Inflammation In Chronic Rhinosinusitis With Nasal Polyps Research Articles

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

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. 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. 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. 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.

Retinoic Acid Treatment Mitigates PM2.5-Induced Type 2 Inflammation: Insights into Modulation of Innate Immune Responses.

Some studies have demonstrated the effects of particulate matter (PM) on chronic rhinosinusitis with nasal polyps (CRSwNP) development, as well as the therapeutic role of retinoic acid (RA) in nasal polypogenesis. However, the immunologic effect of PM in innate lymphoid cells (ILCs) and the exact mechanism of the therapeutic effect of RA remain unclear. Therefore, the present study investigated the effects of fine-dust-induced inflammation in CRSwNP and the mechanisms of the therapeutic effect of RA. PM2.5 exposure exacerbated pathological damage in the nasal mucosa of mice with nasal polyps (NP) via upregulation of type 2 inflammation. Additionally, PM2.5 exposure increased the expression of type 2 cytokines and epithelial-cell-derived cytokines (IL-33 and IL-25) significantly, as well as the ILC populations in human-NP-derived epithelial cells (HNECs). Moreover, RA supplementation significantly increased the expression of ILCreg in Lin-CD45+CD127+ cells, which in turn increased the levels of the anti-inflammatory cytokine IL-10. The findings suggest that PM2.5 exposures could aggravate the CRSwNP type 2 inflammation, and RA treatment may ameliorate fine-dust-induced inflammation by modulating the innate immune response.

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

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. 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/β-catenin signaling-related proteins (Wnt3α and β-catenin) were assayed via western blotting. Pro-inflammatory factors (TNF-α, IL-6, and IL-8) were assessed via ELISA assay. 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/β-catenin activation in CRSwNP mice and SEB-challenged hNECs. In addition, TFF1 inhibition or Wnt/β-catenin activation partially abated EUP-mediated protection against SEB-induced inflammation reaction and EMT events in hNECs. 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/β-catenin signaling, suggesting EUP could be a promising therapeutic agent for CRSwNP.

Type 2 Biomarkers for the Indication and Response to Biologics in CRSwNP.

Three biologics targeting type 2 inflammation have been approved for the treatment of severe and uncontrolled chronic rhinosinusitis with nasal polyps (CRSwNP). Nevertheless, around 40-60% of patients do not respond well to these biological treatments. Selecting appropriate patients is crucial to improve treatment outcome of biologics. This review summarizes the literature data on type 2 biomarkers, with a specific focus on the indication to biologics for severe CRSwNP. No consensus has been reached on how to define mucosal type 2 inflammation in CRSwNP. Clinical markers (e.g., 22-item Sino-nasal Outcome Test (SNOT-22) score, Lund-Mackay CT score (LMS), ethmoid/maxillary sinus CT score, and CT-radiomics), nasal secretion biomarkers (e.g., eosinophil cationic protein and interleukin-5), blood and nasal cytology eosinophil counts, and nasal swab eosinophil peroxidase activity have been reported to be associated with type 2 inflammation in CRSwNP. The time duration since the last surgery, SNOT-22 score at 1 week of treatment, and baseline serum osteoprotegerin levels might indicate the response to dupilumab. LMS and asthma control test scores were found to have moderate predictive value for acceptable improvement after 24-week treatment of omalizumab. High blood eosinophil levels at baseline were associated with treatment response to mepolizumab and benralizumab. Although several clinical and biological markers might be associated with type 2 inflammation and response to biologics in patients with CRSwNP, their validity requires further investigation. Identifying clinically applicable biomarkers for biologic treatment holds significant promise for advancing personalized approaches to biologics and optimizing treatment outcomes for patients with CRSwNP.

Prevalence of type 2 inflammatory signatures and efficacy of dupilumab in patients with chronic rhinosinusitis with nasal polyps from two phase 3 clinical trials: SINUS-24 and SINUS-52.

