The Effect Of PPAR-γ On The Expression Of TSLP In Asthmatic Airway Epithelial Cells

Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The basic pathophysiology of asthma is characterized by chronic airway inflammation, airway hyperresponsiveness, and reversible airflow obstruction. As basic pathologic change, the airway inflammation in asthma is persistent even during adequate therapy and asymptomatic episodes, associated with airway hyperresponsiveness and recurrent episodes. The asthmatic airway inflammation has become a hot point of research around the world. Unlike COPD, the inflammation in asthma is characterized by the infiltration and accumulation of memory-like T-helper (Th2) cells and activated eosinophils, mast cells, overproduction of Th2 type cytokines in the bronchial mucosa. Excessive Th2 cytokine production may partly reflect selective infiltration of T cells with a functional Th2 phenotype. Th2 cells and Th2 type cytokines play a important role in chronic asthmatic airway inflammation.Thymic stromal lymphopoietin (TSLP) has been shown to be one of the factors released by epithelial cells very early following allergen contact and has an important role modulating the maturation and activation of DC.TSLP-induced DC strongly induces the expression of major histocompatibility complex class I and II as well as co-stimulatory molecules, such as CD40, CD80, and CD86, on DCs. It also prolongs DC survival and enhances DC-T cell conjugate formation, resulting in a strong proliferation of CD4+ and CD8+ T cells. As antigen-presenting cells, dendritic cells (DCs) play a critical role in directing T cell responses to an antigen, priming T cells to develop into Thl, Th2, and Thl7 memory/effector cells.DCs activated by thymic stromal lymphopoietin (TSLP) in vitro promote the development of Th2 memory/effector cells. Human TSLP can induce naive CD4+ T cells to generate the proallergic Th2 cytokines, IL-4,-5, and-13, while inhibiting the production of IL-10 and IFN-y. TSLP also has been found to be increased in asthmatic airways. Lung-specific expression of a Tslp transgene induced airway inflammation and hyperreactivity characterized by Th2 cytokines and increased immunoglobulin E. The lungs of Tslp-transgenic mice showed massive infiltration of CD4+ T cell, goblet cell hyperplasia and subepithelial fibrosis, whereas mice lacking the TSLPR exhibit strong Thl responses and fail to develop an inflammatory lung response to Ag. Skin-specific overexpression of TSLP in RBP-j-deficient transgene mice that sustained high systemic levels of TSLP induces bronchial hyper-responsiveness. Antigen-induced allergic challenge to the lung airways of RBP-j-deficient animals resulted in a severe asthmatic phenotype not seen in similarly treated wild-type littermates. The involvement of TSLP in the Th2 inflammatory pathway is suggested by the fact that the expression of TSLP is increased in the bronchial mucosa of asthmatics and its expression is correlated with Th2-attracting chemokine expression and disease severity. These suggest that TSLP is key role involved in the pathogenesis of asthmatic Th2 type inflammation.TSLP is mainly is expressed by airway epithelial cell in asthma. Viral infection in synergy with cytokine production is likely to be a primary driver for TSLPproduction. Rhinovirus and respiratory syncytial virus (RSV) can up-regulate the expression of TSLP in human bronchial epithelial cells (HBECs). Our study suggests that. RSV infection promotes the production of TSLP in OVA sensitized asthmatic mice and aggravates inflammation in mice lung. TSLP expression evoked by rhinoviral double-stranded ribonucleic acid (dsRNA) and polyI:C promotes AEC expressing TSLP. TSLP can promotes the production of Th2 cytokines, and TSLP expression is synergistically enhanced by IL-4 and IL-13. The recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway. Respiratory viral infection and the recruitment of Th2 cytokine-producing cells may amplify Th2 inflammation via the production of TSLP in the asthmatic airway. Stephane Esnault think that TSLP acts as a Bridge between Infection and atopy,and reducing the expression of TSLP may prevent infection inducing the atopy. As a key molecule, TSLP became a new therapy target in asthma.The mechanism of TSLP expression is unclear, some studys suggest that nuclear factor-κB (NF-κB) may be involved.Peroxisome proliferator-activated receptors (PPARs) aremembers of the nuclear receptor superfamily containing transcription factors regulating gene expression. They are ubiquitously expressed through the body, and three subtypes, encoded by separated genes, have been identified, namely PPARα, PPARβ/δ, and PPAR-γ. To date, PPAR-γhas been the most extensively studied receptor among the three PPAR subtypes. PPAR-γwas initially identified as a transcription factor involved in fat cell differentiation and researchers have continued to support its role as a key modulator of adipocyte differentiation. However, accumulating evidence indicates that PPAR-γaffects cell cycle, differentiation, and apoptosis. In addition, PPAR-γactivation downregulates the synthesis and release of immunomodulatory cytokines from various cell types that participate in the regulation of