FGF2 is overexpressed in asthma and promotes airway in airway epithelial cells

Background: Airway inflammation is the core pathological process of asthma, with the key inflammatory regulators incompletely defined. Recently, fibroblast growth factor 2(FGF2) has been reported to be an inflammatory regulator;however, its role in asthma remains elusive. This study aimed to investigate the immunomodulatory role of FGF2 in asthma.Methods: First, FGF2 expression was characterised in clinical asthma samples and the house dust mite(HDM)-induced mouse chronic asthma model. Second, recombinant mouse FGF2(rm-FGF2) protein was intranasally delivered to determine the effect of FGF2 on airway inflammatory cell infiltration. Third, human airway epithelium-derived A549 cells were stimulated with either HDM or recombinant human interleukin-1β(IL-1β) protein combined with or without recombinant human FGF2. IL-1β-induced IL-6 or IL-8 release levels were determined using enzyme-linked immunosorbent assay, and the involved signalling transduction was explored via Western blotting.Results: Compared with the control groups, the FGF2 protein levels were significantly upregulated in the bronchial epithelium and alveolar areas of clinical asthma samples [(6.70±1.79) vs.(16.32±2.40), P=0.0184;(11.20±2.11) vs.(21.00±3.00), P=0.033, respectively] and HDM-induced asthmatic mouse lung lysates [(1.00±0.15) vs.(5.14±0.42),P<0.001]. Moreover, FGF2 protein abundance was positively correlated with serum total and anti-HDM IgE levels in the HDM-induced chronic asthma model(R^(2)=0.857 and 0.783, P=0.0008 and 0.0043, respectively). Elevated FGF2protein was mainly expressed in asthmatic bronchial epithelium and alveolar areas and partly co-localised with infiltrated inflammatory cell populations in HDM-induced asthmatic mice. More importantly, intranasal instillation of rm-FGF2 aggravated airway inflammatory cell infiltration [(2.45±0.09) vs.(2.88±0.14), P=0.0288] and recruited more subepithelial neutrophils after HDM challenge [(110.20±29.43) cells/mm^(2) vs.(238.10±42.77) cells/mm^(2), P=0.0392]without affecting serum IgE levels and Th2 cytokine transcription. In A549 cells, FGF2 was upregulated through HDM stimulation and promoted IL-1β-induced IL-6 or IL-8 release levels [up to(1.41±0.12)-or(1.44±0.14)-fold change vs.IL-1β alone groups, P=0.001 or 0.0344, respectively]. The pro-inflammatory effect of FGF2 is likely mediated through the fibroblast growth factor receptor(FGFR)/mitogen-activated protein kinase(MAPK)/nuclear factor kappa B(NF-κB)pathway.Conclusions: Our findings suggest that FGF2 is a potential inflammatory modulator in asthma, which can be induced by HDM and acts through the FGFR/MAPK/NF-κB pathway in the airway epithelial cells.

Activity of Matrix Metalloproteinase in Airway Epithelial Cells of COPD Patients

Summary:To examine the mRNA expression of matrix metalloproteinase 9 (MMP-9) and the gelatinase activity of its inhibitor, tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) in the primary epithelial cells of patients with COPD, airway epithelial cells were taken from 15 COPD patients and cultured in vitro. The patients were divided into three groups, COPD group, normal smoking control group and non-smoking control group, with 5 subjects in each group, on basis of the smoking history and lung function. The semi-qualitative RT-PCR was employed to determine the mRNA levels of MMP-9 and TIMP-1 and SDS PAGE was used for the determination of the gelatinase activity of MMP-9 and TIMP-1. Our result showed that the mRNA of MMP-9 and TIMP-1 in epithelial cells of the non-smoking subjects was at a low level. The mRNA of MMP-9 and TIMP-1 in COPD patients and smokers was significantly higher than that in non-smokers (P<0.05). No significant difference was found in the levels of MMP-9 and TIMP-1 in epithelial cells between the COPD patients and smokers. The MMP-9/TIMP-1 ratios in COPD patients and smokers were significantly lower than that of non-smokers (P<0.05). The gelatinase activity in the epithelial cells of both COPD patients and normal smokers was increased (P<0.05), but no difference existed in the gelatinase activity in the epithelial cells between COPD patients and normal smokers. It is concluded that the transcription of MMP-9 and TIMP-1 and the gelatinase activity of MMP-9 and MMP-2 in the epithelial cells in COPD patients were increased, which resulted in an imbalance of MMP-9/TIMP-1, thereby causing pulmonary fibrosis. These factors play important roles in the pathogenesis of COPD.