This post hoc analysis of the international SINUS-24/-52 trials (NCT02912468/NCT02898454) aimed to assess dupilumab efficacy in patients with severe chronic rhinosinusitis with nasal polyps (CRSwNP) according to different definitions of type 2 inflammatory signature. Six definitions of type 2 inflammation were used: ≥150 eosinophils/μL or total immunoglobulin E (IgE)≥100IU/mL with a coexisting type 2 condition; ≥150 eosinophils/μL or total IgE≥100IU/mL; ≥150 eosinophils/μL; ≥250 eosinophils/μL or total IgE ≥100IU/mL; coexisting asthma or ≥300 eosinophils/μL; presence of a coexisting type 2 condition. Odds ratios (ORs; dupilumab vs. placebo) for achieving clinically meaningful improvement (≥1 point) from baseline to week 24 (pooled SINUS-24/-52) and week 52 (SINUS-52) were calculated for nasal polyp score (NPS; range 0-8), nasal congestion/obstruction score (NC; 0-3), and loss of smell score (LoS; 0-3). At baseline (n=724), most patients displayed a type 2 inflammatory signature across definitions (64.2%-95.3%). At week 24, ORs for clinically meaningful improvement ranged from 11.9 to 14.9 for NPS across type 2 definitions, 6.5-9.6 for NC, and 12.2-17.8 for LoS (all p<0.0001). OR ranges were similar or greater at week 52: 19.0-36.6, 7.6-12.1, and 9.2-33.5, respectively (all p<0.0001). Most patients with CRSwNP in the SINUS study had type 2 inflammation. Dupilumab demonstrated robust efficacy across definitions of type 2 inflammation, consistent with its profile as an inhibitor of Interleukin-4 and Interleukin-13 signaling, key and central drivers of type 2 inflammation in CRSwNP. This study assessed type 2 inflammation prevalence and dupilumab efficacy in chronic rhinosinusitis with nasal polyps according to algorithm-defined type 2 inflammation Dupilumab efficacy was similar across all type 2 definitions.

SPLA2GIB Promotes PGD2 and IL-13 Production in Eosinophilic Chronic Rhinosinusitis with Nasal Polyps.

Secreted phospholipase A2 Group IB (sPLA2GIB) regulates the release of arachidonic acid, prostaglandins, and other inflammatory lipid mediators. Although it has been well involved in extensive inflammatory diseases, its specific mechanism in chronic rhinosinusitis with nasal polyps (CRSwNP) remains unclear. In this study, we investigated the role of sPLA2GIB in the pathophysiology of CRSwNP. Quantitative PCR, immunofluorescence staining, western blotting, and enzyme-linked immunosorbent assay (ELISA) were used to analyze the expression of sPLA2s, phospholipase A2 receptor (PLA2R), and prostaglandin D2 (PGD2) in nasal samples. Human nasal epithelial cells (HNECs) were cultured at an air-liquid interface (ALI) and stimulated with various cytokines. The human mast cell line HMC-1 was stimulated with sPLA2GIB, and the expression of PGD2 and cytokines in the culture supernatant was detected by ELISA. The mRNA and protein levels of sPLA2GIB were significantly higher in eosinophilic CRSwNP than in control tissues. sPLA2GIB was predominantly expressed in the nasal epithelial cells. PLA2R mRNA and protein levels were upregulated in both eosinophilic and non-eosinophilic CRSwNP compared with the control groups. IL-4, IL-13, TNF-α, and IL-1β upregulated the expression of sPLA2GIB in ALI-cultured HNECs. sPLA2GIB induced PGD2 and IL-13 production in HMC-1 cells in a hydrolytic activity-independent manner. PGD2 protein expression was elevated in tissue homogenates of eosinophilic CRSwNP, and PGD2 upregulated the expression of IL-13 in HMC-1 cells. Increased secretion of sPLA2GIB by epithelial cells may promote eosinophilic inflammation in CRSwNP by enhancing PGD2 and IL-13 production in mast cells via binding to PLA2R. N/A Laryngoscope, 134:1107-1117, 2024.

