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Background: Chronic rhinosinusitis (CRS) is an inflammatory disease affecting more than 10% of the global adult population. It is classified into Th1, Th2, and Th17 endotypes and eosinophilic and non-eosinophilic types. Th2-based inflammation and eosinophilic CRS (ECRS) are associated with tissue remodeling and fibrinolytic system impairment. Objective: To elucidate the role of eosinophils in inducing fibrin deposition in CRS nasal polyp tissues and explore potential regulatory mechanisms. Methods: We analyzed the expression of genes related to the serpin family and fibrinolytic system using Gene Expression Omnibus and Next-generation sequencing data. Differentially expression genes (DEGs) analysis was used to compare control and nasal polyp tissues, followed by KEGG and Gene ontology (GO) analysis. We measured the expression and correlation of plasminogen activator-1 (PAI-1), tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and urokinase plasminogen activator surface receptor (u-PAR) in CRS tissues, and evaluated the effect of eosinophils on the fibrinolytic system using a cytokine array and co-culture. Results: Nasal polyp tissues showed upregulated PAI-1, u-PA, and u-PAR expression and downregulated t-PA expression. Fibrinolytic system-related genes positively correlated with Th2 cytokines, except for t-PA. Eosinophil-derived Chitinase-3-like protein 1 (CHI3L1) increased PAI-1 expression and decreased t-PA levels in fibroblasts and epithelial cells. The inhibition of CHI3L1 suppresses these alterations. Conclusion: CHI3L1 contributes to fibrin deposition by impairing the fibrinolytic system during nasal polyp formation. The regulation of CHI3L1 expression may inhibit fibrin deposition and edema in ECRS, presenting a potential treatment for this condition.
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Proteína 1 Semelhante à Quitinase-3 , Eosinófilos , Fibrinólise , Pólipos Nasais , Inibidor 1 de Ativador de Plasminogênio , Rinite , Sinusite , Humanos , Pólipos Nasais/metabolismo , Pólipos Nasais/imunologia , Sinusite/metabolismo , Sinusite/imunologia , Rinite/metabolismo , Rinite/imunologia , Doença Crônica , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Proteína 1 Semelhante à Quitinase-3/metabolismo , Proteína 1 Semelhante à Quitinase-3/genética , Adulto , Feminino , Masculino , Pessoa de Meia-Idade , Eosinófilos/imunologia , Eosinófilos/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tecidual/genética , Citocinas/metabolismo , RinossinusiteRESUMO
The devastating effects of COVID-19 have highlighted the importance of prophylactic and therapeutic strategies to combat respiratory diseases. Stimulator of interferon gene (STING) is an essential component of the host defense mechanisms against respiratory viral infections. Although the role of the cGAS/STING signaling axis in the innate immune response to DNA viruses has been thoroughly characterized, mounting evidence shows that it also plays a key role in the prevention of RNA virus infections. In this study, we investigated the role of STING activation during Influenza virus (IFV) infection. In both mouse bone marrow-derived macrophages and monocytic cell line THP-1 differentiated with PMA, we found that dimeric amidobenzimidazole (diABZI), a STING agonist, had substantial anti-IFV activity against multiple strains of IFV, including A/H1N1, A/H3N2, B/Yamagata, and B/Victoria. On the other hand, a pharmacological antagonist of STING (H-151) or the loss of STING in human macrophages leads to enhanced viral replication but suppressed IFN expression. Furthermore, diABZI was antiviral against IFV in primary air-liquid interface cultures of nasal epithelial cells. Our data suggest that STING agonists may serve as promising therapeutic antiviral agents to combat IFV.
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Antivirais , Imunidade Inata , Macrófagos , Proteínas de Membrana , Animais , Humanos , Imunidade Inata/efeitos dos fármacos , Camundongos , Antivirais/farmacologia , Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Proteínas de Membrana/agonistas , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Células THP-1 , Replicação Viral/efeitos dos fármacos , Influenza Humana/imunologia , Influenza Humana/virologia , Influenza Humana/tratamento farmacológico , Cães , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/imunologia , Orthomyxoviridae/fisiologia , Benzimidazóis/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Exposure to particulate matter (PM) and house dust mite (HDM) can change the expression patterns of inflammation-, oxidative stress-, and cell death-related genes. We investigated the changes in gene expression patterns owing to PM exposure. OBJECTIVE: This study examined the changes in gene expression patterns following PM exposure. METHODS: We searched for differentially expressed genes (DEGs) following PM exposure using five cell line-based RNA-seq or microarray datasets and six human-derived datasets. The enrichment terms of the DEGs were assessed. RESULTS: DEG analysis yielded two gene sets. Thus, enrichment analysis was performed for each gene set, and the enrichment terms related to respiratory diseases were presented. The intersection of six human-derived datasets and two gene sets was obtained, and the expression patterns following PM exposure were observed. CONCLUSIONS: Two gene sets were obtained for cells treated with PM and their expression patterns were presented following verification in human-derived cells. Our findings suggest that exposure to PM2.5 and HDM may reveal changes in genes that are associated with diseases, such as allergies, highlighting the importance of mitigating PM2.5 and HDM exposure for disease prevention.
