Uncinate Process Tissues Research Articles

Gene transcriptome analysis of nasal epithelial cells in chronic rhinosinusitis with nasal polyps

Objective: To identify the differentially expressed genes in nasal epithelial cells from chronic rhinosinusitis with nasal polyps (CRSwNP), and to analyze related genes which are involved in deficiency of nasal epithelial barrier in CRSwNP patients by analyzing the datasets download from the gene expression omnibus(GEO) database. Methods: The mRNA expression microarray data numbered GSE107624 (7 CRSwNP and 7 controls) and GSE69093 (13 CRSwNP and 11 controls) were downloaded from the publicly available GEO database. These two datasets were jointly analyzed to screen the differentially expressed genes in nasal epithelial cells of controls and CRSwNP patients. In the meanwhile, we further evaluated the function annotation and regulatory pathways of the differentially expressed genes. To further confirmed what we have observed, sinus tissues were collected from patients with CRSwNP (14 cases, 46.8±17.9 years) and uncinate process tissues were collected from patients with nasal septum deviation (7 cases, 23.4±2.3 years) as control group. The primary epithelial cells of nasal mucosa were cultured and the mRNA level of screened genes were measured by Q-PCR. SPSS 22.0 software was used to for statistical analysis. Results: GSE107624 dataset showed that there were 3 856 differentially genes in nasal epithelial cells between CRSwNP and control group, while there were 771 differentially expressed genes in GSE69093 dataset. Finally, 55 up-regulated genes and 3 down-regulated genes were noticed in nasal epithelial cells of CRSwNP patients in the two datasets. GO gene functional annotation analysis showed that SPTBN1, FNBP1L, VAPB and SNX1 were involved in cell adhesion function, MAP1B was participated in the formation of microtubule related complex. KEGG pathway enrichment analysis indicated that BAMBI and SIAH1 were involved in regulation of Wnt pathway, COL6A1 and EIF4E were involved in the regulation of PI3K-AKT pathway. String protein interaction network analysis assumed that MAP1B and VAPB were the core functional proteins. Among top 3 differentially expressed genes COL6A1, MAP1B and BAMBI, only MAP1B gene was increased in nasal epithelial cells of CRSwNP patients in comparison to controls. Conclusion: The increased MAP1B gene in epithelial cells of CRSwNP, as well as abnormal regulation of Wnt and PI3K-AKT signal pathways may mediate the barrier dysfunction in CRSwNP.

The expression and significance of TET gene and 5hmC in chronic rhinosinusitis

Objective:To investigate the expression and clinical significance of TET gene and 5hmC in chronic sinusitis. Methods:Acquiring 20 cases of nasal polyps from chronic sinusitis with polyps（CW）, 20 cases of uncinate process tissues from chronic sinusitis without polyps（CS）, 10 cases of middle turbinate tissues from patients with nasal septum deviated as normal group（N）.The expression of TET gene and 5hmC in chronic sinusitis was measured by immunofluorescence, Western-Blot and Quantitative real-time PCR. Results:Both TET1/2/3 gene and 5 hmC were highly expressed in the CS group, but were lower in the CW group and the N group.There were statistically significant differences between the CS group and the CW group（P<0.05）, and between the CS group and the N group（P<0.05）.The expressions of TET1 and TET3 in the N group were higher than those in the CW group, and the difference was statistically significant（P<0.05）. Conclusion:DNA demethylation plays an important role in the formation of nasal polyps.High expression of TET1, TET2, TET3 and 5hmC May reduce the risk of nasal polyps.Increased DNA demethylation in chronic sinusitis may reduce the risk of nasal polyp formation.When the degree of DNA methylation in chronic sinusitis is high and the degree of DNA demethylation is low, the disease may develop to CRSwNP, and when the degree of DNA methylation is low and the degree of DNA demethylation is high, the disease may develop to CRSsNP.

Human Rhinovirus Infection Enhances the Th2 Environment in Allergic and Non-allergic Patients with Chronic Rhinosinusitis.