Impaired local VD3 metabolism contributes to tissue neutrophilic inflammation in chronic rhinosinusitis with nasal polyps

Abstract Objective: 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 of VD3 function and its effect on local inflammation in CRSwNP. Methods: 25-hydroxyvitamin D3 (25VD3) levels in serum and tissue lysates were detected by ELISA. The expression of VD receptor (VDR), cytochrome P450 family 27 subfamily B member 1 (CYP27B1), the enzymes converting 25VD3 to the active 1,25VD3, and their expression regulation in human nasal epithelial cells (HNECs) were studied by RT-PCR, western blotting, immunofluorescence. HNECs and polyp tissue explants were treated with 1,25VD3. Tissue ECP +eosinophils and MPO +neutrophils were stained and quantified by immunofluorescence. Results: 25VD3 levels in serum and nasal tissue lysates were decreased in CRSwNP patients compared to control subjects. The expression of VDR and CYP27B1 were reduced in CRSwNP. VDR and CYP27B1 expression in HNECs were downregulated by IFN-γ and poly (I:C). 1,25VD3 suppressed neutrophilic inflammation in HNECs and polyp tissues. Tissue 25VD3 levels negatively correlated with neutrophilic inflammation in CRSwNP. Conclusion: Reduced 25VD3 level and decreased CYP27B1 and VDR expression may impair local VD signaling activation, and thus exaggerate neutrophilic inflammation in CRSwNP.

DUPILUMAB IMPROVES CHRONIC RHINOSINUSITIS WITH NASAL POLYPS DISEASE OUTCOMES IRRESPECTIVE OF TYPE 2 SIGNATURE DEFINITION

<h3>Introduction</h3> Chronic rhinosinusitis with nasal polyps (CRSwNP) is a disease of the nasal cavity and paranasal sinuses characterized by type 2 (T2) inflammation. Different algorithms that identify T2 inflammation in CRSwNP have been suggested. This analysis aims to evaluate dupilumab clinical efficacy by these definitions. <h3>Methods</h3> Efficacy was assessed by baseline T2 signature definition (1. ≥150 eosinophils/μL or total ≥IgE 100 IU/mL or any coexisting T2 condition; 2. ≥150 eosinophils/μL or total ≥IgE 100 IU/mL; 3. ≥150 eosinophils/µL; 4. ≥250 eosinophils/μL or total IgE ≥100 IU/mL; 5. asthma or ≥300 eosinophils/μL; 6. any coexisting T2 condition) by least squares (LS) mean differences (dupilumab versus placebo) in change from baseline in nasal polyps score (NPS), nasal congestion/obstruction (NC) score, and loss of smell (LoS) score at Weeks 24 and 52 in the intention-to-treat populations of SINUS-24 and SINUS-52 (NCT02912468/NCT02898454), respectively. <h3>Results</h3> At Week 24, LS mean differences for dupilumab versus placebo by baseline T2 signatures ranged from −2.32 to −2.14 for NPS, −1.10 to −0.92 for NC, and −1.20 to −1.07 for LoS (all <i>P</i> < 0.0001). Similar results were observed at Week 52: −2.64 to −2.40 for NPS, −1.12 to −1.01 for NC, and −1.28 to −1.12 for LoS (all <i>P</i> < 0.0001). <h3>Conclusion</h3> Dupilumab demonstrated robust efficacy across all definitions (combinations and permutations) of T2 inflammation consistent with its profile as an inhibitor of IL-4 and IL-13 signaling, key and central drivers of type 2 inflammation in CRSwNP.

