Single cell RNA sequencing of human eosinophils from nasal polyps reveals eosinophil heterogeneity in chronic rhinosinusitis tissue.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 inflammation in the United States, but the actual roles that eosinophils play in CRSwNP remain largely unclear. OBJECTIVE: To reveal the roles and heterogeneity of eosinophils in nasal polyp (NP) tissue, we performed single cell RNA sequencing (scRNA-Seq) analysis of NP tissue. METHODS: Sinonasal tissues (NP and control sinus tissue) and patient matched peripheral blood (PB) samples were obtained from 5 control patients and 5 patients with CRSwNP. Eosinophils were enriched before processing for scRNA-Seq. The gene expression profiles in eosinophils were determined by microwell-based scRNA-Seq technology (BD Rhapsody platform). We predicted the overall function of NP eosinophils by Gene Ontology (geneontology.org) enrichment and pathway analyses and confirmed expression of selected genes by flow cytometry. RESULTS: After filtering out contaminating cells, we detected 5,542 eosinophils from control PB, 3,883 eosinophils from CRSwNP PB, 101 eosinophils from control sinus tissues (not included in further analyses), and 9,727 eosinophils from NPs by scRNA-Seq. We found that 204 genes were downregulated and 354 genes upregulated in NP eosinophils compared to all PB eosinophils (>1.5-fold, Padj < .05). Upregulated genes in NP eosinophils were associated with activation, cytokine-mediated signaling, growth factor activity, NF-κB signaling, and antiapoptotic molecules. NP eosinophils displayed 4 clusters revealing potential heterogeneity of eosinophils in NP tissue. CONCLUSIONS: Elevated eosinophils in NP tissue appear to exist in several subtypes that may play important pathogenic roles in CRSwNP, in part by controlling inflammation and hyperproliferation of other cells.

The current findings in eosinophilic chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a persistent inflammatory disease of the nasal cavity and paranasal sinuses. Traditional classification is denoted by the presence (CRSwNP) or absence of nasal polyps (CRSsNP). Particularly, CRSwNP is distinguished by the presence of infiltrating cells and inflammatory markers in the nasal mucosa. Patients with CRSwNP in Western countries predominantly display a type 2 endotype, whereas those in Asian regions display a mixed type 2 endotype. Nevertheless, recent transcriptome analyses have revealed two types of nasal polyps - type 2 and non-type 2 polyps, suggesting that geographical differences in endotypes likely resulted from the different proportions of each endotype. Moreover, various endotypes of CRSsNP have been identified, making phenotype a crucial factor for predicting treatment efficacy. Type 2 endotypes, designated as eosinophilic CRS (ECRS) in Japan, are characterized by severe eosinophilic infiltration into the paranasal sinus tissue and are particularly refractory. In this review, we discuss the latest developments in ECRS. We also provide recent findings on the involvement of nasal epithelial cells in pathogenesis.

Middle meatus microbiome in patients with eosinophilic chronic rhinosinusitis in a Japanese population.

BACKGROUND: Chronic rhinosinusitis (CRS) is a common chronic inflammatory disease and is subdivided into eosinophilic and noneosinophilic forms. There are few reports investigating the nasal microbiome and its pathological functions in patients with CRS. OBJECTIVE: We sought to analyze factors contributing to variations of the nasal microbiome in CRS, and on the basis of these factors, to elucidate whether the bacterial metabolites were related to the pathogenesis. METHODS: Nasal swabs were collected, and the V3 to V4 variable region of the 16S ribosomal RNA gene was amplified and sequenced. Factors contributing to variations of the nasal microbiome in patients with CRS were compared. The most influential factor was whether CRS was eosinophilic, and we compared α- and ß-diversity, bacterial species, and predictive bacterial functions between the 2 patient groups. In addition, the metabolites of the key bacteria were extracted, and we evaluated the predicted bacterial functions in airway epithelial cells. RESULTS: In total, 110 patients with CRS and 33 control subjects were enrolled. On the basis of the factors of variation, it was found that patients with eosinophilic CRS (n = 65) had different microbiomes with weighted UniFrac ß-diversity and lower α-diversity compared with those with noneosinophilic CRS (n = 45). A higher abundance of Fusobacterium nucleatum and an increased LPS pathway were observed in patients with noneosinophilic CRS compared with those with eosinophilic CRS. In airway epithelial cells, LPS derived from F nucleatum suppressed the expression levels of ALOX15 induced by TH2 cytokines. CONCLUSIONS: The differences in the nasal microbiome may play a key role in the pathophysiology of CRS.

