Nasal Polyp Fibroblasts Research Articles

Transcriptional analysis of nasal polyps fibroblasts reveals a new source of pro-inflammatory signaling in CRSwNP.

Fibroblasts and others mesenchymal cells have recently been identified as critical cells triggering tissue-specific inflammatory responses. Persistent activation of fibroblasts inflammatory program has been suggested as an underlying cause of chronic inflammation in a wide range of tissues and pathologies. Nevertheless, the role of fibroblasts in the emergence of chronic inflammation in the upper airway has not been previously addressed. We aimed to elucidate whether fibroblasts could have a role in the inflammatory response in chronic rhinosinusitis with nasal polyps (CRSwNP). We performed whole-transcriptome microarray in fibroblast cultured from CRSwNP samples and confirmed our results by qRT-PCR. We selected patients without other associated diseases in upper airway. To investigate shifts in transcriptional profile we used fibroblasts from nasal polyps and uncinate mucosae from patient with CRSwNP, and fibroblasts from uncinate mucosae from healthy subjects as controls. This study exposes activation of a pro-inflammatory and pro-fibrotic transcriptional program in nasal polyps and CRSwNP fibroblasts when compared to controls. Our Gene-set Enrichment Analysis (GSEA) pointed to common up-regulation of several pro-inflammatory pathways in patients-derived fibroblasts, along with higher mRNA expression levels of cytokines, growth factors and extracellular matrix components. Our work reveals a potential new source of inflammatory signaling in CRSwNP. Furthermore, our results suggest that deregulated inflammatory signaling in tissue-resident fibroblasts could support a Type-2 inflammatory response. Further investigations will be necessary to demonstrate the functionality of these novel results.

Nasal polyp fibroblasts (NPFs)-derived exosomes are important for the release of vascular endothelial growth factor from cocultured eosinophils and NPFs

ObjectiveSignificant eosinophil infiltration and tissue remodeling are common characteristics of conditions associated with chronic airway inflammation, such as chronic rhinosinusitis with nasal polyp and bronchial asthma. This study was designed to elucidate the role of eosinophil-fibroblast interactions in tissue remodeling during chronic airway inflammation. MethodsPeripheral blood eosinophils or EoL-1 eosinophilic leukemia cells were cocultured with nasal polyp fibroblasts (NPFs). Coculture-induced release of exosomes, major components of extracellular vesicles (EVs), and a profibrotic cytokine, vascular endothelial growth factor (VEGF), were evaluated by enzyme-linked immunosorbent assay. ResultsEosinophil-NPF interactions stimulated the release of exosomes and VEGF into culture supernatants. Coculture-induced release of exosomes was stimulated earlier than VEGF release, at 3 h of incubation. The average size of the EVs released by NPFs was 133 ± 3.6 nm. NPF-derived EVs (exosome concentration: 25 pg/mL) significantly stimulated VEGF release from EoL-1 cells. Pretreatment of NPFs with exosome inhibitor, GW4869 or DMA attenuated the release of exosomes and VEGF from cocultured EoL-1 cells and NPFs. ConclusionThe results of this study indicate that eosinophil-fibroblast interactions are important in the pathophysiology of tissue remodeling in eosinophil-predominant airway inflammation and that NPF-derived exosomes play a crucial role in the release of VEGF.

Involvement of the extracellular matrix proteins periostin and tenascin C in nasal polyp remodeling by regulating the expression of MMPs.

