塵蟎過敏原Der p 2與脂多醣對呼吸道上皮細胞的發炎反應和上皮-間質轉化現象之相互影響

Abstract

Bronchial asthma is a heterogeneous inflammatory airway disease; it develops a complex pathology that is characterized by reversible expiratory airflow limitation and airway inflammation accompanying with airway remodeling and airway hyper- responsiveness (AHR). Over 50% of individuals who suffered with asthma have been sensitized with environmental allergens such as house dust mites (HDM) and developed a Th2-biased immune response. More than 20 HDM allergen groups have been defined based on sequence and functional homologies. Belonging to group 2 allergens, Der p 2 and Der f 2 are low molecular weight protein without proteolytic activity that can induce specific IgE antibodies production in allergic patients. Epidemiological studies showed that children exposed to an environment with rich microbes may be protected from allergic asthma; however, infections are likely to be connected with asthma exacerbation. The airway is not sterile; therefore, the microbes from the external environment and the microbes residing in the airway may have great influences on asthma development. Recently, others’ studies and ours have demonstrated that activation of the innate immunity also plays a critical role in HDM-induced allergic inflammation, particularly in the combination with environmental endotoxins. The molecular interaction between aeroallergens and LPS in the mucosal membranes of airway is still not clear, and whether this interaction will cause the pathological effect that lead to epithelial-mesenchymal transition (EMT) in the airway epithelial cells is also unknown. Therefore, in this study, we expressed, isolated, and applied recombinant Der p 2 (rDer p 2) with high IgE binding activity from Pichia pastoris to human airway epithelial cell lines to investigate (1) whether the major HDM allergen, Der p 2, could directly induce inflammation and EMT in airway epithelial cells, and if so whether the natural product of propolis could have preventive effects in the TGF-β1-induced EMT in airway epithelial cells; (2) whether the major HDM allergen, Der p2, could augment LPS-induced airway inflammation, and if so, explored the possible molecular mechanisms involved in this augmentation effect. In the first part of our research, the results showed that stimulation of rDer p 2 alone could not induce EMT in airway epithelial cells. Therefore, we used a well-established model, TGF-β1-induced-EMT in A549 cells, to perform our experiments on EMT. Experimental results showed progressive cell morphological change, decreased E-cadherin production, increased N-cadherin production, intracellular F-actin rearrangement, increased ROS production, and increased cell motility with an increase in the concentrations of TGF-1 in A549 cells. Pretreated with propolis and then treated with TGF-1 for 24 h regained epithelial cells morphology and decreased the production of N-cadherin and ROS and decreased cell motility. Propolis prevented the effect of TGF-1-induced Smad2 and Akt activation pathways and Snail expression. Moreover, propolis pretreatment may prevented the TGF-1-induced down-regulation of nuclear hormone receptor and peroxisome proliferator-activated receptor gamma (PPARγ) protein in A549 cells, whose effect was blocked by adding PPARγ antagonist, GW9662. Two active components of propolis, caffeic acid phenethyl ester (CAPE) and pinocembrin (PIN) only had partial effects on TGF-β1-induced EMT in A549 cells. The results of this study suggest that other unidentified components of propolis may be involved in the inhibitory effect on TGF-1-induced EMT in A549 cells. In the follow-up research, the aim was set to explore the pathological roles of Der p 2 in airway epithelia cells, and its effect in LPS-induced airway inflammation. We used rDer p 2 to treat human airway epithelial cell lines, BEAS-2B, A549, or H292 to evaluate the effect of Der p 2 on LPS-induced cytokines or chemokines production. rDer p 2 alone could not induce high levels of IL-6, IL-8 or CCL20. We then added rDer p 2 with LPS. In any case (pre-treatment, co-treatment, and post-treatment), the production levels of IL-6, IL-8 or CCL-20 were higher than those by adding LPS only. This enhancement of inflammatory cytokines production was through JNK signal pathway. These results were further confirmed by using in vivo experiment, which administrated intra-tracheally rDer p 2 and LPS simultaneously into the lungs of mice. These mice produced higher IL-6 and TARC, and the amount of infiltrated cells in their bronchoalveolar lavage fluids (BALF) was larger than mice stimulated with rDer p 2 or LPS alone. In conclusion, our results suggest that recombinant Der p 2 allergen can participate and enhance LPS-induced airway inflammation into eosinophil infiltration and Th2 chemokine predominant, asthma-like inflammation. Propolis is a popular folk medicine with potential pharmacological actions related to immunological reactions. The results of this study suggest that natural propolis extracts may prevent TGF-β1-induced EMT in immortalized type II AECs via multiple inhibitory pathways, which may be clinically applied in the prevention and/or treatment for EMT-related fibrotic diseases as well as airway remodeling in chronic asthma. Der p 2 allergen can participate and enhance LPS-induced airway inflammation. This study also suggested that LPS play an import role in allergy; the phenomenon that Der p 2 and LPS may work together to deteriorate inflammatory reactions reminds us the potential affects from LPS to allergy development cannot be ignored. Appropriate and in-time control over LPS may keep allergy from exacerbation. In the future, further investigations about the effect of LPS in the airway will be continued. There may be novel targets for stopping the progression from chronic inflammation of asthma.