The interleukin-4/interleukin-13 pathway in type 2 inflammation in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is predominantly a type 2 inflammatory disease associated with type 2 (T2) cell responses and epithelial barrier, mucociliary, and olfactory dysfunction. The inflammatory cytokines interleukin (IL)-4, IL-13, and IL-5 are key mediators driving and perpetuating type 2 inflammation. The inflammatory responses driven by these cytokines include the recruitment and activation of eosinophils, basophils, mast cells, goblet cells, M2 macrophages, and B cells. The activation of these immune cells results in a range of pathologic effects including immunoglobulin E production, an increase in the number of smooth muscle cells within the nasal mucosa and a reduction in their contractility, increased deposition of fibrinogen, mucus hyperproduction, and local edema. The cytokine-driven structural changes include nasal polyp formation and nasal epithelial tissue remodeling, which perpetuate barrier dysfunction. Type 2 inflammation may also alter the availability or function of olfactory sensory neurons contributing to loss of sense of smell. Targeting these key cytokine pathways has emerged as an effective approach for the treatment of type 2 inflammatory airway diseases, and a number of biologic agents are now available or in development for CRSwNP. In this review, we provide an overview of the inflammatory pathways involved in CRSwNP and describe how targeting key drivers of type 2 inflammation is an effective therapeutic option for patients.

S100a9 might act as a modulator of the Toll-like receptor 4 transduction pathway in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyp (CRSwNP) is a highly prevalent disorder characterized by persistent nasal and sinus mucosa inflammation. Despite significant morbidity and decreased quality of life, there are limited effective treatment options for such a disease. Therefore, identifying causal genes and dysregulated pathways paves the way for novel therapeutic interventions. In the current study, a three-way interaction approach was used to detect dynamic co-expression interactions involved in CRSwNP. In this approach, the internal evolution of the co-expression relation between a pair of genes (X, Y) was captured under a change in the expression profile of a third gene (Z), named the switch gene. Subsequently, the biological relevancy of the statistically significant triplets was confirmed using both gene set enrichment analysis and gene regulatory network reconstruction. Finally, the importance of identified switch genes was confirmed using a random forest model. The results suggested four dysregulated pathways in CRSwNP, including "positive regulation of intracellular signal transduction", "arachidonic acid metabolic process", "spermatogenesis" and "negative regulation of cellular protein metabolic process". Additionally, the S100a9 as a switch gene together with the gene pair {Cd14, Tpd52l1} form a biologically relevant triplet. More specifically, we suggested that S100a9 might act as a potential upstream modulator in toll-like receptor 4 transduction pathway in the major CRSwNP pathologies.

[Correlation study of NCF2 in chronic rhinosinusitis with nasal polyps].

Objective:After selecting NCF2 based on bioinformatics, clinical experiments were conducted to verify the expression of NCF2 in chronic rhinosinusitis with nasal polyps to study its correlation. Methods:The differentially expressed genes（DEGs） between CRSwNP and non-CRS patients were explored using the CRS-related dataset from the gene expression omnibus GEO database. The weighted gene co-expression network（WGCNA） was used for cluster analysis. The expression and cell distribution of NCF2 in the tissues were determined by single gene enrichment analysis（GSEA）, immune inflammatory infiltration analysis, and principal component（PCA） analysis. The expression degree of NCF2 in the tissues of the subjects was determined by immunohistochemistry, and the percentage of EOS in the peripheral blood of the subjects was detected and the correlation was analyzed. EOS in the tissues of the subjects were counted under a microscope and compared. Results:①The Venn diagram was obtained by crossing the module with the highest correlation between DEGs and WGCNA to determine the core gene NCF2. ②GSEA analysis showed that NCF2 was significantly related to the immunological processes such as allogeneic rejection and asthma. ③The area under the ROC curve was 1, indicating that NCF2 had diagnostic value for CRSwNP. ④NCF2 was highly expressed in nasal polyps, mainly distributed in monocytes and eosinophils. ⑤HE staining showed that the number of EOS in ECRSwNP tissues and the percentage of eosinophils in peripheral blood were higher than those in nonECRSwNP and control groups. ⑥The immunohistochemistry results showed that NCF2 was significantly expressed in the nasal polyps of ECRSwNP patients, which was higher than that in the nasal mucosa of nonECRSwNP group and control group. ⑦The expression of NCF2 in tissues was positively correlated with EOS count in ECRSwNP group and EOS expression in peripheral blood. Conclusion:The expression of NCF2 is increased in eosinophilic chronic rhinosinusitis with nasal polyps, and it is significantly correlated with the expression of eosinophils in peripheral blood and tissues, suggesting that NCF2 may be used as a basis for the intrinsic classification of ECRSwNP and a reference index for clinical diagnosis and treatment.

