Progress on mechanism of airway mucus hypersecretion induced by mycoplasma pneumoniae infection / 国际儿科学杂志

The incidence rate of chronic airway inflammatory diseases caused by airway mucus hypersecretion caused by mycoplasma pneumoniae(MP) infection is increasing year by year.In this paper, the damage of MP on airway mucosa through its adhesion, migration, community acquired respiratory distress syndrome toxin(CARDS Tx)and other virulence factors is described, which directly damages the structure and function of airway epithelium and affects the metabolism of ciated cells.It can also cause the increase of airway pro-inflammatory cytokines and the up regulation of toll-like receptor, resulting in the increase of airway mucus secretion.In addition, when MP infects airway epithelial cells, it will increase mucin(MUC)production through signal transduction pathway, which will increase the secretion of MUC5AC and lead to airway mucus hypersecretion.Controling airway mucus hypersecretion caused by MP infection can reduce the occurrence of chronic airway inflammation and helpful to the treatment of refractory MP infection.

Culture and differentiation of mouse tracheal epithelial cells at air-liquid interface / 中国药理学通报

Aim Air-liquid interface (ALI) cultures of mouse tracheal epithelial cells (MTEC) are a well-established model to study airway epithelial cells. MTEC provides a powerful ap¬proach for the evaluation of the inhalation toxicological in vitro. Methods C57BL/6 mouse tracheal-bronchial epithelial cells were obtained by digestion with protease in cold temperature o- vemight, and the digestion time was optimized to ensure the quantity and viability of the obtained cells. The cells were cul¬tured into collagen coated Transwell inserts. Proliferating phase and air-liquid interface culture were promoted with different cul¬ture media. The expression of tight junction protein and cell trans-epithelial electrical resistance(TEER) were used to evalu¬ate the formation of tight junction between cells and the analysis of cell polarity. The cilia structure was confirmed by electron mi¬ croscopy and immunofluorescence. Results Highly purified and viable primary airway epithelial cells could be harvested and subcultured by our methods, including morphology and immuno- cytochemistry staining confirmed the expression of MUC5AC, a- tubulin, p-tubulin-IV and ZO-1. The development of tight-junc¬tions and epithelium were similar with pseudostratified ciliated columnar epithelium morphology. Conclusions A comprehen¬sive protocol for ALI culture was established, reproducing the characteristic pseudostratified ciliated columnar epithelium mor¬phology and physiological functions in vitro. The MTEC protocol provides a stable and reliable method for the isolation, mucocili¬ary differentiation and reproducing.

Effects of neonatal Streptococcus pneumoniae pneumonia on airway epithelial injury of mice / 解放军医学杂志

Objective To investigate the effects of neonatal Streptococcus pneumoniae pneumonia (S.pp) on airway epithelial injury of mice. Methods Neonatal C57BL/6 (1-week-old) mice were infected intranasally with 2×105 cfu of Streptococcus pneumoniae (S.p) in a volume of 5 μl sterile phosphate buffered saline (PBS) (S.pp group), while the mock-infected controls received the same volume of sterile PBS (control group). Five weeks after the infection, the airway hyperresponsiveness (AHR) was evaluated by invasive body plethysmography system. The lung tissue was harvested, CDH1 and TJP1 mRNA levels were analyzed by RT-PCR. Immunohistochemical method and Western blotting were used to evaluate the expressions of E-cadherin and tight junction protein (ZO-1). Airway inflammation was detected by HE staining, the subcutaneous collagen deposition beneath the airway epithelium was evaluated by Masson staining, and the number of goblet cells was detected by Alcian Blue Periodic Acid Schiff (AB-PAS) staining. Results When the concentration of aerosolized methacholine inhaled by mice ranged from 6.25 to 50 mg/ml, the AHR was obviously higher in S.pp group than that in control group (P<0.001); the levels of CDH1 and TJP1 mRNA in the lung tissue were markedly lower in S.pp group (0.85±0.29 and 0.43±0.16) than those in control group (0.85±0.29 vs. 1.42±0.40, P=0.033; 0.43±0.16 vs. 0.83±0.26, P=0.010, respectively); the expression levels of E-cadherin and ZO-1 in airway epithelium were lower in S.pp group than those in control group (9.66±4.89 vs. 24.52±7.58, P=0.001; 13.54±3.79 vs. 25.53±5.99, P=0.005, respectively); the protein levels of E-cadherin and ZO-1 in lung tissue were remarkably lower in S.pp group than those in control group (0.23±0.06 vs. 0.38±0.06, P=0.019; 0.68±0.12 vs. 0.96±0.16, P=0.032, respectively); the subcutaneous collagen deposition beneath the airway epithelium increased significantly in S.pp group than that in control group (45.54±5.79 vs. 26.3±5.53, P=0.001). The number of goblet cells and inflammatory cell infiltration showed no statistical significance between the two groups. Conclusion Neonatal S.pp may lead to airway epithelial injury and be involved in the formation of AHR.