BackgroundTissue remodeling caused by increased MMPs is involved in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). We previously found higher levels of periostin and tenascin C in CRSwNPs, but whether they are associated with the dysregulation of MMPs is unknown. Therefore, the present study aimed to investigate the regulatory roles of these two ECM proteins in the expression of MMPs in nasal polyps.MethodsThe concentrations of MMP‐2, MMP‐3, MMP‐7, MMP‐8, MMP‐9, MMP‐12, MMP‐13, TIMP‐1, TIMP‐2, TIMP‐3, TIMP‐4, periostin, and tenascin C in tissue homogenates of 51 patients with chronic rhinosinusitis with and without nasal polyps and 15 control subjects were measured and were analyzed by adjusted logistic regression and spearman correlation test. Primary human nasal polyp fibroblasts and epithelial cells were stimulated ex vivo with periostin and tenascin C and the gene expression of MMPs and TIMPs was determined by means of real‐time PCR.ResultsThe protein levels of MMP‐3, MMP‐7, MMP‐8, MMP‐9, TIMP‐1, TIMP‐2, periostin, and tenascin C were significantly higher in patients with CRSwNPs than in healthy control subjects. The adjusted logistic regression analyses showed that MMP‐3, MMP‐7, MMP‐8, MMP‐9, TIMP‐2, periostin, and tenascin C were related to the occurrence of CRSwNP. Spearman correlation test showed periostin was positively correlated with MMP‐3 and TIMP‐2, and tenascin C was positively correlated with MMP‐3, MMP‐7, MMP‐8, MMP‐9, and TIMP‐2. Periostin stimulated the gene expression of MMP‐3, MMP‐7, MMP‐8, and MMP‐9 in fibroblasts and MMP‐9 in epithelial cells ex vivo. Tenascin C stimulated the expression of MMP‐3, MMP‐7, MMP‐8, and MMP‐9 in epithelial cells. The expression of TIMPs in fibroblasts and epithelial cells was affected by neither periostin nor tenascin C.ConclusionsPeriostin and tenascin C might be involved in the remodeling of nasal polyps by regulating the expression of different MMPs in epithelial cells and fibroblasts. Our findings have the potential to identify key factors of tissue remodeling in CRSwNPs.

Salvianolic acidB inhibits myofibroblast differentiation and extracellular matrix accumulation in nasal polyp fibroblasts via the TGF-β1 signaling pathway.

In the process of nasal tissue remodeling, nasal fibroblasts serve an important role via myofibroblast differentiation and the production of extracellular matrix(ECM). Nasal fibroblast abnormalities can lead to conditions such as chronic rhinosinusitis. Salvianolic acidB (SalB), a water-soluble active pharmaceutical compound extract from the root of the traditional Chinese medicine Salviamiltiorrhiza, displays antioxidative, antiproliferative and antifibrosis properties. The present study aimed to investigate the mechanism underlying the effects of SalB on nasal polyp fibroblast (NPF) myofibroblast differentiation and ECM accumulation. Primary NPFs were obtained from nasal polyps of patients with chronic sinusitis. The proliferative and cytotoxic effects of SalB on NPFs were evaluated by performing the Cell Counting Kit-8 assay. The Transwell assay was conducted to assess cell migration. α-smooth muscle actin (α-SMA), TGF-β1 receptor(TβR)-I, TβR-II, Smad2/3 mRNA and protein expression levels and (p)-Smad2/3 phosphorylation levels were measured via reverse transcription-quantitative PCR and western blotting, respectively. TypeIII collagen and fibronectin levels were analyzed by ELISA. The results indicated that SalB significantly downregulated TGF-β1-induced α-SMA, fibronectin and collagenIII expression levels in NPFs. Similarly, SalB significantly decreased TGF-β1-induced TβR-I, TβR-II, p-Smad2/3, MMP-2 and MMP-9 mRNA and protein expression levels in NPFs. Furthermore, SalB significantly decreased TGF-β1-induced NPF migration. Therefore, the present study indicated that SalB inhibited myofibroblast differentiation and ECM accumulation in nasal fibroblasts, suggesting that SalB may inhibit nasal polyp formation via certain mechanisms.

Fibroblasts Regulate Mast Cell Activation in Nasal Polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory sinus disease associated with mast cell (MC) expansion and activation. We find that MCs in the subepithelium of nasal polyps exhibit elevated CD69 expression, a known leukocyte activation marker. However, it is unclear which factors within the polyp drive this activation. As subepithelial MCs colocalize with fibroblasts, cells known to influence MC development and mediator release, we hypothesized that nasal polyp fibroblasts could directly drive MC activation.

Effect of Prostaglandin D2 on mRNA Expression of Three Isoforms of Hyaluronic Acid Synthase in Nasal Polyp Fibroblasts.

