Nasal Fibroblasts Research Articles

Neutrophils in nasal polyps exhibit transcriptional adaptation and proinflammatory role depend on local polyp milieu.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory upper airway disease, divided into eosinophilic CRSwNP (eCRSwNP) and noneosinophilic CRSwNP (neCRSwNP) according to eosinophilic levels. Neutrophils are major effector cells in CRSwNP. but their role in different inflammatory environments remain largely unclear. We performed an integrated transcriptome analysis of polyp-infiltrating neutrophils from CRSwNP patients, using healthy donor blood as a control. Flow cytometry and in vitro studies showed that neutrophils are activated in both CRSwNP type. The scRNA-sequencing analysis demonstrated that neutrophils were classified into five functional subsets, with GBP5+ neutrophils occurring mainly in neCRSwNPs and a high proportion of CXCL8+ neutrophils in both subendotypes. GBP5+ neutrophils exhibited significant IFN-I pathway activity in neCRSwNPs. CXCL8+ neutrophils displayed increased neutrophil activation scores and mainly secrete Oncostatin M (OSM), which facilitates communication with other cells. In vitro experiments revealed that OSM could enhance IL-13- or IL-17-mediated immune responses in nasal epithelial cells and fibroblasts. Our findings revealed that neutrophils exhibited transcriptional plasticity and activation when exposed to polyp tissue and exert their proinflammatory role in the pathogenesis of CRSwNP by releasing OSM to interact with epithelial cells and fibroblasts in a manner dependent on the inflammatory milieu.

The novel thromboxane prostanoid receptor mediates CTGF production to drive human nasal fibroblast self-migration through NF-κB and PKCδ-CREB signaling pathways.

Chronic rhinosinusitis without nasal polyp (CRSsNP) is characterized by tissue repair/remodeling and the subepithelial stroma region in whose nasal mucosa has been reported by us to have thromboxane A2 (TXA2) prostanoid (TP) receptor and overexpress connective tissue growth factor (CTGF). Therefore, this study aimed to investigate the relationship between TP receptor activation and CTGF production/function in human CRSsNP nasal mucosa stromal fibroblasts. We found that TP agonists including U46619 and IBOP ([1S-[1α,2α(Z),3β(1E,3 S*),4α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid) could promote CTGF protein/messenger RNA expression and secretion. The pharmacological intervention and TP activation assay with U46619 identified the possible participation of PKCμ, PKCδ, nuclear factor-κB (NF-κB), and cyclic AMP response element-binding protein (CREB) phosphorylation/activation in the CTGF induction. Moreover, a phorbol ester-phorbol-12-myristate 13-acetate (PMA) exhibited a similar cellular signaling and CTGF production profile to that elicited by TP activation. However, further small interfering RNA interference analysis revealed that only NF-κB and PKCδ-CREB pathways were necessarily required for TP-mediated CTGF production, which could not be completely supported by those findings from PMA. Finally, in a functional assay, although CTGF did not affect fibroblast proliferation, TP-mediated CTGF could drive novel self-migration in fibroblasts both in the scratch/wound healing and transwell apparatus assays. Meanwhile, the overall staining for stress fibers and formation of the lamellipodia and filopodia-like structures was concomitantly increased in the treated migrating cells. Collectively, we provided here that novel TP mediates CTGF production and self-migration in human nasal fibroblasts through NF-κB and PKCδ-CREB signaling pathways. More importantly, we also demonstrated that thromboxane, TP receptor, CTGF, and stromal fibroblasts may act in concert in the tissue remodeling/repair process during CRSsNP development and progression.

Nasal mucosal fibroblasts produce IL-4 to induce Th2 response.