inflammatory processes. Through the anti-inflammatory and immunomodulatory property, PPAR-γis one of the potential candidates in the treatment of inflammatory diseases.PPAR-γis expressed by various cells in the lung. The PPARg receptor has been shown to be up-regulated in asthmatic epithelium, and PPARg agonists in vitro have a range of effects on inflammatory cells, inhibition of T-cell proliferation and cytokine production. Studys suggest that PPAR-γagonist effects in various models of allergic asthma and airway inflammation. In some clinical cases reports, two case subjects whose symptoms related to asthma had remitted during treatment with pioglitazone. Most recently a study in smoking asthmatics showed an improvement in FEV1 after 4 weeks rosiglitazone treatment compared with beclometasone treatment。Positive results in these studies have led to suggestions that PPAR-γagonists might have utility for the treatment of human allergic asthma. The mechanism of the anti-inflammatory in vitro of the PPAR-y agonists has not been fully characterised, and there is considerable study indicates that PPAR-γagonists can inhibit the NF-κB. Whether PPAR-y agonist modulates the expression of TSLP by inhibition of NF-κB and inhibits the Th2 type inflammation by modulating the TSLP expression are unclear.The aim of this study was to measure the concentrations of TSLP in induced sputum and plasma, collected from asthma patients and normal control subjects. To investigate the correlation of TSLP expression to the severity of disease and lung function, and evaluate the clinical significance of TSLP. In 16HBE stimulated by poly I:C, we observed the effect of rosiglitazone and telmisartan on the expression of TLSP and researched its molecular mechanism. Finally, we investigate rosiglitazone and telmisartan effects on expression of TSLP and NF-κB, and effects on airway inflammation in mice.METHODSClinical trial1. The inclusion criteria and exclusion criteria of this experiment.2. Collect clinical datas of patients and complete the lung function test.3. Collect induced sputums and plasma of patients.4. Measure TSLP level of induced sputum and plasma by ELISA assay.5. Statistical analysis.Cell experiments In vitro1. Cell culture of 16HBE.2. Compare the 16HBE Cell viability under different concentration (0.1μM、1μM、5μM、10μM、20μM)of rosiglitazone, telmisartan and GW9662 by Methyltetrazolium (MTT) assay.3. Compare the expression of TSLP mRNA and PPAR-y mRNA in 16HBE after different time of Poly I:C stimulation4. At mRNA and protein levels, we observe the effect of rosiglitazone, telmisartan and GW9662 on TSLP expression at mRNA and protein levels in 16HBE Cell under normal status. 5. At mRNA and protein levels, Observe the effect of rosiglitazone, telmisartan and GW9662 on TSLP expression in 16HBE Cell under Poly I:C stimulation.6. At mRNA levels, Observe the effect of NF-κB inhibitor Bay 11-7082(10μM 20μM) on TSLP expression in 16HBE Cell under Poly I:C stimulation.Animal experiments1. Forty female BALB/c mice were randomly by table of random numbers divided into 5 groups:control group, asthma group, dexamethasone group, rosiglita-zone group, telmisartan group.2. Mice were sensitized and chanllege with OVA, and different drugs were given in different groups.3. Airway hyperresponsiveness to MCh (0,3.125,6.25,12.5,25 and 50 mg/ml in isotonic saline) was assessed by whole body plethysmography, Penh value of different groups were recorded and compared.4. TSLP in mice serum were measured by ELISA assay.5. BAL fluid was isolated, total cells recovered were counted, and the cellular composition of BALF was determined using Hematoxylin-eosin staining. Cytokines (IL-4,IFN-γ,TSLP) in BAL of mice were examined by ELISA assay.6. Pulmonary histopathology:mice lungs were excised and fixed. These tissues were then embedded in paraffin, cut sections and stained with hematoxylin and eosin (H&E); periodic acid—Schiff(PAS)to show mucus production in airway goblet cells.7. The expression of TSLP and p65 in lung specimens was examined by immuno-histochemical staining.Statistical analysisStatistical analysis was performed using SPSS 13.0 software. All data were expressed as mean±SD. One-way analysis of variance (one-way ANOVA) or Welch test and LSD or Dunnett’s T3 methods were used to determine differences between experimental groups according to test of homogeneity of variance. Chi-square test was used to compare the mean percentage and constituent ratio. Spearman’s rank correlation test was used for variables.Significance was accepted when p< 0.05.RESULTSThe results of Clinical trial1. Induced sputum and peripheral blood were collected from 24 healthy control subjects and 39 asthmatic patients.2. The plasma TSLP level (65.15±33.67pg/ml) in asthma group was higher than control subjects(1.07±2.39pg/ml).Compared with control subjects (0.28±0.81pg/ml), TSLP level in induced sputum was elevated in asthma group (17.47±21.16pg/ml)3. The plasma TSLP level in severe asthma patients was higher than mild asthma patients. There are no different between moderate asthma patients and severe asthma patients. The plasma TSLP level in moderate asthma patients was higher than mild asthma patients.4. The induced sputum TSLP level in severe asthma