Effect of Diarylpentanoid Analogues on LipopolysaccharideInduced Interleukin-6 and Interleukin-8 Gene Expression in Airway Inflammation

@#Introduction: Airway inflammation is the pathological hallmark of chronic inflammatory airway diseases, especially asthma and chronic obstructive pulmonary disease (COPD). Airway epithelium plays an indispensable role in these diseases by secreting inflammatory mediators and cytokines in response to foreign substances, such as lipopolysaccharide (LPS). Previous studies have shown that diarylpentanoid analogues, especially 5-(3,4-dihydroxyphenyl)-3-hydroxy-1-(2-hydroxyphenyl)penta-2,4-dien-1-one (DHHPD) and 2-benzoyl-6-(3,4-dihydroxybenzylidene)cyclohexen-1-ol (BDHBC), significantly inhibited nitric oxide (NO) production; suggesting their anti-inflammatory property. However, the therapeutic potential of DHHPD and BDHBC in airway inflammation has not been explored. Thus, this study aims to investigate their effects on interleukin (IL)-6 and IL-8 gene expression in LPS-induced Calu-3 cells, a cellular model of human airway epithelium. Methods: MTT cytotoxicity assay was carried out to identify non-cytotoxic concentrations of DHHPD and BDHBC on Calu-3 cells. RT-PCR was done to determine IL-6 and IL-8 gene expression levels. Results: DHHPD and BDHBC were not cytotoxic on Calu-3 cells up to 200µM. Four non-cytotoxic concentrations were chosen – 6.25, 12.5, 25 and 50µM to determine the effect of both compounds on gene expression. All four concentrations of DHHPD and BDHBC significantly inhibited LPS-induced mRNA expression of IL-6 while all concentrations of BDHBC, except 6.25µM, significantly reduced IL-8 mRNA expression. Similar finding was obtained for DHHPD, except that at 50µM, there was no inhibition of IL-8 mRNA expression. Conclusion: Diarylpentanoid analogues, DHHPD and BDHBC, are proven to be effective in suppressing LPS-induced IL-6 and IL-8 gene expression. However, further studies are required to confirm their inhibitory effects on the production of pro-inflammatory cytokines.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections / 生物医学与环境科学（英文）

OBJECTIVE@#Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.@*METHODS@#A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.@*RESULTS@#Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.@*CONCLUSION@#Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Der p2 Internalization by Epithelium Synergistically Augments Toll-like Receptor-Mediated Proinflammatory Signaling

PURPOSE: House-dust-mite (HDM) major allergen Der p2 shares homology and function with Toll-like receptor (TLR) signaling protein myeloid differentiation-2 (MD2) and may lead to airway inflammation. Should Der p2 be internalized by human airway epithelium, it has the theoretical propensity to potentiate epithelium activation. This study aimed to demonstrate the internalization of Der p2 by airway epithelium and to investigate the effects of Der p2 on MD2 expression and epithelium activation. METHODS: Internalization of recombinant, enhanced green fluorescent protein-labelled Der p2 (rDer p2-EGFP) into human airway epithelium (BEAS-2B) was tracked by laser confocal microscopy and confirmed by immunoblotting. Reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemical staining were used to determine the effect of Der p2 on MD2 expression in vitro and ex vivo. Expression of messenger RNA (mRNA) encoding receptors/cytokines was measured by RT-PCR. Secretion of interleukin-6/interleukin-8 (IL-6/IL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Internalization of Der p2 by BEAS-2B was confirmed by confocal microscopy and immunoblotting using rDer p2-EGFP and rDer p2, respectively. Expression of MD2 protein was increased in BEAS-2B and human nasal polyp airway epithelium cultured with rDer p2. Recombinant Der p2-cultured BEAS-2B caused little spontaneous IL-6/IL-8 secretion but significantly augmented by TLR ligand LPS. IL-6 secretion was up-regulated after MD2 transfection. Internalization of Der p2 was reduced by TLR2 RNA knockdown. Dexamethasone, calcitriol, anti-MD2/anti-TLR2 antibodies, and signalling inhibitors significantly reduced LPS+Der p2-induced IL-6/IL-8 secretion. CONCLUSIONS: Human airway epithelium may internalize Der p2, which potentiates the response to environmental proinflammatory stimuli through MD2 and TLRs. This study highlights a novel mechanism and alleviates IL-6/IL-8 secretion in mite-induced airway inflammation.