Nasal cytology as a reliable non-invasive procedure to phenotype patients with type 2 chronic rhinosinusitis with nasal polyps

BackgroundThe identification of type-2 inflammation in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) acquires a crucial role in the endotypization needed for selecting patients for biological drugs targeting type-2 inflammation: to date, the parameters used include systemic and histological biomarkers. The aim of this study was to investigate whether nasal cytology could identify type-2 inflammation in patients with CRSwNP. MethodologyThirty-three consecutive patients with CRSwNP underwent nasal cytology sampling at the level of the lower nasal turbinate, and of the polypoid tissue, and surgical polyp tissue sample was collected. The cellularity of the 3 collected samples were compared. ResultsMean nasal polyp tissue, nasal polyps cytology and inferior turbinate cytology eosinophils counts were 43.7 ± 39.6 cells/HPF, 32.8 ± 44.7 cells/HPF and 27.6 ± 58.0 cells/HPF respectively with inferior turbinate cytology eosinophils significantly lower than nasal polyp tissue count (p = 0.007). Both mean nasal polyps cytology eosinophils and mean inferior turbinate cytology eosinophils were significantly higher in patients with type-2 CRSwNP (52.5 ± 67.0 cells/HPF vs 12.2 ± 17.3 cells/HPF, p = 0.012, and 32.0 ± 62.1 cells/HPF vs 2.9 ± 2.9 cells/HPF, p = 0.020 respectively). ConclusionsNasal cytology is suitable tool for assessing local biomarkers of type-2 inflammation in CRSwNP.

Calprotectin in nasal secretion: a new biomarker of non-type 2 inflammation in CRSwNP.

SUMMARYObjectiveWe analysed calprotectin in sinonasal secretions of different chronic rhinosinusitis with nasal polyps (CRSwNP) endotypes to assess its role as a biomarker of non-type 2 inflammation.MethodsWe included primary diffuse CRSwNP patients (n = 41) and three different control groups [non-allergic rhinitis (NAR) (n = 13), non-allergic eosinophilic syndrome (NARES) (n = 10) and healthy subjects (n = 12)]. Calprotectin levels were detected in nasal secretions using a chemiluminescent immunoassay (CLIA).ResultsCalprotectin levels in nasal secretions were significantly higher in all non-type 2 endotypes of CRSwNP compared to healthy controls (p < 0.05). In contrast, in type-2 CRSwNP calprotectin was significantly lower compared to controls (p < 0.05). A significant correlation between calprotectin levels and neutrophilic count/HPF was found in CRSwNP (p < 0.01). Clinically, mean levels of calprotectin and neutrophilia were significantly higher in patients who previously underwent 3 or more endoscopic sinus surgeries (p < 0.05).ConclusionsCalprotectin in nasal secretions may be a biomarker of non-type 2 inflammation. Low levels of calprotectin are indicative of a type-2 immune response in both CRSwNP and non-allergic rhinitis. We observed that calprotectin levels significantly increased based on the number of previous surgeries.

Distinct subsets of innate lymphoid cells in nasal polyp

BackgroundGroup 2 innate lymphoid cells (ILC2s) contribute to the pathogenesis of eosinophilic chronic rhinosinusitis with nasal polyps (CRSwNPs). However, the role of other subsets of ILCs and the differentiation of ILCs in CRSwNPs is not well understood. This study aimed to characterize the ILC subsets and evaluate the differentiation of ILCs from ILC precursors (ILCPs) in NP tissue. MethodsILC subsets and ILCPs were evaluated by flow cytometry in fresh sinonasal mucosa from patients with CRSwNPs and control subjects. Subsets were compared based on clinical variables and immunological features of the patients. Sorted ILCPs (Lin−CD127+CD117+CD45RA+IL1R1+) were cultured with cytokines. ResultsThe frequency of ILC1s and IFN-γ-producing ILC1s increased in non-eosinophilic NPs, whereas that of ILC2s and IL-5-producing ILC2s increased in eosinophilic NPs, particularly in patients with comorbid asthma. The frequency of ILC1s and IFN-γ-producing ILC1s, and frequency of ILC2s and IL-5-producing ILC2s positively correlated with that of neutrophils and eosinophils, respectively. The proportion of IFN-γ-producing ILC1s positively correlated with clinical severity and levels of IFN-γ and IL-8. The proportion of IL-5-producing ILC2s positively correlated with levels of IL-5, CCL24, and total IgE. ILCPs were identified in NP tissue and differentiated into IFN-γ-producing or IL-5-producing ILCs in response to increased IL-12 and IL-18 or IL-25 and IL-33 in non-eosinophilic NPs and eosinophilic NPs, respectively. ConclusionsILC1s and ILC2s may be associated with neutrophilic and eosinophilic inflammation in CRSwNPs, respectively. In addition, ILCPs located in the sinus mucosa could differentiate into IFN-γ- or IL-5-producing cells in response to local cytokine stimuli.

