Endotypes of Chronic Rhinosinusitis with Primary and Recurring Nasal Polyps in the Latvian Population.

Chronic rhinosinusitis (CRS) is a complex syndrome with various inflammatory mechanisms resulting in different patterns of inflammation that correlate with the clinical phenotypes of CRS. Our aim was to use detected IL-1, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, Ki 67, HBD-2, HBD-3, and LL-37 to classify specific inflammatory endotypes in chronic rhinosinusitis with the tissue of nasal polyps (CRSwNP). Samples from 35 individuals with primary and recurrent CRSwNP were taken during surgery. The tissues were stained for the previously mentioned biomarkers immunohistochemically. A hierarchical cluster analysis was performed. The clinical parameters were compared between clusters. Five clusters had significantly different biomarkers between groups. There were no significant differences in the clinical parameters, except for the Lund-Mackay score, which was significantly higher in cluster 4 compared to that of cluster 1 (p = 0.024). Five endotypes of (CRSwNP) are characterized by different combinations of type 1, type 2, and type 3 tissue inflammation patterns. In the Latvian population, endotypes associated with neutrophilic inflammation or a combination of neutrophilic inflammation and type 2 inflammation are predominant. Increased proliferation marker Ki 67 values are not associated with more severe inflammation in the tissue samples of chronic rhinosinusitis with nasal polyps.

The oxidant-antioxidant imbalance was involved in the pathogenesis of chronic rhinosinusitis with nasal polyps.

Background: Although oxidative stress is involved in the pathophysiological process of chronic rhinosinusitis with nasal polyps (CRSwNP), the specific underlying mechanism is still unclear. Whether antioxidant therapy can treat CRSwNP needs further investigation. Methods: Immunohistochemistry, immunofluorescence, western blotting and quantitative polymerase chain reaction (qPCR) analyses were performed to detect the distribution and expression of oxidants and antioxidants in nasal polyp tissues. qPCR revealed correlations between oxidase, antioxidant enzymes and inflammatory cytokine levels in CRSwNP patients. Human nasal epithelial cells (HNEpCs) and primary macrophages were cultured to track the cellular origin of oxidative stress in nasal polyps(NPs) and to determine whether crocin can reduce cellular inflammation by increasing the cellular antioxidant capacity. Results: The expression of NOS2, NOX1, HO-1 and SOD2 was increased in nasal epithelial cells and macrophages derived from nasal polyp tissue. Oxidase levels were positively correlated with those of inflammatory cytokines (IL-5 and IL-6). Conversely, the levels of antioxidant enzymes were negatively correlated with those of IL-13 and IFN-γ. Crocin inhibited M1 and M2 macrophage polarization as well as the expression of NOS2 and NOX1 and improved the antioxidant capacity of M2 macrophages. Moreover, crocin enhanced the ability of antioxidants to reduce inflammation via the KEAP1/NRF2/HO-1 pathway in HNEpCs treated with SEB or LPS. Additionally, we observed the antioxidant and anti-inflammatory effects of crocin in nasal explants. Conclusion: Oxidative stress plays an important role in the development of CRSwNP by promoting various types of inflammation. The oxidative stress of nasal polyps comes from epithelial cells and macrophages. Antioxidant therapy may be a promising strategy for treating CRSwNP.

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.