Elevated Serum Leptin Levels in Patients With Eosinophilic Chronic Rhinosinusitis.

Background: Eosinophilic chronic sinusitis (ECRS) is a subtype of CRS with nasal polyps (CRSwNP) that is frequently comorbid with asthma. Notably, ECRS patients often show a high recurrence of NPs after surgical resection. Leptin is a hormone produced by adipocytes that has been implicated in airway inflammatory diseases. However, to date, the role of leptin in ECRS has not been investigated. Objective: To determine whether the serum levels of leptin are altered in patients with ECRS. Methods: In total, 40 patients with ECRS, 15 patients with non-eosinophilic CRS (non-ECRS), and 12 individuals without CRS (control) were included in this study. Patient's serum leptin levels were assessed, and the number of eosinophils in their NPs were measured through a histological evaluation of the three densest areas with cellular infiltrate beneath the epithelial surface. Finally, nasal fibroblast cultures established from NPs were stimulated with varying concentrations of recombinant leptin in vitro to determine whether leptin affects eotaxin-3 (Chemokine (C-C motif) ligand 26 :26: CCL26) expression. Results: The serum leptin levels in both the ECRS and non-ECRS groups were significantly higher than those in the control subjects (p < 0.0001 vs. ECRS; p < 0.05 vs. non-ECRS). Furthermore, ECRS patients displayed significantly elevated serum leptin levels compared to non-ECRS patients (p < 0.001), although there was no difference in body mass index between the groups. Notably, serum leptin levels were correlated with the proportion of eosinophils in peripheral blood (r = 0.3575, p < 0.01) and the number of eosinophils in NPs (r = 0.5109, p < 0.0001). Serum leptin levels were also correlated with eotaxin-3 mRNA expression in NPs (r = 0.5374, p < 0.01). Finally, leptin significantly augmented eotaxin-3 expression in nasal fibroblasts established in vitro from NPs in a leptin receptor-dependent manner (p < 0.05). Conclusion: Leptin levels are elevated in ECRS patients and may both promote and indicate the severity of ECRS as well as systemic type 2-biased inflammatory responses. Combined, these data indicate that circulating leptin may play a significant role in the development of eosinophilic inflammation in NPs.

Enhanced 15-Lipoxygenase 1 Production is Related to Periostin Expression and Eosinophil Recruitment in Eosinophilic Chronic Rhinosinusitis.

BACKGROUND: The pathological features of chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) tissues include an eosinophilic infiltration pattern (eosinophilic CRS (ECRS)) or a less eosinophilic pattern (non-ECRS). Recently, it has been suggested that 15-lipoxygenase 1 (15-LOX-1) may have significant roles in allergic disease; however, the significance of 15-LOX-1 in CRS is not well understood. The objective of this study was to demonstrate the expression of 15-LOX-1 in CRS. METHODS: The mRNA expression levels of 15-LOX-1 and periostin in nasal tissues were measured by quantitative real-time polymerase chain reaction. We also performed an immunofluorescence study of nasal tissues. Cells of the Eol-1 eosinophilic leukemic cell line were stimulated with interleukin-33 to test the induction of 15-LOX-1. RESULTS: The expression level of 15-LOX-1 mRNA in nasal polyps (NPs) was significantly higher in ECRS patients than in non-ECRS patients. The immunofluorescence study revealed that both airway epithelial cells and eosinophils in NPs expressed 15-LOX-1. A significant correlation was seen between the number of eosinophils and the mRNA expression levels of 15-LOX-1 and periostin in nasal polyps. Moreover, interleukin-33 enhanced 15-LOX-1 expression in Eol-1 cells. CONCLUSIONS: 15-LOX-1 was shown to be a significant molecule that facilitates eosinophilic inflammation in ECRS.