Butyrate inhibits type 2 inflammation in eosinophilic chronic rhinosinusitis.

Butyrate and other Short-chain fatty acids (SCFAs) are microbial metabolites from Bacteroides and Clostridium species that may suppress type 2 inflammation. However, the mechanisms of SCFAs in the nasal sinuses are not fully understood. We aimed to clarify the in vitro and in vivo roles of SCFAs in eosinophilic chronic rhinosinusitis (ECRS) pathophysiology. We investigated whether SCFAs induced changes in type 2 cytokines, IgE, and apoptosis and the roles of GPR41, GPR43, and histone deacetylase. Analysis of the control subjects demonstrated that butyrate of SCFAs effectively inhibited type 2 cytokine production in PBMCs, ILC2s, and CD4+ T cells and IgE production in CD19+ B cells. In annexin V analysis, butyrate also induced late apoptosis of PBMCs. The butyrate-induced inhibition of type 2 cytokines appeared involved in histone deacetylase inhibition but not in GPR41 or GPR43. In an analysis of ECRS in humans, butyrate inhibited type 2 cytokine production in PBMCs and nasal polyp-derived cells. The butyrate concentration in nasal lavage fluid was significantly decreased in ECRS patients compared to controls and non-ECRS patients. Our findings confirm that butyrate can inhibit type 2 inflammation and may be a potential therapeutic target for ECRS.

The cyclooxygenase-2 upregulation mediates production of PGE2 autacoid to positively regulate interleukin-6 secretion in chronic rhinosinusitis with nasal polyps and polyp-derived fibroblasts.

Chronic rhinosinusitis (CRS) can be traditionally classified as CRSwNP [with nasal polyps (NPs)] and CRSsNP (without NPs) based on the clinical phenotypes but recently suggested to be classified by the endotypes. We have identified overexpression of the cyclooxygenase-2 (COX-2) gene in NP tissues of Taiwanese CRSwNP patients. Therefore, in this study, we sought to investigate its protein expression/location/distribution in NP specimens and explore its roles in nasal polyposis. The COX-2 protein and mRNA expression was found higher in NPs than that in the control and CRSsNP patients' nasal tissues, mainly located at the epithelium and subepithelial stroma. Consistently, the CRS-related peptidoglycan (PGN) and bradykinin provoked COX-2 mRNA and protein upregulation in the human NP-derived fibroblasts and caused PGE2, thromboxane A2 (TXA2), and interleukin (IL-6) secretion in culture medium. Further analysis revealed that the PI3K/Akt activation and COX-2 induction were necessarily required for PGN-induced IL-6 production/secretion and the induced PGE2, but not TXA2, was speculated to affect IL-6 protein trafficking and production. Finally, the IL-6 increase observed in vitro could also be detected in NP tissues. Collectively, we demonstrated here that COX-2 protein and IL-6 are overexpressed in human NP tissues. In response to PGN challenge, the PI3K/Akt activation and COX-2-mediated PGE2 autacoid correlates with extracellular IL-6 protein trafficking/production in NP-derived fibroblasts, which can additionally contribute to the production of Th17-related cytokines such as IL-17 and TNF-α. This study also suggests COX-2 as a special biomarker for CRSwNP endotyping and may highlight the importance of COX-2 inhibitors in treating CRSwNP.

The effect of nasal douching by hypertonic 2.3 per cent sea water with algae extracts on the concentration of epidermal growth factor, transforming growth factor-α and interleukin-8 in nasal secretions of patients with nasal polyposis following endoscopic surgical treatment.