Increased SERPINB2 potentiates 15LO1 expression via STAT6 signalling in epithelial cells in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: SERPINB2, a biomarker of Type-2 (T2) inflammatory processes, has been described in the context of asthma. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also correlated with T2 inflammation and elevated 15LO1 induced by IL-4/13 in nasal epithelial cells. The aim of this study was to evaluate the expression and location of SERPINB2 in nasal epithelial cells (NECs) and determine whether SERPINB2 regulates 15LO1 and downstream T2 markers in NECs via STAT6 signalling. METHODS: SERPINB2 gene expression in bulk and single-cell RNAseq database was analysed by bioinformatics analysis. SERPINB2, 15LO1 and other T2 markers were evaluated from CRSwNP and HCs NECs. The colocalization of SERPINB2 and 15LO1 was evaluated by immunofluorescence. Fresh NECs were cultured at an air-liquid interface with or without IL-13, SERPINB2 Dicer-substrate short interfering RNAs (DsiRNAs) transfection, exogenous SERPINB2, 15-HETE recombinant protein and pSTAT6 inhibitors. 15LO1, 15-HETE and downstream T2 markers were analysed by qRT-PCR, western blot and ELISA. RESULTS: SERPINB2 expression was increased in eosinophilic nasal polyps compared with that in noneosinophilic nasal polyps and control tissues and positively correlated with 15LO1 and other downstream T2 markers. SERPINB2 was predominantly expressed by epithelial cells in NP tissue and was colocalized with 15LO1. In primary NECs in vitro, SERPINB2 expression was induced by IL-13. Knockdown or overexpression SERPINB2 decreased or enhanced expression of 15LO1 and 15-HETE in NECs, respectively, in a STAT6-dependent manner. SERPINB2 siRNA also inhibited the expression of the 15LO1 downstream genes, such as CCL26, POSTN and NOS2. STAT6 inhibition similarly decreased SERPINB2-induced 15LO1. CONCLUSIONS: SERPINB2 is increased in NP epithelial cells of eosinophilic CRSwNP (eCRSwNP) and contributes to T2 inflammation via STAT6 signalling. SERPINB2 could be considered a novel therapeutic target for eCRSwNP.

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.

Olfactory Loss in Rhinosinusitis: Mechanisms of Loss and Recovery.

Chronic rhinosinusitis (CRS) is a highly prevalent disease and up to 83% of CRS patients suffer from olfactory dysfunction (OD). Because OD is specifically seen in those CRS patients that present with a type 2 eosinophilic inflammation, it is believed that type 2 inflammatory mediators at the level of the olfactory epithelium are involved in the development of this olfactory loss. However, due to the difficulties in obtaining tissue from the olfactory epithelium, little is known about the true mechanisms of inflammatory OD. Thanks to the COVID-19 pandemic, interest in olfaction has been growing rapidly and several studies have been focusing on disease mechanisms of OD in inflammatory conditions. In this paper, we summarize the most recent data exploring the pathophysiological mechanisms underlying OD in CRS. We also review what is known about the potential capacity of olfactory recovery of the currently available treatments in those patients.

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.

An In Vitro Study Evaluating the Safety of Mesalazine on Human Nasoepithelial Cells.

Chronic rhinosinusitis (CRS) is a disease characterised by the inflammation of the nasal and paranasal cavities. It is a widespread condition with considerable morbidity for patients. Current treatment for chronic rhinosinusitis consists of appropriate medical therapy followed by surgery in medically resistant patients. Although oral steroids are effective, they are associated with significant morbidity, and disease recurrence is common when discontinued. The development of additional steroid sparing therapies is therefore needed. Mesalazine is a commonly used therapeutic in inflammatory bowel disease, which shares a similar disease profile with chronic rhinosinusitis. This exploratory in vitro study aims to investigate whether mesalazine could be repurposed to a nasal wash, which is safe on human nasoepithelial cells, and retains its anti-inflammatory effects. CRS patients' human nasal epithelial cells (HNECs) were collected. HNECs were grown at an air-liquid interface (ALIs) and in a monolayer and challenged with mesalazine or a non-medicated control. Transepithelial electrical resistance, paracellular permeability, and toxicity were measured to assess epithelial integrity and safety. The anti-inflammatory effects of mesalazine on the release of interleukin (IL)-6 and tumour necrosis factor alpha (TNF-α) were analysed using human leukemia monocytic cell line (THP-1). mesalazine did not impact the barrier function of HNEC-ALIs and was not toxic when applied to HNECs or THP-1 cells at concentrations up to 20 mM. mesalazine at 0.5 and 1 mM concentrations significantly inhibited TNF-α release by THP-1 cells. mesalazine effectively decreases TNF-α secretion from THP-1 cells, indicating the possibility of its anti-inflammatory properties. The safety profile of mesalazine at doses up to 20 mM suggests that it is safe when applied topically on HNECs.

Functional 2D Nanoplatforms Alleviate Eosinophilic Chronic Rhinosinusitis by Modulating Eosinophil Extracellular Trap Formation.