MicroRNA-21-5p promotes mucosal type 2 inflammation via regulating GLP1R/IL-33 signaling in chronic rhinosinusitis with nasal polyps

It has been known that chronic rhinosinusitis with nasal polyps (CRSwNP) is a type 2 inflammation-dominated disease; however, the reasons causing such type of mucosal inflammation in CRSwNP are not well elucidated. We sought to investigate the role of microRNA-21-5p (miR-21-5p) in regulating mucosal type 2 inflammation in CRSwNP. miR-21-5p expression was detected in nasal mucosa of patients with CRSwNP. Correlations between miR-21-5p and indicators of type 2 inflammation were further analyzed. miR-21 knockout mice were used to explore the role of miR-21-5p in a murine model of eosinophilic (E) CRSwNP. Target gene of miR-21-5p related to type 2 inflammation in CRSwNP was identified. The upregulated miR-21-5p in the nasal mucosa of patients with CRSwNP, compared with control subjects, was expressed higher in patients with ECRSwNP than in patients with nonECRSwNP. miR-21-5p expression was positively correlated with mucosal eosinophil infiltrations and the expression of type 2 inflammatory cytokines. In the CRSwNP mice, miR-21 knockout significantly attenuated type 2 inflammation, as indicated by eosinophil infiltrations and expression of cytokines/chemokines in nasal mucosa and lavage fluid; moreover, genes associated with type 2 inflammation were extensively downregulated at the transcriptome level in miR-21 knockout mice. Glucagon-like peptide-1 receptor, which was negatively correlated with miR-21-5p expression in human nasal mucosa, was identified as the target of miR-21-5p. Overexpression of miR-21-5p induced IL-33 expression, whereas glucagon-like peptide-1 receptor agonist decreased IL-33 production in airway epithelial cells. miR-21-5p aggravates type 2 inflammation in the nasal mucosa of patients with CRSwNP via targeting glucagon-like peptide-1 receptor/IL-33 signaling, which may be a potential therapeutic target for CRSwNP.

Calprotectin in nasal secretion: a new biomarker of non-type 2 inflammation in CRSwNP

Objective. We analysed calprotectin in sinonasal secretions of different chronic rhinosinusitis with nasal polyps (CRSwNP) endotypes to assess its role as a biomarker of non-type 2 inflammation. Methods. We included primary diffuse CRSwNP patients (n = 41) and three different control groups [non-allergic rhinitis (NAR) (n = 13), non-allergic eosinophilic syndrome (NARES) (n = 10) and healthy subjects (n = 12)]. Calprotectin levels were detected in nasal secretions using a chemiluminescent immunoassay (CLIA). Results. Calprotectin levels in nasal secretions were significantly higher in all non-type 2 endotypes of CRSwNP compared to healthy controls (p &lt; 0.05). In contrast, in type-2 CRSwNP calprotectin was significantly lower compared to controls (p &lt; 0.05). A significant correlation between calprotectin levels and neutrophilic count/HPF was found in CRSwNP (p &lt; 0.01). Clinically, mean levels of calprotectin and neutrophilia were significantly higher in patients who previously underwent 3 or more endoscopic sinus surgeries (p &lt; 0.05). Conclusions. Calprotectin in nasal secretions may be a biomarker of non-type 2 inflammation. Low levels of calprotectin are indicative of a type-2 immune response in both CRSwNP and non-allergic rhinitis. We observed that calprotectin levels significantly increased based on the number of previous surgeries.