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Material Particulado , Material Particulado/efeitos adversos , Material Particulado/toxicidade , Humanos , Pyroglyphidae , Transcriptoma , Animais , Linhagem Celular , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
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BACKGROUND: Adipose tissue is known to serve as an abundant and readily accessible source of adipose-derived stem cells (ADSCs) as an alternative to bone marrow. Collagenase is one of the most widely used methods for the isolation of ADSCs from adipose tissue, but it takes a long time, and there are also debates about safety. We propose an ultrasonic cavitation-treated method that can significantly reduce time and avoid the problem of using xenogeneic enzymes in ADSCs isolation. METHODS: ADSCs were isolated from adipose tissue using the enzyme treatment method and the ultrasonic cavitation treatment method. Cell proliferation was measured using cell viability assay. The expression levels of the surface markers of ADSCs were estimated by real-time PCR. After, ADSCs were cultured in chondrogenic, osteogenic, or adipogenic differentiation medium; the differentiation potential of ADCSs was analyzed by Alcian blue, Alizarin Red S, Oil Red O, and real-time PCR. RESULTS: The cells treated with collagenase and ultrasound had similar cell yields and proliferation after isolation. The difference in the expression of surface markers of ADSCs was not statistically significant. ADSCs showed differentiation potential into adipocytes, osteocytes, and chondrocytes, and there was no difference between the enzyme treatment method and the ultrasonic cavitation treatment method. The yield of the ADSC increased in time- and intensity dependently. CONCLUSIONS: Ultrasound certainly serves as a promising method in advancing ADSC isolation technology.
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Células-Tronco Mesenquimais , Células-Tronco , Células-Tronco/metabolismo , Tecido Adiposo , Adipócitos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Proliferação de Células , Células CultivadasRESUMO
BACKGROUND: 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. METHODS: 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-ß1) 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. RESULT: 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. CONCLUSION: 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.
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Miofibroblastos , Pólipos Nasais , Humanos , Células Cultivadas , Miofibroblastos/metabolismo , Fibroblastos/metabolismo , Matriz Extracelular/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Glicólise/fisiologia , Pólipos Nasais/metabolismoRESUMO
The endemic and pandemic caused by respiratory virus infection are a major cause of mortality and morbidity globally. Thus, broadly effective antiviral drugs are needed to treat respiratory viral diseases. Small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (U-exo) have recently gained attention as a cell-free therapeutic strategy due to their potential for safety and efficacy. Anti-viral activities of U-exo to countermeasure respiratory virus-associated diseases are currently unknown. Here, we tested the antiviral activities of U-exo following influenza A/B virus (IFV) and human seasonal coronavirus (HCoV) infections in vitro. Cells were subject to IFV or HCoV infection followed by U-exo treatment. U-exo treatment significantly reduced IFV or HCoV replication and combined treatment with recombinant human interferon-alpha protein (IFN-α) exerted synergistically enhanced antiviral effects against IFV or HCoV. Interestingly, microRNA (miR)-125b, which is one of the most abundantly expressed small RNAs in U-exo, was found to suppress IFV replication possibly via the induction of IFN-stimulated genes (ISGs). Furthermore, U-exo markedly enhanced RNA virus-triggered IFN signaling and ISGs production. Similarly, human nasal epithelial cells cultured at the air-liquid interface (ALI) studies broadly effective anti-viral and anti-inflammatory activities of U-exo against IFV and HCoV, suggesting the potential role of U-exo as a promising intervention for respiratory virus-associated diseases.
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Coronavirus , Exossomos , Vesículas Extracelulares , Células-Tronco Mesenquimais , Antivirais/metabolismo , Antivirais/farmacologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Cordão UmbilicalRESUMO
Background: Eosinophilic 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. Objectives: We 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. Methods: Butyric 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. Results: Treatment 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. Conclusions: Eosinophil-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.
