TSLP Promotes Lung Inflammation Through Activation Of Dendritic Cells In OVA-induced Mice Asthmatic Model

Objectives:Allergic asthma is a chronic inflammatory disease of the airways associated with a predominant Th2 response to inhaled allergens leading to airway infiltration by eosinophils and mast cells, goblet cell hyperplasia, and bronchial hyperreactivity. In addition to Th2-type inflammation, asthma is characterized by features of epithelial stress and injury linked to tissue remodeling A long-recognized property of airway epithelial cells is their function as a complex physical barrier that defends against exposures to potentially harmful inhaled substances and microbial pathogens. It has become more obvious that epithelial cells play important roles in the initiation, maintenance, and regulation of both innate and adaptive immune responses in the airways. TSLP is one of the crucial mediators produced by epithelial cells.TSLP is normally expressed at epithelial cells of the lungs and skin. Recent studies have established that high levels of TSLP are associated with airway inflammatory disease in humans and mice. Dendritic cells (DCs) are professional antigen presenting cells that bridge between innate and adaptive immunity. DCs can be activated directly by pathogens via TLRs or by mediators produced by epithelial cells. Activated dendritic cells express high levels of CD40, CD80 and CD86. TSLP activates DCs to prime naive CD4+T cells and then differentiate into pro-inflammatory Th2 cells.To evaluate the role of TSLP in the development of asthma, we generated a mouse asthma model using OVA, and then identified the expression of TSLP in this process. We also detected the expression of TSLP mRNA after administration of TSLP neutralizing antibody. We found that the expression of TSLP mRNA and protein was significantly elevated in epithelial cells of mice with asthma, while blocking with TSLP neutralizing antibody decreased the expression of TSLP mRNA of lungs, CD40, CD80, CD86 on dendritic cells, and IL-4, IL-5, IL-13 in BALF. TSLP promotes lung inflammation via prime naive CD4+T cells which differentiate into pro-inflammatory Th2 cells.Methods:Eight to ten weeks old BALB/c mice (n=30 weight,20±2g) were obtained from the Animal Center of Shandong University. Mice were randomly divided into three groups:OVA group (n=10), control group (n=10) and TSLP blocking group (TSLP/ OVA, n=10). The model was confirmed by airway hyperresponsiveness, histological analysis of lung tissues. Measuring the concentration of cytokine IL-4, IL-5 and IL-13 in supernatant of BALF by ELISA. The expression of TSLP mRNA in lungs was analyzed by quantitative real-time RT-PCR. The expression of CD40, CD80 and CD86 on dendritic cells was analyzed by Flow Cyto Meter. Furthermore, we measured the expression of TSLP mRNA of lungs, CD40, CD80, CD86 on dendritic cells, and IL-4, IL-5, IL-13 in BALF after treating mice with TSLP neutralizing antibody.Results:1. The Mice Asthma ModelStudies in the OVA-induced model had demonstrated that a subset of overreaction as a major feature of asthma successful model. When compared with mice in control group, the mice of OVA group showed dysphoria, more activity, upper limb raise, urinary and fecal incontinence and so on.2. Analysis of Airway ResponsivenessAirway responsiveness demonstrated the expiratory resistance of OVA-group mice was elevated, which indicates that the OVA-sensitized mice developed marked airway hyperresponsiveness.3. Patho-manifestation by HE in lung tissuesWhen compared with mice in control group, the lung tissue from OVA treated mice showed wall thickening, inflammatory cells infiltration, subepithelial fibrosis, mucous metaplasia. 4. Expression of TSLP in the airway of OVA-induced miceTSLP was highly expressed in epithelial cells of the airway in OVA-induced mice. The smooth muscle cells and inflammatory cells around the larger airways and blood vessels also showed positive TSLP staining. Quantitative analysis showed TSLP was significantly increased in OVA-induced mice.5. The concentration of IL-4,IL-5 and IL-13 in BALFThe mices in OVA group developed eosinophilic inflammation with the concenration of IL-4, IL-5 and IL-13 upregulated compared with the control group (P<0.05)6. Expression of TSLP mRNA in OVA-treated mice lungsWe found that the expression of TSLP mRNA was significantly higher in the lungs of OVA-treated mice than those of the control (P<0.05)7. Expression of TSLP protein in OVA-treated mice lungsWestern blot analysis showed TSLP protein was significantly elevated in OVA mice as compared to the controls (P<0.05)8. Cytokine induction in OVA-induced asthma miceThe levels of CD40, CD80 and CD86 on dendritic cells from BALF were detected by flow cytometry. We found the levels of CD40, GD80 and CD86 on dendritic cells were significant upregulated in BALF of OVA-induced asthma mice (P<0.05)9. Correlational analysis between TSLP mRNA and IL-4, IL-5, IL-13 in BALFThe expression of TSLP mRNA were tightly correlated with the levels of IL-4 (r=0.989), IL-5 (r=0.996) and IL-13 (r=0.998).10. The effect of TSLP neutralizing antibodyThe mice treated with TSLP neutralizing antibody showed a reduction in CD40, CD80, CD86 on dendritic cells; the levels of IL-4, IL-5 and IL-13 in BALF were decreased in mice treated with TSLP neutralizing antibody compared to those OVA-treated mice, but there is no difference between the TSLP/OVA group and the control mice.Conclusions:1. TSLP was highly expressed in epithelial cells of the airway in mice with asthma.2. TSLP can aggravate asthmatic lung inflammation by activating DCs to promote Th2 differentiation.3. TSLP neutralizing antibody can inhibited this process, TSLP signaling is crucial for generation of an inflammatory reaction in asthma.