The Role Of Natural Antibody In Defense Against Staphylococcus Aureus And Its Mechanism

Natural antibodies (NAbs) is defined as a type of antibodies, which circulating in normal individuals, that have been produced in the absence of overt specific antigenic stimulation. Natural antibodies are mainly IgM isotype,coding by germline gene ,the previous data showed that NAbs play important role in the innate immunity,including oncologica,infection,surveillance,autoimmunity and so on.Natural antibodies are the first line of defence in infection, which is the earlist we know among these functions.A feature of natural antibodies is the ability of polyreactive auto-Abs binding both self and nonself antigens such as microbial molecules and thus to defense against infection, such as bacteria,virus,fungus,and parasites infection.B1 cells are the main cells that produce natural antibodies, previous data showed that mature B cells were divided into three subsets: B1 cells, folliclar B cells, and marginal zone B cells.Among them, B1 cell are founded in recent years,located in peritoneal cavity, pleural cavity ,they are playing a very important role in innate immunity,autoimmunity and immunity in mucous. It was reported that B1 cell deficient mice were susceptible to bacteria infection, however,the role and mechanism of NAbs and B1 cells in infection is not clear.Serum NAbs,IgM-/-mice, CD19 transgenic mice and B1 cell transplantation experiments were used in previous NAbs and B1 cell anti-infection studies,and reached many conclusions.However, lack of an purified NAbs and NAbs transgenic mice is an bottleneck of the NAbs and B1 cell study,for example ,in the study of some kind of NAbs and NAbs role in vivo.However, the clinical relevance of staphylococcus aureus infection diseases has increased greatly in recent years, with the occurrence of vancomycin-resistant staphylococcus aureus strain and increasing population of immunocompromised hosts, the fatality from staphylococcus aureus infections is serious in china.Natural immunity gained more and more interests among scientists. People are paying more and more attention to it, so ,to know exactly the mechanism and how to apply natural antibodies to staphylococcus aureus infections disease became an urgent issue.In our previous study , We got 3 hybridomas which secret natural antibody and 3B4 is best among these hybridomas,it is showed that 3B4 binds staphylococcus aureus and regulate the phagocytosis of the bacteria.These findings demonstrated the anti-infection function of 3B4.Furthermore, with the IgH transgenic mice with elevated serum level of 3B4 we had established to investigate the role of natural anti-keratin autoantibody (AK auto Ab) in defense against staphylococcus aureus infection in vivo,which has a priority over other models for the special elevated antibody level to analyze the mechanism in anti-infection process.We studied the role of 3B4 in defense against staphylococcus aureus in vitro and in vivo.The following work will be done in this research:1. IgM 3B4's role in defense against S. aureus in vitro.(1) Both ELISA and flow cytometry proved that 3B4 binds to Staphylococcus aureus.(2) In vitro bacterial internalization Assays. Macrophages eluded from mice peritoneal cavity were exposed to staphylococcus aureus .It is showed that IgM 3B4 may modulate the phagocytosis of staphylococcus aureus by FCM.2. IgM 3B4's role in defense against S.aureus in vivo (1) After inoculation , Survival rate of the transgenic mice were higher than the control mice.(2) Bacteria burden,the neutrophils, inflammatory factor assay. Bacteria burden in the peritoneal lavage fluid, kidney were analyzed by colony forming assay, and the neutrophils, inflammatory factor phagocytic index poliferation level of B cell subsets were analyzed by flow cytometry. Colony forming assay showed that littermate control displayed significantly higher bacterial burdens than transgenic mice (P<0.01). Neutrophil proportion, inflammatory factor concentration in transgenic mice were lower than the control(P<0.01)as indicated by flow cytometry. These findings demonstrate that natural anti-keratin IgM may protect the host against S. aureus in vivo,and it may served as the basis for further research on the function and mechanism of natural antibody.3. Anti-infection mechanism of 3B4 and B1 cell.(1) Anti-keratin, Anti-staphylococcus aureus and total IgM antibody detection.Peritoneal cavity fluid were eluted from peritoneal cavity of transgenic and control mice and the antibody level were measured by ELISA.We found that transgenic mice displayed higher anti-staphylococcus aureus and anti-keratin IgM production. (2) Phagocytic index, B1 cell proliferation level and plasma cell proportion assay by FCM. Phagocytic index ,B1a cell proliferation level and plasma cell proportion are higher in Tg mice than that of littermate control (P<0.01).However, we found that B cells in transgenic mice have the phagocytic ability,while,this results need to be verify in later experiments.We analyzed the NAbs 3B4's role in binding S.aureus and regulating the of S.aureus.Through the peritoneal cavity infection model,we detected the bacteria burden,the neutrophils, and inflammatory factor concentration after infection.To know the mechanism,We analyzed the anti-keratin IgM, anti-staphylococcus aureus IgM antibodies and B1a cell proliferation level and plasma cell proportion. We found that the clearance of the S.aureus and immune response in transgenic mice are more intensive than that of control mice.These results NAbs 3B4's role in S.aureus infection, and may serve as a basis for further study.