OBJECTIVE: To investigate epidermal growth factor, transforming growth factor-α and interleukin-8 production in nasal mucosa irrigated with hypertonic 2.3 per cent solution with algae extracts, in comparison to 0.9 per cent NaCl during the first two weeks after surgery for nasal polyposis, in relation to symptoms and local findings. METHODS: This prospective study included 20 nasal polyposis patients postoperatively irrigated with hypertonic solution and 20 nasal polyposis patients postoperatively irrigated with isotonic solution. We evaluated nasal symptom score, endoscopic score and mediator levels in nasal secretions before and after irrigation. RESULTS: Following treatment, nasal symptom score and endoscopic score were significantly lower in the hypertonic solution group (p = 0.023; p < 0.001, respectively). The increase in the epidermal growth factor and the decrease in the transforming growth factor-α and interleukin-8 concentration were higher in the hypertonic group (p < 0.001 for all mediators). CONCLUSION: Irrigation with a hypertonic solution was found to be more effective than an isotonic solution in nasal mucosa reparation.

Nasal polyps show decreased mucociliary transport despite vigorous ciliary beating.

OBJECTIVE: Mucociliary transport function in the airway mucosa is essential for maintaining a clean mucosal surface. This function is impaired in upper and lower airway diseases. Nasal polyps are a noticeable pathological feature that develop in some of the patients with chronic rhinosinusitis. Like ordinary nasal mucosae, nasal polyps have a ciliated pseudostratified epithelium with vigorous ciliary beating. We measured ex vivo Mucociliary Transport Velocity (MCTV) and Ciliary Beat Frequency (CBF) and explored the expressions of Planar Cell Polarity (PCP) proteins in nasal polyps in comparison with turbinate mucosae. METHODS: Inferior turbinates and nasal polyps were surgically collected from patients with chronic rhinosinusitis. Ex vivo MCTV and CBF were measured using a high-speed digital imaging system. Expressions of PCP proteins were explored by fluorescence immunohistochemistry and quantitative RT-PCR. RESULTS: The MCTV of nasal polyps was significantly lower than that of the turbinates (7.43 ±â€¯2.01 vs. 14.56 ±â€¯2.09 µm/s; p = 0.0361), whereas CBF did not differ between the two tissues. The MCTV vector was pointed to the posteroinferior direction in all turbinates with an average inclination angle of 41.0 degrees. Immunohistochemical expressions of Dishevelled-1, Dishevelled-3, Frizzled3, Frizzled6, Prickle2 and Vangl2 were lower in the nasal polyps than in the turbinates. Confocal laser scanning microscopy showed that Frizzled3 was localized along the cell junction on the apical surface. The expression levels of mRNAs for Dishevelled-1, Dishevelled-3 and Frizzled3 in the nasal polyps were also decreased in comparison with the turbinates. CONCLUSION: These results indicate that muco ciliary transport in nasal polyps is impaired although vigorous ciliary beating is maintained, and that the impairment may be caused by a decrease in Dishevelled/Frizzled proteins and resultant PCP disarrangement. LEVEL OF EVIDENCE: Level 3.

Aberrant expressions of TAM receptors are associated with postoperative recurrence in chronic rhinosinusitis with nasal polyps.

OBJECTIVES: TAM receptors (TYRO3, AXL, and MER) play important roles in inflammatory responses, but their effects in chronic rhinosinusitis with nasal polyps (CRSwNP) remain elucidated. We aim to evaluate the values of TAM receptors in disease severity and postoperative recurrence of CRSwNP. METHODS: We initially enrolled 160 patients with CRSwNP who were treated with functional endoscopic sinus surgery (FESS) and postoperative recurrence was evaluated during the follow-up period. Circulating TAM receptor levels were detected by enzyme-linked immunosorbent assay (ELISA), and tissue expressions were measured by real-time polymerase chain reaction (RT-PCR) and immunohistochemical (IHC). The relationships between TAM receptor levels and postoperative recurrence were examined. RESULTS: A total of 150 patients completed the follow-up schedule, 49 patients experienced postoperative recurrence and the remaining 101 patients were non-recurrent. In recurrent CRSwNP patients, serum levels of TAM receptors were increased compared to those in non-recurrent patients and were positively correlated with disease severity scores (P < 0.05). Circulating TYRO3 and MER were identified as potential predictors of postoperative recurrence based on receiver operating characteristics (ROC) and Kaplan-Meier plots (P < 0.05). Furthermore, tissue TAM receptor levels, as determined by both RT-PCR and IHC, were enhanced in the recurrent group than in the non-recurrent group (P < 0.05) and were predictive of postoperative recurrence (P < 0.05). Interestingly, circulating TYRO3 and MER concentrations, as well as tissue TYRO3 expression, were found to be significantly increased in patients who experienced postoperative recurrence (P < 0.05). IHC images from the same patients revealed that TAM expressions were enhanced in the recurrent tissues compared to their baseline tissue levels. CONCLUSIONS: Our laboratory results demonstrated that TAM receptors were increased in recurrent CRSwNP patients and associated with postoperative recurrence. Moreover, the new laboratory findings suggested that measuring circulating levels of TAM receptors might serve as a promising new approach to assess disease progression and predict the risk of postoperative recurrence.

