Effects Of ANP Signaling On CD4~+T Cells And Its Role In Allergic Asthma

BackgroundBronchial asthma (hereinafter referred to asthma), is a complex chronic airway disease which is characterized by persistent airway inflammation and hyperresponsiveness. A variety of cells and cytokines such as eosinophils, mast cells, T lymphocytes, neutrophils, and so on, play an important role in the process of airway inflammatory reaction in asthma, in which CD4+T cells have been considered as major effector cells in the immunological pathogenesis of asthma. As we know, CD4+T cells comprise several subsets such as the most classic Thl/Th2cells which are all differentiated from the same progenitor cells-ThO cells (also named naive CD4+T cells). Th2cell dominance has been considered to be the significant mechanism of allergic asthma for a long time, because activation and dominance of Th2cells can produce lots of cytokines such as IL-4, IL-5which play a dominant role in inducing an eosinophilic airway inflammation of allergic asthma.Th17cells, recently discovered, a new important member different from Th1/Th2cells in CD4+T cells family, have been recognised to play a significant role in inflammatory and immune response especially in autoimmune reaction. In CD4+T cells family, similar to Thl/IFN-γ and Th2/IL-4, IL-17is an identifying and the representative cytokine of Thl7cells. Previous studies have confirmed that Th17/IL-17is also involved in the course of allergic asthma, but the mechanism is still unclear.Atrial natriuretic peptide (ANP), a multifunctional peptide cleaved from pro-ANP hormone, was firstly isolated from tissue of heart atrium and famous for its biological actions on cardiovascular system including a stimulation of natriuresis, diuresis, vasorelaxation, and so forth. Actually, lung is also an important organ which can produce ANP besides the cardiovascular system, and meanwhile lung is one of the target organs of ANP signaling.Many investigations show ANP is localized in peripheral lungtissue, tracheal epithelial cells, respiratory epithelium, human fetal lung tissue, etc; effecting receptor-natriuretic peptide receptor A (NPRA) and its clearance receptor-natriuretic peptide receptor C (NPRC) of ANP are all expressed in bronchial and alveolar cells via radionuclide location and immunocytochemical analyses; NPRA and NPRC are also discovered in the lungtissue of humans, pigs and mice; in addition, the lung homogenates could degrade pro-ANP with neutral endopeptidases to a greater degree compared with the kidney and liver homogenates, and so on. ANP, receoptors system with high affinity and masses of peptidases for ANP metabolism exist in the lung suggest that lung is not only the target organ of ANP, but it also owns an integrated metabolic system for ANP, in which effects of ANP are not only dependent on ANP production from circulation. These data indicate that ANP signaling may play a nonnegligible role in the physiological and pathological responses of the lung. However, so far, little has been known about it.In fact, accumulating evidence suggest ANP signaling is closely related with the inflammatory and immune reactions besides its regulation in the homeostasis of volume-pressure. For example, pro-ANP is all found in the immune organs such as thymus, spleen, lymph nodes from pigs, mice and rats; ANP mRNA also can be detected in the thymus and tonsil of human and mouse; ANP signaling can attenuate the proliferation and differentiation of thymocytes; a study discovered that ANP could dose-dependently induce the release of histamine from mast cells of peritoneum; ANP signaling also affects the activities of neutrophilis, macrophages, dendritic cells. Moreover, NPRA also can be expressed on T lymphocyes. These data demonstrate that ANP signaling is indeed implicated in the inflammatory and immune responses.Some observations from clinic found levels of ANP in peripheral blood of asthmatics in exacerbation were significantly higher than those of asthmatics in remission and the healthy, which suggests ANP signaling is correlated with asthma, but the effect and mechanism are still unknow. Based on the above, we have a hypothesis:now that abnormal change of ANP levels in peripheral blood of asthmatics were happened and ANP signaling is closed related with immune and inflammatory response, whether ANP signaling may play a role in allergic asthma via regulating the activities of CD4+T cells?Mouse splenic CD4+T cells and mouse models with allergic asthma are performed in this study. One purpose of this study is to define whether ANP signaling could regulate the activities of CD4+T cells and to explore the mechanism; the other purpose is to observe whether ANP signaling is implicated in allergic asthma by influencing the activities of CD4+T cells. Firstly, we are going to investigate what ANP signaling's effect on the differentiation and function of CD4+T cells in vitro which isolated from spleens by immune magnetic beads sorting. Then, we will observe the effects of ANP signaling on airway inflammation of mouse models with allergic asthma and changes of transcription factors and related cytokines of CD4+T cells after activating or preventing ANP signaling. By studying in vitro and in vivo, we want to initially elucidate ANP signaling indeed plays an important role in allergic asthma via modulating CD4+T cells and meanwhile to provide an evidence for further recognising the role of ANP signaling in other pulmonary diseases. Part I Effects of ANP signaling on the