Hyaluronan is one of the major extracellular matrixes in chronic rhinosinusitis (CRS) associated with tissue remodeling. Prostaglandin D2 (PGD2) is also associated with the pathogenesis of CRS. However, little is known about whether PGD2 regulates hyaluronan production by human airway fibroblasts. We sought to determine the effect of PGD2 on the mRNA expression of three isoforms of membrane-bound hyaluronic acid synthase (HAS1, HAS2 and HAS3) in fibroblasts, the major source of hyaluronan production, derived from CRS patients. Nasal polyp-derived fibroblasts (NPDF) and uncinate tissue-derived fibroblasts (UTDF) were established from CRS patients with nasal polyps and those without, respectively. These fibroblasts were stimulated with PGD2 or PGD2 receptor (DP/CRTH2)-selective agonists in the presence or absence of receptor-selective antagonists. mRNA levels for HAS1, HAS2 and HAS3 were determined by real-time quantitative PCR. PGD2 (1 µM) significantly enhanced HAS1 but not HAS2 or HAS3 mRNA expression by NPDF. Enhanced HAS1 mRNA expression was also obtained by stimulation with a DP receptor-selective agonist, but not with a CRTH2 receptor-selective agonist. In addition, PGD2-induced HAS1 mRNA expression was significantly inhibited by pre-treatment with DP receptor-selective antagonists. Similar induction of PGD2-induced HAS1 mRNA expression was seen in UTDF. PGD2 selectively stimulates HAS1 mRNA expression in local fibroblasts in CRS via DP, but not CRTH2, receptors.

Modification of Collagen Derivatives with Water-Soluble Polymers for the Development of Cross-Linked Hydrogels for Controlled Release.

Novel cross-linked hydrogels were synthesized as potential materials for the development of smart biofertilizers. For this purpose, hydrogels were prepared using collagen hydrolysate recovered from tannery waste. The water-soluble polymer poly(sodium 4-styrenesulfonate-co-glycidyl methacrylate) (P(SSNa-co-GMAx)) was among others used for the cross-linking reaction that combined hydrophilic nature with epoxide groups. The synthetic procedure was thoroughly investigated in order to ensure high percentage of epoxide groups in combination with water-soluble behavior. The copolymer did not show cytotoxicity against normal lung, skin fibroblasts, or nasal polyps fibroblasts. Through the present work, we also present the ability to control the properties of cross-linked hydrogels by altering copolymer’s composition and cross-linking parameters (curing temperature and time). Hydrogels were then studied in terms of water-uptake capacity for a period up to six days. The techniques Proton Nuclear Magnetic Resonance (1H NMR), Thermogravimetric Analysis (TGA), Size Exclusion Chromatography (SEC), and Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) were applied for the characterization of the synthesized copolymers and the cross-linked hydrogels. Three samples of biofertilizers based on collagen hydrolysate functionalized with P(SSNa-co-GMAx) and starch and having nutrients encapsulated (N, P, K) were prepared and characterized by physical–chemical analysis and Energy Dispersive X-ray analysis-Scanning Electron Microscope (EDAX-SEM) in terms of microstructure. Preliminary tests for application as fertilizers were performed including the release degree of oxidable organic compounds.

Effect of Acacia Honey on Transforming Growth Factor-Beta-1-Induced Myofibroblast Differentiation and Matrix Metalloproteinase-9 Production in Nasal Polyp Fibroblasts

BackgroundAcacia honey is known to have antioxidant, immune-modulatory, and antiproliferative properties. Nasal fibroblasts participate in local immune responses that control the recruitment of inflammatory cells and the production of extracellular matrix.ObjectivesThe aim of this study was to determine the effect of acacia honey on myofibroblast differentiation and matrix metalloproteinase-9 (MMP-9) production in nasal polyp fibroblasts.MethodsPrimary nasal fibroblasts were isolated from nasal polyps and treated with transforming growth factor-beta 1 (TGF-β1). Reverse transcription-polymerase chain reaction and Western blot analysis were then performed to determine α-smooth muscle actin (α-SMA), tissue inhibitors of matrix metalloproteinase-1, and MMP-9 mRNA expression and protein production in nasal polyp fibroblasts. Phosphorylated Smad ( pSmad) 2/3 and phosphorylated adenosine monophosphate-activated protein kinase ( pAMPK) were then determined by Western blotting.ResultsTGF-β1 stimulation increased α-SMA and MMP-9 mRNA expression and protein production in nasal polyp fibroblasts. Acacia honey effectively suppressed α-SMA and MMP-9 mRNA expression and protein production. It also prevented phosphorylation of Smad 2/3 and AMPK.ConclusionAcacia honey can inhibit TGF-β1-induced myofibroblast differentiation and MMP-9 production in nasal fibroblasts. These results suggest that acacia honey might be useful for inhibiting nasal polyp formation.