Single-cell RNA sequencing reveals the epithelial cell, fibroblast, and key gene alterations in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the nasal mucosa, and epithelial-mesenchymal transition (EMT) is thought to be an essential process in the pathogenesis of CRSwNP. However, the mechanisms of epithelial and fibroblastic changes at the single-cell level are unclear. In this study, we investigated the epithelial cell, fibroblast, and key gene alterations in the development of CRSwNP. We revealed major cell types involved in CRSwNP and nasal mucosal inflammation formation, then mapped epithelial and fibroblast subpopulations. We showed that the apical and glandular epithelial cells and the ADGRB3+ and POSTN+ fibroblasts were the key cell subtypes in the progression of CRSwNP. Pseudotime and cell cycle analysis identified dynamic changes between epithelial cells and fibroblasts during its development. WFDC2 and CCL26 were identified as the key marker genes involved in the development of CRSwNP and were validated by IHC staining, which may provide a potential novel target for future CRSwNP therapy. ScRNA-seq data provided insights into the cellular landscape and the relationship between epithelial cells and fibroblasts in the progression of CRSwNP. WFDC2 and CCL26 were identified as the key genes involved in the development of CRSwNP and may be the potential markers for gene therapy.

Impaired local Vitamin D3 metabolism contributes to IL-36g overproduction in epithelial cells in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Vitamin D (VD) possesses immunomodulatory properties, but its role in chronic rhinosinusitis with nasal polyps (CRSwNP) remains poorly studied. Herein, we aim to explore the regulation and function of VD3 in CRSwNP. METHODS: 25-hydroxyvitamin D3 (25VD3) levels in serum and tissue lysates were detected by ELISA. The expression of VD receptor (VDR) and cytochrome P450 family 27 subfamily B member 1 (CYP27B1), the enzyme that converts 25VD3 to the active 1,25-hydroxyvitamin D3 (1,25VD3), and their expression regulation in human nasal epithelial cells (HNECs) were studied by RT-PCR, western blotting, immunofluorescence, and flow cytometry. RNA sequencing was performed to identify genes regulated by 1,25VD3 in HNECs. HNECs and polyp tissue explants were treated with 1,25VD3, 25VD3, and dexamethasone. RESULTS: 25VD3 levels in serum and nasal tissue lysates were decreased in patients with eosinophilic and noneosinophilic CRSwNP than control subjects. The expression of VDR and CYP27B1 were reduced in eosinophilic and noneosinophilic CRSwNP, particularly in nasal epithelial cells. VDR and CYP27B1 expression in HNECs were downregulated by interferon y and poly (I:C). Polyp-derived epithelial cells demonstrated an impaired ability to convert 25VD3 to 1,25VD3 than control tissues. 1,25VD3 and 25VD3 suppressed IL-36y production in HNECs and polyp tissues, and the effect of 25VD3 was abolished by siCYP27B1 treatment. Tissue 25VD3 levels negatively correlated with IL-36y expression and neutrophilic inflammation in CRSwNP. CONCLUSION: Reduced systemic 25VD3 level, local 1,25VD3 generation and VDR expression result in impaired VD3 signaling activation in nasal epithelial cells, thereby exaggerating IL-36y production and neutrophilic inflammation in CRSwNP.