Airway allergy and viral infection.

There are complex interactions between airway allergy and viral infection. Available evidence suggests that viral respiratory infection can initiate, maintain and activate exacerbation of allergic conditions in respiratory tract. Innate and inflammatory responses to acute viral infection play important roles in its relationship to allergic reactions. On the other hand, biased immune responses toward Th2 caused by an allergic reaction may make the immune response ineffective in combating viral infection. It was previously shown that allergy can increase the expression level of rhinovirus receptors on mucosal epithelial cells. This suggests that airway allergy may increase the risk of rhinovirus infection. We have recently shown that allergy may also increase the expression level of influenza virus receptors. This suggests that airway allergy and viral infection may have a reciprocal interaction. The effect of allergy on the risk and outcome of viral infection needs to be further confirmed in clinical studies and its potential implication for clinical practice should be considered.

Short-term exposure of mice to cigarette smoke and/or residual oil fly ash produces proximal airspace enlargements and airway epithelium remodeling

Chronic obstructive pulmonary disease (COPD) is associated with inflammatory cell reactions, tissue destruction and lung remodeling. Many signaling pathways for these phenomena are still to be identified. We developed a mouse model of COPD to evaluate some pathophysiological mechanisms acting during the initial stage of the disease. Forty-seven 6- to 8-week-old female C57/BL6 mice (approximately 22 g) were exposed for 2 months to cigarette smoke and/or residual oil fly ash (ROFA), a concentrate of air pollution. We measured lung mechanics, airspace enlargement, airway wall thickness, epithelial cell profile, elastic and collagen fiber deposition, and by immunohistochemistry transforming growth factor-β1 (TGF-β1), macrophage elastase (MMP12), neutrophils and macrophages. We observed regional airspace enlargements near terminal bronchioles associated with the exposure to smoke or ROFA. There were also increases in airway resistance and thickening of airway walls in animals exposed to smoke. In the epithelium, we noted a decrease in the ciliated cell area of animals exposed to smoke and an increase in the total cell area associated with exposure to both smoke and ROFA. There was also an increase in the expression of TGF-β1 both in the airways and parenchyma of animals exposed to smoke. However, we could not detect inflammatory cell recruitment, increases in MMP12 or elastic and collagen fiber deposition. After 2 months of exposure to cigarette smoke and/or ROFA, mice developed regional airspace enlargements and airway epithelium remodeling, although no inflammation or increases in fiber deposition were detected. Some of these phenomena may have been mediated by TGF-β1.

Suppression of CFTR-mediated Cl- Secretion of Airway Epithelium in Vitamin C-deficient Mice

Hyperoxic ventilation induces detrimental effects on the respiratory system, and ambient oxygen may be harmful unless compensated by physiological anti-oxidants, such as vitamin C. Here we investigate the changes in electrolyte transport of airway epithelium in mice exposed to normobaric hyperoxia and in gulonolacton oxidase knock-out (gulo[-/-]) mice without vitamin C (Vit-C) supplementation. Short-circuit current (Isc) of tracheal epithelium was measured using Ussing chamber technique. After confirming amiloride-sensitive Na+ absorption (DeltaIsc,amil), cAMP-dependent Cl- secretion (DeltaIsc,forsk) was induced by forskolin. To evaluate Ca2+-dependent Cl- secretion, ATP was applied to the luminal side (DeltaIsc,ATP). In mice exposed to 98% PO2 for 36 hr, DeltaIsc,forsk decreased, DeltaIsc,amil and DeltaIsc,ATP was not affected. In gulo(-/-) mice, both DeltaIsc,forsk and DeltaIsc,ATP decreased from three weeks after Vit-C deprivation, while both were unchanged with Vit-C supplementation. At the fourth week, tissue resistance and all electrolyte transport activities were decreased. An immunofluorescence study showed that the expression of cystic fibrosis conductance regulator (CFTR) was decreased in gulo(-/-) mice, whereas the expression of KCNQ1 K+ channel was preserved. Taken together, the CFTR-mediated Cl- secretion of airway epithelium is susceptible to oxidative stress, which suggests that supplementation of the antioxidant might be beneficial for the maintenance of airway surface liquid.