Cystatin SN is a potent upstream initiator of epithelial-derived type 2 inflammation in chronic rhinosinusitis

Cystatin SN (CST1) and cystatin SA (CST2) are cysteine protease inhibitors that protect against allergen, viral, and bacterial proteases. Cystatins are overexpressed in the setting of allergic rhinitis and chronic rhinosinusitis with nasal polyps (CRSwNP); however, their role in promoting type 2 inflammation remains poorly characterized. The purpose of this study was to use integrated poly-omics and a murine exposure model to explore the link between cystatin overexpression in CRSwNP and type 2 inflammation. In this institutional review board- and institutional animal care and use committee-approved study, we compared tissue, exosome, and mucus CST1 and CST2 between CRSwNP and controls (n= 10 per group) by using matched whole exome sequencing, transcriptomic, proteomic, posttranslational modification, histologic, functional, and bioinformatic analyses. C57/BL6 mice were dosed with 3.9 μg/mL of CST1 or PBS intranasally for 5 to 18 days in the presence or absence of epithelial ABCB1a knockdown. Inflammatory cytokines were quantified by using Quansys multiplex assays or ELISAs. Of the 1305 proteins quantified, CST1 and CST2 were among the most overexpressed protease inhibitors in tissue, exosome, and mucus samples; they were localized to the epithelial layer. Multiple posttranslational modifications were identified in the polyp tissue. Exosomal CST1 and CST2 were strongly and significantly correlated with eosinophils and Lund-Mackay scores. Murine type 2 cytokine secretion and TH2 cell infiltration increased in a time-dependent manner following CST1 exposure and was abrogated by epithelial knockdown of ABCB1a, a regulator of epithelial cytokine secretion. CST1 is a potent upstream initiator of epithelial-derived type 2 inflammation in CRSwNP. Therapeutic strategies targeting CST activity and its associated posttranslational modifications deserve further interrogation.

Distinct Gene Set Enrichment Profiles in Eosinophilic and Non-Eosinophilic Chronic Rhinosinusitis with Nasal Polyps by Bulk RNA Barcoding and Sequencing.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease with a high symptom burden, including nasal congestion and smell disorders. This study performed a detailed transcriptomic analysis in CRSwNP classified as eosinophilic CRS (ECRS), nonECRS according to the Japanese Epidemiological Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC) criteria, and a group of ECRS with comorbid aspirin intolerant asthma (Asp). Gene expression profiles of nasal polyps and the uncinate process in CRSwNP patients and normal subjects (controls) were generated by bulk RNA barcoding and sequencing (BRB-seq). A differentially expressed genes (DEGs) analysis was performed using DESeq2 software in iDEP to clarify any relationship between gene expression and disease backgrounds. A total of 3004 genes were identified by DEGs analysis to be associated with ECRS vs control, nonECRS vs control, and Asp vs control. A pathway analysis showed distinct profiles between the groups. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) showed distinct phenotype-specific pathways of expressed genes. In the specific pathway of “cytokine–cytokine receptor interaction”, the differentially expressed genes were widely distributed. This study indicates that transcriptome analysis using BRB-seq may be a valuable tool to explore the pathogenesis of type 2 inflammation in CRSwNP.

Necroptosis Underlies Neutrophilic Inflammation Associated with the Chronic Rhinosinusitis with Nasal Polyps (CRSwNP).