Eupatilin alleviates inflammation and epithelial-to-mesenchymal transition in chronic rhinosinusitis with nasal polyps by upregulating TFF1 and inhibiting the Wnt/ß-catenin signaling pathway.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a multifactorial inflammatory disease characterized by high prevalence and morbidity. However, its pathogenesis is still obscure. This work focuses on the effects of Eupatilin (EUP) on inflammation reaction and the epithelial-to-mesenchymal transition (EMT) process in CRSwNP. METHODS: In vivo and in vitro CRSwNP models were established based on BALB/c mice and human nasal epithelial cells (hNECs) to investigate the effects of EUP on EMT and inflammation in CRSwNP. Protein levels of TFF1, EMT-related factors (E-cadherin, N-cadherin, and Vimentin), and Wnt/ß-catenin signaling-related proteins (Wnt3α and ß-catenin) were assayed via western blotting. Pro-inflammatory factors (TNF-α, IL-6, and IL-8) were assessed via ELISA assay. RESULTS: EUP treatment significantly reduced the number of polyps, epithelial thickness, and mucosal thickness in CRSwNP mice. Besides, EUP treatment also suppressed inflammation reaction and EMT events in CRSwNP mice and SEB-challenged hNECs in a dose-dependent manner. Also, EUP treatment dose-dependently upregulated TFF1 expression and inhibited Wnt/ß-catenin activation in CRSwNP mice and SEB-challenged hNECs. In addition, TFF1 inhibition or Wnt/ß-catenin activation partially abated EUP-mediated protection against SEB-induced inflammation reaction and EMT events in hNECs. CONCLUSIONS: Taken together, our findings highlighted the inhibitory role of EUP on the inflammation and EMT processes in CRSwNP in vivo and in vitro via upregulating TFF1 and inhibiting the Wnt/ß-catenin signaling, suggesting EUP could be a promising therapeutic agent for CRSwNP.

Lipocalin-2 promotes neutrophilic inflammation in nasal polyps and its value as biomarker.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common chronic inflammatory disease of the nasal cavity and paranasal sinuses. The role of neutrophils in the pathogenesis of CRSwNP has attracted more attention in recent years, due to its association with more severe disease and reduced steroid responsiveness. Lipocalin-2 (LCN2) has been found to modulate neutrophils infiltration in other neutrophilic inflammation including inflammatory bowel disease, rheumatoid arthritis, and psoriasis. The aim was to evaluate the expression and regulator role of LCN2 in neutrophilic inflammation in CRSwNP, and its role as a potential biomarker predicting non-eosinophilic CRSwNP (neCRSwNP). METHODS: Bioinformatic analysis, immunostainings, real-time PCR and ELISA were used to analyze the expression and location of LCN2 in nasal tissues. The expression of proinflammatory mediators were assessed in nasal tissues and secretions. LCN2 production in human nasal epithelial cells (HNECs) and neutrophils, as well as its role in neutrophilic inflammation was evaluated by in vitro experiments. RESULTS: LCN2 was mainly located in neutrophils and HNECs of nasal polyps. LCN2 expression was also significantly higher in the polyp tissue and nasal secretions from patients with neCRSwNP. The LCN2 levels were positively correlated with type 3 inflammation markers, including G-CSF, IL-8, and IL-17. LCN2 expression could be upregulated by IL-17 A and TNF-α in HNECs, and LCN2 could also promote the expression of IL-8 in dispersed polyp cells and HNECs. CONCLUSIONS: LCN2 could serve as a novel biomarker predicting patients with neCRSwNP, and the increased expression of LCN2 may participate in the pathogenesis of neCRSwNP.

IgE directly affects eosinophil migration in chronic rhinosinusitis with nasal polyps through CCR3 and predicts the efficacy of omalizumab.

