The Effect Of β-arrestin2on Interleukin-17Expression And Production Of CD4~+T Lymphocytes And Associated Mechanisms In A Murine Asthma Model

PrefaceBronchial asthma is chronic airway inflammation with the characteristics of reversible airway obstruction, bronchial hyperresponsiveness and airway submucosal infiltration of lymphocytes and eosinophil. Regarding the induction of airway inflammation, it was widely recognized that it is a result of immune response in the bronchial airway. Many experts believe that imbalance of two types of CD4+T-helper cells, Thl and Th2, is the underlying cause for asthma. Specifically, the increased activity of Th2cells is a key event in asthma induction.In recent years, the roles of another type of CD4+T-helper cells, Th17, in airway inflammation are receiving increasing attention. Th17is characterized by the secretion of Interleukin-17(IL-17). It has been recognized as a novel pro-inflammatory CD4+T effector cell. TGF-β and IL-6are essential for Th17cell differentiation. IL-17is a cytokine that discovered recently, belongs to a unique type of multi-functional cytokines, which coordinates the local inflammation of tissues by inducing the release of proinflammatory factors and mobilizing neutrophils, so it plays an important role in allergic airway inflammation in asthma.β-arrestins, members of the arrestin family of proteins, are designated consist of (3-arrestin1and β-arrestin2. They are initially known as negative regulators of GPCR-mediated signaling. We now know that they also serve as scaffolds and adapters in receptor endocytosis and signal transduction. One study had shown that β-arrestin2was essential to the development of allergic asthma and it exerted its regulatory effect at a proximal step in the inflammatory cascade.β-arrestins recruit endocytic proteins and a variety of signaling molecules to the receptors, thus activate mitogen-activated protein kinase (MAPK) cascades including extracellular signal-regulated kinase1/2 (ERK1/2), Jun N-terminal kinase (JNK), one of the p38kinases, and Src family kinases. ERK1/2is the one of the most important signaling pathways. One study had shown that IL-17production and expression in murine lymph node cells can regulated by utilization of MAPK (ERK1/2, p38and JNK) signaling.Since that β-arrestin2was essential to the development of allergic asthma and it exerted its regulatory effect at a proximal step in the inflammatory cascade, whether β-arrestin2take effect by regulating IL-17production and expression? Recently one study has demonstrated that β-arrestin2positively mediated IL-6production which is very important for the IL-17production and this was mediated, in part, by the ERK1/2signaling pathway.We hade known that (3-arrestins activated mitogen-activated protein kinase (MAPK) cascades and IL-17production and expression in murine lymph node cells can regulated by utilization of MAPK (ERK1/2, p38and JNK) signaling, So,we propose a hypothesis that β-arrestin2may regulate IL-17expression and production partly through ERK1/2signaling pathway in allergic asthma.To confirm this hypothesis, our research was divided into three parts:(1) establish acute mouse model of asthma and confirm reliability of the model with modified evaluation criterion.(2) investigate the change of β-arrestin2expression and IL-17expression in acute mouse model of asthma and elucidate the effect of β-arrestin2on IL-17expression and production in spleen CD4+T lymphocytes from acute mouse model of asthma.(3) investigate whether (3-arrestin2stimulated IL-17expression and production of spleen CD4+T lymphocytes in a murine asthma model mediated by ERK1/2activation to confirm underlying mechanisms of β-arrestin2stimulating Interleukin-17expression and production of Spleen CD4+T lymphocytes in a murine asthma model. Through this study, we hope to provide further experimental evidence for therapeutic treatment of asthma. Part I Establish acute mouse model of asthma and confirm reliability of the modelObjective:To establish acute mouse model of asthma and confirm reliability of the model with modified evaluation criterion.Methods:20SPF level female BABL/c mice were randomly divided into normal control group and asthma group, with15mice in asthma group and5mice in normal control group. The asthma model was established by sensitization with intraperitoneal injection of ovalbumin (OVA) and aerosol challenge with repeated inhalation of OVA in the asthma group. The control group received PBS as the substitution of OVA. After24hours of the last inhalation, asthmatic symptoms were observed; The changes in airway response were determined by lung resistance(RL) and Lung dynamic compliance (Cdyn) stimulated by Methacholine(Mch); Lung tissue sections were staininged for general pathology; The white cell count, cell counts of neutrophils, eosinophils, lymphocytes and macrophages of bronchoalveolar lavage fluid (BALF) were measured; The cell counts of spleen CD4+T lymphocytes was detected.Results:(1) Asthma symptoms were more severe in asthma group compared with normal group.(2) The dose-response curves for RL was obviously shifted upward in asthma group compared with normal group(p<0.01);while the dose-response curves for Cdyn was significantly shifted downward in asthma group (p<0.01).(3) Total white cell counts, cell counts of neutrophils, eosinophils, lymphocytes and macrophages of bronchoalveolar lavage fluid (BALF) significantly increased in asthmatic mice than those of normal control group (respectively p<0.01).(4) There were more extensive inflammatory cells infiltration around the bronchi, and mucus excretion in airway lumen was found in asthma group compared with normal control group(p<0.01). (5) The cell counts of spleen CD4+T lymphocytes in asthma group were obviously higher than those in normal control group(p<0.01).Conclusions:(1)The reliability of the established acute mouse model of asthma was successfully confirmed.