Effect of Lithium on Cell Cycle Progression of Pig Airway Epithelial Cells

To investigate the effect of lithium on cell cycle progression of airway epithelial cells, primary pig tracheobronchial epithelial cells were incubated with lithium chloride (LiCl) at different concentrations (0, 5 mmol/L, and 10 mmol/L) and time (12 h, 16 h and 24 h). After the treatment, cells were counted, cell cycle profile was measured by BrdU labeling and flow cytometry, and expression of cyclin D1 and cyclin B1 were detected by Western blotting. The results showed that after 24h of 10mmol/L but not 5mmol/L LiCl treatment, proliferation of cells was slowed down as manifested by delayed confluence and cell number accumulation (P<0.05). Lithium did not change the percentage of cells in S phase (P>0.05), but 24 h incubation with 10 mmol/L LiCl induced a G 2/M cell cycle arrest. Furthermore, 10mmol/L LiCl elevated cyclin D1 expression after 12h treatment, while expression of cyclin B1 increased more significantly after 24h incubation. These data demonstrate that lithium inhibits proliferation of pig airway epithelial cells by inhibiting cell cycle progression, and suggest that lithium-sensitive molecule(s) such as glycogen synthase kinase 3 may have a role in the regulation of growth of airway epithelial cells.

Inhibitory Effect of Dexamethasone on Expression of Cysteine-rich 61 Protein in Airway Epithelial Cells of Allergic Mouse Models

Summary： In order to study whether cysteine-rich 61 protein （cyr61） is involved in the pathogenesis of asthma and its relation to airway inflammation, the effect of dexamethasone （Dxm） on the expression of cyr61 in the lung tissues of asthmatic mice was investigated. Forty BALB/c mice were divided into asthma group （n=15）, control group （n=10） and Dxm group （n=15）. The asthma group was sensitized and challenged by ovalbumin （OVA）. The mice in Dxm group were intraperitoneally administered with Dxm after OVA challenge. The expression of cyr61 in the lung tissues was detected by using immuno- histochemistry, and that of eotaxin protein in the bronchoalveolar lavage fluid （BALF） by using en- zyme-linked immunosorbent assay （ELISA）. The number of inflammatory cells in BALF was also ana- lyzed. The results showed that the cyr61 expression was highest in asthma group （P〈0.05）, followed by Dxm group （P〈0.05） and control group. The cyr61 had a positive correlation with the total nucleated cells （r=0.867, P〈0.05）, especially eosinophils （r=0.856, P〈0.05）, and eotaxin level （r=0.983, P〈0.05） in the BALF. Our findings suggested that cyr61 is expressed in airway epithelial cells and has a positive correlation with eotaxin and number of airway infiltrating eosinophils.

Effects of Cigarette Smoke Extract on E-cadherin Expression in Cultured Airway Epithelial Cells

Summary: To investigate whether the change of E-cadherin (ECD) expression plays a role in the injury and repair of airway epithelial cells (AEC) caused by smoking, porcine AECs were cultured by using an enzyme-dispersed method. After exposure of the AECs to cigarette smoke extract (CSE), the ECD expression in the cells was detected by using immunocytochemistry and in situ hybridization. The results showed that ECD was distributed on the plasma membrane at the cell junctions of AECs. After exposure to 20 % CSE, the membranous ECD expression was decreased, the cytoplasmic ECD expression was increased (P<0.01) as the exposure time went on. But the content of ECD mRNA in the AECs did not chang. It suggests that the change of ECD ex- pression is regulated at the posttranslational level and plays a role in the injury and repair of AEC caused by smoking.