Objectives. This study was conducted to determine whether patients with allergic rhinitis might be more susceptible to human rhinovirus (HRV) infection and whether the effects of infection on the elicited immune responses are different in allergic and non-allergic patients with chronic rhinosinusitis (CRS).Methods. Uncinate process tissues were obtained from 61 CRS patients (of whom 39 had allergies and 22 did not) and were infected with HRV-16 using an air-liquid interface organ culture system. The expression levels of programmed cell death-ligand (PD-L)1, PD-L2, intracellular adhesion molecule 1, interferon-gamma (IFN-γ), interleukin (IL)-4, IL-5, and IL-10 were evaluated in the infected nasal mucosa.Results. The HRV infection rates were not significantly different between the allergy (74.4%) and non-allergy (72.7%) groups. In the allergy group, the expression of PD-L1 (P=0.013) and IL-10 (P=0.040) was significantly elevated in the HRV-infected tissues, and there was a strong correlation between PD-L1 and IL-10 (r=0.868, P<0.001). In contrast, infected tissues from the non-allergy group displayed increased levels of IL-4 (P=0.039), IL-5 (P=0.023), and IFN-γ (P=0.031), as well as an increased IL-4/IFN-γ ratio, after HRV infection (P=0.043).Conclusion. This study showed that HRV infection rates were similar in the nasal mucosa of patients with CRS regardless of the presence of allergic rhinitis. HRV infection enhanced the Th2 environment by modulating PD-L1 and PD-L2 expression levels in allergic mucosa and by increasing the IL-4/IFN-γ ratio in non-allergic mucosa.

Reduced Local Response to Corticosteroids in Eosinophilic Chronic Rhinosinusitis with Asthma.

Eosinophilic chronic rhinosinusitis (ECRS), a subgroup of chronic rhinosinusitis with nasal polyps, is recognized as a refractory eosinophilic disorder characterized by both upper and lower airway inflammation. In some severe cases, disease control is poor, likely due to local steroid insensitivity. In this study, we focused on protein phosphatase 2A (PP2A), a key factor regulating glucocorticoid receptor (GR) nuclear translocation, and examined its association with local responses to corticosteroids in eosinophilic airway inflammation. Our results indicated reduced responses to corticosteroids in nasal epithelial cells from ECRS patients with asthma, which were also associated with decreased PP2A mRNA expression. Eosinophil peroxidase stimulates elevated PP2A phosphorylation levels, reducing PP2A protein expression and activity. In addition, mRNA levels of inflammatory mediators (TSLP, IL-25, IL-33, CCL4, CCL5, CCL11, and CCL26) associated with eosinophilic airway inflammation in epithelial cells were increased in nasal polyps (eosinophil-rich areas) compared with those in uncinate process tissues (eosinophil-poor areas) from the same patients. PP2A reduction by siRNA reduced GR nuclear translocation, whereas PP2A overexpression by plasmid transfection, or PP2A activation by formoterol, enhanced GR nuclear translocation. Collectively, our findings indicate that PP2A may represent a promising therapeutic target in refractory eosinophilic airway inflammation characterized by local steroid insensitivity.

