Inflammation And Immune Stress Of Airway Epithelium Modulated By Adipokines

Epidemiologic data indicate an increasing incidence of asthma in the obese individuals recent decades, very little is known about the possible association between them. Systemic inflammation status was observed in obesity, while asthma is also considered as a chronic airway inflammation in nature. It is speculated that obesity provides an appropriate immunological foundation which increased susceptibility to asthma. Adipokines from adipocytes involved in many obesity-related conditions such as type2diabetes and atherosclerosis, which account as the hinge between energy metabolism and inflammatory immune response. Investigation about the role of adipokines in obesity asthma will provide a new theoretical basis and research tools for treatment of asthma. Damage of airway epithelial structure and function may result in susceptibility to asthma, which could be a priming process in this disease. Bronchial epithelial cells (HBECs) interact with lymphocytes, constituting the airway mucosal immune system. Therefore, the assumption we brought up is that adipokines mediate relevance between obesity and asthma as a result of regulating airway epithelial function.Objectives:To verify the hypothesis mentioned above, this study based on:①Setting up an obesity-asthma model and look into the expression of the three adipokines:leptin, adiponectin and resistin. Analyze the relationship between adipokines and airway reactivity, as well as the inflammatory factor. ②Investigating the effect of adipokines on the proliferation, wound repair and apoptosis in HBECs.③Observing the role of adipokines on immune inflammatory signals transfer in HBECs.Methods and results:1. The effect of adipokines on airway inflammation in obesity-asthma model.(1) The establishment and identify of obesity-asthma model: Die-induced C57BL/6J obese mice were sensitized and challenged with ovalbumin (OVA) to establish obesity-asthmatic models. Body weight, Lee’s index and oil Red O staining in liver were used to assess levels of obesity. Pulmonary function, pulmonary tissue sections HE staining, and the differential counts of inflammatory cells in bronchial alveolar lavage fluid (BALF) were used to evaluate pulmonary inflammation. Result shows that, obese asthma mice had higher airway resistance and airway inflammation compared with asthma alone.(2) Detection the protein expression level of adipokines, Th cytokines in plasma and BALF, performing correlation analysis:ELISA was used to exam the adipokines and Th cytokines IFN-γ、IL-4、IL-17content. Pearson analysis was used to evaluate the correlativity. Data shows that increases in the BALF leptin also significantly correlated with increases in the plasma. Significant negative correlation was found between BALF adiponectin and IFN-γ (R2=0.987, P<0.05)、airway resistance (R2=0.9146,P<0.05)。2. The effect of adipokines on proliferation, wound repair and apoptosis in HBECs.(1) The role of adipokines on proliferation and wound repair in HBECs:MTT was used to test the cell proliferation activity and flow cytometry (FCM) was carried out to detect the cell cycle. The repair of mechanical wound was dynamic monitored within24hours. Data shows that compared with leptin and resistin, adiponectin seems to promote the proliferation activity and the S+G2phase cell fraction (from32.29%±1.78%to43.19%±3.81%, P<0.05). The wound repair was also accelerated by adiponectin. In order to explore the possible mechanism, we found adiponectin could enhance the intracellular free calcium concentration in HBECs, and calmodulin (CaM) inhibitor W7could inhibit the adiponectin induced repair acceleration. Suggesting the Ca2+/CaM pathway involved in the regulatory effect of adiponectin in HBECs.(2) Function of adipokines on the oxidizing apoptosis in HBECs: Utilize the Annexin V/PI to detect the apoptosis in ozone stressed HBECs. FCM results showed that adiponectin significantly rescued the ozone induced apoptosis (decreased the apoptosis rate from58.76%±3.06%to3.76%±1.31%, and increased the living cell from28.66%±2.42%to82.98%±3.96%, P<0.05). This effect maybe attributable to reduce the production of the reactive oxygen and the feedback regulation function between adiponectin and its receptor adipoRl.3. The role of adipokines on immune inflammatory signals transfer in HBECs.(1) The influence of adipokines on proliferation of HBECs co-cultured lymphocytes:Lymphocytes were isolated from human blood and labeled by CFSE probe, then co-cultured with HBECs with or without adipokines for48hours. FCM shows that in LPS stimulated cells, cell proliferation was promoted by letpin but inhibited by adiponectin (the first generation of lymphocytes was promoted by adiponectin from LPS induced17.47%±2.99%to26.04%±3.80%, P<0.05). There is no effect was observed in OVA stimulated cells.(2) The role of adipokines on CD4、CD8T cell subsets in HBECs: CD4-FITC/CD8-PE/CD3-PE-Cy5monoclonal antibody and flow cytometry was used in this study. We found that the ratio of CD4subsets was down-regulated in co-cultured suspended lymphocytes. No effect was observed in adipokines pre-incubated group (P>0.05).(3) The role of adipokines on adhesive attraction between HBECs and lymphocyte:Adhesive attraction between HBECs and FITC-CD3marked lymphocyte was observed by light microscope and fluorescence microscope. Adherence rate was assessed by microplate reader and FCM. Results showed that lymphocytes, particular the CD4T cell adhere to HBECs specifically. Adipokines have no remarkable impact here (P>0.05).(4) Impact of adipokines on Th cytokines release:We detected the IL-4、IFN-γ and IL-17by ELISA in supernatant of co-cultured system. In LPS stimulated groups, leptin as well as resistin promoted the release of IFN-y and IL-17, adiponectin reduced the IFN-y but increased the IL-17release. This is various form OVA stimulated groups.(5) The influence of adipokines on OVA antigen intake ability and TLR4protein expression in HBECs:The HBECs intake of FITC-OVA in30min,60min and90min was observed by fluorescence microscope. Leptin, adiponectin and resistin have no the effect on OVA antigen intake in HBECs (P>0.05), which is detected by FCM. Adiponectin decreased the up-regulated TLR4induced by LPS (from26.54±3.62to19.9±1.97). (6) The effect of adipokines on the B7molecular profiles:There is no apparent difference on B7molecular mRNA expression in HBECs with or without adipokines incubation (P>0.05), data was measured by Real-time PCR.(7) Impact of adipokines on TSLP release from HBECs:ELISA was used to exam the TSLP released from HBECs. It can be observed that TSLP could be stimulated by OVA, but no influence on TSLP release was observed under adipokines treatment (P>0.05). Suggesting other mechanism participate in the immune modulating function of adipokines.Conclusion:1. Obesity aggravates the airway responsiveness and local inflammation. Adiponectin in BALF is closely related to airway resistance and Thl type inflammation, possessing an anti-inflammatory effect.2. Adiponectin maybe a potential protector to human bronchial epithelial cell for regulating proliferation, wound repair and apoptosis, compared with leptin and resistin.3. Adipokines act on different aspect of immune inflammatory signals transfer in HBECs, participating in the regulation of airway mucosal immunity.This paper embraces40figures,10tables and216references.