TSLP promotes lung Inflammation via activating dendritic cells in OVA-induced mice asthmatic model / 中华微生物学和免疫学杂志

Objective To study the expression of thymic stromal lymphopoietin(TSLP) and the activation of DCs in OVA-induced murine asthma model, and investigate the effects and underlying mecha-nisms of TSLP on lung inflammation. Methods Thirty BALB/c mice were randomly divided into control group, OVA group and TSLP neutralizing antibody treated group. The asthma model was evaluated by airway responsiveness and histological analysis of lung tissues ; The levels of TSLP mRNA in lungs were determined by quantitative real-time PCR; The expression of TSLP in lungs were determined by immunohistochemistry and Western blot; The expression of CD40, CD80, CD86 in BALF was detected by FACS. Results Both the histological analysis of lung tissues and the airway responsiveness were all consistent with the characteris-tic of murine asthma model. The expression of TSLP and TSLP mRNA in the OVA group was significantly in-creased compared with blank group. The expression of CD40, CD80, CD86 in BALF from OVA group was increased significantly compared with the control group. Furthermore, treating mice with TSLP neutralizing antibody reduced the expression of CD40, CD80, CD86 on dendritic cells, and IL-4, IL-5, IL-13 in the OVA group. Conclusion Our study indicate that TSLP was highly expressed in the bronchial epithelia of murine asthma model, via upregulation of CD40, CD80, CD86, induce DCs to active CD4~+ T cells and pro-duce type 2 responses, so that aggravating the lung inflammation of asthma. Blocking TSLP is capable of in-hibiting the production of Th2 cytokines, thus presents a promising strategy for the treatment of asthma.

Construction of recombinant adenovirus vector Ad-elafin for nature antibiotic elafin gene and its expression in primary airway epithelium / 第三军医大学学报

Objective To construct the recombinant adenovirus for nature antibiotic elafin protein and observe its expression in primary airway epithelia.Methods The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183.The candidate clone was further analyzed by restriction endonuclease digestion,PCR,and sequencing.Then the recombinant adenovirus plasmid was digested with PacⅠ and transfected into 293 cells for packaging and amplification.Infection titer and rate were monitored by green fluorescent protein(GFP)expression.Finally the primary airway epithelium was infected with Ad-elafin,and the GFP expression was observed by fluorescence microscope in epithelial cells 24 h after transfection.The elafin protein was detected by ELISA in the supernatant.Results Restriction endonuclease and PCR confirmed that elafin gene was cloned into the adenovirus vector successfully.Twenty-four hours after transfection,the GFP expression was seen by fluorescence microscopy.The concentration of elafin protein was(2.2?0.4)ng/ml in supernatant of transfected group,while that of control group was(1.9?0.3)ng/ml,P

The expression of MMP-9 in bronchial biopsy mucosal tissue in asthmatic subjects / 中国实用内科杂志

Objective To investigate the expression of Matrix Metalloproteinase 9 (MMP 9) in bronchial biopy mucosal tissue and its relation to airway eosinophilic infiltration,lung function and airway hyperresponsiveness.Method 10 subjects with asthma were enrolled,meanwhile,10 healthy subjects and 6 subjects with COPD were as controls.At the same time,immunohistochemistry was employed.Results Immunohistochemistry for MMP 9 was positive in all asthmaticus,and just as such in 2/6 subjects with COPD.On the contrary,immunohistochemistry for MMP 9 was negative in all healthy subjects.The amount of MMP 9 expression was positively correlated with the number of eosinophil infiltrated to airway (r=0 62,P