differentiation and capacity of Th17cellsObjectives(1) To observe effects of ANP signaling on the differentiation and capacity of Th17cells in vitro;(2) To probe the signaling pathway of ANP to regulate Th17cells.Methods(1) To aseptically excise and grind the spleens of BALB/c mice and to prepare the splenic mononuclear cells suspension;(2) CD4+T cells were isolated from splenic mononuclear cells by positive selection with immuno-magnetic beads (MACS technology) and then counting and detecting the vitality of CD4+T cells;(3) To define the expression of NPRA in CD4+T cells by WB;(4) CD4+T cells were cultured for4days as control group (C), directional differentiation group (D), ANP addition group (ANP), A71915plus ANP group (A7), KT5823plus ANP group (KT);(5) To detect the changes of the second messenger cGMP after intervening ANP signaling; cultured for4days, to measure the rate of IL-17-producing CD4+T cells by FACS; to detect the expression of RORγt and BATF (specific transcription factors for Th17cells) by WB; to confirm the changes of IL-17in supernatant. Results(1) Compared with the control group, the rate of IL-17-producing CD4+T cells could be significantly augmented in the cytokines microenvironment for directional differentiation comprising anti-IFN-y, anti-IL-4, TGF-β1, IL-6and IL-23, meanwhile along with increased production of IL-17in supernatant (P<0.01);(2) Compared with differentiation group, addition of ANP could decrease the proportion of IL-17-producing CD4+T cells (P<0.01). Accordingly, the expression of RORyt and BATF (P<0.05) and the levels of IL-17in supernatant were also reduced (P<0.05); and the inhibitory effects showed dose-dependent (10-8-10-6M ANP);(3) When using the ANP/NPRA signaling antagonist-A71915, the inhibitory effects of ANP on the differentiation and capacity of Th17cells were mostly reversed (respectively P<0.05);(4) Similarly, KT5823were performed in advance, the inhibitory effects of ANP on the differentiation and capacity of Th17cells were also mostly recovered (respectively P<0.05);(5) Changes of cGMP coupled NPRA were consistent following the intervention of ANP signaling(respectively P<0.01).Conclusions(1) ANP signaling could suppress the differentiation and capacity of Th17cells in vitro and displayed a dose-dependent effect; (2) NPRA/PKG pathway played an important role in the process of ANP effecting on Thl7cells. Part Ⅱ Effects of ANP signaling on Th17/IL-17and its role in mouse models with allergic asthmaObjectives(1) To establish and evaluate mice models with allergic asthma;(2) To observe changes of ANP and NPRA in mice with allergic asthma;(3) To observe effects of ANP signaling on airway inflammation of mice models with allergic asthma, local expression of transcription factor for Th17cells and changes of IL-17in lung.Methods40SPF female BALB/c mice were randomly divided into five groups according to the study design:normal group, asthma group, ANP inhalation group (ANP), A71915intraperitoneal injection(i.p) plus ANP inhalation group (ANP+A71915), A71915(i.p) alone group. Every group contains8mice. As the scheme, mice models with allergic asthma were established by sensitized with10μg OVA/mouse+0.2ml A1(OH)3/mouse by i.p and then aerosol challenge with5%OVA via repeated inhalation in asthma group while the same volume of PBS were performed as the substitution of OVA in the normal group. In addition, aerosol nebulization of solution with ANP dry powder (dose1μg/g mouse) at30min before every OVA-challenge in ANP group and similarly, A71915 (dose0.5μg/g mouse) was used by i.p in A71915group at30min before every ANP inhalation. Other operations were same as the asthma group and behaviours of mice in every group should be observed during establishing the models. After24hours of the last challenge, operations were carried out as following:(1) after anesthesia, mice were implemented with tracheal intubation and changes of airway response were determined by recording lung resistance(RL) and lung dynamic compliance (Cdyn) stimulated with Methacholine of different doses (Mch);(2) total cells count, differential counts and production of ANP, IL-4, IL-17in bronchoalveolar lavage fluid (BALF) were detected;(3) observation and analysis of bronchial tube and lungtissue were completed by pathological staining;(4) expressions of NPRA for ANP signaling in lungtissue and splenic CD4+T cells from normal and asthma group were quantified by Western-blot;(5) RORyt and BATF proteins were also detected by WB in lungtissue and changes of ANP, IL-4, IL-17levels in lung homogenates.Results(1) Asthmatic symptoms of varying degrees were emerged in mice of asthma group, ANP group and A71915group such as tremble, unease, breathlessness, incontinence and so on while the normal group showed basically normal. In addition, no death of mice appeared during establishing model of asthma and drug treatment in vivo; (2) Compared with the normal group, the dose-reponse curve of RL was significantly shifted up (P<0.01) while the curve of Cdyn was apparently shifted down (P<0.01) in asthma group, which suggests airway hyperresponsiveness (AHR) was successfully established.