Th2 polarization is essential for the pathogenesis of allergic rhinitis (AR). Th2 polarization's mechanism requires further understanding. IL-4 is the primary cytokine involved in Th2 response. Fibroblasts play a role in immune regulation. This study aims to elucidate the role of nasal mucosal fibroblast-derived IL-4 in the induction of Th2 responses. Nasal mucosal tissues were obtained from surgically removed samples from patients with nasal polyps, whether with or without AR. Fibroblasts were isolated from the tissues by flow cytometry cell sorting, and analyzed by RNA sequencing (RNAseq). The data from RNAseq showed that nasal fibroblasts expressed genes of GATA3, CD80, CD83, CD86, STAT6, IL2, IL4, IL5, IL6, IL13 and costimulatory factor. The data were verified by RT-qPCR. The level of gene activity was positively correlated with those of AR-related cytokines present in nasal secretions. Nasal fibroblasts release IL-4 upon activation. Nasal fibroblasts had the ability to transform naive CD4+ T cells into Th2 cells, which can be eliminated by inhibiting IL-4 receptor or CD28 in CD4+ T cells. To sum up, nasal mucosal fibroblasts produce IL-4, which can induce Th2 cell development. The data implicate that nasal fibroblasts are involved in the pathogenesis of nasal allergy.

IL-4Rα signaling promotes barrier-altering oncostatin M and IL-6 production in aspirin-exacerbated respiratory disease

Aspirin-exacerbated respiratory disease (AERD) is a severe disease involving dysregulated type 2 inflammation. However, the role other inflammatory pathways play in AERD is poorly understood. We sought to broadly define the inflammatory milieu of the upper respiratory tract in AERD and to determine the effects of IL-4Rα inhibition on mediators of nasal inflammation. Twenty-two AERD patients treated with dupilumab for 3 months were followed over 3 visits and compared to 10 healthy controls. Nasal fluid was assessed for 45 cytokines and chemokines using Olink Target 48. Blood neutrophils and cultured human mast cells, monocytes/macrophages, and nasal fibroblasts were assessed for response to IL-4/13 stimulation invitro. Of the nasal fluid cytokines measured, nearly one third were higher in AERD patients compared to healthy controls, including IL-6 and the IL-6 family-related cytokine oncostatin M (OSM), both of which correlated with nasal albumin levels, a marker of epithelial barrier dysregulation. Dupilumab significantly decreased many nasal mediators, including OSM and IL-6. IL-4 stimulation induced OSM production from mast cells and macrophages but not from neutrophils, and OSM and IL-13 stimulation induced IL-6 production from nasal fibroblasts. In addition to type 2 inflammation, innate and IL-6-related cytokines are also elevated in the respiratory tract in AERD. Both OSM and IL-6 are locally produced in nasal polyps and likely promote pathology by negatively affecting epithelial barrier function. IL-4Rα blockade, although seemingly directed at type 2 inflammation, also decreases mediators of innate inflammation and epithelial dysregulation, which may contribute to dupilumab's therapeutic efficacy in AERD.

Effect of Air Pollutants on Allergic Inflammation in Structural Cells of the Nasal Mucosa.

Air pollution is an increasing global concern, and its effect on allergic inflammation has attracted the attention of many researchers. Particulate matter (PM) is a major component of ambient air pollution, and heavy metals are the primary toxic constituents of PM. As previous studies on the impact of air pollutants on allergic inflammation did not adequately mimic real-world atmospheric exposure, we developed an experimental model to investigate the effects of aerosolized air pollutants on nasal epithelial cells and fibroblasts. We collected particulate matter 2.5 (PM2.5) samples from ambient 24-hour air samples obtained in Seoul from August 2020 to August 2022, and then conducted component analysis for metallic constituents. Primary nasal epithelial cells and nasal fibroblasts, obtained and cultured from the turbinate tissues of human participants, were treated with PM2.5. The associations of heavy metals identified from the component analysis with cytokine expression were investigated. A three-dimensional (3D)-hybrid culture model, consisting of co-culture of an air-liquid interface and nasal fibroblast spheroids, was constructed to observe the impact of aerosolized air pollutants. Among the heavy metals, Si was the predominant component of PM2.5, and Zn showed the highest correlation with the concentration of PM2.5 in Seoul. PM2.5, Zn, and Si increased the production of epithelial cell-derived cytokines, and PM2.5 and Zn exhibited similar trends with one another. Exposure of the 3D-hybrid model to aerosolized PM2.5 and Zn resulted in elevated periostin, alpha-smooth muscle actin, and fibronectin expression in fibroblast spheroids, and those without an epithelial barrier exhibited a similar increase in periostin expression. Ambient air pollutants in the form of aerosols increase the expression of allergic inflammatory cytokines in both nasal epithelial cells and fibroblasts. Regulations on air pollution will help reduce the global burden of allergic diseases in the future.

