Drift Of Th Subsets Induced By RSV Infected Bronchial Epithelial Cells

Objectives:Aberrant activation and imbalance of immune system constitute the base for airway hyperresponsive disease occurring or persistting. When insulted by exogeneous organnism, antigen presenting cells induce lymphocytes to proliferate and differentiate, activating immuno-reaction. Drift of help T lymphocytes (Th) subsets usually leads prominent expression of cytokines which are mainly released by Th cells, subsequently resulting in asthma. In recent years, it is well accepted that bronchial epithelial cells form not only a physical barrier to the environment but also contribute to the earliest antiviral immune response to foreign antigens. It involves in maintenance and regulation of homeostasis in respiratory micro-environment. The immune response of epithelia to infection and antigen exposure includes presenting antigens to lymphocytes and releasing chemokines and cytokines into submucosa, which initiates inflammatory reaction. Damage of airway epithelial structure and function may result in susceptibility to asthma, which could be a priming process in this disease.Respiratory syncytial virus (RSV) is an important respiratory pathogen that produces an annual epidemic of respiratory illness which is seen primarily in infants, but also in adults, worldwide. And this virus infection may cause a predisposition to the development of asthma, which stems from clinical and epidemiological evidences. However, the mechanisms of RSV-induced asthma are incompletely understood. Respiratory epithelial cells are the first and main target of RSV. We hypothesize that bronchial epithelial cells, infected by RSV, have an important regulatory effect on immune activation by presenting antigen signals and releasing inflammatory factors, thereby induce abnormal drift of Th subsets and finally form an immune state like asthma. The aim of this study was to observe the immune activation and imbalance of lymphocytes in vitro stimulated by RSV infected human bronchial epithelial cells (HBECs). And then investigate the molecular mechanism and find out the target points in it.Therefore, this study focused on:(1) Establish the model of human bronchial epithelial cells (HBECs) persistently infected with RSV and observe activation of lymphocytes and distribution of Th subsets induced by the model.(2) Prepare the gene chips of normal HBECs and RSV infected HBECs, observe the differences in mRNA expression and filter out the genes encoding secreted proteins with differences.(3) Validate the significantly up-regulated genes which encode secreted proteins, observe regulating effect of the encoded protein on differentiation of Th subsets and explore its signal transduction mechanism.Methods and results:1. Activation of lymphocytes induced by RSV persistently infected human bronchial epithelial cells (HBECs)1) Establish the model of HBECs persistently infected with RSVThe virulence of RSV strains determined by TCID50 was 1.4×106 pfu/mL. Set the multiplicity of infection (MOI) was 0.0001 and build the RSV persistently infected model. The model had following characteristics:?infected cells were still able to split and passage;?the persistent existence of RSV was detected in every passage of infected cells. Enlarged fusion cells were seen in later period under phase contrast microscope, virus particles of various sizes which were indicated by bright yellow green fluorescence were seen under fluorescence microscope. Edema of mitochondrias, expansion of endoplasmic reticulum, fissure around nucleus, generous lysosomes in cytoplasm and intracellular virus particles were all observed under electron microscope.2) Activation of lymphocytes induced by RSV persistently infectedHBECsLymphocytes isolated from human peripheral blood were co-cultured with RSV infected-HBECs. Four groups were set up, they were lymphocytes merely (Group L), lymphocytes interfered by RSV (Group RL), co-culture of lymphocytes with normal HBECs (Group HL), and co-culture of lymphocytes with infected HBECs (Group HRL). After co-cultured with HBECs for 24 hours, lymphocytes were collected and examined for cell cycle and apoptosis rate. The levels of IL-4, IFN-y and IL-17 in supernatants were measured. Cell cycle analysis for lymphocytes showed a significant increase of S phase (mitotic phase) cells, decrease of G1 phase and higher apoptosis rate in Group HRL compared with other three groups, then the proportion of S phase cells in group HL was next to group HRL and more than the two rested groups. The data suggested that accelerated proliferation and apoptosis of lymphocytes were induced by RSV persistently infected HBECs. Normal HBECs also stimulated proliferation of lymphocytes in a certain extent. RSV alone had no effect on them. ELISA results showed that in group HRL, the levels of IL-4, IFN-?and IL-17 in supernatant were also higher than other groups. The level of IFN-y in group HL was higher than group L and RL3) Supernatants from RSV infected HBECs altered the distribution of ThsubsetsThe distribution of Th subsets was analyzed by flow cytometer after individually treated with cell culture fluid, supernatants from normal HBECs and RSV infected HBECs for 24 h. The results showed that Th2 and Thl7 differentiation were induced by the treatment of supernatant from RSV infected HBECs significantly and normal HBECs to certain extent. But Treg differentiation was suppressed after being treated with supernatant secreted from RSV infected HBECs.2. The differences of mRNA expression in RSV infected human bronchial epithelial cells (HBECs)Prepare the gene chips of normal HBECs and RSV persistently infected HBECs, analyze the differences of mRNA expression. Microarray results showed that, compared with normal HBECs, there were 349 up-regulated and 154 down-regulated genes genes in RSV infected HBECs.12 genes with different mRNA expression encode secreted proteins,3 down-regulated genes and 9 up-regulated genes. Among them, the expression of CYR61 gene was obviously reduced (ratio<0.2) and GVIN1, LEP, IL8 genes were obviously increased (ratio>5).HBECs persistently infected with RSV led to significant changes in mRNA expression, then led to changes of local micro-environment.3. The involvement of leptin and IL-8 in regulation of RSV infected bronchial epithelial cells on Th subsets driftAccording to the results of gene chip,12 genes encoding secreting protein in RSV infected HBECs showed differential expression, in which 2 genes named IL8 and LEP raised the highest level. To ensure the result from gene chip, expression of IL-8 and leptin mRNA were detected by fluorescent quantitation PCR. The results were consistent with gene chip, that expression of IL-8 and leptin mRNA was significantly up-regulated in HBECs after infected with RSV. The levels of IL-8 and leptin in supernate from normal HBECs and RSV infected HBECs were measured with ELISA. The results showed that levels of IL-8 and leptin in RSV infected group were higher than control. So, RSV infected HBECs could over secret IL-8 and leptin.Refer to concentration of IL-8 and leptin in supernate from RSV persistently infected HBECs, lymphocytes isolated from peripheral blood were treated by human recombinant IL-8 and leptin for 24h, then distribution of Th2, Th17 and Treg subsets in lymphocytes were examined by flow cytometry. The results showed that both IL-8 and leptin contributed to differentiation of Th2 and Th17 subsets, especially leptin. Moreover, IL-8 and leptin had a synergetic effect on Th2 differentiation. But they had no effect on Treg differentiation. The protein level of extracellular signal-regulated kinasel/2 (ERK1/2) and phosphorylated ERK1/2 in lymphocytes were detected by western blotting and immunofluorescence. The results showed that IL-8 and leptin promoted ERK1/2 phosphorylation.These results suggested that HBECs after infected with RSV secreted IL-8 and leptin excessively. And IL-8 and leptin may promote differentiation of Th2 and Thl7 subsets by activating the signal molecule ERK1/2. Conclusion:Persistent infection of human bronchial epithelial cells (HBECs) with RSV induced activation of lymphocytes and abnomal drift of Th subsets. IL-8 and leptin which were over-secreted by RSV infected HBECs were involved in the Th subsets drift by activating the signal molecule ERK1/2.