Effect of Prostaglandin D2 on VEGF Release by Nasal Polyp Fibroblasts

Effect of Prostaglandin D2 on VEGF Release by Nasal Polyp Fibroblasts

Effect of salvianolic acid B on proliferation and apoptosis of human nasal polyps fibroblasts

Objective:The aim of this study is to discuss the effects and mechanism of salvianolic acid B (SAB) on the proliferation and apoptosis of human nasal polyps fibroblasts (NPFs). Method:The NPFs were treated with SAB at different concentrations (5, 10, 20, 40 μmol/L). CCK-8 assay was used to determine the inhibition of SAB on the proliferation of NPFs and determined the optimum concentration for the following experiments. The NPFs were divided into four groups: a) control group: the cells were cultured in DMEM without TGF-β1 or salvianolic acid B; b) the TGF-β1 group: the cells were cultured in DMEM containing 5 ng/ml TGF-β1; c) the SAB group: the cells were cultured in DMEM containing SAB at the optimum concentration by CCK-8 assay; d) the TGF-β1+SAB treatment groups: the cells were cultured in DMEM containing 5 ng/ml TGF-β1 and SAB at the optimum concentration by CCK-8 assay. MTT assay was used to determine the proliferation of NPFs in four groups. The apoptosis rate was analyzed by Annexin-V FITC/PI double staining. The activities of Caspase-3 were detected with spectrophotometric method. Apoptosis related proteins Bcl-2 and Bax were measured by western blot. Result:CCK-8 assay showed that SAB could inhibite NPFs proliferation in a dose-dependent pattern (P<0.05). The following experiments were carried out with the concentration of salvianolic acid B at 10 μmol/L. MTT results showed that SAB could inhibit the proliferation of NPFs stimulated by TGF-β1 (P<0.01). Annexin-V FITC/PI double staining showed that SAB could induce apoptosis of NPFs (P<0.05). The activities of Caspase-3 were increased with SAB (P<0.05). Western blot results showed that SAB reduced the Bcl-2 protein expression, and increased the Bax protein expression and the ratio of Bax/Bcl-2 (P<0.05). Conclusion:Salvianolic acid B could inhibit proliferation and induce apoptosis of NPFs. Its mechanism may be associated with increasing the activities of Caspase-3 by inhibiting the expression of Bcl-2 protein and promoting the expression of Bax protein.

The Effects of Melittin and Apamin on Airborne Fungi-Induced Chemical Mediator and Extracellular Matrix Production from Nasal Polyp Fibroblasts.

Melittin and apamin are the main components of bee venom and they have been known to have anti-inflammatory and anti-fibrotic properties. The aim of this study was to evaluate the effect of melittin and apamin on airborne fungi-induced chemical mediator and extracellular matrix (ECM) production in nasal fibroblasts. Primary nasal fibroblasts were isolated from nasal polyps, which were collected during endoscopic sinus surgery. Nasal fibroblasts were treated with Alternaria and Aspergillus. The effects of melittin and apamin on the production of interleukin (IL)-6 and IL-8 were determined with enzyme linked immunosorbent assay. ECM mRNA and protein expressions were determined with the use of quantitative RT-PCR and Western blot. Alternaria-induced IL-6 and IL-8 production was significantly inhibited by apamin. However, melittin did not influence the production of IL-6 and IL-8 from nasal fibroblasts. Melittin or apamin significantly inhibited collagen type I, TIMP-1, and MMP-9 mRNA expression and protein production from nasal fibroblasts. Melittin and apamin inhibited Alternaria-induced phosphorylation of Smad 2/3 and p38 MAPK. Melittin and apamin can inhibit the fungi-induced production of chemical mediators and ECM from nasal fibroblasts. These results suggest the possible role of melittin and apamin in the treatment of fungi induced airway inflammatory diseases.