Effect of Peroxiredoxin 1 on the biological function of airway epithelial cells and epithelial-mesenchymal transition

Peroxiredoxin 1(PRDX1)participates in tumor cell proliferation,apoptosis,migration,invasion,and the epithelial-to-mesenchymal transition(EMT).This study aimed to investigate the effect of PRDX1 on the EMT of airway epithelial cells stimulated with lipopolysaccharide(LPS)and transforming growth factor-beta 1(TGF-β1).PRDX1 overexpression significantly increased the proliferation and migration of human bronchial epithelial(BEAS-2B)cells,reduced cell apoptosis(p<0.01),and induced EMT and collagen deposition by upregulating the expression of the matrix metallopeptidase(MMP)2,MMP9,α-smooth muscle actin(α-SMA),N-cadherin,vimentin and twist proteins and inhibiting E-cadherin expression(p<0.05).PRDX1 overexpression promoted TGF-β1-mediated inhibition of cell proliferation and migration and significantly enhanced the TGF-β1-induced EMT and collagen synthesis(p<0.05).Knockdown of PRDX1 inhibited cell proliferation,migration,EMT,and collagen synthesis(p<0.01),reversed LPS-mediated inhibition of cell proliferation and migration,and significantly suppressed LPS-induced EMT and collagen synthesis(p<0.01).The result indicating that PRDX1 may be involved in LPS/TGF-1-induced EMT and collagen synthesis in human bronchial epithelial cells.

Effect of Cigarette Smoke Extract on the Proliferation of Human Airway Epithelial Cells and Expression and Activation of FAK

The effect of cigarette smoke extract (CSE) on the proliferation of human airway epithelial cells and the possible mechanism was studied. After airway epithelial cells were treated with different concentrations of CSE for 24 h, the cell proliferation was measured by MTT and the distribution of different cell cycles by flow cytometry. The FAK expression level was detected by Western blot and the degree of tyrosine phosphorylation by immunoprecipitation. The results showed that CSE could inhibit the proliferation of human airway epithelial cells, arrest the epithelial cells in G1 phase of cell cycle, dramatically decrease the number of epithelial cells in S and G2 phases; Meanwhile CSE could decrease the expression level of FAK and the degree of its tyrosine phosphorylation. The above effects of CSE were concentration-dependent. The expression of FAK and the degree of its phosphorylation was positively correlated to the increased number of epithelial cells in G1 phase, and negatively to the number of epithelial cells in S and G2 phases. It was concluded that the mechanism by which CSE could inhibit the proliferation of human epithelial cells was contributed to the increased expression and activation of FAK.

β-catenin/Tcf Signaling in Squamous Differentiation of Porcine Airway Epithelial Cells

For a preliminary study of the role of β-catenin/Tcf signaling in squamous differentiation of airway （tracheobronchial） epithelial cells, a stable mutant of β-catenin was transfected into primarily cultured porcine airway epithelial cells. Western blotting revealed that exogenous protein was observed in large quantity in cytoplasm and nucleus, When co-transfected with Tcf luciferase reporter plasmids, β-catenin mutant increased the reporter＇s transcriptional activities. However, mRNA expression of a squamous differentiation marker, small proline-rich protein （SPRP）, was not elevated, as shown by reverse transcription-polymerase chain reaction. These findings suggest that β-catenin/Tcf signaling may not be directly involved in the squamous differentiation of porcine airway epithelial cells.

Exposure to ephedrine attenuates Th1/Th2 imbalance underlying OVA-induced asthma through airway epithelial cell-derived exo-somal lnc-TRPM2-AS

Although various anti-inflammatory medications,such as ephedrine,are employed to manage cough-variant asthma,their underlying mechanisms are yet to be fully understood.Recent studies suggest that exosomes derived from airway epithelial cells(AECs)contain components like messenger RNAs(mRNAs),micro-RNAs(miRNAs),and long noncoding RNA(lncRNA),which play roles in the occurrence and progression of airway inflammation.This study investigates the influence of AEC-derived exosomes on the efficacy of ephedrine in treating cough-variant asthma.We established a mouse model of asthma and measured airway resist-ance and serum inflammatory cell levels.Real-time polymerase chain reaction(RT-qPCR),Western blotting,and enzyme-linked im-munosorbent assay(ELISA)analyses were used to assess gene and protein expression levels.Exosomes were isolated and character-ized.RNA immunoprecipitation(RIP)and RNA pull-down assays were conducted to examine the interaction between hnRNPA2B1 and lnc-TRPM2-AS1.In the ovalbumin(OVA)-challenged mouse model,ephedrine treatment reduced inflammatory responses,air-way resistance,and Th1/Th2 cell imbalance.Exosomes from OVA-treated AECs showed elevated levels of lnc-TRPM2-AS1,which were diminished following ephedrine treatment.The exosomal lnc-TRPM2-AS1 mediated the Th1/Th2 imbalance in CD4^(+)T cells,with its packaging into exosomes being facilitated by hnRNPA2B1.This study unveils a novel mechanism by which ephedrine ameli-orates OVA-induced CD4^(+)T cell imbalance by suppressing AEC-derived exosomal lnc-TRPM2-AS1.These findings could provide a theoretical framework for using ephedrine in asthma treatment.

