by repeated exposure to natural airborne allergens. These findings provide mechanistic insights into the development of type 2 immunity to airborne allergens and recognize airway UA as a key player that regulates the process in respiratory mucosa.

Increase in the prevalence of follicular regulatory T cells correlates with clinical efficacy of sublingual immunotherapy with house dust mites.

BACKGROUND: Allergic rhinitis (AR) impairs quality of life and affects nearly 40% of the Japanese population. Sublingual immunotherapy (SLIT) is the disease-modifying treatment for AR, but requires the selection of a biomarker associate with clinical efficacy in patients with AR who are treated with SLIT. The present study sought to examine objective biomarkers used for assessing the clinical efficacy of SLIT. METHODS: The authors examined the effects of 1 year of SLIT treatment with house dust mites (HDMs) using peripheral blood mononuclear cells (PBMCs) and serum from patients with AR. The prevalences of follicular regulatory T (Tfr), type 2 follicular helper T (Tfh2), type 2 helper T (Th2), conventional regulatory T (Treg), and type 1 regulatory T (Tr1) cells were examined by flow cytometry. Serum concentrations of HDM-specific IgA, IgE, and IgG4 antibodies, and HDM-induced production of interleukin (IL) 5 and IL-10 from cultured PBMCs were evaluated by enzyme-linked immunosorbent assay. RESULTS: Following 1 year of SLIT, the prevalences of Tfr, conventional Treg, and Tr1 cells were significantly increased, whereas that of Th2 cells and Tfh2 cells were significantly decreased; the serum concentration of HDM-specific IgG4 was significantly increased; and HDM-induced production of IL-5 from PBMCs was significantly decreased, while that of IL-10 was significantly increased. The increase in the prevalence of Tfr cells after SLIT correlated positively with the improvement of clinical symptom scores. CONCLUSION: An increase in Tfr cells may play an important role in SLIT, and may be a useful indicator for the clinical efficacy of SLIT.

SARS-CoV-2 induces inflammation and intracranial infection through the olfactory epithelium-olfactory bulb pathway in non-human primates.

We examined the histopathological changes in the olfactory mucosa of cynomolgus and rhesus macaque models of SARS-CoV-2 infection. SARS-CoV-2 infection induced severe inflammatory changes in the olfactory mucosa. A major histocompatibility complex (MHC) class II molecule, HLA-DR was expressed in macrophage and supporting cells, and melanocytes were increased in olfactory mucosa. Supporting cells and olfactory neurons were infected, and SARS-CoV-2 N protein was detected in the axons of olfactory neurons and in olfactory bulbs. Viral RNA was detected in olfactory bulbs and brain tissues. The olfactory epithelium-olfactory bulb pathway may be important as a route for intracranial infection by SARS-CoV-2.

Practical guide for the diagnosis and management of primary ciliary dyskinesia.

OBJECTIVE: Primary ciliary dyskinesia (PCD) is a relatively rare genetic disorder that affects approximately 1 in 20,000 people. Approximately 50 genes are currently known to cause PCD. In light of differences in causative genes and the medical system in Japan compared with other countries, a practical guide was needed for the diagnosis and management of Japanese PCD patients. METHODS: An ad hoc academic committee was organized under the Japanese Rhinologic Society to produce a practical guide, with participation by committee members from several academic societies in Japan. The practical guide including diagnostic criteria for PCD was approved by the Japanese Rhinologic Society, Japanese Society of Otolaryngology-Head and Neck Surgery, Japanese Respiratory Society, and Japanese Society of Pediatric Pulmonology. RESULTS: The diagnostic criteria for PCD consist of six clinical features, six laboratory findings, differential diagnosis, and genetic testing. The diagnosis of PCD is categorized as definite, probable, or possible PCD based on a combination of the four items above. Diagnosis of definite PCD requires exclusion of cystic fibrosis and primary immunodeficiency, at least one of the six clinical features, and a positive result for at least one of the following: (1) Class 1 defect on electron microscopy of cilia, (2) pathogenic or likely pathogenic variants in a PCD-related gene, or (3) impairment of ciliary motility that can be repaired by correcting the causative gene variants in iPS cells established from the patient's peripheral blood cells. CONCLUSION: This practical guide provides clinicians with useful information for the diagnosis and management of PCD in Japan.

Transcription of interleukin-25 and extracellular release of the protein is regulated by allergen proteases in airway epithelial cells.