Role of IL-17A in Chronic Rhinosinusitis With Nasal Polyp

PurposeTh17-associated inflammation is increased in chronic rhinosinusitis with nasal polyp (CRSwNP), and is associated with disease severity and steroid resistance. Overexpressed interleukin (IL)-17A affects CRSwNP by tissue remodeling, eosinophilic accumulation, and neutrophilic infiltration. We aimed to identify the role of IL-17A in CRSwNP and to evaluate the effects of anti-IL-17A blocking antibody on nasal polyp (NP) formation using a murine NP model. Moreover, we sought to investigate whether the inhibition of mechanistic target of the rapamycin (mTOR) signal pathway could suppress IL-17A expression and NP formation.MethodsHuman sinonasal tissues from control subjects and patients with chronic rhinosinusitis (CRS) were analyzed using immunohistochemistry (IHC) and immunofluorescence staining. The effects of IL-17A neutralizing antibody and rapamycin were evaluated in a murine NP model. Mouse samples were analyzed using IHC, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay.ResultsIL-17A+ inflammatory cells were significantly increased in number in NP from patients with CRSwNP compared to that in uncinate process tissues from control subjects and patients with CRS without NP or CRSwNP. CD68+ M1 macrophages dominantly expressed IL-17A, followed by neutrophils and T helper cells, in NP tissues. Neutralization of IL-17A effectively reduced the number of NPs, inflammatory cytokines, and IL-17A-producing cells, including M1 macrophages. Inhibition of IL-17A via the mTOR pathway using rapamycin also attenuated NP formation and inflammation in the murine NP model.ConclusionsIL-17A possibly plays a role in the pathogenesis of CRSwNP, the major cellular source being M1 macrophage in NP tissues. Targeting IL-17A directly or indirectly may be an effective therapeutic strategy for CRSwNP.

JESREC score and mucosal eosinophilia can predict endotypes of chronic rhinosinusitis with nasal polyps

ObjectiveRecently, JESREC score and mucosal eosinophil count have been used to diagnose eosinophilic chronic rhinosinusitis (ECRS) in Japan. However, it remains unknown whether the subtypes of CRS diagnosed by these criteria have different endotypes. In the present study, we investigated whether JESREC score and mucosal eosinophil count were appropriate for classification of CRS subgroups into endotypes. MethodsA cross-sectional study involving 71 consecutive patients with CRS with nasal polyps (CRSwNP) and 13 control patients was performed. Nasal polyp tissues from CRSwNP patients and uncinate process tissues from control patients were collected for analysis of inflammatory cells by immunohistochemistry and measurement of cytokines and chemokines by ELISA and quantitative real-time PCR. We compared the differences between subtypes according to JESREC score and mucosal eosinophil count and investigated the subgroups with different endotypes by cluster analysis and principal component analysis. ResultsIn the 71 CRSwNP patients, 9 patients had JESREC score <11 and mucosal eosinophil count <70/HPF (Group A), 20 patients had JESREC score ≥11 and mucosal eosinophil count <70/HPF (Group C), and 42 patients had JESREC score ≥11 and mucosal eosinophil count ≥70/high-power field (HPF) (Group D). Semiquantitative analysis of inflammatory cells showed that eosinophils, neutrophils, macrophages, mast cells, and basophils differed significantly between the subgroups. At the mRNA level, CLC, IL5, IL13, CCL11, CCL24, CCL26, POSTN, CSF3, and IL8 showed significant differences. At the protein level, eotaxin-2/CCL24, eotaxin-3/CCL26, and G-CSF had significant differences. Cluster analysis using gene expression levels in 55 CRS patients and 11 control patients revealed that the patients could be classified into five clusters. Cluster 1 (n=27) contained all patients with Group D. Cluster 2 (n=11) comprised all control patients. Cluster 3 (n=4) included mixed subtypes: one with Group A and three with Group D. Cluster 4 (n=7) and Cluster 5 (n=17) contained all patients with Groups A and C, respectively. Furthermore, the principal component analysis revealed that the subtypes had different characteristics. ConclusionCRS subtypes based on JESREC score and mucosal eosinophil count showed different inflammatory patterns, and unsupervised statistical analyses supported the classification that can predict endotypes. From these results, we concluded that the classification based on JESREC score and mucosal eosinophil count was useful for predicting CRS endotypes.

Local IL-25 contributes to Th2-biased inflammatory profiles in nasal polyps.