The therapeutic outcomes of patients with eosinophilic chronic rhinosinusitis (ECRS) remain unsatisfactory, largely because the underlying mechanisms of eosinophilic inflammation are uncertain. Here, it is shown that the nasal secretions of ECRS patients have high eosinophil extracellular trap (EET) and cell-free DNA (cfDNA) levels. Moreover, the cfDNA induced EET formation by activating toll-like receptor 9 (TLR9) signaling. After demonstrating that DNase I reduced eosinophilic inflammation by modulating EET formation, linear polyglycerol-amine (LPGA)-coated TiS2 nanosheets (TLPGA) as functional 2D nanoplatforms with low cytotoxicity, mild protein adsorption, and increased degradation rate is developed. Due to the more flexible linear architecture, TLPGA exhibited higher cfDNA affinity than the TiS2 nanosheets coated with dendritic polyglycerol-amine (TDPGA). TLPGA reduced cfDNA levels in the nasal secretions of ECRS patients while suppressing cfDNA-induced TLR9 activation and EET formation in vitro. TLPGA displayed exceptional biocompatibility, preferential nasal localization, and potent inflammation modulation in mice with eosinophilic inflammation. These results highlight the pivotal feature of the linear molecular architecture and 2D sheet-like nanostructure in the development of anti-inflammation nanoplatforms, which can be exploited for ECRS treatment.

Proteomic and metabolomic proof of concept for unified airways in chronic rhinosinusitis and asthma.

BACKGROUND: The pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) with comorbid asthma remains unclear. OBJECTIVE: To assess upper and lower airway unity and identify a possible common pathogenesis in CRSwNP with asthma. METHODS: This study analyzed the expression of proteins and metabolites in nasal lavage fluid cells (NLFCs) and induced sputum cells (ISCs). Differentially expressed proteins and their function-related metabolites in the upper and lower airways of patients having CRSwNP with or without asthma were identified; relevant signaling pathways were analyzed, and key pathway-related proteins were identified. Parallel reaction monitoring was used to verify these target proteins. RESULTS: Protein or metabolite expression between NLFCs and ISCs was highly correlated and conservative on the basis of expression profiles and weighted gene coexpression network analysis. There were 17 differentially coexpressed proteins and their function-related 13 metabolites that were identified in the NLFCs and ISCs of CRSwNP, whereas 11 proteins and 11 metabolites were identified in CRSwNP with asthma. An asthma pathway was involved in the copathogenesis of upper and lower airways in whether CRSwNP or CRSwNP with asthma. The asthma pathway-related proteins proteoglycan 2 and eosinophil peroxidase, as the core of the protein-metabolism interaction networks between the upper and lower airways, were both highly coexpressed in NLFCs and ISCs in patients having either CRSwNP or CRSwNP with asthma by parallel reaction monitoring validation. CONCLUSION: Proteomics and metabolomics reveal upper and lower airway unity. Asthma pathway-related proteins proteoglycan 2 and eosinophil peroxidase from the upper airway could be used to assess the potential risk of lower airway dysfunction in CRSwNP.

Nasal hyperreactivity in allergic rhinitis and chronic rhinosinusitis with polyps: a role for neuronal pathways.

BACKGROUND: Nasal hyperreactivity (NHR) is prevalent in all chronic upper airway inflammatory phenotypes, including allergic rhinitis (AR) and chronic rhinosinusitis with nasal polyps (CRSwNP). Although NHR in patients with non-allergic rhinitis is mediated by neuronal pathways, AR and CRSwNP are mainly characterized by type 2 inflammation. METHODS: Eighteen healthy controls and 45 patients with symptomatic AR/CRSwNP underwent a cold, dry air (CDA) provocation test for objective diagnosis of NHR. Before and after, questionnaires were filled out and nasal secretions and biopsies were collected. Markers for neurogenic inflammation (substance P, calcitonin gene-related peptide, neurokinin A), epithelial activation (IL-33), and histamine were measured in secretions by ELISA; and expression of neuronal markers PGP9.5, TRPV1, and TRPM8 was studied in biopsies by RT-q-PCR. Effects of histamine on TRPV1/A1 were studied with Ca2+-imaging using murine trigeminal neurons. RESULTS: CDA-provocation reduced peak nasal inspiratory flow (PNIF) of patients with subjective NHR but not of non-NHR controls/patients CDA-provocation reduced peak nasal inspiratory flow (PNIF) of patients with subjective NHR but not of non-NHR controls/patients. Subjective (subjectively reported effect of CDA) and objective (decrease in PNIF) effects of CDA were significantly correlated. Levels of neuropeptides and histamine in nasal secretions and mRNA expression of PGP9.5, TRPV1, and TRPM8 correlated with CDA-induced PNIF-reduction. CDA-provocation induced an increase in IL-33-levels. Both TRPV1 and TRPA1 expressed on afferent neurons were sensitized by exposure to histamine. CONCLUSION: NHR is not an on/off phenomenon but spans a continuous spectrum of reactivity. A neurogenic inflammatory background and increased histamine-levels are risk factors for NHR in AR/CRSwNP.

