Research On The Mechanism Of Dopamine D1Receptors Participating In Th17Cell Differentiation And Acute Asthma

BackgroundBronchial asthma is a chronic inflammation of the airway, involving a combination of inflammatory cells, and characterized by reversible airway obstruction, hyperresponsiveness and persistent inflammation of the airway. Th2cell dominance in Th1/Th2imbalance is conventionally considered the immunological mechanism of asthma. The dominance of Th2cells leads to the onset of eosinophilic asthma. Glucocorticoid and (or) P receptor agonists as first-line drugs are able to treat the vast majority of patients with eosinophilic asthma and improve their quality of life. But some patients show glucocorticoid resistance. Most of these patients are suffering from severe neutrophilic asthma, which has become a difficult clinical problem. Therefore, the physiological and pathological characteristics of these patients, the pathogenesis of the disease, new therapeutic targets and methods have become a hot research field in recent years.Newly discovered Th17cells can elucidate some experimental and clinical phenomena which can not be explained by the Th2cell theory. Th17cells secrete interleukin-17(IL-17). Signaling pathways mediated by IL-17can induce target cells to produce a variety of inflammatory factors stimulating tissues, can result in recruitment and activation of neutrophils in the airway, followed by tissue infiltration and tissue damage. Glucocorticoid not only fail to inhibit the neutrophils, but can increase the activity and lifetime of the neutrophils. In addition, IL-17is also involved in airway remodeling. It is confirmed that Th17/IL-17axis is correlated with the severity of asthma. In summary, it is merited to carry further exploration on the molecular level Th17/IL-17axis, especially in the upstream of the axis, to find new therapeutic targets, which is expected to achieve a breakthrough in the treatment of asthma.Dopamine receptors can be divided into the D1and D2receptors. A lot of research has been carried out on the relationship between D1receptors and neurological and mental disorders. The Dl receptor is an important target for the treatment of these mental disorders. Recently, it has been found that D1receptor is also involved in the pathogenesis of autoimmune diseases. The antagonist of D1receptor can inhibit Th17and Th2cell differentiation and promote Thl cell differentiation, thereby playing the role in prevention and treatment of experimental autoimmune encephalomyelitis (EAE), diabetes in non-obesity diabetic (NOD) mice; the antagonist of D1receptor can inhibit the formation of crescent in mice with nephrotoxic serum nephritis(NTN); it can reduce inflammation in rheumatoid arthritis and reduce joint damage. Since over-activation of Th2and Th17cells and inhibition of Th1cells are involved in the pathogenesis of asthma, we hypothesized that the D1receptors also play an important role in the pathogenesis of asthma, and looking forward to find new targets for asthma treatment.The Dl receptor antagonist, agonist and saline is administrated intraperitoneally by three groups of mice with asthma respectively. We observed behavior of the mice in each group, tested pulmonary function, carried out bronchoalveolar lavage fluid (BALF) cell count and its IL-17levels, made and read lung pathological sections, detected Th17cell proportion in splenic CD4+T cells by flow cytometry. We evaluate the impact of the D1receptor antagonists and agonists on mice model of asthma and on Th17cells in vivo through these indicators.Then we explored the possible signaling pathway conducted by the D1receptors to affect Thl7cells. Nakagome K et al think that maybe the D1receptors activation increases cAMP in naive CD4+T cells, thereby promotes the Th17cell differentiation. In addition, the D1receptors affecting the secretion of IL-23in DCs may also be involved in the signaling pathway. Why cAMP can promote naive CD4+T cells differentiating to Thl7cells? However, there are currently no related reports. In this study, we will explore the mechanism involved in cAMP promoting naive CD4+T cells differentiating to Th17cells.The D1receptor is a G protein-coupled receptor (GPCR), mediating classic Gs/AC/cAMP signaling pathway. Zhen et al found that exciting D1receptors increased intracellular cAMP levels, and then activate the p38mitogen-activated