Thrombin and activated coagulation factor X stimulate the release of cytokines and fibronectin from nasal polyp fibroblasts via protease-activated receptors.

Nasal epithelial cells and infiltrating eosinophils express tissue factor, and high thrombin activity and excess fibrin deposition are found in nasal secretion and in nasal polyp from patients with chronic rhinosinusitis with nasal polyp (CRSwNP). Activated coagulation factors play important roles not only in thrombosis but also in inflammation through interaction with protease-activated receptors (PAR). However, little is known about the effects of activated coagulation factors on the release of cytokines and extracellular matrix from nasal polyp fibroblasts (NPF). The purpose of this study was to analyze the expression of PARs, which are receptors for activated coagulation factors, on NPFs and to determine the roles of thrombin and activated coagulation factor X (FXa) in the release of cytokines and fibronectin from NPFs. NPFs were obtained from patients with CRSwNP, and the messenger RNA (mRNA) and protein expression of PARs in these NPFs were examined. We then investigated whether thrombin or FXa stimulates the release of transforming growth factor (TGF) beta 1, fibronectin, eotaxin-1, interleukin (IL) 6, or IL-8 from cultured NPFs. The effects of PAR agonists on the release of cytokines and fibronectin were also examined. NPFs expressed the mRNA and proteins of all four PARs: PAR-1, PAR-2, PAR-3, and PAR-4. Both thrombin and FXa significantly stimulated the release of TGF beta 1, fibronectin, eotaxin-1, IL-6, and IL-8 from cultured NPFs. PAR-1 and PAR-2 agonists stimulated the secretion of TGF beta 1, fibronectin, eotaxin-1, IL-6, and IL-8. PAR-3 agonist stimulated the release of TGF beta 1, fibronectin, and eotaxin-1. PAR-4 agonist did not induce the release of these molecules. NPFs play important roles in the pathophysiology of CRSwNP such as in nasal polyp formation and inflammatory cell infiltration by releasing cytokines and extracellular matrix proteins. Activated coagulation factors, thrombin and FXa, stimulate the release of these cytokines and fibronectin from NPFs via PARs.

Effect of eosinophils activated with Alternaria on the production of extracellular matrix from nasal fibroblasts

BackgroundEosinophils and fibroblasts are known to play major roles in the pathogenesis of nasal polyps. Fungi are commonly found in nasal secretion and are associated with airway inflammation. ObjectiveTo investigate whether activated eosinophils by airborne fungi can influence the production of extracellular matrix (ECM) from nasal fibroblasts. MethodsInferior turbinate and nasal polyp fibroblasts were stimulated with Alternaria or Aspergillus, respectively, for 24 hours and ECM messenger RNA (mRNA) and protein expressions were measured. Eosinophils isolated from healthy volunteers were stimulated with Alternaria or Aspergillus for 4 hours then superoxide, eosinophil peroxidase, and transforming growth factor β1 were measured. Then activated eosinophils were cocultured with nasal fibroblasts for 24 hours, and ECM mRNA expressions were measured. ResultsAlternaria strongly enhanced ECM mRNA expression and protein production from nasal fibroblasts. Alternaria also induced the production of superoxide, eosinophil peroxidase, and transforming growth factor β1 from eosinophils, and activated eosinophils enhanced ECM mRNA expression when they were cocultured without the Transwell insert system. ConclusionEosinophils activated with Alternaria enhanced ECM mRNA expression from nasal polyp fibroblasts. Alternaria plays an important role in tissue fibrosis in the pathogenesis of nasal polyps by directly or indirectly influencing the production of ECM from nasal fibroblasts.

Sirtuin 6 modulates hypoxia-induced autophagy in nasal polyp fibroblasts via inhibition of glycolysis.