(3) Compared with the normal group, airway hyperresponsiveness of A71915+ANP and A71915group was also obvious (respectively P<0.05) but reduced in contrast with asthma group (respectively P<0.05); while AHR of ANP group was not evident which was similar to normal group (respectively P>0.05);(4) Total cells counts, differential cells counts of eosinophils, etc in BALF of asthma group were increased than those in BALF of normal group (respectively P<0.01); however, those in ANP group were not only significantly more than normal group (P<0.01) but even more seriuos than asthma group (P<0.05); while those in A71915group were obviously reduced compared with asthma (P<0.05); those in A71915+ANP group were also decreased compared with ANP group(P<0.05);(5) Compared with normal group, levels of IL-4, IL-17in BALF of asthma group were apparently augmented (respectively P<0.01); while increased production of IL-4in BALF of ANP group (P<0.01) was more outstanding and the mean value of ANP group was even more higher than that of asthma group (P<0.05), but the levels of IL-17showed no obvious decline (P>0.05); in A71915group, the levels of IL-4, IL-17in BALF were all decreased compared with asthma group (P<0.05); in A71915+ANP group, levels of IL-4decreased (P<0.05) but change of IL-17was not significant compared with normal group (P>0.05);(6) Production of ANP in BALF and lungtissue homogenates of asthma group were significantly higher than those in normal group (respectively P<0.01);(7) Levels of NPRA protein in lungtissue of mice with asthma were much more than the normal group (P<0.05) and those in splenic CD4T cells were similar (P<0.05);(8) Numerous inflammatory cells infiltrated and diffused around the bronchus and vessel, and lots of mucus secreted around airway compared with the normal group(respectively P<0.01); when inhalation of ANP, the above became more serious compared with asthma group (respectively P<0.05); and A71915+ANP group showed relaxative than ANP group (respectivelyP<0.05); while this situation in A71915group was attenuated compared with asthma group (respectively P<0.05);(9) Expression of RORyt and BATF of Th17cells in mice lungtissue of asthma group were increased than the normal group (respectively P<0.05); while the RORyt and BATF were decreased after inhalation of ANP (respectively P<0.05) and expression of RORyt and BATF became much less in A71915+ANP group (P<0.05); (10) The changes of IL-4, IL-17in mice lung homogenates of every group were similar to those of in BALF.Conclusions(1) Mice models with allergic asthma were successfully established based on the data;(2) ANP signaling plays a role in the course of allergic asthma;(3) ANP signaling could remit AHR of mice with allergic asthma;(4) Not completely consistent with experimental data in vitro, ANP signaling could reduce expression of transcription factors of Thl7cells in lungtissue not along with significantly decreased IL-17in vivo, which also may be one of mechanisms;(5) ANP signaling aggravate airway inflammation of allergic asthma. Part III Effects of ANP signaling on functional balance of Th1/Th2cells and its role in airway inflammation of allergic asthmaObjectives(1) To investigate the effects of ANP signaling on functional balance of Thl/Th2cells in vitro;(2) To explore ANP signaling aggravating airway inflammation of allergic asthma by promoting dominance of Th2cells based on study in vivo.MethodsTo aseptically excise and grind the spleens of BALB/c mice with allergic asthma and to prepare the splenic mononuclear cells suspension. Then, CD4+T cells were isolated through positive selection with immuno-magnetic microbeads. CD4+T cells were cultured as the design after counting and detecting the vitality. All experiments in vitro were divided into control group, ANP group, A71915+ANP group according to the different intervention. Cells were harvested after24h by stimulated ConA (end concentration-5μg/ml). Then, for the sake of defining the effects of ANP signaling on the functional balance of Thl/Th2cells, to detect changes of T-bet/GATA3expression by WB and to measure levels of IFN-y/IL-4in supernatant were performed. In vivo, detection of T-bet/GATA3for Thl/Th2cells in lungtissue of mice via WB and measurement of IFN-γ levels in BALF and lung homogenates were finished, and other experiments were processed in Part Ⅱ.Results(1) Compared with noraml group, expressions of GATA3in asthma group were obviously more higher; meanwhilie, expression of GATA3showed much more in ANP inhalation group than asthma group while T-bet was decreased (respectively P<0.05); when blockade of ANP signaling, expression of GATA3was decreased while that of T-bet was increased (respectively P<0.05);(2) IFN-y levels in lung homogenates decreased compared with asthma group which was consistent with changes of T-bet (P<0.05) but not in BALF (P>0.05); and no obvious change of IFN-y levels happened in ANP+A71915group (P>0.05);(3) In vitro, expression of GATA3in splenic CD4+T cells from mice with asthma was higher than control group after stimulated by ANP for24h along with reduced expression of T-bet (respectively P<0.05), in which the trend of changes were basically consistent with the results from in vivo; whlie A71915performed in advance, effects of ANP signaling on expressions of T-bet/GATA3were neutralized (respectively P<0.05);(4) Level of IFN-y in supernatant was decreased while IL-4was increased in ANP group compared with the control (respectively P<0.05); When ANP signaling inhibitor was used, the changes of IFN-y/IL-4were also reversed (respectively P<0.05).Conclusions(1) In vitro, ANP signaling could affect functional balance of Thl/Th2cells, that is promoting Th2-type response while inhibiting Th1-type response;(2) In vivo, ANP signaling could aggravate Th2cells response in lung, which is probably one of the mechanisms for ANP signaling to exacerbate airway inflammation of allergic asthma.