High-Yield Production of the Major Birch Pollen Allergen Bet v 1 With Allergen Immunogenicity in Nicotiana benthamiana.

Type I allergy is an immunological disorder triggered by allergens and causes significant health problems. The major allergen of birch pollen is Bet v 1, which belongs to the pathogen-related protein 10 (PR-10) family. Here, we established a rapid and robust method for the production of Bet v 1 in Nicotiana benthamiana leaves, with binding activity to allergic patients' IgE. The Bet v 1 allergen was expressed in N. benthamiana using a strong agroinfiltration-based transient protein expression system, which consists of a deconstructed geminiviral vector system with a double terminator. Five days post-infiltration, the allergen concentration in N. benthamiana leaves was 1.2 mg/g of fresh mass, being this the maximum yield of Bet v 1 in plants reported up to now. A part of plant-derived Bet v 1 was glycosylated. Bet v 1 purified from N. benthamiana or Brevibacillus brevis was used to carry out enzyme-linked immunoassays; both recombinant allergens were found to have comparable binding properties to the IgE of allergic patients. These results suggest that our plant expression system allows rapid and robust production of the allergen, which keeps the immunogenicity.

Serum IgG4 as a biomarker reflecting pathophysiology and post-operative recurrence in chronic rhinosinusitis.

BACKGROUND: Type 2 chronic rhinosinusitis (CRS), especially eosinophilic CRS (ECRS), is an intractable upper airway inflammatory disease. Establishment of serum biomarkers reflecting the pathophysiology of CRS is desirable in a clinical setting. As IgG4 production is regulated by type 2 cytokines, we sought to determine whether serum IgG4 levels can be used as a biomarker for CRS. METHODS: Association between the serum IgG4 levels and clinicopathological factors was analyzed in 336 CRS patients. Receiver operating characteristics (ROC) analysis was performed to determine the cut-off value of serum IgG4 levels that can be used to predict the post-operative recurrence. RESULTS: Serum IgG4 levels were significantly higher in patients with moderate to severe ECRS versus those with non to mild ECRS. The levels were also significantly higher in asthmatic patients and patients exhibiting recurrence after surgery compared to controls. ROC analysis determined that the best cut-off value for the serum IgG4 level to predict the post-operative recurrence was 95 mg/dL. The corresponding sensitivity and specificity were 39.7% and 80.5%, respectively. When we combined the two cut-off values for the serum IgG4 and periostin, patients with high serum levels of either IgG4 or periostin exhibited a high post-operative recurrence (OR: 3.95) as compared to patients having low serum levels of both IgG4 and periostin. CONCLUSIONS: The present results demonstrate that the serum IgG4 level is associated with disease severity and post-operative course in CRS. In particular, the combination of serum IgG4 and periostin could be a novel biomarker that predicts post-operative recurrence.

Eosinophilic chronic rhinosinusitis.

Eosinophilic chronic rhinosinusitis (ECRS) is a subgroup of chronic rhinosinusitis with nasal polyps (CRSwNP), which is associated with severe eosinophilic infiltration and intractable. Its symptoms include dysosmia, nasal obstruction, and visous nasal discharge. The cause of ECRS is not clear, although it is thought that Staphylococcus aureus and its enterotoxins are involved in stimulating the Th2 system to promote IgE production and eosinophil infiltration through various pathways. While, the coagulation system is activated and the fibrinolytic system is suppressed, leading to deposition of fibrinous networks in nasal polyps. Therefore, a fibrin-degrading agent could be a new treatment for ECRS. Genetic analysis of nasal polyp cells using next-generation sequencing has identified some of the factors involved in ECRS, including periostin, which can be used as a biomarker of this condition. A protease inhibitor could be a therapeutic agent for ECRS. Regarding the role of eosinophils, many researchers have been interested in the mechanism of ETosis. However, the mechanism leading to development of nasal polyps is unknown. In Japan (as well as in East Asia), the incidence of non-ECRS is decreasing and that of ECRS is increasing, but the reason is also unknown. Thanks to the development of biologics therapy, it is thought that there will be a shift to precision medicine in the future.