Effect of Retinoic Acid on the Differentiation and Lysozyme Expression in Human Airway Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Retinoic acid (RA)-deprived cultures of normal human tracheobronchial epithelial (NHTBE) cells became squamous metaplastic, failed to produce mucin and instead secreted or released large amounts of lysozyme (LZ). The purpose of this study was to elucidate the relationship between RA-deficiency induced squamous metaplasia and increased LZ as a function of time. MATERIALS AND METHOD:The change of lysozyme protein and lysozyme mRNA was investigated over time in cultures using passage-2 normal human tracheobronchial epithelial (NHTBE) cells and passage-2 normal human keratinocytes (NHK). The amount of lysozyme and mucin was measured with dot blot, message of lysozyme with RT-PCR, and cornifin mRNA with Northern blot. RESULTS: Lysozyme message levels were consistently higher in RA-sufficient than RA-deficient cultures. Intracellular and extracellular LZ increased to a peak on the day 16 and thereafter decreased in the RA-deficient cultures. LZ gene expression in the RA-deficient cultures was barely detectable on the day 7 but was clearly expressed between days 10 and 14, but thereafter message levels decreased markedly. On day 12, large numbers of cells began to exfoliate in the RA-deficient cultures. Extracellular LZ appeared simultaneously at the apical surface, presumably released from the exfoliated cells, which contained high concentrations of LZ. Intracellular LZ levels were more than 11 fold less in NHK cells compared to NHTBE cells. CONCLUSION: This study suggests that cellular accumulation of lysozyme protein is a unique feature of metaplastic squamous differentiation. Further studies are needed to find out what mechanisms are involved.

Regulation of Mucin and Non-Mucin Secretions and Gene Expression by Retinoic Acid in Human Airway Epithelium / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Airway hypersecretion is a frequent feature of several respiratory tract diseases including rhinitis, sinusitis, and otitis media. Efforts are being made in several laboratories to elucidate mechanisms involved in the regulation of secretion. There are several factors which modulate expression of the secretory phenotype, such as retinoic acid (RA), triiodothyronine, steroid, and extracellular matrix. We have been interested in elucidating the role of retinoids in regulating differentiation of mucin and non-mucin secretions. MATERIALS AND METHODS: Retinoic acid was removed from the culture media of normal human tracheobronchial epithelial cells grown in the air-liquid interface cultures. The effects on cell phenotype and mucin, lysozyme (LZ), and the secretory leukocyte protease inhibitor (SLPI) secretion and gene expression were examined. RESULTS: Removal of RA from the media induced squamous differentiation and caused a drastic decrease in mucin secretion and a decrease in expression of the mucin genes, MUC2 and MUC5AC. Lysozyme and SLPI secretions were increased in RA-depleted cultures. Paradoxically, LZ mRNA was decreased, while the SLPI mRNA levels were increased. A most intriguing finding was the paradoxical response of LZ to RA-depletion. The reason for this apparant incongruity between mRNA and protein levels is currently under investigation. CONCLUSION: Our studies show that RA is an important factor for mucous differentiation.

Roles of NF-?B in expression of hBD-2 mRNA in human primary epithelial cells induced by TNF-? / 第三军医大学学报

Objective To explore the roles of NF-?B in expression of human ?-defensin-2(hBD-2) mRNA induced by TNF-? in the human airway primary epithelial cells.Methods After the human bronchial primary epithelial cells were stimulated with TNF? or first with NF-?B inhibitor PDTC,then TNF-?,the expression of hBD-2 mRNA was detected by RT-PCR.The I?B-? protein level in the cytoplasm was detected by Western blotting and the nuclear factor-kappa B(NF-?B) binding activity was analyzed by electrophoretic mobility shift assays.Results The hBD-2 mRNA could be detected after 2.5 h of TNF-? stimulation and expressed in a dose-dependent manner.The NF-?B could be activated after 0.5 h of TNF-? stimulation.The supershifts assays indicated that the p65-p50 heterodimer formed complexes of NF-?B were involved in the activated of NF-?B.Conclusion TNF-? can induce the expression of hBD-2 mRNA in a dose-dependent manner.The p65-p50 heterodimer formed complexes of NF-?B play an important role in the regulation of hBD-2 gene expression in response to TNF-?.