The Inhibition Effect and Mechanism of Staurosporine Isolated from Streptomyces sp. SNC087 Strain on Nasal Polyp.

This study aims to explore the potential inhibition effects of staurosporine isolated from a Streptomyces sp. SNC087 strain obtained from seawater on nasal polyps. Staurosporine possesses antimicrobial and antihypertensive activities. This research focuses on investigating the effects of staurosporine on suppressing the growth and development of nasal polyps and elucidating the underlying mechanisms involved. The experimental design includes in vitro and ex vivo evaluations to assess the inhibition activity and therapeutic potential of staurosporine against nasal polyps. Nasal polyp-derived fibroblasts (NPDFs) were stimulated with TGF-β1 in the presence of staurosporine. The levels of α-smooth muscle actin (α-SMA), collagen type-I (Col-1), fibronectin, and phosphorylated (p)-Smad 2 were investigated using Western blotting. VEGF expression levels were analyzed in nasal polyp organ cultures treated with staurosporine. TGF-β1 stimulated the production of Col-1, fibronectin, and α-SMA and was attenuated by staurosporine pretreatment. Furthermore, these inhibitory effects were mediated by modulation of the signaling pathway of Smad 2 in TGF-β1-induced NPDFs. Staurosporine also inhibits the production of VEGF in ex vivo NP tissues. The findings from this study will contribute to a better understanding of staurosporine's role in nasal polyp management and provide insights into its mechanisms of action.

PM2.5 activates IL-17 signaling pathway in human nasal mucosa-derived fibroblasts

Fine particulate matter (PM2.5) represents a prevalent environmental pollutant in the atmosphere, capable of exerting deleterious effects on human health. Numerous studies have indicated a correlation between PM2.5 exposure and the development of chronic upper airway inflammatory diseases. The objective of this study was to investigate the impact of PM2.5 on the transcriptome of fibroblasts derived from nasal mucosa. Initially, nasal mucosa-derived fibroblasts were isolated, cultured, and subsequently stimulated with PM2.5 (100 μg/mL) or an equivalent volume of normal culture medium for a duration of 24 h. Following this, total RNA from these cells was extracted, purified, and subjected to sequencing using next-generation RNA sequencing technology. Differentially expressed genes (DEGs) were then identified and utilized for functional enrichment analysis. A protein–protein interaction (PPI) network of DEGs was constructed, and validation of key genes and proteins was carried out using quantitative real-time PCR and ELISA methods. Results revealed 426 DEGs, comprising 276 up-regulated genes and 150 down-regulated genes in nasal mucosa-derived fibroblasts treated with PM2.5 compared to control cells. Functional enrichment analysis indicated that DEGs were predominantly associated with inflammation-related pathways, including the IL-17 signaling pathway. In alignment with this, PPI analysis highlighted that hub genes were primarily involved in the regulation of the IL-17 signaling pathway. Subsequent validation through quantitative real-time PCR and ELISA confirmed significant alterations in the relative expressions of IL-17 signaling pathway-related genes and concentrations of IL-17 signaling pathway related proteins in nasal mucosa-derived fibroblasts treated with PM2.5 compared to control cells. In conclusion, PM2.5 intervention substantially altered the transcriptome of nasal mucosa-derived fibroblasts. Furthermore, PM2.5 has the potential to exacerbate the inflammatory responses of these fibroblasts by modulating the expression of key genes in the IL-17 signaling pathway.

Inhibitory effect of doxycycline conjugated with deoxycholic acid and polyethylenimine conjugate on nasal fibroblast differentiation and extracellular production.

