Regulatory Effects Of CGAS On B Cell Development And Humoral Immune Response

ObjectiveCyclic GMP-AMP synthase(cGAS)is an important cellular double-stranded DNA sensor,which senses the double-stranded DNA in the cytoplasm,initiates the transcription of type I interferon,and mediates antiviral immunity.At present,the research on cGAS mainly focuses on the field of antiviral immunity,and whether cGAS affects the B cell development has not been reported in the literature.The database shows that cGAS is highly expressed on bone marrow,suggesting that cGAS may affect B cell development,thus we focuse on the investigation of cGAS and B cell development.The main aim of this study is to clarify the role of cGAS in the B cell development,and to explore the regulatory effect of cGAS on B cell development and humoral immune response.Methods1.Investigation of the effect and mechanism of cGAS on B cell developmentFlow cytometry was used to detect the amount of B cells in bone marrow,peripheral blood and spleen of the wild type(WT)mice and Cgas knockout(Cgas-/-)mice to explore the effect of Cgas on the amount of B cells.Flow cytometry was used to detect the amount of B cells in the bone marrow of the WT mice and Cgas-/-mice at different developmental stages to define the specific stage of B cell development affected by cGAS.D-J junction and V-DJ junction fragments of bone marrow B cells were amplified by PCR,and the effect of cGAS on BCR gene rearrangement of B cells was detected.Flow cytometry was used to detect the effect of cGAS on apoptosis and cell cycle progression of B cells.2.Investigation of the effect of cGAS on humoral immune responseBone marrow sections of WT mice and Cgas-/-mice were used to detect the differentiation status of bone marrow cells,and a mouse systemic lupus erythematosus model was further established to explore whether the B cells of Cgas-/-mice had autoreactivity.WT mice and Cgas-/-mice were immunized intraperitoneally with NP-conjugated chicken gamma globulin(NP-CGG)antigen to stimulate the immune response of the mice,and the effect of Cgas knockout on the production of IgG antibody in mice was explored.WT and Cgas-/-mice were infected with staphylococcus aureus(5×106 CFU/20g body weight)to establish a mouse model of staphylococcus aureus infection and to explore the effect of cGAS on the immune response to staphylococcus aureus infection in mice.Results1.The amount of B cells in the bone marrow,peripheral blood and spleen of the Cgas-/mice was significantly higher than that of the WT mice.The data of the screening of B cell development stages in bone marrow showed that the up-regulation effect of Cgas knockout on the amount of B cell number started from the Pre-B stage.2.Overexpression of cGAS resulted in a significant increase in cell apoptosis and the increased proportion of cells in G2/M phase of the cell cycle.3.Cgas-/-mice immunized with NP-CGG antigen could produce higher level of IgG antibody and have stronger humoral immune response.4.Cgas-/-mice were able to produce higher levels of IgG antibodies and eliminate the extracellular bacteria more effectively after infection with S.aureus.The Cgas-/-mice had longer survival time after the infection with lethal doses of staphylococcus aureus.ConclusionKnockout of Cgas up-regulated the amount of B cells in bone marrow,spleen and peripheral blood,and the regulatory effect of cGAS on B cell amount started from the Pre-B stage.Further investigation of the mechanism showed that cGAS may regulate the cell cycle progression of B cells in development in the bone marrow,induce B cells arrested at G2/M phase,and then induce apoptosis of cells in G2/M phase,thus to control the amount of B cells.Knockout of Cgas increased the number of B cells in mice,produced higher levels of IgG antibodies after immunization with NP-CGG,and cleared bacteria more effectively after infection with Staphylococcus aureus.Innovation1.It revealed that cGAS was involved in the physiological process of B cell development and affects the humoral immune response.The function and mechanism investigation of cGAS were improved.2.The negative regulatory effect of cGAS on the immune response of staphylococcus aureus was revealed,which provided new ideas for the precise regulation of the staphylococcus aureus induced immune response in clinical practice.