IL-25 has been proposed to play a key role in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to evaluate the association of IL-25 with the Th2-biased inflammatory profiles in CRSwNP. Nasal polyp (NP) tissues and control uncinate process tissues were collected from 92 patients with CRSwNP, 20 patients with chronic rhinosinusitis without nasal polyps (CRSsNP), and 16 normal control subjects. IL-25 expression was examined using immunohistochemistry and immunofluorescence staining, flow cytometry, RT-qPCR, and ELISA. The inflammatory profiles and clinical characteristics of 2 NP subtypes (IL-25high and IL-25low ) were evaluated, and the effects of IL-25 on Th2 cytokine production in cultured dispersed polyp cells were examined invitro. The mRNA and protein levels of IL-25 were significantly increased in the polyp tissues compared with the control uncinate process tissues. The IL-25high subtype showed greater computed tomography scores, endoscopic scores, and Th2 response. Exposure to IL-25 activated type 2 innate lymphoid cells and Th2 cells in NP simultaneously which further increased Th2 cytokine production invitro. Local IL-25 plays a crucial role in promoting Th2-biased inflammatory profiles in NP and may serve as a promising therapeutic target in CRSwNP patients.

Expression of cysteinyl leukotriene receptor GPR17 in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps.

The cysteinyl leukotrienes (cysLTs) are proinflammatory lipid mediators that act on the type 1 cysLT receptor (CysLT1R) in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). GPR17, a G protein-coupled orphan receptor with homology to the cysLT receptors, has been proposed as a damage sensor during inflammation. However, the expression and correlation of GPR17 and CysLT1R in eosinophilic CRSwNP (ECRS) and non-eosinophilic CRSwNP (non-ECRS) have not been well investigated. To evaluate the expression of GPR17 and its correlation with CysLT1R in the 2 CRSwNP subsets. Polyp tissues were collected from CRSwNP subjects (15 ECRS and 14 non-ECRS), and uncinate processes were collected from 12 CRSsNP subjects and 13 control subjects. The mRNA and protein levels of GPR17 and CysLT1R were examined using qRT-PCR, immunohistochemistry, and western blotting. Additionally, the correlation between GPR17 and CysLT1R at the mRNA and protein levels was evaluated. All assays were performed in a blinded manner. Polyp tissues exhibited significantly increased GPR17 expression relative to uncinate process tissues from CRSsNP patients, or healthy controls (P=0.0012 and P<0.0001, respectively). Compared with the non-ECRS subset, the ECRS subset showed significantly increased GPR17 expression. Moreover, the GPR17 expression was positively correlated with CysLT1R in nasal polyps. The increased expression of GPR17 in nasal polyps and the differential expression between eosinophilic and non-eosinophilic CRSwNP subsets suggest that these subsets may have distinct pathogenic mechanisms. The positive correlation between GPR17 and CysLT1R in polyp tissues might imply substantial regulatory mechanisms that must be elucidated.

Expression of heme oxygenase-1 in nasal polyps and regulation by glucocorticoid

To evaluate the expression and possible modulation of heme oxygenase-1 (HO-1) in nasal polyps of patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Nasal polyps and uncinate process tissues were collected from 25 CRSwNP patients and 19 healthy controls with nasal septal deviation. HO-1 expression was examined using qRT-PCR, immunohistochemistric staining and Western blot analysis. Moreover, additional uncinate process mucosal samples of 15 healthy controls with nasal septal deviation were harvested for nasal explant culture experiments. HO-1 expression was measured in cultured nasal explant in response to specific inflammatory and glucocorticoid stimulation. SPSS 20.0 software was used to analyze the data. The mRNA and protein expression of HO-1 was significantly increased in polyp tissues, 1.220±0.397 in mRNA and 1.409±0.701 in protein, compared with healthy controls 0.464±0.318 in mRNA and 0.017±0.1147 in protein (U=22.00 in mRNA and U=1.00 in protein, both P< 0.05). The immunohistochemical results showed that HO-1 was mainly distributed in the epithelial layer, submucosal glands and inflammatory cells in nasal tissues. Nasal explant culture experiments demonstrated that HO-1 mRNA was upregulated by IL-17A. The HO-1 mRNA level before the stimulation was 1.000, and 17.264±4.275 after the stimulation of 1 ng/ml IL-17A (U=0, P<0.05), 19.128±4.605 after the stimulation of 10 ng/ml IL-17A (U=0, P<0.05), but was significantly suppressed after stimulation with glucocorticoids (dexamethasone, DEX). The mRNA level after the glucocorticoids stimulation was 0.370±0.101 (U=0, P<0.05) and 0.316±0.167 (U=0, P<0.05) respectively. Furthermore, the HO-1 mRNA was inhibited by TGF-β1, the mRNA level was 0.217±0.322 (U=0, P<0.05), 0.070±0.070 (U=0, P<0.05), respectively. Increased HO-1 expression may play a role in the pathogenesis of CRSwNP, which may be considered as the therapeutic target.