Chronic rhinosinusitis (CRS) is an inflammatory disease affecting the sinuses or nose. Persistent inflammatory responses can lead to tissue remodeling, which is a pathological characteristics of CRS. Activation of fibroblasts in the nasal mucosal stroma, differentiation and collagen deposition, and subepithelial fibrosis have been associated with CRS. We aimed to assess the inhibitory effects of doxycycline and deoxycholic acid-polyethyleneimine conjugate (DA3-Doxy) on myofibroblast differentiation and extracellular matrix (ECM) production in nasal fibroblasts stimulated with TGF-β1. To enhance efficacy, we prepared DA3-Doxy using a conjugate of low-molecular-weight polyethyleneimine (PEI) (MW 1800) and deoxycholic acid (DA) and Doxy. The synthesis of the DA3-Doxy polymer was confirmed using nuclear magnetic resonance, and the critical micelle concentration required for cationic micelle formation through self-assembly was determined. Subsequently, the Doxy loading efficiency of DA3 was assessed. The cytotoxicity of Doxy, DA3, PEI, and DA-Doxy in nasal fibroblasts was evaluated using the WST-1 assay. The anti-tissue remodeling and anti-inflammatory effects of DA3-Doxy and DA3 were examined using real-time polymerase chain reaction (Real-time PCR), immunocytochemistry, western blot, and Sircol assay. Both DA3 and DA3-Doxy exhibited cytotoxicity at 10 μg/ml in nasal fibroblasts. Doxy partially inhibited α-smooth muscle actin, collagen types I and III, and fibronectin. However, DA3-Doxy significantly inhibited α-SMA, collagen types I and III, and fibronectin at 5 μg/ml. DA3-Doxy also modulated TGF-β1-induced changes in the expression of MMP 1, 2, and 9. Nonetheless, TGF-β1-induced expression of MMP3 was further increased by DA3-Doxy. The expression of TIMP 1 and 2 was partially reduced with 5 μg/ml DA3-Doxy. Although initially developed for the delivery of genetic materials or drugs, DA3 exhibits inhibitory effects on myofibroblast differentiation and ECM production. Therefore, it holds therapeutic potential for CRS, and a synergistic effect can be expected when loaded with CRS treatment drugs.

Role of Fibroblasts in Chronic Inflammatory Signalling in Chronic Rhinosinusitis with Nasal Polyps-A Systematic Review.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory disease of the nose and paranasal sinuses characterized by the presence of nasal polyps. The symptoms produced by the presence of nasal polyps such as nasal obstruction, nasal discharge, facial pain, headache, and loss of smell cause a worsening in the quality of life of patients. The source of the nasal polyps remains unclear, although it seems to be due to a chronic inflammation process in the sinonasal mucosa. Fibroblasts, the main cells in connective tissue, are intimately involved in the inflammation processes of various diseases; to this end, we carried out a systematic review to evaluate their inflammatory role in nasal polyps. Thus, we evaluated the main cytokines produced by nasal polyp-derived fibroblasts (NPDF) to assess their involvement in the production of nasal polyps and their involvement in different inflammatory pathways. The results of the review highlight the inflammatory role of NPDF through the secretion of various cytokines involved in the T1, T2, and T3 inflammatory pathways, as well as the ability of NPDF to be stimulated by a multitude of substances. With these findings, the fibroblast is positioned as a new potential therapeutic target in the treatment of CRSwNP.

Trial of thromboxane receptor inhibition with ifetroban: TP receptors regulate eicosanoid homeostasis in aspirin-exacerbated respiratory disease

