Vagal-α7nAChR Signaling Regulates Platelet Biogenesis And Immune Function Of Megakaryocytes And Platelets In Lung

Megakaryocyte is a precursor cell that produces platelets.It is mainly developed from hematopoietic stem cells through megakaryocyte lineage cells under the action of thrombopoietin（Thrombopoietin,TPO）.The maturation of megakaryocytes will undergo unique cellular activities,including intranuclear mitosis,the formation of plasma membrane boundary system and the formation and release of precursor platelets.Studies have shown that the lung has become a place to provide the largest hematopoietic microenvironment except bone marrow,and as the first capillary bed after megakaryocytes leave bone marrow,it can produce nearly 50%of platelets;Megakaryocytes in lung tissue manifested a unique immune characteristics.Platelets are involved in many important physiological processes such as hemostasis,angiogenesis and innate immunity.Platelets play a dual role in acute lung injury.On the one hand,they maintain the integrity of alveolar capillary barrier.On the other hand,they can also participate in pathogen clearance and inflammatory injury by recruiting inflammatory cells.With the in-depth understanding of the mutual regulation between various systems of the body,researchers have found an ongoing dialogue between the nervous system and the blood system.For example,the adrenergic and cholinergic branches of the sympathetic nerve maintain the homeostasis and mobilization of the circadian rhythm of hematopoietic stem cells（HSCs）through innervating the bone marrow sinuses in collaborative manner;Central parasympathetic cholinergic signals reduce the mobilization and release of hematopoietic stem cells by inhibiting sympathetic noradrenergic tension.The sympathetic neurotransmitters epinephrine and norepinephrine can promote the maturation of megakaryocytes and the production of platelets;GABA signaling plays a positive regulatory role in the development of megakaryocyte lineage cells.Most organs of the body are dominated by the autonomic nervous system composed of sympathetic nerve and parasympathetic nerve,which belongs to dual innervation.The postganglionic parasympathetic nerve fibers innervating the lung take shape in the tenth pair of cerebral nerves（vagus nerve）.Vagus nerve endings can control the microenvironment of the distal lung and regulate the physiological and pathological process of the lung by releasing acetylcholine neurotransmitters.In recent years,the role of vagus cholinergic signal in acute lung injury has been deeply studied.However,its regulatory role in hematopoiesis has not been clarified.ObjectivesTo clarify the regulation mode and specific mechanism of vagal-α7nAChR signaling on platelet production and immune function of pulmonary megakaryocytes on the basis of neurophysiology,molecular biology and immunology.Methods1.Detect the platelet content in left and right ventricular blood and megakaryocytes in lung tissue of mice,compare the difference of platelet content,and determine the existence,spatial location and expression of cholinergic receptor of megakaryocytes in lung by flow cytometry and immunofluorescence technique;2.Construct vagectomy and Chrnα7^fl/fl/PF4-cre^tg/wt conditional knockout mouse model,the changes of peripheral blood platelet content and megakaryocyte lineage cells in lung,bone marrow and spleen were detected and analyzed by animal automatic blood cell analyzer and flow cytometry;3.To establish MEG01 cell line in vitro culture and cell maturation induction system,and explore the possible mechanism of cholinergic signal activation on cell activity at the genomic and proteomic levels by using genomic high-throughput sequencing and protein mass spectrometry.Flow cytometry,pull-down and immunofluorescence were used to analyze the effects of cholinergic signal on the expression of glycoprotein molecules in megakaryocytes,the release of platelet like particles,polyploidy and the formation of DMS.4.To establish MEG01 cell line in vitro immune response system,and the acute lung injury model（sterile and bacterial）was constructed on Chrnα7^fl/fl/PF4-cre^tg/wt mice.To evaluate the effect of cholinergic signal on the innate immune response of megakaryocytes/platelets and the ability of platelets to recruit inflammatory cells.5.To establish in vitro culture of inflammatory cells（neutrophils）and induced immune response model,and the acute lung injury model（LPS）was constructed on the basis of vagotomy and agonist treatment.To evaluate the effects of cholinergic signal on neutrophil recruitment and extracellular trap net formation.Results1.Platelets in the left ventricle of mice are higher than those in the right ventricle.Megakaryocytes in the lung are spatially located in the space of distal pulmonary epithelial cells.Megakaryocytes and platelets both expressedα7nAChR.2.The platelet content,the percentage and cell count of MKP in lung tissue increased in mice after vagectomy,and there were no significant changes in bone marrow and spleen.At the same time,the percentage of megakaryocytes in lung tissue increased,and the proportion of mature cells was higher.And the platelet count in peripheral blood of Chrnα7^fl/fl/PF4-cre^tg/wt mice was higher than that of Chrnα7^fl/fl/PF4-cre^wt/wt,but there was no significant change in megakaryocyte lineage progenitor cells;3.Α7nAChR activation changed the actin cytoskeleton signaling and GTPase activity of megakaryocytes,and significantly inhibit the expression of glycoprotein CD41,the release of platelet like particles and the formation of polyploidy.The expression level of active form Cdc42 protein was significantly down regulated by agonists,which seriously damaged the formation of DMS.4.LPS can significantly mobilize the immune response ability of MEG01 cells,which is further enhanced by the intervention of agonists;Chrnα7^fl/fl/PF4-cre^tg/wt mice showed mild inflammatory injury,decreased inflammatory cell recruitment and significantly increased CFU in lung tissue;5.In vitro experiments showed that cholinergic signal significantly weakened the immune response of neutrophils,which was manifested in the decrease of cytokine expression level,the down regulation of signal pathway phosphorylated protein and the formation of extracellular trap net.After vagotomy,neutrophil recruitment and the formation of extracellular trap net was increased,and the inflammatory injury was aggravated;On the contrary,the intervention of agonists significantly reduced the above inflammatory response.ConclusionVagal-α7nAChR affect platelet production by regulating the differentiation and maturation of megakaryocytes,which is mediated by F-actin cytoskeleton rearrangement regulated by GTPase.The immunological function of cholinergic signaling（α7nAChR）on megakaryocytes/platelets is different from neutrophils.It mainly plays an important role in recruiting inflammatory cells to the site of pathogen invasion to eliminate pathogens.Our study provided a new insight for the regulation of nerve in the blood system;and also revealed the immunological function of cholinergic signaling（α7nAChR）on Mks and platelet.