Periostin as a novel biomarker for postoperative recurrence of chronic rhinosinitis with nasal polyps.

We previously reported that chronic rhinosinusitis with nasal polyps (CRSwNP) was subdivided into four chronic rhinosinusitis (CRS) subtypes using the JESREC scoring system. We sought to identify the gene expression profile and biomarkers related with CRSwNP by RNA-sequence. RNA-sequencing was performed to identify differentially expressed genes between nasal polyps (NPs) and inferior turbinate mucosa from 6 patients with CRSwNP, and subsequently, quantitative real-time PCR was performed to verify the results. ELISA was performed to identify possible biomarkers for postoperative recurrence. In the RNA-sequencing results, periostin (POSTN) expression was the highest in NP. We focused on POSTN and investigated the protein level of POSTN by immunohistochemistry and ELISA. POSTN was diffusely expressed in moderate and severe eosinophilic CRS using immunohistochemistry, and its staining pattern was associated with the severity of the phenotype of the CRSwNP (P < 0.05). There was a significant difference between the POSTN high/low groups for postoperative recurrence when the cutoff point was set at 115.5 ng/ml (P = 0.0072). Our data suggests that the protein expression level of POSTN was associated with the severity of CRSwNP, and serum POSTN can be a novel biomarker for postoperative recurrence of CRSwNP.

DNA Methylation of Proximal PLAT Promoter in Chronic Rhinosinusitis With Nasal Polyps.

Background Nasal polyps (NP) are characterized by pseudocysts derived from stromal tissue edema and cause persistent infections in patients with chronic rhinosinusitis (CRS). A low level of tissue-type plasminogen activator (gene name PLAT) is considered a cause of stromal tissue edema because of insufficient plasmin activation in NP; however, the mechanism regulating PLAT gene expression levels is still unclear. The epigenetic mechanism regulating the PLAT gene expression has been studied in other tissues. Objective We aimed to investigate the methylation levels in the proximal PLAT promoter and their effects on gene expression in NP tissue. Methods We investigated the methylation levels at 3 CpG sites in the proximal PLAT promoter regions (-618, -121, and -105 with respect to the transcription initiation site) by bisulfite pyrosequencing and their effects on the gene expression by quantitative real-time polymerase chain reaction (qPCR) in 20 paired samples of NP and inferior turbinate tissue (IT) from patients with CRS. Results The DNA methylation levels at all CpG sites were higher ( P < .01), and the PLAT expression was lower ( P < .001) in NP compared with IT. The methylation changes at the -618 site showed a negative correlation with the gene expression changes between NP and IT ( r = -.65, P < .01). Conclusions Hypermethylation of PLAT promoter may downregulate the gene expression in NP, leading to excessive fibrin deposition by aberrant coagulation cascade. DNA methylation of proximal PLAT promoter may contribute to NP growth and have a potential as a new therapeutic target.

Large cell neuroendocrine carcinoma of the submandibular gland: case report and literature review.

Large cell neuroendocrine carcinoma (LCNEC) of the salivary gland is extremely rare. We report on a case of LCNEC in the submandibular gland. A 58-year-old male had a four-month history of an enlarging mass in his left submandibular region. He underwent lymph node resection and metastasis of LCNEC was suspected. Magnetic resonance imaging of the neck showed a solid submandibular gland tumor with marginal blurring. Positron-emission tomography and upper gastrointestinal endoscopy showed no evidence of malignancy other than in the left submandibular gland. He underwent left submandibular gland resection and left upper neck dissection. The final diagnosis was LCNEC of the submandibular gland; surgical margin was negative. Fourteen months later he is free of tumors. This is the first report of LCNEC of the submandibular gland. LCNEC of the salivary gland shows high-grade malignancy like that of the lung. According to past reports, two of four patients died despite multidisciplinary treatments. There are no standard treatments for LCNEC of the salivary glands. More studies are needed to define prognostic factors and establish therapeutic methods.