Critical link between glycogen synthase kinase 3β and forkhead box P3 in patients with chronic rhinosinusitis with nasal polyps

Critical link between glycogen synthase kinase 3β and forkhead box P3 in patients with chronic rhinosinusitis with nasal polyps

Expression of leukotriene and its receptors in eosinophilic chronic rhinosinusitis with nasal polyps.

Cysteinyl leukotriene (LT) has been proposed in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). This study sought to examine the expression of the LT receptor (LTR) in CRSwNP patients and evaluate the potential role of LTR antagonist (LTRA) in the management of eosinophilic CRSwNP (ECRS) patients. Nasal polyps and uncinate process tissues were collected from 18 ECRS patients, 13 non-eosinophilic CRSwNP (non-ECRS) patients, and 16 control subjects. The messenger RNA (mRNA) and protein expression of LTR (cysteinyl leukotriene receptor 1 [CysLT1R] and cysteinyl leukotriene receptor 2 [CysLT2R]) was examined using quantitative reverse-transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, and Western blot analysis. Moreover, the effects of LTRA and steroids on total nasal symptom scores (TNSS) of uncontrolled ECRS patients were evaluated. The mRNA and protein expression of CysLT1R and CysLT2R was significantly increased in polyp tissues compared with healthy controls (p < 0.05). Compared with the non-ECRS subset, the ECRS subset showed significantly increased expression of CysLT1R and CysLT2R, as well as leukotriene C4 (LTC4) and leukotriene D4 (LTC4) levels (p < 0.05). Moreover, combined LTRA and steroids significantly decreased TNSS more than steroids alone in uncontrolled ECRS patients (p < 0.01). Our findings indicate that LTR was differentially expressed between ECRS and non-ECRS patients, and that LTRA may be used as an additional therapy for ECRS patients.

Expression of heme oxygenase-1 in eosinophilic and non-eosinophilic chronic rhinosinusitis with nasal polyps: modulation by cytokines.

Oxidative stress is characteristic of chronic airway inflammatory diseases such as asthma, chronic obstructive pulmonary disease and chronic rhinosinusitis with nasal polyps (CRSwNP). Heme oxygenase (HO)-1 has been proposed to be a cytoprotective enzyme against oxidative stress in CRSwNP. However, the expression and regulation of HO-1 in eosinophilic CRSwNP (ECRS) and non-eosinophilic CRSwNP (non-ECRS) subsets has not been well documented. Nasal polyps and uncinate process tissues were enrolled from 40 CRSwNP patients (ECRS, 17; non-ECRS, 23) and 20 control subjects, respectively. The messenger RNA (mRNA) and protein expression of HO-1 was examined using qRT-PCR, immunohistochemistry, and Western blot staining. Moreover, the stimulatory effects of several cytokines (interferon γ [IFN-γ], interleukin [IL]-5, and IL-13, etc.) on HO-1 mRNA expression in cultured nasal explants were evaluated. The mRNA and protein expression of HO-1 was significantly increased in polyp tissues compared with healthy controls (p < 0.05), and the non-ECRS subset showed significantly increased HO-1 expression compared with the ECRS subset (p < 0.05). Moreover, in cultured nasal explant, HO-1 mRNA was significantly upregulated in the presence of IFN-γ, IL-27, IL-5, IL-13, and IL-17A, but was significantly inhibited by transforming growth factor β1 (TGF-β1) (p < 0.05). Our findings indicate that HO-1 was differentially expressed and regulated in ECRS and non-ECRS patients.