To elucidate the interaction between hypoxia-induced autophagy and glycolysis in nasal polyp fibroblasts, and the regulatory role of Sirtuin 6 (SIRT6) in the pathogenesis of nasal polyp. Through examining the expressions of lactate dehydrogenase (LDH), microtubule-associated protein II light chain 3 (LC3II) (an autophagy marker), and production of lactate under hypoxia, the interaction between autophagy and glycolysis was investigated. The role of SIRT6 on the hypoxia-induced autophagy and glycolysis was also examined. Nasal polyp specimens were used to examine the expressions of hypoxia-inducible factor (HIF) 1α, LDH, and LC3II by Western blot analysis, and primary cultures of nasal polyp fibroblasts were established from resected nasal polyps to measure hypoxia-induced LDH and LC3II expression by Western blot analysis and lactate production by colorimetry. Forced expression of SIRT6 with a lentiviral-based technique was used to evaluate its suppressive effect on autophagy and glycolysis. Immunohistochemical staining was performed to detect the expressions of SIRT6, LDH, and beclin (another autophagy marker) in nasal polyps. Expression of HIF-1α, LDH, and an autophagy marker, LC3II, are increased in nasal polyp specimens, and forced expression of SIRT6 in nasal polyp fibroblasts inhibited LDH expression, lactate production under hypoxia, and SIRT6. An immunohistochemistry study of nasal polyp showed that SIRT6 expression was reduced and LDH and beclin were enhanced. Analysis of these data indicated that hypoxia may contribute to the formation of nasal polyp by promoting autophagy in nasal polyp fibroblasts. Through the antiglycolytic activity of SIRT6, the autophagy was suppressed, which was beneficial to nasal polyp formation. Modulation of glucose metabolism through SIRT6-based strategy may possess therapeutic potential for nasal polyposis in the future.

Effect of prostaglandin D2 on VEGF release by nasal polyp fibroblasts

BackgroundVascular endothelial growth factor (VEGF) is known to be associated with the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). VEGF is produced by a variety of cells including fibroblasts. It was recently reported that prostaglandin (PG) E2 induces VEGF release by nasal polyp fibroblasts. However, little is known regarding possible regulation of VEGF by other PGs. We have reported that molecules that regulate PGD2 metabolism play roles in the pathogenesis of CRS including in local eosinophilia and type 2 cytokine production. In the present study, we sought to determine whether PGD2 regulates VEGF release by nasal polyp fibroblasts. MethodsNasal polyp fibroblasts were established from nasal polyps. These fibroblasts were stimulated with serial dilutions of PGD2 or PGD2 receptor (DP/CRTH2)-selective agonists in the presence or absence of receptor-selective antagonists. The concentration of VEGF in the culture supernatants was determined using ELISA. Results5 μM of PGD2 significantly induced VEGF release by nasal polyp fibroblasts. VEGF release was also obtained by stimulation with a DP receptor-selective, but not with a CRTH2 receptor-selective agonist. In addition, PGD2-induced VEGF release was significantly inhibited by pre-treatment with DP receptor-selective antagonists. In contrast, pre-treatment with a CRTH2 receptor-selective antagonist significantly enhanced PGD2-induced VEGF release. ConclusionsPGD2 stimulates VEGF production via DP but not CRTH2 receptors in nasal polyp fibroblasts.

Fatty Acid Composition of Cultured Fibroblasts Derived from Healthy Nasal Mucosa and Nasal Polyps

Background: Fibroblasts from nasal polyps (NP) of asthma patients have reduced expression of cyclooxygenase 2 (COX-2) and production of prostaglandin E2 (PGE2). We hypothesized that the reported alterations are due to alterations in the availability of arachidonic acid (AA). Objective: The objective was to determine the fatty acid composition of airway fibroblasts from healthy subjects and from asthma patients with and without aspirin intolerance. Methods: We analyzed the fatty acid composition of cultured fibroblasts from non-asthmatics (n = 6) and from aspirin-tolerant (n = 6) and aspirin-intolerant asthmatics (n = 6) by gas chromatography-flame ionization detector. Fibroblasts were stimulated with acetyl salicylic acid (ASA). Results: The omega-6 fatty acids dihomo-gamma-linolenic acid (C20:3) and AA (C20:4), and omega-3 fatty acids docosapentaenoic acid (DPA) (C22:5) and docosahexaenoic acid (DHA) (C22:6) were significantly higher in NP fibroblasts than in fibroblasts derived from nasal mucosa. The percentage composition of the fatty acids palmitic acid (C16:0) and palmitoleic acid (C16:1) was significantly higher in fibroblasts from patients with NP and aspirin intolerance than in fibroblasts derived from the nasal NP of aspirin-tolerant patients. ASA did not cause changes in either omega-3 or omega-6 fatty acids. Conclusions. Our data do not support the hypothesis that a reduced production of AA in NP fibroblasts can account for the reported low production of PGE2 in nasal polyps. Whether the increased proportion of omega-3 fatty acids can contribute to reduced PGE2 production in nasal polyps by competitively inhibiting COX-2 and reducing the amount of AA available to the COX-2 enzyme remains to be elucidated.