Aspirin-exacerbated respiratory disease (AERD) is the triad of asthma, nasal polyposis, and respiratory reactions to COX-1 inhibitors. Overproduction of cysteinyl leukotrienes and underproduction of prostaglandin E2 (PGE2) are hallmarks of AERD. Amouse model predicted a key role for the thromboxane-prostanoid (TP) receptor in AERD. Our aim was to determine whether ifetroban, a TP receptor antagonist, attenuates aspirin-induced respiratory symptoms in patients with AERD. A total of 35 patients with AERD completed a 4-week double-blinded, placebo-controlled trial of ifetroban and underwent an oral aspirin challenge. The primary outcome was change in the provocative dose of aspirin that caused a 2-point increase in Total Nasal Symptom Score. Changes in lung function, eicosanoid levels, and platelet and mast cell activation were assessed. Cultured human nasal fibroblasts were stimulated with or without the TP agonist U46619 and assayed for prostanoid production. Ifetroban was well tolerated in AERD and did not change the mean 2-point increase in Total Nasal Symptom Score (P= .763). Participants taking ifetroban had greater aspirin-induced nasal symptoms and a greater decline in FEV1 value than did participants receiving placebo (-18.8%± 3.6% with ifetroban vs -8.4%± 2.1% with placebo [P= .017]). Four weeks of ifetroban significantly increased urinary leukotriene E4 levels and decreased nasal PGE2 levels compared with placebo. Peak aspirin-induced urinary thromboxane levels correlated with peak urinary leukotriene E4 and prostaglandin D2 metabolite levels in participants taking ifetroban. U46119 significantly potentiated the production of PGE2 by cultured nasal fibroblasts from subjects with AERD but not by cultured nasal fibroblasts from controls without polypoid sinusitis. Contrary to our hypothesis, TP receptor blockade worsened aspirin-induced reactions in AERD, possibly by exacerbating dysregulation of the eicosanoid system. TP signaling on stromal cells may be critical to maintaining PGE2 production when COX-2 function is low.

Glycolytic reprogramming is involved in tissue remodeling on chronic rhinosinusitis.

Glycolytic reprogramming is a key feature of chronic inflammatory disease. Extracellular matrix (ECM) produced by myofibroblasts plays an important role in tissue remodeling of nasal mucosa in chronic rhinosinusitis (CRS). This study aimed to determine whether glycolytic reprogramming contributes to myofibroblast differentiation and ECM production in nasal fibroblasts. Primary nasal fibroblasts were isolated from the nasal mucosa of patients with CRS. Glycolytic reprogramming was assessed by measuring the extracellular acidification and oxygen consumption rates in nasal fibroblast, with and without transforming growth factor beta 1 (TGF-β1) treatment. Expression of glycolytic enzymes and ECM components was measured by real-time polymerase chain reaction, western blotting, and immunocytochemical staining. Gene set enrichment analysis was performed using whole RNA-sequencing data of nasal mucosa of healthy donors and patients with CRS. Glycolysis of nasal fibroblasts stimulated with TGF-B1 was upregulated along with glycolytic enzymes. Hypoxia-inducing factor (HIF)-1α was a high-level regulator of glycolysis, and increased HIF-1α expression promoted glycolysis of nasal fibroblasts, and inhibition of HIF-1α down-regulated myofibroblasts differentiation and ECM production. This study suggests that inhibition of the glycolytic enzyme and HIF-1α in nasal fibroblasts regulates myofibroblast differentiation and ECM generation associated with nasal mucosa remodeling.

Role of Nasal Fibroblasts in Airway Remodeling of Chronic Rhinosinusitis: The Modulating Functions Reexamined.

Chronic rhinosinusitis (CRS) is a multifactorial inflammatory disease of the nose and sinuses that affects more than 10% of the adult population worldwide. Currently, CRS is classified into endotypes according to the inflammatory response (Th1, Th2, and Th17) or the distribution of immune cells in the mucosa (eosinophilic and non-eosinophilic). CRS induces mucosal tissue remodeling. Extracellular matrix (ECM) accumulation, fibrin deposition, edema, immune cell infiltration, and angiogenesis are observed in the stromal region. Conversely, epithelial-to-mesenchymal transition (EMT), goblet cell hyperplasia, and increased epithelial permeability, hyperplasia, and metaplasia are found in the epithelium. Fibroblasts synthesize collagen and ECM, which create a structural skeleton of tissue and play an important role in the wound-healing process. This review discusses recent knowledge regarding the modulation of tissue remodeling by nasal fibroblasts in CRS.

Secretome Analysis of Human Nasal Fibroblast Identifies Proteins That Promote Wound Healing.