protein kinase (p38MAPK). p38MAPK is the intersection point of intracellular information transferring, and can activate the nuclear transcription factor kB (NF-kB). NF-kB is a ubiquitous transcription regulator and can control the transcription of the BATF gene.BATF (B cell activating transcription factor) is one of the AP-1(activator of protein1) family members recently discovered. BATF is one of the indispensable nuclear transcription factors in Th17cells, involved in the maintenance of RORa and RORγt expression, and induce IL-17expression synergisticly with RORyt by direct binding to the IL-17gene promoter induction of. Thus, we boldly speculate that the D1receptors promote Th17cell differentiation by regulating BATF.In this study, we explore whether the D1receptors promote Th17cell differentiation by regulating BATF in vitro. First, normal mouse spleen CD4+T cells were disposed with D1receptor antagonist and agonist respectively. Simultaneously Th17-promoting environment was provided. BATF and RORyt expression was detected by western blot (WB); IL-17levels in supernatants were measured by ELISA; the proportion of Th17cells was test by flow cytometry. After exploring the impact of the D1receptors on Thl7cell differentiation and BATF expression, we transfected two groups of mouse spleen CD4+T cells with BATF small interfering RNA (siRNA) and control siRNA respectively. Simultaneously Th17-promoting environment and D1receptor agonist were provided. BATF and RORyt expression was detected by western blot (WB); IL-17levels in supernatants were measured by ELISA; the proportion of Th17cells was test by flow cytometry. We are aimed to compare Th17cell differentiation after the activation of the D1receptors, accompanied by silence of part BATF gene or normal expression of BATF gene, to clarify the mechanism of D1receptors participating Th17cell differentiation, to provide cues for the treatment of asthma. Chapter1Dopamine D1receptors’ effect on airway inflammation in acute asthma and Th17/IL-17axisPurpose(1) Establish a mouse model of acute asthma, and identify the model;(2) Observe the effect of D1receptor antagonists and agonists for airway inflammation and Th17/IL-17axis in asthma.Method24SPF BABL/c female mice were randomly divided into four groups:the control group (CK), the asthma group (AS), the D1receptor antagonist SCH23390treatment asthma group (SCH treatment group) and the D1receptor agonist SKF83959treatment asthma group (SKF treatment group). OVA-sensitized and aoralized to establish acute asthma model and control group use an equal volume of PBS instead of. Intraperitoneal injection of respective chemicals30minutes was carried out before each atomization in SCH treatment group and SKF treatment group.24hours after the last challenge, all mice were disposed:(1) the general conduct of activities in mice were observed;(2) to collect the BALF and carry out the cell count and cell classification, detect IL-17concentration in BALF;(3) made lung pathological slices and observe inflammatory cell infiltration and mucus secretion;(4) grind all the mice spleen, prepare mononuclear cell suspension, use MACS to isolate spleen CD4+T cells, calculate the spleen-derived CD4+T cells and test their viability;(5) determine the proportion of Thl7cells in the spleen-derived CD4+T cells and test IL-17concentration in the culture supernatant;(6) after methacholine challenge,airway hyperresponsiveness was determined by invasive pulmonary impedance method.Result(1) Mice of control group behaved normally; asthmatic mice appeared typical asthma-like symptoms; the SCH treatment group mice showed less asthma-like symptoms significantly; the SKF treatment group mice’s symptoms were somewhat heavier;(2) Compared with the control group, the total number of leukocytes, amount of Lym, Neu and Eos, and IL-17levels in BALF from asthma group significantly increased (all P<0.05); compared with asthma group, the total number of leukocytes, amount of Lym, Neu and Eos, and IL-17levels in BALF from SCH treatment group statistically decreased(P<0.05); compared with asthma group, the total number of leukocytes, amount of Lym, Neu and Eos, and IL-17levels in BALF from SKF treatment group statistically increased (P<0.05);(3) Compared with control group, inflammatory cell infiltration, goblet cell hyperplasia and mucus secretion in lung were far more obvious in asthmatic