BACKGROUND: Whether IgE affects eosinophil migration in chronic rhinosinusitis with nasal polyps (CRSwNP) remains largely unclear. Moreover, our understanding of local IgE, eosinophils, and omalizumab efficacy in CRSwNP remains limited. OBJECTIVE: We investigated whether IgE acts directly on eosinophils and determined its role in omalizumab therapy. METHODS: Eosinophils and their surface receptors were detected by hematoxylin and eosin staining and flow cytometry. IgE and its receptors, eosinophil peroxidase (EPX), eosinophilic cationic protein, and CCR3 were detected by immunohistochemistry and immunofluorescence. Functional analyses were performed on blood eosinophils and polyp tissues. Logistic regression was performed to screen for risk factors. Receiver operating characteristic curve was generated to evaluate the accuracy. RESULTS: Both FcεRI and CD23 were expressed on eosinophils. The expression of FcεRI and CD23 on eosinophil in nasal polyp tissue was higher than in peripheral blood (both P < .001). IgE and EPX colocalized in CRSwNP. IgE directly promoted eosinophil migration by upregulating CCR3 in CRSwNP but not in healthy controls. Omalizumab and lumiliximab were found to be effective in restraining this migration, indicating CD23 was involved in IgE-induced eosinophil migration. Both IgE+ and EPX+ cells were significantly reduced after omalizumab treatment in those who experienced response (IgE+ cells, P = .001; EPX+ cells, P = .016) but not in those with no response (IgE+ cells, P = .060; EPX+ cells, P = .151). Baseline IgE+ cell levels were higher in those with response compared to those without response (P = .024). The baseline local IgE+ cell count predicted omalizumab efficacy with an accuracy of 0.811. CONCLUSIONS: IgE directly promotes eosinophil migration, and baseline local IgE+ cell counts are predictive of omalizumab efficacy in CRSwNP.

Detection of dynorphin 1-17 biotransformation fragments in human nasal polyps by UPLC-QTOF-MS.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a persistent inflammation of the sinonasal mucosa. CRSwNP treatments are associated with inconsistent efficacy and recurrence of symptoms. Dynorphin 1-17 (DYN 1-17) and its fragments have been shown to modulate the immune response in various inflammatory conditions. This study aimed to investigate the effect of different pH and degrees of inflammation on DYN 1-17 metabolism in human CRSwNP tissues. DYN 1-17 was incubated with grade 3 and grade 4 inflamed tissues of CRSwNP patients at pH 5.5 and pH 7.4 over a range of incubation periods. The resulting fragments were identified using an ultra-performance liquid chromatography (UPLC) system coupled to quadrupole-time of flight (QTOF) mass spectrometry based on their accurate mass. The rate of DYN 1-17 fragmentation was slower at pH 5.5 in comparison to pH 7.4. The extent and rate of metabolism of DYN 1-17 were much lower in grade 3 inflamed tissue (31-32 fragments) than in grade 4 (34-41 fragments). N-Terminal fragments (DYN 1-15, 1-11, 1-10, and 1-6) were metabolized slower at pH 5.5 as compared to pH 7.4. DYN 1-12, 1-8, 2-10, 4-10, 5-10, and 8-14 were only observed under the inflammatory pH while DYN 5-17 and 6-17 were only identified upon incubation with grade 4 CRSwNP tissues. DYN 1-17 metabolism was significantly affected by the pH level and the severity of the inflammation of CRSwNP tissues, indicating the potential roles of DYN 1-17 and its fragments in modulating the inflammatory response and their avenue as therapeutics in future studies.

Staphylococcus aureus lysate induces an IgE response via memory B cells in nasal polyps.

BACKGROUND: Locally increased IgE levels plays a pathologic role in chronic rhinosinusitis with nasal polyps (CRSwNP). OBJECTIVE: This study aimed to investigate whether Staphylococcus aureus could induce aberrant IgE synthesis in CRSwNP and the potential mechanisms involved. METHODS: Total IgE, IL-4, IL-5, and IL-13 concentrations in the supernatants of the cultures stimulated with S aureus lysate were assessed by ELISA. S aureus-induced cellular responses were investigated by single-cell RNA sequencing. Flow cytometry and quantitative reverse transcription PCR were used to analyze B-cell subsets and stimulated cell ε-germline transcript expression, respectively. IgE-positive B-cell and germinal center localization were assessed by immunohistochemistry and immunofluorescence. RESULTS: S aureus lysate induced IgE production in the supernatants of nasal polyp (NP) tissues but not in those of healthy nasal mucosa. Moreover, IgE levels increased from days 2 to 4 after stimulation, paralleling the enhanced ε-germline transcript, IL-5, and IL-13 expression. Single-cell RNA sequencing revealed that there were increased IL-5 and IL-13 in group 2 innate lymphoid cells and identified a clonal overlap between unstimulated memory B cells and S aureus-stimulated plasma cells. The enriched IgE within NPs was mainly produced by IgE-negative memory B cells. Cellular evidence indicated that the IgE memory response to S aureus might also exist in the peripheral blood of CRSwNP patients. The S aureus-induced IgE memory response was associated with elevated IgE levels in NPs, asthma, and postoperative CRSwNP recurrence. CONCLUSIONS: S aureus induced an IgE response via IgE-negative memory B cells in CRSwNP patients, possibly contributing to CRSwNP development.

