The Mechanism Of Glutamine Metabolism Regulating IgM~+B Cell Immune Process Of Tilapia

B-cell is an important component of adaptive immunity,playing an essential role in defending against pathogens and maintaining immune homeostasis,as well as providing the body with specific,long-lasting immune protection.Once recognized with the pathogens,B cells are rapidly activated,proliferate and different into plasma cells that secrete a large number of antibodies,providing a strong humoral immune defence.Although there are significant differences in body structure,immune organs and the location of B-cell production and maturation in teleost compared to mammals,fish already have a well-established adaptive immune system and B-cell-mediated immune responses.The role of IgM~+and Ig T~+B cells in the regulation of antimicrobial immune response and mucosal homeostasis has been elucidated in a variety of important aquaculture fish.However,the activation program of B cells and their immune regulatory mechanisms are unclear.Therefore,in this study,we took Nile tilapia(Oreochromis niloticus)as a model to elucidate the immune response of B cells against pathogenic infections in teleost with revealing the signaling mechanism of IgM~+B cell activation.In addition,we explored the molecular mechanism of glutamine metabolism regulating the immune effect of IgM~+B cells in Tilapia from the perspective of immunometabolism.In the Edwardsiella piscicida bacterium infection model,we found that both the proportion and number of IgM~+B cells were significantly increased during the adaptive immune phase,and a large amount of IgM antibodies were secreted by the B cells.The secondary infection could trigger the up-regulation of antibody transcription level and serum immunoglobulin content more quickly.Thus,in response to reinfection,Tilapia B cells are able to trigger a more rapid and effective immune memory response.In addition,our results showed that the expansion of IgM~+B cell lineage was the result of clonal proliferation,and this process was accompanied by programmed cell apoptosis.On the other hand,IgM antibodies secreted by tilapia B cells have an extremely high affinity for specific antigens and are able to adhere,aggregate and neutralise pathogenic bacteria and enhance cytophagocytosis through opsonization.Immunofluorescence showed that tilapia serum IgM antibody had a stronger binding ability to bacteria after 28 days of infection.Further results showed that IgM antibody could significantly enhance the phagocytic activity of tilapia macrophages and B cells,thereby enhancing the ability of immune cells to destroy pathogenic microorganisms.Onset of the B cell immune response is initiated by the antigen-linked BCR,which transmits and amplifies the activation signal through its downstream molecular events and pathways.We used monoclonal antibody to establish an IgM~+B cell activation model in tilapia in which IgM cross-linking with the BCR triggers proximal signaling and downstream signaling pathway activation,induced by intranuclear transcriptional regulation,to reveal the strategy of B cell activation in teleost.B cell activation triggers upregulation of the expression of the BCR complex,including IgM and CD79,and its coupled protein tyrosine kinases SYK,Lyn,and Btk.Through the recruitment of subsequent effector molecules,the p65 NF-κB,m TORC1 and MAPK/ERK signalling pathways are activated,which endows B cells with appropriate strategies to respond to antigen and make activities such as clonal proliferation,enhancement of survival,antibody secretion and differentiation arrest.Immune cell activation is a highly energy-consuming event.Cell growth,rapid proliferation and production of large amounts of antibodies requiring a huge supply of material and energy.So,the antigen-induced B cell activation drives a comprehensive layout of metabolism,transforming it into a highly metabolic and energy-using state.Our results show that B-cell activation induces the expression of the glutamine transporters ASCT2 and SNAT2,as well as the metabolism-critical enzymes GLS1and GLUD,and resulted in increased transient accumulation of intracellular glutamine.Through withdrawal or oversupply glutamine as well as GLS1 inhibition,we further clarified that glutamine metabolism is an important intrinsic pathway supporting immune responses in B-cell activation,proliferation and antibody secretion in tilapia.More importantly,we identified a set of regulatory strategies for the induction of glutamine metabolism in tilapia B cells.During the anti-microbial response and IgM antibody-induced B cell activation,c-Myc expression was upregulated and induced B cell proliferation and antibody secretion through regulation of glutamine uptake and metabolism.In addition,m TORC1 signaling pathway regulated the immune response of tilapia B cells,and inhibition of rapamycin or trametinib was able to directly impair IgM~+B cell proliferation and antibody secretion.Downstream of BCR signaling,m TORC1 pathways regulated B cell immune response by promoting c-Myc-mediated glutamine metabolism.In summary,this study elucidated the anti-bacterial immune function of IgM~+B cells in tilapia and the activation mechanism.In addition,we discussed the relationship between B cells and glutamine metabolism from the perspective of metabolic program supporting and regulating cellular immune effects.It aims to explore in depth the mechanisms of B cell immune regulation in teleost,and provide valuable evidence for exploring B cell-mediated adaptive immunity in teleost.It will further provide theoretical and technical support for the anti-disease immune mechanism and immune capacity enhancement of aquaculture animals.