Epithelial cells at mucosal surfaces are integral components of innate and adaptive immunity. IL-25 is reportedly produced by epithelial cells and likely plays vital roles in regulating type-2 immune responses. However, little is known regarding the mechanisms that control production and extracellular releases of IL-25. We hypothesized that proteases from the multiple allergens may induce IL-25 production in airway epithelial cells. In this study, we found that IL-25 is constitutively produced and detectable in cytoplasm of resting normal human bronchial epithelial (NHBE) cells. When exposed to airborne allergens such as house dust mite (HDM), stored IL-25 was released rapidly to the extracellular space. IL-25 release was not accompanied by cell death, suggesting involvement of active secretory mechanism(s). HDM also enhanced IL-25 mRNA transcription, which was dependent on their protease activities. Furthermore, activation of NHBE cells with authentic proteases, such as trypsin and papain, or with a peptide agonist for protease-activated receptor 2 was sufficient to enhance IL-25 mRNA transcription and protein. Protease-driven increase in mRNA transcription and allergen-driven extracellular release of IL-25 protein was also observed in primary nasal epithelial cells from healthy individuals. These findings suggest that IL-25 production by airway epithelial cells is regulated by the transcription and protein release levels and that allergen proteases likely play pivotal roles in both biological processes.

Therapeutic Potential of Valproic Acid for Postviral Olfactory Dysfunction: A Single-Arm Pilot Study.

OBJECTIVES: Although some patients with postviral olfactory dysfunction (PVOD) recover spontaneously, many others are left with the degree of smell loss and there are no established drugs for the treatment of patients with PVOD. Valproic acid (VPA) has been widely used for the treatment of epilepsy. Its potential neuroregenerative effects have been shown via animal studies. This is the first study to treat PVOD patients with VPA. This open-label, single-arm, phase II study was conducted to investigate the effects of VPA in patients with PVOD. METHODS: The patients received oral tablets of VPA 200 mg twice a day for 24 weeks. In total, 11 patients with PVOD were recruited. Oder scores of recognition and detection threshold (measured with a T&T olfactometer), and visual analog scale were examined during the treatment. RESULTS: All odor scores significantly improved over time. Although the mean duration of olfactory dysfunction in this study was 11.5 months, both odor recognition threshold and odor detection threshold scores significantly improved 4 weeks after treatment initiation compared to the pre-treatment threshold scores. The olfactory recovery rates in patients treated with VPA were clearly better than those we previously reported in PVOD patients who received Toki-shakuyaku-san, the traditional treatment in Japan. The olfactory recovery rates of patients with PVOD at 12 weeks and 24 weeks of VPA treatment were both 77.8%, and the olfactory cure rates at 12 weeks and 24 weeks of VPA treatment were 33.3% and 44.4%, respectively. No serious adverse events were observed. CONCLUSIONS: VPA seems to be a safe treatment option in patients with PVOD. The effects of VPA treatment for PVOD patients should be studied with a controlled study design in the future.

A decreased prevalence of group 2 innate lymphoid cells in blood is associated with good postoperative outcomes in patients with chronic rhinosinusitis.

OBJECTIVE: Due to the high postoperative recurrence rate in eosinophilic chronic rhinosinusitis (eCRS) patients, there is a need for an index to predict the postoperative outcomes. Group 2 innate lymphoid cells (ILC2s) are important effector cells for type 2 immune responses in eosinophilic airway inflammation. The aim of this study was to investigate whether the prevalence of ILC2s in sinonasal tissues or in peripheral blood is associated with the postoperative outcome in CRS patients. METHODS: Twelve patients with eCRS and ten patients with non-eCRS were recruited. We examined the ILC2 prevalence in sinonasal tissues and in peripheral blood before and after endoscopic sinus surgery (ESS). Pre- and postoperative blood eosinophil counts were also examined. Lund-Mackay computed tomography (LMK-CT) scores were used to evaluate the disease severities and the postoperative outcomes; cases with more than 50% improvement were categorized into the good outcome group, and cases with less than 50% improvement were categorized into the poor outcome group. RESULTS: The ILC2 prevalence in sinonasal tissues was correlated with that in preoperative blood in eCRS and non-eCRS patients. The ILC2 prevalence in sinonasal tissues and in preoperative blood was not correlated with the pre- or postoperative LMK-CT scores. Postoperatively, the ILC2 prevalence in blood was decreased in eCRS and non-eCRS patients, and blood eosinophil count was also decreased in eCRS patients but not in non-eCRS patients. The ILC2 prevalence in postoperative blood was decreased in the good outcome group but not in the poor outcome group. Blood eosinophil counts were not decreased postoperatively in both good and poor outcome groups. CONCLUSION: The decreased ILC2 prevalence in postoperative blood may be a predictive biomarker for evaluating postoperative outcomes in eCRS and non-eCRS patients.