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Pólipos Nasais , Sinusite , Ácido Butírico/farmacologia , Doença Crônica , Eosinófilos/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Pólipos Nasais/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Sinusite/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Chronic rhinosinusitis (CRS) pathogenesis is closely related to tissue remodeling, including epithelial-mesenchymal transition (EMT). Epigenetic mechanisms play key roles in EMT. DNA methylation, mediated by DNA methyltransferases (DNMTs), is an epigenetic marker that is critical to EMT. The goal of this study was to determine whether DNMTs were involved in TGF-ß1-induced EMT and elucidate the underlying mechanisms in nasal epithelial cells and air-liquid interface cultures. Global DNA methylation and DNMT activity were quantified. DNMT expression was measured using real-time PCR (qRT-PCR) in human CRS tissues. mRNA and protein levels of DNMTs, E-cadherin, vimentin, α-SMA, and fibronectin were determined using RT-PCR and Western blotting, respectively. DNMT1, DNMT3A, and DNMT3B gene expression were knocked down using siRNA transfection. MAPK phosphorylation and EMT-related transcription factor levels were determined using Western blotting. Signaling pathways were analyzed using specific inhibitors of MAPK. We demonstrated these data in primary nasal epithelial cells and air-liquid interface cultures. Global DNA methylation, DNMT activity, and DNMT expression increased in CRS tissues. DNMT expression was positively correlated with Lund-McKay CT scores. TGF-ß1 dose-dependently induced DNMT expression. Further, 5-Aza inhibited TGF-ß1-induced DNMT, Snail, and Slug expression related to EMT, as well as p38 and JNK phosphorylation in A549 cells and TGF-ß1-induced DNMT expression and EMT in primary nasal epithelial cells and air-liquid interface cultures. TGF-ß1-induced DNMT expression leads to DNA methylation and EMT via p38, JNK, Snail, and Slug signaling pathways. Inhibition of DNMT suppressed the EMT process and therefore is potentially a CRS therapeutic strategy.
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Transição Epitelial-Mesenquimal , Sinusite , Células A549 , Caderinas/metabolismo , Metilação de DNA , Células Epiteliais/metabolismo , Humanos , Sinusite/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Tissue remodeling contributes to ongoing inflammation and refractoriness of chronic rhinosinusitis (CRS). During this process, epithelial-mesenchymal transition (EMT) plays an important role in dysregulated remodeling and both microRNA (miR)-29b and heat shock protein 47 (HSP47) may be engaged in the pathophysiology of CRS. This study aimed to determine the role of miR-29b and HSP47 in modulating transforming growth factor (TGF)-ß1-induced EMT and migration in airway epithelial cells. Expression levels of miR-29b, HSP47, E-cadherin, α-smooth muscle actin (α-SMA), vimentin and fibronectin were assessed through real-time PCR, Western blotting, and immunofluorescence staining. Small interfering RNA (siRNA) targeted against miR-29b and HSP47 were transfected to regulate the expression of EMT-related markers. Cell migration was evaluated with wound scratch and transwell migration assay. miR-29b mimic significantly inhibited the expression of HSP47 and TGF-ß1-induced EMT-related markers in A549 cells. However, the miR-29b inhibitor more greatly induced the expression of them. HSP47 knockout suppressed TGF-ß1-induced EMT marker levels. Functional studies indicated that TGF-ß1-induced EMT was regulated by miR-29b and HSP47 in A549 cells. These findings were further verified in primary nasal epithelial cells. miR-29b modulated TGF-ß1-induced EMT-related markers and migration via HSP47 expression modulation in A549 and primary nasal epithelial cells. These results suggested the importance of miR-29b and HSP47 in pathologic tissue remodeling progression in CRS.