(2) The cell counts of spleen CD4+T lymphocytes can become one of evaluation criterions of acute asthmatic mouse model. Part Ⅱ The effect of β-arrestin2on interleukin-17expression and production in spleen CD4+T lymphocytes from acute mouse model of asthmaObjective:To investigate the change of β-arrestin2expression and IL-17expression and production in acute mouse model of asthma, and to elucidate the effect of β-arrestin2on IL-17expression and production in spleen CD4+T lymphocytes from acute mouse model of asthma.Methods:Grinding spleens of mice from asthmatic group and normal group into spleen mononuclear cells suspension. After removing red blood cells from the suspension, CD4+T lymphocytes were isolated from spleen mononuclear cells with immuno-magnetic beads, and then were cultivated in RPMI-1640with10%Fetal Bovine Serum and stimulated by ConA and PMA. After24hours, the expression of β-arrestin2protein in CD4+T lymphocytes was detected by Western blot, the expression of β-arrestin2mRNA in CD4+T lymphocytes was detected by Realtime PCR. Next, Splenic CD4+T lymphocytes from acute mouse model of asthma were transfected with three chemosynthesis β-arrestin2siRNA sequences and negative control sequences. The best efficient siRNA-β-arrestin2was chosed by detecting the β-arrestin2mRNA expression by RT-PCR. β-arrestin2protein of the best efficient transfected group and the negative control group were measured by Western blot. Then the best siRNA-β-arrestin2was transfected into splenic CD4+T lymphocytes from asthma group, the CD4+T lymphocytes from normal group,asthma group and transfected group were stimulated by ConA and PMA for24hours. the expression of IL-17protein in CD4+T lymphocytes was detected by Western blot, the expression of IL-17mRNA in CD4+T lymphocytes was detected by Realtime PCR, the accumulation of IL-17in supernatants from cultures of CD4+T lymphocytes were measured by ELISA.Results:(1) The expression of β-arrestin2mRNA and protein in spleen CD4+ T lymphocytes from asthma group was increased compared with those from normal control group(respectively p<0.01);(2) siRNA-β-arrestin2-1123had the best silencing effect among all siRNA sequences(p<0.01);(3) The expression of IL-17mRNA and protein in spleen CD4+T lymphocytes from asthma group was increased compared with those from normal control group(respectively p<0.01); The accumulation of IL-17in supernatants from cultures of CD4+T lymphocytes from asthma group was increased compared with those from normal control group(p<0.01);(4) The expression of IL-17mRNA and protein in spleen CD4+T lymphocytes from transfected group was lower in comparison with those from asthma group(respectively p<0.01); The accumulation of IL-17in supernatants from cultures of CD4+T lymphocytes from transfected group was lower in comparison with those from asthma group(p<0.01).Conclusions:(1) β-arrestin2expression was increased in spleen CD4+T lymphocytes from acute mouse model of asthma.(2) IL-17expression and production was increased in spleen CD4+T lymphocytes from acute mouse model of asthma.(3) β-arrestin2increased IL-17expression and production of spleen CD4+T lymphocytes in a murine asthma model. Part Ⅲ The underlying mechanism of β-arrestin2stimulating interleukin-17expression and production of spleen CD4+T lymphocytes in a murine asthma modelObjective:To investigate whether β-arrestin2stimulated IL-17expression and production of spleen CD4+T lymphocytes in a murine asthma model mediated by ERK1/2activation.Methods:(1) Firstly to elucidate the effect of β-arrestin2on activated ERK1/2protein(phosphorylated-ERK1/2) expression in spleen CD4+T lymphocytes from acute mouse model of asthma, CD4+T lymphocytes from normal group,asthma group and transfected group were stimulated by ConA and PMA for24hours. Then, phosphorylated-ERK1/2(p-ERK1/2) protein expression of CD4+T lymphocytes was detected by Western blot.(2) Next to elucidate the effect of activated ERK1/2on IL-17expression and production in spleen CD4+T lymphocytes from acute mouse model of asthma, CD4+T lymphocytes from acute mouse model of asthma were pretreated with the ERK1/2inhibitor, PD98059. CD4+T lymphocytes from normal group, asthma group and PD98059group were stimulated by ConA and PMA for24hours. Then, the expression of IL-17protein in CD4+T lymphocytes was detected by Western blot, the expression of IL-17mRNA in CD4+T lymphocytes was detected by Realtime PCR, the accumulation of IL-17in supernatants from cultures of CD4+T lymphocytes were measured by ELISA.Results:(1) The expression of activated ERK1/2protein in spleen CD4+T lymphocytes from asthma group was increased compared with those from normal control group(p<0.01);(2) The expression of activated ERK1/2protein in spleen CD4+T lymphocytes from transfected group was lower in comparison with those from asthma group(p<0.01);(3) The expression of IL-17mRNA and protein in spleen CD4+T lymphocytes from PD98059group was lower in comparison with those from asthma group(respectively p<0.05); The accumulation of IL-17in superatants from cultures of CD4+T lymphocytes from PD98059group was lower in comparison with those from asthma group(p<0.05).Conclusions:(1) Activated ERK1/2expression was increased in spleen CD4+T lymphocytes from acute mouse model of asthma;(2) Activated ERK1/2increased IL-17expression and production of spleen CD4+T lymphocytes in a murine asthma model;(3) β-arrestin2increased activated ERK1/2expression of spleen CD4+T lymphocytes in a murine asthma model; Activating ERK1/2expression is one of the mechanisms of β-arrestin2increasing IL-17expression and production of CD4+T lymphocytes in a murine asthma model.