Comparing Protein and Gene Expression Signature between Nasal Polyps and Nasal Fluids in Chronic Rhinosinusitis.

INTRODUCTION: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a serious inflammatory condition. Nasal fluids (NFs) present a noninvasive alternative to nasal biopsy for studying CRSwNP pathogenesis. We aimed to compare the protein and mRNA inflammation signature between nasal polyps (NPs) and NFs. METHOD: The performance of polyvinyl alcohol (PVA) sponges and NFs absorbable device (NFAD) for collecting NFs from 20 patients with CRSwNP was compared using the Luminex assay. The other group consisted of four healthy controls and an additional 21 CRSwNP patients (including eosinophilic CRSwNP [ECRSwNP] and non-eosinophilic CRSwNP [NECRSwNP]) for protein quantification by Olink platform and gene expression evaluation by RNA-sequencing. Spearman's analysis was performed to detect correlations between protein expression levels in NFs and clinical assessment variables. RESULTS: NFAD-collected NFs contained at least a 2-fold higher concentration of cytokines than that obtained using PVA sponge, and these cytokines levels are significantly associated with NPs (ρ > 0.45, p < 0.05). Differentially expressed proteins between NFs and NPs were significantly correlated in the ECRSwNP subgroup compared with controls (ρ = 0.41, p < 0.01). Levels of Th2/IL-13, MCP4, and CCL4, characteristic of eosinophilic infiltration, were increased in ECRSwNP patients. A significant correlation between gene and protein expression was observed (ρ = 0.34, p < 0.01). PDL2 levels in NFs were positively correlated with ECRSwNP postoperative recurrence, the nasal VAS, and SNOT-22 scores (ρ > 0.68, p < 0.05 for all). CONCLUSION: Our study revealed similarities and discrepancies in inflammatory signatures between NPs and NFs in the same CRSwNP patient.

MZB1-expressing cells are essential for local immunoglobulin production in chronic rhinosinusitis with nasal polyps.

BACKGROUND: The expression of MZB1 genes is significantly elevated in patients who have chronic rhinosinusitis with nasal polyp (CRSwNP) disease compared with healthy controls. OBJECTIVE: To characterize MZB1-positive B cells in CRSwNP and to estimate the contribution of distinct subsets of B cells to the local overproduction of immunoglobulins. METHODS: Single-cell RNA-sequencing with Cellular Indexing of Transcriptomes and Epitopes by Sequencing technology, Switching Mechanism At the 5' end of RNA Template sequencing, flow cytometry, immunohistochemistry and immunofluorescence staining, Western blot, QuantiGene Plex assay, B-cell ImmunoSpot assay, Luminex assay, and enzyme-linked immunosorbent assay were performed. RESULTS: Significantly higher mRNA expression of MZB1 and HSP90B1 was found in type 2 CRSwNP compared with controls. In CRSwNP, MZB1 expression correlated with the local production of IgE. MZB1 could be colocalized with plasma and mature B cells, especially marginal zone (MZ) B cells. Single-cell transcriptome and epitope studies revealed prominent populations of B cells in type 2 CRSwNP with unexpectedly high MZB1 gene expression. The MZ B-cell population was significantly increased in CRSwNP compared with healthy controls in both peripheral blood mononuclear cells and nasal tissue single-cell suspensions. When those single cells were cultured overnight, the MZ B-cell numbers were positively correlated with local IgE production but negatively correlated with local IgM production. In vitro, MZB1 stimulation up-regulated the mRNA expression of IgE. CONCLUSION: MZB1 was primarily expressed by plasma and mature B cells in nasal mucosa. MZB1 expression level was increased in CRSwNP compared with controls. MZB1 contributed to the local IgE production in type 2 CRSwNP.