Conditioned medium from cultured fibroblast cells is recognized to promote wound healing and growth through the secretion of enzymes, extracellular matrix proteins, and various growth factors and cytokines. The objective of this study was to profile the secreted proteins present in nasal fibroblast conditioned medium (NFCM). Nasal fibroblasts isolated from human nasal turbinates were cultured for 72h in Defined Keratinocytes Serum Free Medium (DKSFM) or serum-free F12: Dulbecco's Modified Eagle's Medium (DMEM) to collect conditioned medium, denoted as NFCM_DKSFM and NFCM_FD, respectively. SDS-PAGE was performed to detect the presence of protein bands, followed by MALDI-TOF and mass spectrometry analysis. SignalP, SecretomeP, and TMHMM were used to identify the secreted proteins in conditioned media. PANTHER Classification System was performed to categorize the protein according to protein class, whereas STRING 10 was carried out to evaluate the predicted proteins interactions. SDS-PAGE results showed the presence of various protein with molecular weight ranging from ~10kDa to ~260kDa. Four protein bands were identified using MALDI-TOF. The analyses identified 104, 83, and 7 secreted proteins in NFCM_FD, NFCM_DKSFM, and DKSFM, respectively. Four protein classes involved in wound healing were identified, namely calcium-binding proteins, cell adhesion molecules, extracellular matrix proteins, and signaling molecules. STRING10 protein prediction successfully identified various pathways regulated by secretory proteins in NFCM. In conclusion, this study successfully profiled the secreted proteins of nasal fibroblasts and these proteins are predicted to play important roles in RECs wound healing through various pathways.

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 irrigation with licorice extract (Glycyrrhiza glabra) in treating nasal polyps by reducing fibroblast differentiation and extracellular matrix production in TGF-β1-stimulated nasal polyp-derived fibroblasts by inhibiting the MAPK/ERK-1/2 pathway – an in vitro and in clinic study

BackgroundTo date, treating nasal polyps (NPs) is still a medical challenge. However, we have developed an innovative therapy using licorice extract (LE: Glycyrrhiza glabra) to treat rhinitis and sinusitis via nasal irrigation and have discovered that it significantly affects treatment of NPs.Hypothesis/purposeThis study investigated the mechanism of LE on NPs.Study designNPs were collected from three patients using tissue biopsies before and 2 weeks after nasal irrigation with licorice for histopathological analysis. Additionally, NPs from two patients were collected, and nasal polyp-derived fibroblasts (NPDF) were isolated and cultured.MethodsThe TGF-β1-stimulated NPDF model was used to examine the effect of LE on fibroblast differentiation (biomarker: α-SMA), the consequent production of extracellular matrix (ECM; biomarkers: fibronectin, FBN), and the functional signaling pathway.ResultsImmunohistochemistry (IHC) revealed that the number of eosinophils and the expression of α-SMA and interstitial collagen of polyps after licorice treatment significantly decreased. Additionally, RT-PCR, western blotting, and immunofluorescence (IF) showed that α-SMA and FBN expressions were significantly increased in the NPDF, which was stimulated by TGF-β1, and LE dose-dependently could effectively reduce this effect. Furthermore, western blotting showed that LE could attenuate α-SMA and FBN expressions by preventing the signaling pathway of MAPK/ERK-1/2, which IHC and IF further confirmed. In addition, LE effectively suppressed the cell migration of NPDF, which is related to polyp expansion.ConclusionLE is clinically used to treat sinusitis with NPs through nasal irrigation, which significantly reduces the size of NPs. This effect could attenuate fibroblast differentiation, ECM production and cell migration, and one of the functional mechanisms may be through inhibition of the MAPK/ERK-1/2 signaling pathway.Trial registrationISRCTN (No. 51425529) registered on 17/04/2020 (retrospectively registered) - http://www.isrctn.com/ISRCTN51425529

Interleukin-19 upregulates fibronectin and collagen I expression via the NF-κB-Smad2/3 pathway in fibroblasts of patients with chronic rhinosinusitis.

