Studies On The Biological Changes Of Megakaryocytes And Platelets In The Mouse Peritonitis Model

Objectives:Traditionally,megakaryocytes and platelets are regarded as the professional cells performing an important role in hemostasis and thrombosis.However,recent studies have shown that megakaryocytes and platelets can also participate in immune response through various ways,and have important immunomodulatory effects.With the development of single cell transcriptome sequencing technology,more and more attention has been paid to the concept of cell subpopulations.But due to the properties,scarcity and fragility,it is difficult to obtain megakaryocytes,resulting in few single cell transcriptome sequencing studies in related field,thus an accurate understanding of megakaryocyte subpopulations has not been uncovered.In order to study the changes in the biological characteristics,transcriptome subpopulations and immune regulatory role of megakaryocytes and platelets in acute infection,heat-inactivated Escherichia coli(E.coli)was injected intraperitoneally in mice to establish the peritonitis model in this research.Based on this model,dynamic changes of immune cells in peripheral blood and abdominal cavity were detected firstly;secondly,the megakaryocytes in the bone marrow of normal mice and peritonitis model mice were analyzed by single cell transcriptome sequencing to reveal the changes in their subpopulations and functions;finally,the platelets in the peripheral blood of normal mice and peritonitis model mice were analyzed by transcriptome sequencing to re veal the changes in their transcriptome and platelet transfusion experiment was used for functional verification.To summarize,this study investigates the changes of megakaryocyte and platelet transcriptomes under the condition of acute infection,and validates them with in vitro and in vivo functional experiments,which expands our understanding of the immunomodulatory function and mechanism of megakaryocytes and platelets and helps to provide new insights for the diagnosis and treatment of acute infectious diseases in clinic.Methods:(1)Mice were injected intraperitoneally with heat-inactivated Escherichia coli(E.coli)to construct an acute infection model of peritonitis.(2)The number and size of peripheral blood cells,peritoneal immune cells and bone marrow megakaryocytes in mice were dynamically detected by blood routine,flow cytometry and other experimental methods after infection.(3)Megakaryocytes in the bone marrow of normal mice and peritonitis model mice were analyzed by Smart-Seq2 single cell transcriptome sequencing,and bioinformatics technology was used to reveal the changes in megakaryocyte subpopulations and functions before and after infection,which was verified by using single-cell qPCR,immunofluorescence,flow cytometry,and other experimental methods.(4)Platelets in the peripheral blood of normal mice and peritonitis model mice were analyzed by transcriptome sequencing,and bioinformatics technology was used to reveal the changes in platelet transcriptome and functions before and after infection.(5)Based on the mouse peritonitis model,platelet transfusion in vivo was used to study the dynamic changes of platelets and its influence on immune response and inflammation process.Results:(1)In the mouse peritonitis model,macrophages in the abdominal cavity were the first to play an important role in bactericidal effect in the early stage of infection,and then neutrophils from peripheral blood migrated quickly to the abdominal cavity to participate in the inflammatory response(2)During the acute infection,the proportion of CD41+CD42d+megakaryocytes in the mouse bone marrow was significantly increased,but the size of megakaryocytes was significantly reduced.(3)The single cell transcriptome sequencing analysis showed that mouse bone marrow megakaryocytes could be divided into 5 subgroups,including subgroup 1 with high expression of mitochondrial-related genes,subgroups 2 and 3 with the feature of thrombopoiesis,subgroup 4 with the enrichment of genes related to DNA replication and cell cycle,and subgroup 5 expressing a large number of immune response-related genes.(4)Compared with normal mice,the proportion of immune subpopulations marked by CD48 in the bone marrow megakaryocytes was significantly increased after infection.The bioinformatics analysis and single cell qPCR showed that compared with CD148+CD48-megakaryocytes,CD148+CD48+immune megakaryocytes highly expressed multiple immune receptors and immune mediator molecules.(5)In situ bone marrow staining showed that compared with CD48-megakaryocytes,CD48+megakaryocytes were closer to blood vessels and had more adherent neutrophils.(6)During the acute infection,the number and size of peripheral blood platelets were changed significantly.Among them,the number of platelets showed a dynamic change that first decreased and then increased;Mean Platelet Volume(MPV)and Platelet Distribution Width(PDW)were significantly increased after acute infection.(7)The transcriptome sequencing analysis of platelets showed that the platelet transcriptome was significantly changed after infection.Strikingly,immune responserelated genes were significantly up-regulated,and the expression of platelet surface adhesion molecules was also significantly increased.(8)Platelet transfusion could promote rapid migration of neutrophils to the abdominal cavity.Compared with unstimulated platelets,transfusion of platelets after immune stimulation could promote more neutrophils to migrate to the abdominal cavity.Conclusions:This study used the mouse peritonitis model to reveal the dynamic changes of peripheral blood and abdominal cavity immune cells,bone marrow megakaryocytes,and peripheral blood platelets under the condition of acute infection,and preliminarily clarify the immunomodulatory function and mechanism of megakaryocytes and platelets in response to acute infection,and expand our understanding of the biological characteristics and functions of megakaryocytes and platelets.Through single cell transcriptome sequencing of mouse bone marrow megakaryocytes,multiple cell subpopulations in megakaryocytes were found,including CD48-labeled immune subpopulations.Compared with CD48-megakaryocytes,CD48+megakaryocytes had stronger immunomodulatory function.Through transcriptome sequencing of mouse peripheral blood platelets,we found that the platelet transcriptome was changed significantly and the immunomodulatory function of platelets was enhanced significantly after infection.In conclusion,this study revealed the changes of megakaryocyte and platelet transcriptomes under the condition of acute infection of mouse peritonitis,provided new evidence for the immunomodulatory function of megakaryocytes and platelets and was of great significance for the diagnosis and treatment of acute infection in clinic.