Severe Type 2 Inflammation Leads to High Platelet-Activating-Factor-Associated Pathology in Chronic Rhinosinusitis with Nasal Polyps-A Hierarchical Cluster Analysis Using Bulk RNA Barcoding and Sequencing.

Platelet-activating factor (PAF) is a phospholipid-derived inflammatory mediator that triggers various inflammatory conditions, including eosinophil activation and recruitment. This study aimed to evaluate the expressions of PAF-metabolism-associated genes, namely genes coding the enzymes involved in PAF synthesis (LPCAT1, LPCAT2, LPCAT3, and LPCAT4), PAF degradation (PAFAH1B2, PAFAH1B3, and PAFAH2), and the gene for the PAF receptor (PTAFR) in subtypes of CRSwNP classified by clinical- or hierarchal-analysis-based classifications. Transcriptomic analysis using bulk RNA barcoding and sequencing (BRB-seq) was performed with CRSwNP, including eosinophilic CRS (ECRS) (n = 9), nonECRS (n = 8), ECRS with aspirin-exacerbated respiratory disease (Asp) (n = 3), and controls with a normal uncinate process mucosa (n = 6). PTAFR was only upregulated in ECRS and nonECRS. In the hierarchical cluster analysis with clusters 1 and 2 reflecting patients with low-to-moderate and high levels of type 2 inflammation, respectively, cluster 1 exhibited a significant downregulation of LPCAT2 and an upregulation of PTAFR expression, while cluster 2 showed an upregulation of LPCAT1, PAFAH1B2, and PTAFR and downregulation of PAFAH2 expression. Understanding this strong PAF-associated pathophysiology in the severe type 2 inflammation group could provide valuable insights into the treatment and management of CRSwNP.

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.

Metabolic syndrome facilitates histopathological changes and the risk of postoperative recurrence in chronic rhinosinusitis with nasal polyps.

BACKGROUND: The relationship between metabolic syndrome (MS) and chronic rhinosinusitis with nasal polyps (CRSwNP) remains unclear. This study aimed to examine the effects of MS on histopathological features and postoperative recurrence in patients with CRSwNP. METHODS: We recruited 529 patients with CRSwNP who underwent functional endoscopic sinus surgery. They were divided into MS and non-MS groups and followed up for 2 years to evaluate postoperative recurrence. Clinical characteristics, histopathological features, the immunoactivity of signature cytokines, and the risk of postoperative recurrence were compared between the two groups. RESULTS: In total, 490 patients with CRSwNP were included in the study, 145 of whom experienced postoperative recurrence. The recurrence rate, tissue eosinophil count and percentage, and expression levels of IL-5 and IL-17A were significantly higher in the MS group compared to the non-MS group. Furthermore, within the MS group, patients who experienced recurrence exhibited higher tissue eosinophil counts and IL-5 and IL-17A levels than those in the non-MS group. Notably, the eosinophil count and IL-5 and IL-17A levels were higher in tissues collected during revision surgery than in those collected during primary surgery, particularly in patients with MS. Binary logistic regression analysis and Kaplan-Meier survival curves consistently indicated that MS independently increased the risk of postoperative recurrence in patients with CRSwNP. Furthermore, the risk increased with the number of MS components presented. CONCLUSION: MS promoted tissue eosinophil infiltration, and IL-5 and IL-17A expression, and increased the risk of postoperative recurrence in patients with CRSwNP. MS was identified as an independent risk factor for postoperative recurrence, and the risk increased with an increase in the number of MS components.

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.

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.

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.

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.

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.