Yurong Tan

Wound repair and proliferation of bronchial epithelial cells enhanced by bombesin receptor subtype 3 activation.

The present study was designed to investigate the role of bombesin receptor subtype 3 (BRS-3) in airway wound repair. The results showed that: (1) There was few expression of BRS-3 mRNA in the control group. In contrast, the expression of BRS-3 mRNA was gradually increased in the early 2 days, and peaked on the fourth day, and then decreased in the ozone-stressed AHR animal. BRS-3 mRNA was distributed in the ciliated columnar epithelium, monolayer columnar epithelium cells, scattered mesenchymal cells and Type II alveolar cells; (2) The wound repair and proliferation of bronchial epithelial cells (BECs) were accelerated in a concentration-dependent manner by BRS-3 activation with P3513, which could be inhibited by PKA inhibitor H89. The study demostrated that activation of BRS-3 may play an important role in wound repair of AHR.

Expression of Nodal on Bronchial Epithelial Cells Influenced by Lung Microbes Through DNA Methylation Modulates the Differentiation of T-Helper Cells

Background/Aims: The previous study in our lab showed that Nodal molecule on bronchial epithelial cells (BECs) was modulated by all kinds of lung microbes. The present study was designed to determine the effects of Nodal on proliferation of BECs and BECs-induced differentiation of T-helper (Th) cells. The epigenetic mechanisms of Nodal expression following treatments of different lung microbes were also identified. Methods: Real-time polymerization chain reaction (PCR) and western blot were used to determine the expression of Nodal. Flow cytometry was used to observe the effects of proliferation of BECs and subsequent BECs-induced differentiation of Th cells. Methylation levels of CpG islands in Nodal promoters were also analyzed by time of flight mass spectrometry. Results: The results showed that Nodal promoted proliferation of BECs and BECs-induced differentiation of Th cell from Th1 to Th2 and Th17. Nodal promoter showed a hyper-methylation in normal BECs. Through methylation modification in the promoter, P. aeruginosa or A.baumanni inhibited the expression of Nodal while RSV promoted the expression of Nodal. Conclusions: Our data showed that Nodal promoted Th2 and Th17 differentiation and inhibited Th1 differentiation which may cause imbalance of airway microenvironment. P. aeruginosa or A.baumanni may be hopeful for the treatment of airway hyperresponsveness by inhibition Nodal expression through DNA methylation modification in the promoter.

Wound repair and proliferation of bronchial epithelial cells regulated by CTNNAL1

Adhesion molecules play vital roles in airway hyperresponsiveness (AHR) or airway inflammation. Our previous study indicated that adhesion molecule catenin alpha‐like 1 (CTNNAL1) is relevant closely to asthma susceptibility, but its biological function or significance is still unclear. In the present study, we observed the temporal and spatial distribution of CTNNAL1 expression in mouse lung tissue with the OVA‐sensitized asthma model and found that the level of CTNNAL1 mRNA showed a prominent negative correlation with pulmonary resistance (RL). To study the function of CTNNAL1 in airway, effects of CTNNAL1 on proliferation and wound repair activity of human bronchial epithelial cells (HBEC) was investigated with antisense oligonucleotide (ASO) technique. The results showed that: (1) CTNNAL1 ASO could decelerate the repairing velocity and proliferation of HBEC; (2) CTNNAL1 expression was increased on the edge cells of mechanic wounded area in culture; (3) extracellular matrix component fibronectin (Fn) obviously promoted wound repair activity and proliferation of HBEC, which could be blocked by CTNNAL1 ASO; (4) Western blot showed that Fn could promote FAK phosphorylation, which also be inhibited by CTNNAL1 ASO. In conclusion, the level of CTNNAL1 mRNA expression is highly correlated to airway resistance; CTNNAL1 may contribute to the wound repair and proliferation of HBEC. Furthermore, it may serve to Fn mediated cell‐extracellular adhesion and its signal transduction. J. Cell. Biochem. 103: 920–930, 2008.

Pulmonary peptidergic innervation remodeling and development of airway hyperresponsiveness induced by RSV persistent infection.

