Regulation Of MTORC1 Signaling Pathway On B Cell And Granulocyte Immune Function In Fish

As the critical immune cell types in adaptive and innate immunity,B cells and granulocytes play an extremely important role in the defense against pathogenic invasion.Fish were the first vertebrates to possess B cells and immunoglobulins(Ig),and have similar mammalian adaptive immune response mechanisms against infections.B cells represent important effector cells in humoral immunity,capable of secreting specific Ig molecules upon infection by pathogens,and performing immune functions such as neutralizing viruses,controlling parasites,inhibiting bacterial adhesion,and producing agglutination by binding to pathogens.Granulocytes are an important immune cell type in innate immunity,mainly through phagocytosis to remove pathogens.Once fish expose to pathogen infection,granulocytes can be recruited to the site of infection,recognize and phagocytose the pathogen,and kill the pathogen by combining cytotoxic mechanisms.With the development of fish immunology,the immune functions of fish B cells and granulocytes have been gradually clarified,but the regulatory mechanisms regarding the immune functions performed by fish B cells and granulocytes are still unclear.The mechanistic target of rapamycin(m TOR)signaling pathway is one of the central signaling pathways that regulates proliferation and cell growth in the organism.In mammals,the m TORC1 signaling pathway has been shown to regulate B cells and granulocytes to exercise anti-infection immune functions,so whether this regulatory role has emerged in teleost fish?What is the mechanism of action?It is not clear.In this study,we systematically explored the regulatory mechanism of m TORC1signaling pathway on the immune function of fish B cells and granulocytes,and found that m TORC1 signaling pathway plays an important function in the proliferation,apoptosis and phagocytosis of B cells and granulocytes.More importantly,the m TORC1 signaling pathway has a regulatory role in the formation of pathogen-specific Igs in B cells after infection by pathogens.First,in this study,we used rainbow trout(Oncorhynchus mykiss)as a research model to explore the effect of m TORC1 on B cell immune function,the specific findings of this study are as follows:Based on phylogenetic analysis of different vertebrate m TOR molecules,it was found that fish m TORC1 molecules are highly conserved with other vertebrates,hypothesizing that the m TORC1 signaling pathway may play similar regulatory functions as mammals.In this study,we first treated B cells with rapamycin(RAPA)in vitro and found that the phosphorylation levels of the proteins of the components in the m TORC1 signaling pathway were significantly inhibited.Meanwhile,at the transcript level,it was found that inhibition of the m TORC1 signaling pathway affected the expression of phagocytosis-related genes,apoptosis-related genes,and genes related to B cell proliferation and immunoglobulin secretion in both Ig T~+and Ig M~+B cells,hypothesizing that the m TORC1 signaling pathway may be involved in the regulation of and B cell immune function such as phagocytosis,apoptosis,proliferation and Ig secretion.Furthermore,In vitro inhibition of the m TORC1signaling pathway in B cells revealed a significant decrease in the proportion of both phagocytic Ig T~+and Ig M~+B cells,and phagocytosis of Ig T~+and Ig M~+B cells was also significantly suppressed,indicating that the m TORC1 signaling pathway can affect the phagocytic ability of B cells.Through in vitro and in vivo experiments,inhibition of the m TORC1 signaling pathway in B cells was followed by increased apoptosis and decreased proliferationa of both Ig T~+and Ig M~+B cells,leading to a significant decrease in thier final proportion.In addition,both in vitro and in vivo assays demonstrated that the production of Igs was also regulated by the m TORC1signaling pathway in B cells,with a significant inhibited the secretion of Ig T,Ig D and Ig M.More importantly,GFP-labeled F.columnare induced the production of large amounts of bacterial-specific Ig M and Ig T in rainbow trout serum and gill mucus,respectively,and produced agglutination against pathogenic bacteria.However,once the fish m TORC1 signaling pathway was inhibited by RAPA,Ig M in serum and Ig T titers in gill mucus decreased significantly,which significantly affected the agglutination and growth of specific Igs against F.columnare.When reinfected with F.columnare,the susceptibility of fish increased significantly and mortality intensified.The above findings suggest that the m TORC1 signaling pathway plays an essential role in the regulation of adaptive immunity of fish B cells.In addition,we also used largemouth bass(Micropterus salmoides)as a research model to analyze the effect of m TORC1 on granulocyte immune function,the specific findings of this study are as follows:By flow-sorting largemouth black bass head kidney granulocytes and treating with RAPA,the phosphorylation levels of the proteins of the components in the m TORC1 signaling pathway were significantly inhibited in granulocytes.Transcriptome analysis revealed that m TOR,cell cycle,apoptosis,autophagy,endocytosis and phagosome signaling pathways were significantly affected by RAPA treatment of granulocytes.Meanwhile,during in vitro culture of granulocytes,inhibition of the m TORC1 signaling pathway resulted in decreased proliferation and increased apoptosis of granulocytes.This results demonstrated that the m TORC1signaling pathway is involved in the regulation of cellular life activities.Interestingly,in contrast to the results in B cells,inhibition of the m TORC1 signaling pathway in granulocytes in vitro revealed a significant increase in the proportion of phagocytic granulocytes with enhanced phagocytic capacity.This suggested that the m TORC1signaling pathway may play different roles in regulating cellular phagocytosis in different cells.In summary,this study demonstrated that the m TORC1 signaling plays a critical role in the regulation of B cell and granulocyte survival,proliferation,and phagocytosis.More importantly,the m TORC1 signaling pathway is indispensable for the production of specific Igs during B cell resistance to bacterial infection.The results elucidate the conservation of the vertebrate m TORC1 signaling pathway in adaptive immune cells(B cells)and innate immune cells(granulocytes),providing an important theoretical basis for the study of fish immunology.