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Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Proteínas de Choque Térmico HSP47/antagonistas & inibidores , Proteínas de Choque Térmico HSP47/genética , Fator de Crescimento Transformador beta1/metabolismo , Células A549 , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Movimento Celular/fisiologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/metabolismo , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Rinite/genética , Rinite/metabolismo , Sinusite/genética , Sinusite/metabolismo , Sinusite/patologia , Fator de Crescimento Transformador beta1/administração & dosagem , Fator de Crescimento Transformador beta1/genéticaRESUMO
BACKGROUND: Chronic rhinosinusitis is involved in myofibroblast differentiation and extracellular matrix (ECM) accumulation. High mobility group box chromosomal protein 1 (HMGB-1) is known to stimulate lung fibroblast to produce ECM in lung fibrosis. The aim of this study was to investigate whether HMGB-1 induces myofibroblast differentiation and ECM production in nasal fibroblasts and to identify the signal pathway. METHODS: Human nasal fibroblasts were cultured. After stimulation with HMGB-1, expressions of α-smooth muscle actin (α-SMA) and fibronectin were determined by real-time PCR and western blot. Total collagen was measured by Sircol assay. To investigate signal pathway, various signal inhibitors and RAGE siRNA were used. RESULTS: HMGB-1 increased α-SMA and fibronectin in mRNA and protein levels. It also increased collagen production. RAGE siRNA inhibited HMGB-1-induced α-SMA and fibronectin, and production of collagen. Furthermore, the inhibitors of RAGE downstream molecules such as p38, JNK and AP-1 also blocked the HMGB-1-induced effects. CONCLUSIONS: HMGB-1 induces myofibroblast differentiation and ECM production in nasal fibroblast, which is mediated by RAGE, p38, JNK and AP-1 signal pathway. These results suggest that HMGB-1 may play an important role in tissue remodeling during chronic rhinosinusitis progression.
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Matriz Extracelular , Proteína HMGB1 , Miofibroblastos/citologia , Actinas/metabolismo , Diferenciação Celular , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Proteína HMGB1/genética , Humanos , Transdução de Sinais , Fator de Transcrição AP-1RESUMO
BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory disease of the sinonasal mucosa. Thymic stromal lymphopoietin (TSLP) is associated with T-helper 2 (Th2) response and induced by pathogen, allergen, toll-like receptor (TLR) ligands, and cytokines. Fibroblasts are known to be modulators of wound-healing, from inflammation to tissue remodeling. We examined effect of lipopolysaccharide (LPS) on TSLP production and the underlying mechanisms. We aimed to determine whether the effects of commonly used medications in CRS, namely corticosteroids, and macrolides, are related to LPS-induced TSLP in nasal fibroblasts. METHODS: Fibroblasts were isolated from inferior turbinate tissues of CRS patients. TSLP and TLR4 expressions were determined by reverse transcript-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunoassay, and immunofluorescence staining. Mitogen-activated protein kinase (MAPK), protein kinase B (Akt), and nuclear factor-kappaB (NF-κB) phosphorylation was determined by Western blot and/or luciferase assay. RESULTS: LPS increased TSLP expression in a dose- and time-dependent manner. LPS antagonist and corticosteroids inhibited TLR4 expression in LPS-stimulated fibroblasts. LPS-RS, macrolides, corticosteroids, and specific inhibitors suppressed LPS-induced alterations. Ex vivo culture showed similar results. CONCLUSION: LPS induces TSLP production via the TLR4, MAPK, Akt, and NF-κB pathways. The effects of corticosteroids and macrolides are related to LPS-induced TSLP expression. We explored new treatment modalities targeting LPS-induced TSLP production that could replace the currently used corticosteroid and macrolides for treatment of CRS.
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Lipopolissacarídeos , Proteínas Proto-Oncogênicas c-akt , Corticosteroides/farmacologia , Células Cultivadas , Citocinas , Fibroblastos , Humanos , Macrolídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno , NF-kappa B , Receptor 4 Toll-Like/genética , Linfopoietina do Estroma do TimoRESUMO
We hypothesized that differences in the microbiome could be a cause of the substantial differences in the symptoms of and treatment options for adult and pediatric patients with chronic rhinosinusitis (CRS). First, we characterized the differences in the nasal microbiomes of pediatric and adult CRS patients. Swabs were obtained from 19 patients with chronic rhinosinusitis (9 children and 10 adults). The bacterial 16S rRNA gene was pyrosequenced to compare the microbiota of the middle meatus. No significant differences were found in species richness and alpha-diversity indices between the two groups. However, in the comparison of diversity between groups using the unweighted pair group method with arithmetic mean (UPGMA) clustering of microbiome taxonomic profiles, we observed a relatively clear separation between the adult and pediatric groups. Actinobacteria had a significantly higher relative abundance in the adult group than in the pediatric group at the phylum level. At the genus level, Corynebacterium showed significantly higher relative abundance in the adult group than in the pediatric group. This is a comparative study between the microbiomes of adult and pediatric CRS patients. We expect this study to be the first step in understanding the pathogenesis of CRS in adults and children using microbiome analysis.