Increased IL-21 expression in chronic rhinosinusitis with nasalpolyps.

IL-21 is a key cytokine for regulating B cell immunity, which is involved in several inflammatory conditions. This study sought to define a role for IL-21 in activated B lymphocytes and enhanced tissue eosinophilia in NP tissues during the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). NP and uncinate process tissues were collected from 64 CRSwNP patients, 25 CRSsNP patients, and 29 control subjects. IL-21 expression was examined using IHC staining, qRT-PCR, flow cytometry, and ELISA, and its clinical implication was evaluated. Moreover, the effects of IL-21 on B cell differentiation and Ig production in cultured NP cells were examined in vitro. The mRNA and protein levels of IL-21 were significantly increased in polyp tissues compared with control tissues (P < 0.05). Polyp IL-21 level was significantly associated with polyp size, tissue eosinophilia and asthma comorbidity, and recurrence after surgery (P < 0.05). Both Th1 and Th17 cells were the main cellular sources of IL-21 in polyp tissues. The percentage of IL-21(+) CD4(+) cells was significantly higher in polyp tissues compared with control tissues and matched PBMCs (P < 0.01). Accordingly, the percentage of CD19(+) CD20(+/-) CD38(high) cells was significantly higher in polyp tissues compared with control tissues (P < 0.01). Moreover, recombinant IL-21 significantly increased the percentage of CD19(+) CD20(+/-) CD38(high) cells (plasmablasts) and IgG and IgA production in cultured NP cells in vitro (P < 0.05). Increased IL-21 level in polyp tissues was associated with disease severity, local B cell activation, and immunoglobulin production, suggesting that IL-21 might play an important role in promoting persistent mucosal inflammation in CRSwNP patients.

The antimicrobial protein short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is differentially modulated in eosinophilic and noneosinophilic chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a highly heterogeneous disease with aberrant host defense responses. However, whether innate immunity is similarly impaired in patients with eosinophilic and those with noneosinophilic CRSwNP remains unclear. We sought to evaluate the expression and possible modulation of short palate, lung, and nasal epithelium clone 1 (SPLUNC1), an innate immune molecule, in the 2 CRSwNP subsets. Polyp tissue and uncinate processes were collected from 40 patients with CRSwNP, 27 patients with chronic rhinosinusitis without nasal polyps (CRSsNP), and 22 control subjects. Expression of SPLUNC1; Toll-like receptor (TLR) 2, TLR3, and TLR4; and the proinflammatory cytokines IL-1α, IL-4, IL-13, IL-17A, and IFN-γ was examined in nasal tissues. Additionally, SPLUNC1 expression in response to specific inflammatory stimulation was measured in cultured polyp epithelial cells and A549 cells. Polyp tissues exhibited significantly decreased expression of SPLUNC1 and other innate immune molecules compared with uncinate process tissues from patients with CRSwNP (P < .05), patients with CRSsNP, and healthy control subjects. Moreover, the eosinophilic CRSwNP subset exhibited significantly decreased SPLUNC1 expression and numbers of submucosal glands, as well as significantly increased IL-4 and IL-13 mRNA levels, compared with the noneosinophilic subset (P < .05). Accordingly, SPLUNC1 expression in polyp epithelial cells was significantly inhibited by IL-4 and IL-13 stimulation in vitro but was significantly upregulated after stimulation with TLR agonists and glucocorticoids (P < .05). Differential SPLUNC1 suppression between the eosinophilic and noneosinophilic CRSwNP subsets suggests that they possess distinct pathogenic mechanisms. This finding might benefit the design of appropriate therapeutic interventions targeted to each subset.