patients was higher than mild asthma patients and moderate asthma patients. There are no different between moderate asthma patients and mild asthma patients.5. There are no different between eosinophilic asthma or non-eosinophilic asthma on plasma and induced sputum TSLP levels.6. The plasma TSLP levels showed significantly positive correlations with induced sputum TSLP levels(r=0.453).7. The induced sputum TSLP levels showed significantly negative correlations with FEV1(r=-0.379),FEV1%predicted (r=-0.504),FEV1/FVC (r=-0.353), and positive correlations with ACQ5 score(r=0.330).8. The plasma TSLP levels showed significantly negative correlations with FEV1(r=-0.370),FEV1%predicted (r=-0.513),FEV1/FVC (r=-0.347), and positive correlations with day symptoms score(r=0.365).9. The induced sputum TSLP levels showed significantly negative correlations with percentages and absolute cell counts of EOS in sputum (r=0.337, r=0.365). Results of Cell experiments In vitro1. Under different concentration (0.1μM、1μM、5μM、10μM、20μM)of rosiglitazone, telmisartan and GW9662, no effects on 16HBE Cell viability were observed.2. The expressions of TSLP mRNA in 16HBE on different time after Poly I:C stimulation were induced to peak levels at 3h, corresponding copies of TSLP mRNA was increased to 4.57±0.66 fold compared to Oh group.3. No changes were observed on the expression of PPAR-y mRNA in 16HBE after different time of Poly I:C stimulation.4. At mRNA and protein levels, No effects of rosiglitazone, telmisartan and GW9662 were observed on TSLP expression in 16HBE Cell under normal status.5. At mRNA and protein levels, rosiglitazone and telmisartan inhibited TSLP expression in 16HBE Cell under Poly I:C stimulation alone. The effect of rosiglita-zone and telmisartan on TSLP expression was blocked or diminished in the presence of GW9662.6. At mRNA levels, NF-κB inhibitor Bay 11-7082(20μM) on TSLP expression in 16HBE Cell under Poly I:C stimulation.Results of animal experiments1. Airway responsiveness to MCh increased along with its concentration in different groups. Mice in the asthma group have the most high %baseline Penh value compared to mice in other groups.2. H&E staining and PAS staining showed that epithelial cells hypertrophy and hyperplasia, mucus hypersecretion, tremendous eosinophil and lymphocytes infiltration were observed in asthma group. All kinds of treatment inhibited the airway inflammation to some degree. The airway inflammation score of asthma group were higher than other groups (χ2= 1.686, P<0.001)3. Immunohistochemical staining showed the most significant TSLP expression in AEC of asthma group mice.Other treatment groups showed decreased expression of TSLP. 4. Immunohistochemical staining showed the most significant TSLP expression in AEC of asthma group mice.Other treatment groups showed decreased expression of TSLP.5. The numbers of total cells in BAL in asthma group were higher than other groups (P<0.001). The numbers and proportion of eosinophils in asthma group were higher than other groups (P<0.001).6. The mice serum TSLP levels in asthma group (35.13±5.94pg/ml) were highest in all groups. The mice serum TSLP levels decreased in other treatment.7. The BAL TSLP levels in asthma group (27.84±6.08 pg/ml) were highest in all groups. The mice serum TSLP levels decreased in other treatment.8. The BAL IL-4 levels in asthma group were highest in all groups. The mice serum TSLP levels decreased in other treatment.9. The BAL INF-y levels in rosiglitazone group were higher than asthma group.CONCLUSIONS1. The plasma and induced sputum TSLP level in asthma group was higher than control subjects. The plasma and induced sputum TSLP level were inversely correlated with pulmonary function and disease severity.2. The plasma TSLP levels showed significantly positive correlations with induced sputum TSLP levels. It indicates that TSLP levels in blood may be a new biomarker of asthma.3. The induced sputum TSLP levels showed significantly negative correlations with percentange and absolute cell counts of EOS in sputum.4. The expressions of TSLP mRNA in 16HBE on different time after Poly I:C stimulation were induced to peak levels at 3h.5. No effects of rosiglitazone, telmisartan and GW9662 were observed on TSLP expression in 16HBE Cell under normal status. At mRNA and protein levels, rosiglitazone and telmisartan inhibited TSLP expression in 16HBE Cell under Poly I:C stimulation alone. The effect of rosiglitazone and telmisartan on TSLP expression was blocked or diminished in the presence of GW9662.It suggests that rosiglitazone and telmisartan inhibited TSLP expression via PPAR-y activation.6. At mRNA levels, NF-κB inhibitor Bay 11-7082(20μM) inhibited TSLP expression in 16HBE Cell under Poly I:C stimulation. It indicates that inhibition of NF-κB can suppress the expression of TSLP7. The expression of TSLP is increased in AEC in asthma mice. PPAR-y agonists can inhibit the expression of TSLP in AEC, and inhibit the inflammation and decrease airway hyperresponsiveness in asthma mice. It suggests that rosiglitazone and telmisartan inhibit asthmatic airway inflammation by modulating the TSLP expression.8. Rosiglitazone and telmisartan may modulates the expression of TSLP by inhibition of NF-κB.