Nasal polyp antibody-secreting cells display proliferation signature in aspirin-exacerbated respiratory disease.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) causes nasal obstruction and olfactory dysfunction. Aspirin-exacerbated respiratory disease (AERD) is the triad of CRSwNP, asthma, and respiratory reactions to COX-1 inhibitors. Patients with AERD have elevated nasal IL-5 levels and high numbers of antibody-secreting cells (ASCs), including plasma cells and plasmablasts, in their polyp tissue; in addition, their nasal polyp (NP) IgE levels are correlated with disease severity and recurrence of nasal polyposis. OBJECTIVE: We sought to explore differences in the transcriptomic profile, activation markers, and IL-5Rα expression and function of NP ASCs from patients with AERD and CRSwNP. METHODS: NP tissue was collected from patients with AERD and CRSwNP and digested into single-cell suspensions. NP cells were analyzed for protein expression by mass cytometry. For IL-5Rα functional studies, plasma cells were purified and cultured in vitro with or without IL-5 and analyzed by bulk RNA sequencing. RESULTS: Compared with polyp tissue from patients with CRSwNP, polyp tissue from patients with AERD contained significantly more ASCs and had increased ASC expression of IL-5Rα. ASCs from patients with AERD expressed higher protein levels of B-cell activation and regulatory markers (CD40, CD19, CD32, and CD38) and the proliferation marker Ki-67. ASCs from patients with AERD also expressed more IL5RA, IGHE, and cell cycle- and proliferation-related transcripts (CCND2, MKI67, CDC25A, and CDC25B) than did ASCs from patients with CRSwNP. Stimulation of plasma cells from patients with AERD with IL-5 induced key cell cycle genes (CCND2 and PTP4A3), whereas IL-5 stimulation of ASCs from patients with CRSwNP induced few transcriptomic changes. CONCLUSION: NP tissue ASCs from patients with AERD express higher levels of functional IL-5Rα and markers associated with cell cycling and proliferation than do ASCs from patients with aspirin-tolerant CRSwNP.

Multiple cytokine analyses identify CSF1 as a robust biomarker for predicting postoperative recurrence in chronic rhinosinusitis with nasal polyps.

OBJECTIVE: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous disease with a high rate of postoperative recurrence. This study aimed to discover potential biomarkers by analyzing multiple cytokine profiles in serum to predict postoperative recurrence in CRSwNP and to explore the underlying mechanisms. METHODS: In this prospective study, we enrolled 18 healthy controls (HC) and 60 CRSwNP patients and analyzed the baseline serum cytokine profiles using the Luminex assay. Patients were followed up for more than 2 years and divided into non-recurrence and Recurrence groups. The differentially expressed cytokines were validated in the serum and tissue samples in a validation cohort, and their predictive values for recurrence were evaluated. RESULTS: Fifty-four CRSwNP patients completed the follow-up schedule, including 37 patients in the non-Recurrence group and 17 patients in the Recurrence group. Multiple cytokine analyses showed that serum CD40, CD40L, IL-18, IL-8, MCP1, and CSF1 levels were elevated in the CRSwNP group, especially in the Recurrence group, compared to the HC group. Receiver operating characteristic curves (ROC) and Kaplan-Meier survival analysis showed that serum levels of CD40, CD40L, and CSF1 were closely associated with the risk of postoperative recurrence. Further validation results showed that both serum and tissue mRNA levels of CD40, CD40L, and CSF1 were significantly higher in the Recurrence group in comparison with the non-recurrence and HC groups, and tissue CSF1 mRNA expression exhibited a robust value for predicting the CRSwNP recurrence. Immunofluorescence results revealed that CSF1 was enhanced in the recurrent CRSwNP patients, especially in the epithelial cell area, and CSF1 expressions were augmented when patients suffered postoperative recurrence. CONCLUSIONS: Circulating cytokine profiles may affect the risk of postoperative recurrence in CRSwNP patients. Our discovery-validation results suggested that CSF1 might serve as a robust biomarker for predicting CRSwNP recurrence.