Tissue remodeling is a prominent characteristic of chronic rhinosinusitis (CRS). Excess deposition of fibronectin (FN) and collagen (Col) I by fibroblasts is crucial for the pathologic tissue remodeling in CRS without nasal polyps (CRSsNP). Increased interleukin (IL)-19 level in patients with CRS had been demonstrated in our previous studies. Here, we aimed to evaluate the role of IL-19 in mediating FN and Col I expression in CRS. Nasal mucosal tissue samples were collected from patients with CRS with nasal polyps (CRSwNP), CRSsNP, and controls. The expression of IL-19, vimentin, FN, and Col I were detected using immunohistochemistry and immunofluorescence. Primary human nasal fibroblasts were treated with IL-19, then the activation of Smad2/3, NF-κB and relevant pathways, and the expression of FN and Col I were measured. Expression levels of vimentin, FN, and Col I were significantly increased in nasal tissues from patients with CRSsNP compared with CRSwNP and control subjects. Moreover, IL-19 co-localized with FN and Col Ι in nasal tissues. IL-19-treated fibroblasts had increased production of FN and Col I, which was associated with the activated Smad2/3 and NF-κB pathways. Moreover, Smad2/3 activation was mediated by the NF-κB pathway in IL-19-treated fibroblasts. IL-19 promotes FN and Col I production via the activated NF-κB-Smad2/3 pathway in fibroblasts, leading to fibrosis and collagen deposition in patients with CRS.

Bone Marrow Derived Mesenchymal Stromal Cells Promote Vascularization and Ciliation in Airway Mucosa Tri-Culture Models in Vitro

Patients suffering from irresectable tracheal stenosis often face limited treatment options associated with low quality of life. To date, an optimal tracheal replacement strategy does not exist. A tissue-engineered tracheal substitute promises to overcome limitations such as implant vascularization, functional mucociliary clearance and mechanical stability. In order to advance a tracheal mucosa model recently developed by our group, we examined different supporting cell types in fibrin-based tri-culture with primary human umbilical vein endothelial cells (HUVEC) and primary human respiratory epithelial cells (HRE). Bone marrow-derived mesenchymal stromal cells (BM-MSC), adipose-derived mesenchymal stromal cells (ASC) and human nasal fibroblasts (HNF) were compared regarding their ability to promote mucociliary differentiation and vascularization in vitro. Three-dimensional co-cultures of the supporting cell types with either HRE or HUVEC were used as controls. Mucociliary differentiation and formation of vascular-like structures were analyzed by scanning electron microscopy (SEM), periodic acid Schiff’s reaction (PAS reaction), two-photon laser scanning microscopy (TPLSM) and immunohistochemistry. Cytokine levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-6 (IL6), interleukin-8 (IL8), angiopoietin 1, angiopoietin 2, fibroblast growth factor basic (FGF-b) and placenta growth factor (PIGF) in media supernatant were investigated using LEGENDplex™ bead-based immunoassay. Epithelial morphology of tri-cultures with BM-MSC most closely resembled native respiratory epithelium with respect to ciliation, mucus production as well as expression and localization of epithelial cell markers pan-cytokeratin, claudin-1, α-tubulin and mucin5AC. This was followed by tri-cultures with HNF, while ASC-supported tri-cultures lacked mucociliary differentiation. For all supporting cell types, a reduced ciliation was observed in tri-cultures compared to the corresponding co-cultures. Although formation of vascular-like structures was confirmed in all cultures, vascular networks in BM-MSC-tri-cultures were found to be more branched and extended. Concentrations of pro-angiogenic and inflammatory cytokines, in particular VEGF and angiopoietin 2, revealed to be reduced in tri-cultures compared to co-cultures. With these results, our study provides an important step towards a vascularized and ciliated tissue-engineered tracheal replacement. Additionally, our tri-culture model may in the future contribute to an improved understanding of cell-cell interactions in diseases associated with impaired mucosal function.

Effect of Thymoquinone and Transforming Growth Factor-β1 on the Cell Viability of Nasal Polyp-Derived Fibroblast

BACKGROUND: Nasal polyps are benign masses in the nasal cavity and the abnormal growth of sinonasal tissue due to a chronic inflammatory process. Many fibroblasts populate the nasal polyp stroma release cytokines such as Transforming Growth Factor (TGF) and producing a variety of cytokines resulting in inflammatory cell infiltration. Thymoquinone (TQ) is the main active component in Nigella sativa oil and has the ability to reduces cell viability in many cancer cell line. AIM: The purpose of this study was to determine the effect of TQ and TGF-β1 on cell viability of Nasal Polyp-Derived Fibroblast. MATERIALS AND METHODS: Nasal polyp-derived fibroblasts were isolated from nasal polyp specimen and treated with various concentrations of TQ at 1–1000 μM and TGF-β1 at 5 ng/ml to determine the cell viability using the Cell Counting Kit-8 assay after 48 h incubation. RESULTS: TQ significantly reduced the viability of nasal polyp fibroblast cells to 72.49% at 20 μM and reduced to 5% at 50 μM until 1000 μM with IC50 at 21.93 μM. TGF-β1 at 5 ng/ml significantly reduced the viability of nasal polyp fibroblast cells to 81.96% and TGF-β1 appears to have a dual effect that depends on the concentration of TQ. CONCLUSION: This study proved that TQ and TGF-β1 were able to reduce the viability of nasal polyp fibroblast cells.