Alternaria Induces Production of Thymic Stromal Lymphopoietin in Nasal Fibroblasts Through Toll-like Receptor 2.

PurposeChronic rhinosinusitis with nasal polyps is a chronic inflammatory disease with markedly increased eosinophils, Th2-type lymphocytes, fibroblasts, and goblet cells. Fungi are commonly associated with airway inflammatory diseases, and thymic stromal lymphopoietin (TSLP) is important in the development of Th2 inflammatory responses. The aim of this study was to investigate the interaction between airborne fungi and nasal fibroblasts in TSLP mRNA and protein expression.MethodsInferior turbinate and nasal polyp fibroblasts were stimulated with Alternaria and Aspergillus, respectively, for 48 hours, and TSLP mRNA and protein expressions were measured. The reverse transcriptase polymerase chain reaction was performed for the Toll-like receptor (TLR) mRNA expression of the nasal fibroblasts. To determine the role of TLR in the induction of TSLP, the fibroblasts were transfected with siRNA against TLR2 and TLR5.ResultsAlternaria induced TSLP mRNA and protein expression in both inferior turbinate and nasal polyp fibroblasts. The nasal polyp fibroblasts responded more strongly to the fungi. TLR2 and TLR5 mRNA expressions were significantly increased with fungal stimulation and TSLP production was significantly inhibited by siRNA against TLR2.ConclusionsThe results of this study show that TSLP expression could be induced in nasal fibroblasts by exposure to Alternaria and that TLR2 may be involved in the process. The promotion of TSLP production in nasal fibroblasts by airborne fungi may facilitate the development or exacerbation of Th2-type nasal inflammation, especially in CRS with nasal polyps.

Role of TLRs in the production of chemical mediators in nasal polyp fibroblasts by fungi.

Fibroblasts are major supporting cells in nasal mucosa and can induce inflammatory process with recruitment of inflammatory cells. Airborne fungi have been suggested as an etiologic factor of chronic rhinosinusitis (CRS). The aim of this study was to investigate the interaction between airborne fungi and pattern recognition receptors (PRRs) in nasal fibroblasts. Primary nasal polyp fibroblasts were cultured with Alternaria and Aspergillus for 48h. To determine the production of chemical mediators interleukine-6 (IL-6), IL-8, granulocyte-macrophage colony stimulating factor (GM-CSF), eotaxin, and regulated on activation normal T expressed and secreted (RANTES) were measured with enzyme immunoassay methods. PRRs for toll-like receptors (TLRs) and protease-activated receptors (PARs) mRNA were determined with reverse transcription polymerase chain reaction (RT-PCR). To determine the role of PRRs, fibroblasts were treated with small interfering RNA (siRNA). IL-6 and IL-8 productions were significantly increased by 50 and 100μg/ml of Alternaria. However, GM-CSF, eotaxin, and RANTES productions did not change. Aspergillus did not influence the production of chemical mediators from nasal polyp fibroblasts. TLR2 and TLR5 mRNA expressions were significantly increased by fungi and these two TLRs were associated with the production of IL-6 and IL-8. Alternaria interacts as a pathogen-associated molecular pattern with the PRRs, such as TLR2 and TLR5, which induce the production of inflammatory chemical mediators from nasal polyp fibroblasts. Airborne fungi enhance the innate immune defense mechanism and may be associated with the pathogenesis of nasal inflammatory diseases.

Effect of lipopolysaccharide on glucocorticoid receptor function in control nasal mucosa fibroblasts and in fibroblasts from patients with chronic rhinosinusitis with nasal polyps and asthma.

BackgroundChronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the upper airways frequently associated with asthma. Bacterial infection is a feature of CRSwNP that can aggravate the disease and the response to glucocorticoid treatment.ObjectiveWe examined whether the bacterial product lipopolysaccharide (LPS) reduces glucocorticoid receptor (GR) function in control nasal mucosa (NM) fibroblasts and in nasal polyp (NP) fibroblasts from patients with CRSwNP and asthma.MethodsNP (n = 12) and NM fibroblasts (n = 10) were in vitro pre-incubated with LPS (24 hours) prior to the addition of dexamethasone. Cytokine/chemokine secretion was measured by ELISA and Cytometric Bead Array. GRα, GRβ, mitogen-activated protein-kinase phosphatase-1 (MKP-1) and glucocorticoid-induced leucine zipper (GILZ) expression was measured by RT-PCR and immunoblotting, GRα nuclear translocation by immunocytochemistry, and GRβ localization by immunoblotting. The role of MKP-1 and GILZ on dexamethasone-mediated cytokine inhibition was analyzed by small interfering RNA silencing.ResultsPre-incubation of nasal fibroblasts with LPS enhanced the secretion of IL-6, CXCL8, RANTES, and GM-CSF induced by FBS. FBS-induced CXCL8 secretion was higher in NP than in NM fibroblasts. LPS effects on IL-6 and CXCL8 were mediated via activation of p38α/β MAPK and IKK/NF-κB pathways. Additionally, LPS pre-incubation: 1) reduced dexamethasone’s capacity to inhibit FBS-induced IL-6, CXCL8 and RANTES, 2) reduced dexamethasone-induced GRα nuclear translocation (only in NM fibroblasts), 3) did not alter GRα/GRβ expression, 4) decreased GILZ expression, and 5) did not affect dexamethasone’s capacity to induce MKP-1 and GILZ expression. MKP-1 knockdown reduced dexamethasone’s capacity to suppress FBS-induced CXCL8 release.ConclusionThe bacterial product LPS negatively affects GR function in control NM and NP fibroblasts by interfering with the capacity of the activated receptor to inhibit the production of pro-inflammatory mediators. This study contributes to the understanding of how bacterial infection of the upper airways may limit the efficacy of glucocorticoid treatment.

Vitamin D decreases the secretion of eotaxin and RANTES in nasal polyp fibroblasts derived from Taiwanese patients with chronic rhinosinusitis with nasal polyps

Eosinophils are important inflammatory cells involved in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). Vitamin D and its derivatives, in addition to their classic role as regulators of electrolytes homeostasis, have modulatory effects in immunological and inflammatory responses. Such properties suggest that vitamin D might also play a role in inflammatory airway diseases such as CRSwNP. In this study, we investigated the effect of vitamin D derivatives (calcitriol and tacalcitol) on the secretion of eotaxin and Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES), the two major eosinophil chemoattractants, in fibroblasts derived from the polyps of Taiwanese CRSwNP patients. Patients diagnosed with eosinophilic CRSwNP but without malignancies or asthma and undergoing elective endoscopic sinus surgery were recruited. Three primary fibroblast cultures were established using the polyp specimens obtained from these patients. The third to eighth passages of the fibroblasts were used for in vitro studies. Nasal polyp-derived fibroblasts were stimulated with IL-1β (10 ng/mL) for 24 hours, followed by replacement with media alone or with calcitriol or tacalcitol (10μM) and incubation for another 24 hours. After the treatments, the levels of secreted eotaxin and RANTES were evaluated by ELISA assays. The results showed that IL-1β could substantially stimulate the secretion of eotaxin (p < 0.01) and RANTES (p < 0.01) in nasal polyp-derived fibroblasts. More importantly, this stimulatory effect was significantly suppressed by adding calcitriol (p ≤ 0.002 for eotaxin and p ≤ 0.008 for RANTES) or tacalcitol (p ≤ 0.009 for eotaxin and p ≤ 0.02 for RANTES). Therefore, the inhibitory effect of vitamin D derivatives on eotaxin and RANTES secretion might shed light not only on the disease mechanism, but also on the potential use of vitamin D in pharmacotherapy of Taiwanese patients with CRSwNP.