mice(P<0.05); airway inflammation and mucus hypersecretion of SCH treatment group were significantly less than asthma group (P<0.05); airway inflammation and mucus hypersecretion in the SKF treatment group increased compared with asthma group (P <0.05);(4) Compared with control group, the number of splenic CD4+T cells obviously increased in Asthma group (P<0.01); the number of CD4+T cells significantly reduced in SCH treatment group (P<0.01), while the number of CD4+T cells significantly increased in SKF treatment group(P<0.01);(5) Compared with control group, the ratio of Th17cells in splenic CD4+T cells and IL-17levels in culture supernatant obviously increased in Asthma group (P<0.01); the SCH treatment group had a lower level of ratio of Thl7cells and IL-17levels than that in Asthma group (P<0.01), while the SKF treatment group had a higher level of ratio of Th17cells and IL-17levels than that in Asthma group (P<0.01);(6) Compared with control group, airway responsiveness statistically increased in asthma group (P<0.05); airway hyperresponsiveness (AHR) was eased in SCH treatment group compared with that in asthma group (P<0.05), while AHR was aggravated in SKF treatment group compared with that in SCH treatment group (P<0.05).Conclusion(1) Mouse model of acute asthma established in this experiment was successful, and Th17/IL-17axis was involved in the pathogenesis of asthma;(2) The D1receptor was involved in the pathogenesis of asthma, and the regulation of Th17/IL-17axis was one of the mechanisms. Chapter2Dopamine Dl receptors promote Th17cell differentiation through BATF activationPurpose(1) To explore the effect of the D1receptors on Th17cell differentiation and BATF in vitro;(2) To confirm that the D1receptors promote Th17cell differentiation by regulating BATF.MethodExperiments in vitro included two parts:during the first part, normal mice splenic CD4+T cells separated and purified by MACS were devided into three groups:control group, the D1receptor antagonist SCH23390treatment group and the D1receptor agonist SKF83959treatment group. Every group were provided with Th17-promoting environment and corresponding reagent. After cultivation, the expression of BATF and RORyt were detected by western blot (WB); IL-17levels in supernatants were measured by ELISA; the proportion of Th17cells was tested by flow cytometry.In the second part normal mice splenic CD4+T cells were divided into two groups:BATF-siRNA group and control siRNA group. First of all, two groups of mice splenic CD4+T cells were transfected with BATF-specific siRNA and control siRNA respectively. Simultaneously Th17cell-promoting environment and the D1receptor agonist SKF83959were added. After cultivation, the expression of BATF and RORyt were detected by western blot (WB); IL-17levels in supernatants were measured by ELISA; the proportion of Th17cells was tested by flow cytometry.Result(1) Compared with control group, the proportion of Th17cells in SCH23390treatment group significantly decreased(P<0.01), while the proportion of Th17cells in SKF83959treatment group significantly increased(P<0.01);(2) Compared with control group, RORγt expression in SCH23390treatment group significantly decreased(P<0.01), while RORyt expression in SKF83959treatment group significantly increased(P<0.01);(3) Compared with control group, IL-17levels in culture supernatant from SCH23390treatment group significantly decreased(P<0.01), while IL-17levels in culture supernatant from SKF83959treatment group significantly increased(P<0.01);(4) Compared with control group, BATF expression in SCH23390treatment group significantly decreased(P<0.01), while BATF expression in SKF83959treatment group significantly increased(P<0.01);(5) BATF expression in BATF-siRNA group was less than that in control siRNA group (P<0.01);(6) The proportion of Th17cells in BATF-siRNA group was less than that in control siRNA group (P<0.01);(7) RORyt expression in BATF-siRNA group was less than that in control siRNA group (P<0.01);(8) IL-17levels in culture supernatant from BATF-siRNA group were lower than that from control siRNA group (P<0.01).Conclusion(1) In vitro the D1receptor antagonists can down-regulate BATF expression and suppress Th17cell differentiation; the D1receptor agonists can increase BATF expression and promote Th17cell differentiation;(2) The D1receptors promote Th17cell differentiation through regulation of BATF.