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Microbiota , Rinite/microbiologia , Sinusite/microbiologia , Adulto , Bactérias/metabolismo , Biodiversidade , Criança , Doença Crônica , Feminino , Humanos , MasculinoRESUMO
Cigarette smoke exposure has been shown to be associated with chronic rhinosinusitis and tissue remodeling. The present study aimed to investigate the effects of cigarette smoke extract (CSE) on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) production in nasal fibroblasts and to determine the underlying molecular mechanisms. Primary nasal fibroblasts from six patients were isolated and cultured. After the exposure of fibroblasts to CSE, the expression levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured by real-time PCR, ELISA, and immunofluorescence staining. The enzymatic activities of MMP-2 and MMP-9 were measured by gelatin zymography. Reactive oxygen species (ROS) production was analyzed using dichloro-dihydro-fluorescein diacetate and Amplex Red assays. PI3K/Akt phosphorylation and NF-κB activation were determined by Western blotting and luciferase assay. CSE significantly increased MMP-2 expression and inhibited TIMP-2 expression but did not affect MMP-9 and TIMP-1 expression. Furthermore, CSE significantly induced ROS production. However, treatment with ROS scavengers, specific PI3K/Akt inhibitors, NF-κB inhibitor, and glucocorticosteroids significantly decreased MMP-2 expression and increased TIMP-2 expression. Our results suggest that steroids inhibit CSE-regulated MMP-2 and TIMP-2 production and activation through the ROS/ PI3K, Akt, and NF-κB signaling pathways in nasal fibroblasts. CSE may contribute to the pathogenesis of chronic rhinosinusitis by regulating MMP-2 and TIMP-2 expression.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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(1) Background: Tissue remodeling and extracellular matrix (ECM) accumulation contribute to the development of chronic inflammatory diseases of the upper airway. Endoplasmic reticulum (ER) stress is considered to be the key signal for triggering tissue remodeling in pathological conditions. The present study aimed to investigate the role of ER-stress in TGF-ß1-stimulated nasal fibroblasts and inferior turbinate organ cultures; (2) Methods: Fibroblasts and organ cultures were pretreated with 4-phenylbutyric acid (PBA) and stimulated with TGF-ß1 or thapsigargin (TG). Expression of ER-stress markers, myofibroblast marker, and ECM components was measured by Western blotting and real-time PCR. Reactive oxygen species (ROS) were quantified using 2',7'-dichlorofluorescein diacetate. Cell migration was evaluated using Transwell assays. Contractile activity was measured by collagen contraction assay; (3) Results: 4-PBA inhibited TGF-ß1 or TG-induced ER-stress marker expression, phenotypic changes, and ECM. Pre-treatment with ROS scavengers inhibited the expression of TGF-ß1-induced ER-stress markers. Migration and collagen contraction of TGF-ß1 or TG-stimulated fibroblasts were ameliorated by 4-PBA treatment. These findings were confirmed in ex vivo organ cultures; (4) Conclusions: 4-PBA downregulates TGF-ß1-induced ER-stress marker expression, migration, and collagen contraction via ROS in fibroblasts and organ cultures. These results suggest that ER-stress may play an important role in progression of chronic upper airway inflammatory diseases by aiding pathological tissue remodeling.
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Estresse do Retículo Endoplasmático , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Conchas Nasais/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Fenilbutiratos/farmacologia , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Conchas Nasais/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/antagonistas & inibidores , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
BACKGROUND: All-trans retinoic acid (ATRA), a derivative of vitamin A, is known to have anti-fibrogenic effects and regulates cell proliferation and differentiation. Therefore, these abilities of ATRA may influence tissue remodeling in the upper airway. The aims of the present study were to investigate the effects of ATRA on the myofibroblast differentiation, extracellular matrix (ECM) production, cell migration, and collagen gel contraction and to determine the molecular mechanisms of ATRA in TGF-ß1-induced nasal polyp-derived fibroblasts (NPDFs). METHODS: NPDFs were isolated from nasal polyp. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. TGF-ß1-induced fibroblasts were pretreated with ATRA. The expression levels of alpha-smooth muscle actin (α-SMA), collagen type 1, fibronectin, phospho-mitogen-activated protein kinase, and p-p50 (nuclear factor-kappaB [NF-κB]) were measured by Western blot analysis, real-time polymerase chain reaction, and/or immunofluorescence staining. Cell migration was analyzed with cell migration scratch assay and Transwell migration assay. Collagen contractile activity was measured using a collagen gel contraction assay. RESULTS: ATRA had no significant cytotoxic effect in NPDFs. Expression levels of α-SMA, collagen type 1, and fibronectin stimulated by TGF-ß1 were significantly downregulated in the ATRA-pretreated fibroblasts. TGF-ß1-induced cell migration and collagen gel contraction were significantly inhibited by ATRA pretreatment. ATRA also significantly inhibited phosphorylation of c-Jun N-terminal kinase (JNK), p38, and p50 in TGF-ß1-induced NPDFs, but did not inhibit phosphorylation of extracellular signal-related kinase (ERK). CONCLUSION: ATRA downregulated myofibroblast differentiation, ECM production, cell migration, and collagen gel contraction via p38, JNK-dependent NF-κB-signaling pathways in TGF-ß1-induced NPDFs. The findings suggest that ATRA could serve as a novel therapeutic agent to ameliorate nasal polyp development.