Airway Fibroblast Secretory Products Enhance Cell Migration

Background: The nasal fibroblast secretome, which includes various cytokines, chemokines, and growth factors, promotes cell migration. Currently, the proteomics of Airway Fibroblast (AF) Conditioned Medium (AFCM) are being actively studied. Objective: This study was aimed at profiling and identifying the AF secreted proteins that can enhance wound healing of the airway epithelium and predict the potential pathway involved. Methods: Airway Epithelial Cells (AECs) and AFs were isolated from redundant human nasal turbinate and cultured. AFCM was collected by culturing the AFs either with serum-free airway epithelium basal medium (AECM) or with serum-free F12:DMEM (FDCM). For evaluating cell migration, the AECs were supplemented with airway epithelium medium and defined keratinocyte medium (1:1; AEDK; control), or with AEDK supplemented with 20% AECM or 20% FDCM. The mass spectrometry sample was prepared by protein precipitation, followed by gel electrophoresis and in-gel digestion. Results : AECM promoted better cell migration compared to the FDCM and the control medium. Bioinformatics analysis identified a total of 121, and 92 proteins from AECM and FDCM, respectively: 109 and 82 were identified as secreted proteins, respectively. STRING® analysis predicted that 23 proteins from the AECM and 16 proteins from the FDCM are involved in wound healing. Conclusion: Conditioned medium promotes wound healing by enhancing cell migration, and we successfully identified various secretory proteins in a conditioned medium that play important roles in wound healing.

Eosinophil-Derived Osteopontin Induces the Expression of Pro-Inflammatory Mediators and Stimulates Extracellular Matrix Production in Nasal Fibroblasts: The Role of Osteopontin in Eosinophilic Chronic Rhinosinusitis.

BackgroundEosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis (CRS) and is a refractory or intractable disease. However, a reliable clinical marker or an effective treatment strategy has not yet been established. ECRS is accompanied by excessive eosinophil infiltration and Th2 inflammatory response, which is closely related to tissue remodeling in the upper airways.ObjectivesWe sought to investigate the effect of eosinophils on tissue remodeling in ECRS. The purpose of this study was to identify the effects of eosinophils on the expression of pro-inflammatory mediators and extracellular matrix (ECM) in nasal fibroblasts and the key mediators that stimulate them.MethodsButyric acid was used to differentiate EOL-1 cells into eosinophils. We co-cultured differentiated EOL-1 cells and fibroblasts to measure the expression of pro-inflammatory mediators and ECM in fibroblasts. Among the cytokines secreted from the differentiated EOL-1 cells, factors that induced tissue remodeling of fibroblasts were identified.ResultsTreatment with butyric acid (BA) differentiated EOL-1 cells into eosinophils. Differentiated EOL-1 cells induced fibroblasts to produce pro-inflammatory mediators, IL-6 and IL-8, and tissue remodeling factor, VEGF. It also induced myofibroblast differentiation and overexpression of ECM components. Differentiated EOL-1 cells overexpressed osteopontin (OPN), and recombinant OPN increased the expression of IL-6, IL-8, VEGF, and ECM components in nasal fibroblast. OPN was overexpressed in the nasal tissue of patients with ECRS and was associated with the severity of CRS.ConclusionsEosinophil-derived OPN stimulated nasal fibroblasts and contributed to inflammation and tissue remodeling in ECRS. Moreover, the expression level of OPN was proportional to the severity of ECRS. Therefore, OPN regulation is a potential treatment for ECRS.