17,18-Epoxyeicosatetraenoic Acid Inhibits TNF-α-Induced Inflammation in Cultured Human Airway Epithelium and LPS-Induced Murine Airway Inflammation.

BACKGROUND: 17,18-Epoxyeicosatetraenoic acid (17,18-EpETE), an eicosapentaenoic acid metabolite, is generated from dietary oil in the gut, and antiinflammatory activity of 17,18-EpETE was recently reported. OBJECTIVE: To evaluate the inhibitory effects of 17,18-EpETE in airway inflammation, we examined in vitro and in vivo effects on mucus production, neutrophil infiltration, and cytokine/chemokine production in airway epithelium. METHODS: Nasal tissue localization of G protein-coupled receptor 40 (GPR40), a receptor of 17,18-EpETE, was determined by immunohistochemical staining. Expression of GPR40 mRNA in nasal mucosa of chronic rhinosinusitis (CRS) patients and control subjects was determined by reverse transcription-polymerase chain reaction (RT-PCR). The in vitro effects on airway epithelial cells were examined using normal human bronchial epithelial cells and NCI-H292 cells. To examine the in vivo effects of 17,18-EpETE on airway inflammation, we induced goblet cell metaplasia, mucus production, and neutrophil infiltration in mouse nasal epithelium by intranasal lipopolysaccharide (LPS) instillation. RESULTS: GPR40 is mainly expressed in human nasal epithelial cells and submucosal gland cells. RT-PCR analysis revealed that the expression of GPR40 mRNA was increased in nasal tissues from CRS patients compared with those from control subjects. 17,18-EpETE significantly inhibited tumor necrosis factor (TNF)-α-induced production of interleukin (IL)-6 , IL-8, and mucin from cultured human airway epithelial cells dose dependently, and these antiinflammatory effects on cytokine production were abolished by GW1100, a selective GPR40 antagonist. Intraperitoneal injection or intranasal instillation of 17,18-EpETE significantly attenuated LPS-induced mucus production and neutrophil infiltration in mouse nasal epithelium. Inflammatory cytokine/chemokine production in lung tissues and bronchoalveolar lavage fluids was also inhibited. CONCLUSION: These results indicate that 17,18-EpETE plays a regulatory role in mucus hypersecretion and neutrophil infiltration in nasal inflammation. Local or systemic administration may provide a new therapeutic approach for the treatment of intractable airway disease such as CRS.

Nasal polyp fibroblasts (NPFs)-derived exosomes are important for the release of vascular endothelial growth factor from cocultured eosinophils and NPFs.

OBJECTIVE: Significant eosinophil infiltration and tissue remodeling are common characteristics of conditions associated with chronic airway inflammation, such as chronic rhinosinusitis with nasal polyp and bronchial asthma. This study was designed to elucidate the role of eosinophil-fibroblast interactions in tissue remodeling during chronic airway inflammation. METHODS: Peripheral blood eosinophils or EoL-1 eosinophilic leukemia cells were cocultured with nasal polyp fibroblasts (NPFs). Coculture-induced release of exosomes, major components of extracellular vesicles (EVs), and a profibrotic cytokine, vascular endothelial growth factor (VEGF), were evaluated by enzyme-linked immunosorbent assay. RESULTS: Eosinophil-NPF interactions stimulated the release of exosomes and VEGF into culture supernatants. Coculture-induced release of exosomes was stimulated earlier than VEGF release, at 3 h of incubation. The average size of the EVs released by NPFs was 133 ± 3.6 nm. NPF-derived EVs (exosome concentration: 25 pg/mL) significantly stimulated VEGF release from EoL-1 cells. Pretreatment of NPFs with exosome inhibitor, GW4869 or DMA attenuated the release of exosomes and VEGF from cocultured EoL-1 cells and NPFs. CONCLUSION: The results of this study indicate that eosinophil-fibroblast interactions are important in the pathophysiology of tissue remodeling in eosinophil-predominant airway inflammation and that NPF-derived exosomes play a crucial role in the release of VEGF.