Assuntos
Matriz Extracelular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Pólipos Nasais/patologia , Fator de Crescimento Transformador beta1/farmacologia , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Pólipos Nasais/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoAssuntos
Aerossóis/uso terapêutico , Fibroblastos/fisiologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Nariz/patologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Interleucina-6/genética , Interleucina-8/genética , MAP Quinase Quinase 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
BACKGROUND: Fibroblast migration plays a significant role in wound healing after endoscopic sinonasal surgery. Cigarette smoke extract (CSE) is a potent inhibitor of fibroblast functions including cell proliferation and migration. The purpose of the study was to determine the influence of CSE on migration and collagen gel contraction in nasal fibroblasts and investigate its underlying mechanisms. METHODS: Fibroblast migration was evaluated using wound healing assay and transwell migration assay. Contractile activity was assessed by collagen gel contraction assay. Reactive oxygen species (ROS) were quantified by 2',7'-dichlorofluorescein diacetate. Fibroblasts were treated with CSE and N-acetylcysteine (NAC), metformin, compound C, or transfected with small interfering RNA (siRNA) to suppress adenosine monophosphate-activated protein kinase (AMPK) expression. AMPK activation was determined by Western blot. RESULTS: CSE and metformin were found to significantly reduce the migration and collagen gel contraction activity of nasal fibroblasts. Conversely, pretreatment with NAC and compound C significantly enhanced the migration and collagen gel contraction activity of fibroblasts. ROS production and AMPK phosphorylation were found to be significantly induced by CSE treatment, whereas the activity was inhibited on treatment with NAC, metformin, compound C, or AMPK siRNA. Silencing of AMPK expression was found to significantly reverse the suppressive effect of CSE in nasal fibroblasts. CONCLUSION: CSE has an inhibitory effect on cell migration and collagen gel contraction activity via the ROS/AMPK signaling pathway in nasal fibroblasts.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Movimento Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Nicotiana , Espécies Reativas de Oxigênio/metabolismo , Fumaça/efeitos adversos , Adulto , Células Cultivadas , Colágeno/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Nicotiana/química , Conchas Nasais/patologia , Conchas Nasais/cirurgiaRESUMO
HSP47 is required for the production of collagen and serves an important role in tissue remodeling, a pathophysiologic mechanism of chronic rhinosinusitis (CRS). We investigated the relationship between HSP47 expression and tissue remodeling in CRS. We also determined the underlying molecular mechanisms of TGF-ß1-induced HSP47 and extracellular matrix (ECM) production in nasal fibroblasts. HSP47, α-SMA, fibronectin, and collagen type I expression levels were measured using real-time PCR, western blotting, and immunofluorescence staining. Fibroblast migration was analyzed using scratch and transwell migration assays. Contractile activity was measured with a collagen gel contraction assay. HSP47 is increased in patients with CRS without nasal polyps. TGF-ß1 induced HSP47 expression in nasal fibroblasts. Myofibroblast differentiation and ECM production, which are induced by TGF-ß1, were inhibited by siHSP47. We also confirmed that the Smad2/3 signaling pathway is involved in TGF-ß1-induced HSP47 expression in nasal fibroblasts. In a functional assay, TGF-ß1-enhanced migration and contraction ability were inhibited by HSP47 knockout. Glucocorticoid reversed the stimulatory effects of TGF-ß1 on HSP47 expression and ECM production in nasal fibroblasts and ex vivo organ cultures. HSP47 expression is involved in TGF-ß1-induced myofibroblast differentiation and ECM production through the Smad2/3 signaling pathway, which might contribute to tissue remodeling in chronic rhinosinusitis.