BackgroundNecroptosis is an inflammatory cell death associated with a variety of chronic diseases. Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease accompanied by eosinophil and neutrophil infiltration. The role of necroptosis in the pathogenesis of CRSwNP remains elusive.MethodsCell death, including apoptosis, pyroptosis and necroptosis in control sinonasal mucosa and CRSwNP, were analyzed by immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF) staining for cleaved caspase 3, cleaved gasdermin D and p-MLKL, respectively. Correlations between necroptosis, inflammatory cytokines and neutrophil infiltration were assessed and a possible role of necroptosis in CRSwNP was evaluated. Primary nasal polyp cells (DNPCs) were stimulated with damage-associated molecular patterns (DAMPs) including ATP or IL-1α and their expression of inflammatory cytokines was analyzed using RT-PCR. The expression of TNF-α and IFNs in nasal polyps was measured by ELISA; human monocyte THP-1 cells were treated with TNF-α or IFN-γ and cell death was measured by LDH release.ResultsNecroptosis, rather than apoptosis or pyroptosis, was overtly activated in both eosinophilic and non-eosinophilic CRSwNP as evidenced by the presence of prominent phosphorylation of MLKL compared to controls. The abundance of DAMPs (IL-1α, HMGB1), inflammatory cytokines (IL-6) and chemokines (IL-8, CXCL-1) were all increased especially in non-eosinophilic CRSwNP. The extent of necroptosis was positively correlated with the abundance of DAMPs and cytokines, and neutrophil infiltration in CRSwNP. In DNPCs, ATP and IL-1α induced the expression of IL-8 and CXCL-1. Macrophage was found to be the predominant cell type positive for p-MLKL in CRSwNP. Concomitant treatment with TNF-α and IFN-γ, which were abundantly present in CRSwNP, triggered marked necroptosis in THP-1 cells.ConclusionNecroptosis induced by TNF-α and IFN-γ may facilitate the production and release of a myriad of proinflammatory cytokines and entailed neutrophil infiltration to exacerbate inflammation in CRSwNP.

Upregulation of Basonuclin1 Is Associated with p63-Involved Epithelial Barrier Impairment and Type-2 Helper T-cell Inflammation in Chronic Rhinosinusitis with Nasal Polyps

Background: Tumor protein p63 has been shown to be important for epithelial dysfunction, including epithelial barrier defects and mucosal inflammation, in the development of chronic rhinosinusitis with nasal polyps (CRSwNP). Basonuclin1 (BNC1), an epithelial-specific transcriptional factor, is a direct downstream target of p63 and thus might be involved in the pathogenesis of CRSwNP. Objective: We sought to investigate whether BNC1 was associated with p63-mediated epithelial barrier defects and nasal mucosal inflammation in CRSwNP. Methods: Nasal tissue biopsies were obtained from 91 patients to CRSwNP, 49 chronic rhinosinusitis without nasal polyps (CRSsNP) patients, and 28 control subjects. Immunohistochemistry and immunofluorescence staining were used to determine the distribution of BNC1 in tissues and localization in cells, respectively. Quantitative PCR was performed to detect the expression levels of BNC1, TP63, epithelial barrier proteins, and type-2 helper T-cell inflammation-related genes. Results: BNC1 mRNA expression was significantly elevated in the tissues in CRSwNP patients compared with CRSsNP (1.96-fold, p = 0.0003) and control groups (2.40-fold, p < 0.0001). BNC1 staining was strongly positive in the nasal epithelium and co-localized with p63-positive epithelial cells. The expression of BNC1 mRNA was strongly correlated with TP63 mRNA level both in tissue biopsies (r = 0.78, p < 0.0001) and epithelial scrapings (r = 0.97, p < 0.0001). BNC1 expression was also positively correlated with epithelial barrier protein genes (CDH1, CLDN1, CLDN4, TJP1, and TJP2) and epithelial genes involved in T_H2 inflammation (IL33, CCL26, CLC, and ALOX15). Conclusions: Overexpression of BNC1 may be associated with increased expression of TP63, and possibly contribute to the epithelial barrier defects and T_H2 inflammation in CRSwNP.

Multidisciplinary consensus on a stepwise treatment algorithm for management of chronic rhinosinusitis with nasal polyps.

Multidisciplinary consensus on a stepwise treatment algorithm for management of chronic rhinosinusitis with nasal polyps.