Human galectin-9 potently enhances SARS-CoV-2 replication and inflammation in airway epithelial cells

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a global economic and health crisis. Recently, plasma levels of galectin-9 (Gal-9), a β-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis, were reported to be elevated in the setting of severe COVID-19 disease. However, the impact of Gal-9 on SARS-CoV-2 infection and immunopathology remained to be elucidated. In this study, we demonstrate that Gal-9 treatment potently enhances SARS-CoV-2 replication in human airway epithelial cells (AECs), including immortalized AECs and primary AECs cultured at the air–liquid interface. Gal-9–glycan interactions promote SARS-CoV-2 attachment and entry into AECs in an angiotensin-converting enzyme 2 (ACE2)-dependent manner, enhancing the binding of the viral spike protein to ACE2. Transcriptomic analysis revealed that Gal-9 and SARS-CoV-2 infection synergistically induced the expression of key pro-inflammatory programs in AECs, including the IL-6, IL-8, IL-17, EIF2, and TNFα signaling pathways. Our findings suggest that manipulation of Gal-9 should be explored as a therapeutic strategy for SARS-CoV-2 infection.

Involvement and therapeutic implications of airway epithelial barrier dysfunction in type 2 inflammation of asthma

Type 2 inflammation is a complex immune response and primary mechanism for several common allergic diseases including allergic rhinitis,allergic asthma,atopic dermatitis,and chronic rhinosinusitis with nasal polyps.It is the predominant type of immune response against helminths to prevent their tissue infiltration and induce their expulsion.Recent studies suggest that epithelial barrier dysfunction contributes to the development of type 2 inflammation in asthma,which may partly explain the increasing prevalence of asthma in China and around the globe.The epithelial barrier hypothesis has recently been proposed and has received great interest from the scientific community.The development of leaky epithelial barriers leads to microbial dysbiosis and the translocation of bacteria to inter-and sub-epithelial areas and the development of epithelial tissue inflammation.Accordingly,preventing the impairment and promoting the restoration of a deteriorated airway epithelial barrier represents a promising strategy for the treatment of asthma.This review introduces the interaction between type 2 inflammation and the airway epithelial barrier in asthma,the structure and molecular composition of the airway epithelial barrier,and the assessment of epithelial barrier integrity.The role of airway epithelial barrier disruption in the pathogenesis of asthma will be discussed.In addition,the possible mechanisms underlying the airway epithelial barrier dysfunction induced by allergens and environmental pollutants,and current treatments to restore the airway epithelial barrier are reviewed.

Airway Epithelial Cell Function and Respiratory Host Defense in Chronic Obstructive Pulmonary Disease

INTRODUCTIONOur lungs have a vital role in mediating the exchange of oxygen and carbon dioxide between the air we breathe and the body.This function is under constant pressure as inhaled air contains numerous particles,gasses,and microorganisms that may cause injury and infection to the lungs.Removal and neutralization of potential harmful substances from inhaled air is a main function of the airway epithelium.