Respiratory syncytial virus (RSV) infection causes bronchiolitis in infants and children, which is an important risk factor for the development of chronic asthma. To probe the underlying mechanisms that RSV infection increases the susceptibility of asthma, this present study was designed to establish a RSV persistent infection animal model by cyclophosphamide (CYP) pretreatment that more closely mimic human RSV infection. CYP is an immunosuppressant, which induced deficiency in cellular and humoral immunity. Pulmonary RSV titers, airway function and peptidergic innervation were measured on 7d, 28 d, 42 d and 60 d postinfection. The results showed that during RSV persistent infection, the lungs of RSV-inoculated animals pretreated with CYP showed higher RSV titers and exhibited obvious chronic inflammation. The results also showed that protein gene product 9.5 (PGP9.5), substance P (SP) and calcitonin gene-related peptide (CGRP)-immunoreactive fibers increased and vasoactive intestinal peptide (VIP)-immunoreactive fibers decreased during RSV persistent infection. These results demonstrate that RSV persistent infection induces significant alterations in the peptidergic innervation in the airways, which may be associated with the development of altered airway function.

Ozone stress down-regulates the expression of cystic fibrosis transmembrane conductance regulator in human bronchial epithelial cells.

To investigate abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR) expression in chronic inflammatory airway diseases and its regulation mechanisms, the present study was designed to observe the expression of CFTR, CFTR chloride current and the possible relevant signal pathways in in vitro and in vivo bronchial epithelium by using real-time PCR, immunofluorescence, Western blot and whole cell patch-clamp. The results demonstrated that CFTR staining was decreased in rat airway epithelium under ozone stress. Ozone stress also down-regulated CFTR protein and mRNA expression and CFTR chloride current in cultured human bronchial epithelial cells (HBEC). STAT1 signal pathway was checked to investigate the signal mechanism. It was found that pretreatment with STAT1 inhibitor attenuated the down-regulated CFTR expression induced by ozone stress. We also observed that ozone stress accelerated the phosphorylation of STAT1 in HBEC, which could be influenced by some signaling molecules related to the early transduction of cellular stress. Furthermore, reactive oxygen species inhibitors N-acetylcysteine and nitric oxide synthase inhibitor aminoguanidine increased the expression of CFTR. Ozone stress could down-regulate the expression of CFTR and decrease CFTR chloride current in HBEC. The signal mechanism which referred to cascade events in cells included early oxidative stress signal transmission molecules, and subsequently transcription modulator STAT1.

Infection with respiratory syncytial virus alters peptidergic innervation in the lower airways of guinea-pigs

To probe the mechanisms by which respiratory syncytial virus (RSV) infection in early life forms an important risk factor for the development of chronic asthma, an airway hyper‐responsiveness (AHR) animal model of guinea‐pigs with persistent RSV infection was established by intranasal instillation of 2 × 105 plaque‐forming units RSV. On days 0, 7, 28, 42 and 60 postinoculation, the RSV copy numbers, airway function and peptidergic innervation were measured in the peripheral airways. The results showed that the virus was persistent in the lungs. During persistent infection (days 42 and 60), the lung resistance and the total cells, neutrophils and eosinophils of infected guinea‐pigs increased significantly; the airway showed signs of chronic inflammation; and the substance P‐ and calcitonin gene‐related peptide‐positive fibres increased, but vasoactive intestinal polypeptide‐positive fibres decreased. These results suggest that persistent RSV infection can cause long‐term chronic airway inflammation and persistent airway neural network abnormality, which may be related to the occurrence of AHR.

BRS-3 activation transforms the effect of human bronchial epithelial cells from PGE2 mediated inhibition to TGF-β1 dependent promotion on proliferation and collagen synthesis of lung fibroblasts

Airway re‐modelling in asthma usually results in an irreversible weakness of pulmonary ventilation, however, its initiating or controlling mechanism remains unclear. In this study, we hypothesize that signal communication between airway epithelial cells and sub‐mucosal fibroblast cells may play an important role in the maintenance of structure homeostasis in a physiologic condition and in initiation of airway remodelling in a stressed condition. To test the hypothesis, a co‐cultured system of human bronchial epithelial cells (BEC) and human lung fibroblasts (HLF) were designed to observe the effects of BEC, in the normal state or in a BRS‐3 activated state, on the proliferation and collagen synthesis of HLF. The results showed that the proliferation activities of both BEC and HLF inhibited each other under the normal state. BRS‐3‐activated BEC can transform the reciprocal inhibition into promoting effects. The secretion of TGF‐β1 increased and the synthesis of PGE2 decreased from BRS‐3‐activated BEC, which were correlated with the proliferation and collagen synthesis of HLF. The proliferation activities of HLF were weakened by co‐culture with TGF‐β1 antisense oligonucleotides (ASO) treated BEC. It was concluded that, in the normal state, BEC inhibits the activities of fibroblasts through release of PGE2 to maintain the airway homeostasis; however when stressed, for example by BRS‐3 activation, BEC promote the activities of fibroblasts mediated by TGF‐β1, thereby facilitating the airway re‐modelling.