State-of-the-art overview on biological treatment for CRSwNP.

The majority of patients with uncontrolled severe CRSwNP, asthma and atopic dermatitis share a similar T helper 2 type inflammation linked to their underlying phenotype. This discovery has triggered new research around treatments targeting specific cytokines driving inflammation in CRSwNP like IL-4, IL-13, IL-5 and IgE. Biologicals are increasingly tested as additional tre- atment for patients suffering from severe chronic rhinosinusitis with nasal polyps (CRSwNP). Their efficacy has been demonstrated in multiple studies. All studies differ in terms of baseline characteristics of included patients and outcome parameters analysed. A comparative analysis of the efficacy of reported biologicals for CRSwNP based on the published data for phase 2 and 3 studies. The aim was to provide a comprehensive overview across the different biologicals and outcome parameters. In a first step we critically selected out of all available phase 2 and 3 clinical trials the ones containing the most rigorous and compatible study designs. Meaning studies that comply with a need for a clear definition of CRSwNP, at least two administra- tion doses, comparable timeframes and the same outcome parameters studied. This assessment was performed using a PRISMA search. We retained 7 studies with significant data for dupilumab, mepolizumab and omalizumab. In a second step the effect-sizes of treatment with those biologicals were compared for the most important outcome parameters both patient relevant (nasal con- gestion, smell loss and SNOT-22 scores) and patient irrelevant (CT scan Lund-Mackay, smell test and nasal polyp scores). Therapy duration of 16 to 25 weeks was chosen for evaluation of efficacy. A direct comparison of efficacy between dupilumab, mepolizumab and omalizumab is challenging given differences in inclusion criteria, outcome parameters and time-points of analyses. We have been able to conclude that effect sizes of dupilumab, mepolizumab and omalizumab seem large enough to reflect a major reduction in symptom burden as experienced by patients suffering from refractory CRSwNP. The effect size of dupilumab on both patient relevant and patient irrelevant parameters of smell loss are clearly significant and reflect the clinical experience of major reduction of smell impairment in treated patients. Despite the heterogeneity of protocols, dosages and time-points of analyses of biological trials in CRSwNP, this over- view highlights outcomes of biological treatment in CRSwNP in a comprehensive way. Real-life registries, comparative trials and/ or endotype-driven treatment plans are needed to provide the answers to the multiple questions that are still open today.

Endoplasmic reticulum stress exacerbates inflammation in chronic rhinosinusitis with nasal polyps via the transcription factor XBP1

Endoplasmic reticulum (ER) stress results in the activation of the unfolded protein response (UPR), a process that is involved in the pathogenesis of many inflammatory diseases. However, the role of ER stress in chronic rhinosinusitis with nasal polyps (CRSwNP) has yet to be elucidated. In this study, we found that the protein expression levels of a range of ER stress regulators, including p-PERK, ATF4, ATF6 and XBP1s, were significantly increased in CRSwNP compared to controls. Importantly, the expression of ATF4 and XBP1s was positively correlated with heightened inflammation in CRSwNP. In human nasal epithelial cells, the ER stress inducer tunicamycin (TM) could potentiate Toll-like receptors (TLRs) induced proinflammatory cytokines production. Furthermore, we found that the silencing of XBP1, but not ATF4 or ATF6, abrogated the proinflammatory effect of TM. Mechanistically, ER stress did not affect the NF-κB, MAPK or IRF3 signaling pathways. However, the ER stress regulator XBP1s was able to bind directly to the promoter region of inflammatory genes to modulate gene transcription. Besides, the commensal bacteria Staphylococcus aureus and several inflammatory factors, such as IL4, IL13, IL17 and IFNγ, could induce ER stress in epithelial cells. Collectively, ER stress plays a crucial role in facilitating TLR-induced inflammation. Targeting XBP1 can inhibit the inflammatory response, thus offering a potential approach to treat CRSwNP.