Dexamethasone protects airway epithelial cell line NCI-H292 against lipopolysaccharide induced endoplasmic reticulum stress and apoptosis

Background Endoplasmic reticulum （ER） stress and ER stress-mediated apoptosis were reported to be involved in the pathogenesis of several diseases. In a recent study, it was reported that the ER stress pathway was activated in the lungs of lipopolysaccharide （LPS）-treated mice. It was also found that the C/EBP homologous protein （CHOP）, an apoptosis-related molecule, played a key role in LPS-induced lung damage. The aim of this study was to verify whether LPS could activate the ER stress response in airway epithelial cells and which molecule was involved in the pathway. This study was also aimed at finding new reagents to protect the airway epithelial cells during LPS injury. Methods ER stress markers were observed in LPS-incubated NCI-H292 cells. SiRNA-MUC5AC was transfected into NCI-H292 cells. The effects of dexamethasone and erythromycin were observed in LPS-induced NCI-H292 cells. Results LPS incubation increased the expression of ER stress markers at the protein and mRNA levels. The knockout of MUC5AC in cells attenuated the increase in ER stress markers after incubation with LPS. Dexamethasone and erythromycin decreased caspase-3 activity in LPS-induced NCI-H292 cells. Conclusions LPS may activate ER stress through the overexpression of MUC5AC. Dexamethasone may protect human airway epithelial cells against ER stress-related apoptosis by attenuating the overload of MUC5AC.

Galectin-7 overexpression destroys airway epithelial barrier in transgenic mice

When the integrity of airway epithelium is destroyed,the ordered airway barrier no longer exists and increases sensitivity to viral infections and allergens,leading to the occurrence of airway inflammation such as asthma.Here,we found that galectin-7 transgenic(+)mice exhibited abnormal airway structures as embryos and after birth.These abnormalities included absent or substantially reduced pseudostratified columnar ciliated epithelium and increased monolayer cells with irregular arrangement and widening of intercellular spaces.Moreover,airway tissue from galectin-7 transgenic(+)mice showed evidence of impaired cell–cell junctions and decreased expression of zonula occludens-1(ZO-1)and E-cadherin.When treated with respiratory syncytial virus(RSV)or ovalbumin(OVA),galectin-7 transgenic(+)mice developed substantially increased bronchial epithelial detachment and apoptosis,airway smooth muscle and basement membrane thickening,and enhanced airway responsiveness.We found that Galectin-7 localized in the cytoplasm and nucleus of bronchial epithelial cells,and that increased apoptosis was mediated through mitochondrial release of cytochrome c and upregulated JNK1 activation and expression of caspase-3 in galectin-7 Tg(+)mice.These findings suggested that Galectin-7 causes airway structural defects and destroys airway epithelium barrier,which predispose the airways to RSV or OVA-induced epithelial apoptosis,injury,and other asthma responses.

Overexpression of mclca3 in airway epithelium of asthmatic murine models with airway inflammation

Asthma is a worldwide prevalent disease that is a .considerable health burden in many countries. In recent years, the airway epithelium is increasingly recognized as a central contributor to the pathogenesis of asthma. One of the most highly induced genes in epithelial ceils in experimental allergic airway disease is the third murine calcium-activated chloride channel homologue （mclca3, alias gob-5）. Its human homology protein is hCLCA, which has been identified as clinically relevant molecules in diseases with secretory dysfunctions including asthma and cystic fibrosis.

Plasmacytoid dendritic cell deficiency in neonates enhances allergic airway inflammation via reduced production of IFN-α

Allergic asthma,a chronic inflammatory airway disease associated with type 2 cytokines,often originates in early life.Immune responses at an early age exhibit a Th2 cell bias,but the precise mechanisms remain elusive.Plasmacytoid dendritic cells(pDCs),which play a regulatory role in allergic asthma,were shown to be deficient in neonatal mice.We report here that this pDC deficiency renders neonatal mice more susceptible to severe allergic airway inflammation than adult mice in an OVA-induced experimental asthma model.Adoptive transfer of pDCs or administration of IFN-αto neonatal mice prevented the development of allergic inflammation in wild type but not in IFNAR1−/−mice.Similarly,adult mice developed more severe allergic inflammation when pDCs were depleted.The protective effects of pDCs were mediated by the pDC-/IFN-α-mediated negative regulation of the secretion of epithelial cell-derived CCL20,GM-CSF,and IL-33,which in turn impaired the recruitment of cDC2 and ILC2 cells to the airway.In asthmatic patients,the percentage of pDCs and the level of IFN-αwere lower in children than in adults.These results indicate that impairment of pDC-epithelial cell crosstalk in neonates is a susceptibility factor for the development of allergeninduced allergic airway inflammation.