Differentiation of Th Subsets Inhibited by Nonstructural Proteins of Respiratory Syncytial Virus Is Mediated by Ubiquitination

Human respiratory syncytial virus (RSV), a major cause of severe respiratory diseases, constitutes an important risk factor for the development of subsequent asthma. However, the mechanism underlying RSV-induced asthma is poorly understood. Viral non-structural proteins NS1 and NS2 are critically required for RSV virulence; they strongly suppress IFN-mediated innate immunity of the host cells. In order to understand the effects of NS1 and NS2 on differentiation of Th subsets, we constructed lentiviral vectors of NS1 or NS2 to infect 16 HBE and analyzed the expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 by Flow Cytometric Analysis and real-time PCR. The results showed that NS1 inhibited expression of HLA-DR, CD80 and CD86 and differentiation of Th1, Th2 and Th17 lymphocytes, which could be reversed by deleting elongin C binding domain. NS2 inhibited the differentiation of Th2 and Th17, which was reversed by proteasome inhibitors of PS-341. Our results indicated that NS1 inhibited the differentiation of T lymphocytes through its mono-ubiquitination to interacted proteins, while NS2 inhibited differentiation of Th2 and Th17 through ubiquitin-proteasome pathway, which may be related with the susceptibility to asthma after RSV infection.

An inactivated Pseudomonas aeruginosa medicament inhibits airway allergic inflammation and improves epithelial functions

The features of asthma are airway hyperresponsiveness (AHR), excess production of Th2 cytokines, and eosinophil accumulation in the lungs. To investigate the antiasthmatic potential of an inactivated Pseudomonas aeruginosa medicament (PPA), as well as the underlying mechanism involved, we studied the effects of PPA on airway and epithelial functions. Airway resistance, cell enumeration, and IL-4, IFN-γ, and IL-17 secretion in bronchoalveolar lavage fluid were assayed on an OVA-sensitized AHR animal model. Flow cytometry was used to observe the effects of PPA on cell proliferation, real-time PCR was used to test the expressions of toll like receptor 4 and 5, and Th17 signal molecules Act1, NF-kB negative regulator A20, and western blot were used to detect NF-kB expression on cultured human bronchial epithelial cells (BECs). PPA-treated animals had suppressed airway resistance, eosinophil and lymphocytes infiltration, and IL-4 and IL-17 secretion. PPA can stimulate toll-like receptor-4 and 5 expressions, promote cell proliferation in normal and OVA-treated BECs, significantly decrease Act1 and NF-kB, and increase A20 expression in BECs treated by OVA. Our data suggest the therapeutic mechanism by which PPA effectively treats allergic inflammation on reductions of airway responsiveness, eosinophil infiltration, IL-4 and IL-17 secretion, and improvements of epithelial functions.

Wound repair and anti-oxidative capacity is regulated by ITGB4 in airway epithelial cells

Integrin beta 4 (ITGB4) is a structural adhesion molecule which engages in maintaining the integrity of airway epithelial cells. Its specific cytomembrane structural feature strongly indicates that ITGB4 may engage in many signaling pathways and physiologic processes. However, in addition to adhesion, the specific biologic significance of ITGB4 in airway epithelial cells is almost unknown. In this article, we investigated the expression and functional properties of ITGB4 in airway epithelial cells in vivo and in vitro. Human bronchial epithelial cell line (16HBE14O-cells) and primary rat tracheal epithelial cells (RTE cells) were used to determine ITGB4 expression under ozone tress or mechanical damage, respectively. An ovalbumin (OVA)-challenged asthma model was used to investigate ITGB4 expression after antigen exposure in vivo. In addition, an ITGB4 overexpression vector and ITGB4 silence virus vector were constructed and transfected into RTE cells. Then, wound repair ability and anti-oxidation capacity was evaluated. Our results demonstrated that, on the edge of mechanically wounded cell areas, ITGB4 expression was increased after mechanical injury. After ozone stress, upregulation expression of ITGB4 was also detected. In the OVA-challenged asthma model, ITGB4 expression was decreased on airway epithelial cells accompanying with structural disruption and damage of anti-oxidation capacity. Besides, our study revealed that upregulation of ITGB4 promotes wound repair ability and anti-oxidative ability, while such abilities were blocked when ITGB4 was silenced. Taken together, these results showed that ITGB4 was a new interesting molecule involved in the regulation of wound repair and anti-oxidation processes for airway epithelial cells.