The Regulatory Effect Of Toll-like Receptor 3 On Regulating The Proliferation Of Haemocytes In Crassostrea Gigas

Haemocytes are the main immune cells of invertebrates,which can eliminate invasive pathogens through phagocytosis and play an important role in immune defense.In the immune response of the body,the proliferation of haemocytes is a key step to rapidly improve the immune defense ability of the body,but its specific regulatory mechanism is still unclear.Immune recognition is the first step of the organism’s immune defense.The Toll-like receptors（TLRs）,as the typical pattern recognition receptors in the innate immune response,play an important role in activating various immune defense functions and directly regulate cell proliferation and survival.Compared with vertebrates,the number of TLR signal related genes in invertebrate genome was significantly expanded.In particular,83 TLR genes were predicted in Crassostrea gigas genome,which provided the possibility to study the structural and functional diversity of TLR molecules.At present,studies on immune defense of C.gigas have shown that pathogen stimulation can significantly induce haemocytes proliferation and increase the total number of haemocytes,but the regulatory mechanism of haemocytes proliferation remains unclear,and the potential role of TLR signaling in regulating haemocytes proliferation remains unclear.In this study,invertebrate C.gigas as the research object,toll-like receptor 3 was identified by bioinformatics analysis,RNA interference,fluorescence quantitative PCR,flow cytometry and other technical means and methods,and the regulation effect of Cg TLR3 of C.gigas on haemocytes proliferation was preliminarily explored.The main results were as follows:1.The Cg TLR3 gene of Crassostrea gigas was identified and its conserved molecular structure was clarifiedCg TLR3 gene was screened from the genome of C.gigas.The length of the open reading frame of Cg TLR3 gene was 2166 bp,encoding a polypeptide composed of 721 amino acid residues.The predicted molecular weight of the protein was 83.55 k Da,and the theoretical isoelectric point was 9.00.The Cg TLR3 sequence contains conserved domains of the TLR family,including five extracellular LRR domains,one transmembrane domain and one TIR intracellular domain.The phylogenetic tree was constructed by NJ（Neighbor-Joining）method of MEGAX software to analyze the molecular evolutionary relationship of TLR.The results showed that Cg TLR3 joined with the TLR of Ostrea edulis and other invertebrates,indicating that Cg TLR3 was relatively conserved in evolution.2.The recognition characteristics of Cg TLR3 for a variety of ligands were clarified,and the binding activity of Cg TLR3 with adaptor proteins was detectedThe extracellular ligand-binding domain（r Cg TLR3-LRR）and the intracellular domain（r Cg TLR3-TIR）of Cg TLR3 were obtained by prokaryotic expression and protein purification techniques,and polyclonal antibodies were prepared by immunizing mice with the intracellular segment recombinant protein.ELISA was used to detect the binding activity of r Cg TLR3-LRR to PGN,MAN,LPS and poly（I:C）.The results showed that the binding activity of r Cg TLR3-LRR to LPS increased in a dose-dependent manner,and the binding activity of r Cg TLR3-LRR to the other three PAMPs increased first and then stabilized.Western blotting was used to detect the binding activity of the extracellular segment recombinant protein of Cg TLR3（r Cg TLR3-LRR）with four bacteria,V.splendidus,E.coli,M.luteus,S.aureus and the fungus P.splenoris.The results showed that recombinant Cg TLR3（r Cg TLR3-LRR）could bind to Gram-negative bacteria,Gram-positive bacteria and fungi,and the binding activity of the recombinant Cg TLR3（r Cg TLR3-LRR）was from large to small.P.pastoris>E.coli>M.luteus>S.aureus>V.splendidus.The interaction between Cg TLR3 and Cg My D88-2 was detected by biofilm interference technology,and it was found that r Cg TLR3-TIR could combine r Cg My D88-2 and fit its combined curve.The binding constant（K_D）of r Cg TLR3-TIR and r Cg My D88-2 is 7.22×10^-7 M.3.It was found that Cg TLR3 was mainly expressed in granulosa cells of C.gigas,and the expression level increased significantly after pathogen stimulationq RT-PCR was used to detect the expression level of Cg TLR3 in different tissues of C.gigas（hepatopancreas,gills,adductor muscles,haemocytes,labial palps,gonad and mantles）,and it was found that the m RNA expression level of Cg TLR3 was the highest in haemocytes and 87.34-fold that in gonads（the lowest expression level）（p<0.05）.The distribution of Cg TLR3 in haemocytes cells was detected by immunohistochemistry,and it was found that Cg TLR3 was mainly distributed on the cell membrane of haemocytes cells.Flow cytometry was used to detect the distribution of Cg TLR3 in three types of haemocytes subsets,and it was found that the distribution of Cg TLR3 was the most in granulocytes,and the proportion of FITC⁺cells in granulocytes was2.57-fold that in semi-granulocytes（p<0.05）,4.45-fold that of agranulocytes（p<0.01）.The expression of Cg TLR3 m RNA in granulocyte was 7.27-fold higher than that in semi-granulocytes（p<0.05）,8.51-fold the expression level of agranulocytes（p<0.01）.The expression of Cg TLR3in blood lymphocytes detected after stimulation of V.splendidus showed that the expression of Cg TLR3 began to increase gradually at 6 h after stimulation,and the expression of Cg TLR3 was the highest at 12 h,which was 2.93-fold of that of control group,respectively（p<0.05）and 4.15-fold（p<0.01）,decreased gradually after 24 h,and the expression level at 24 h was 2.73-fold that of control group（p<0.05）.4.It was revealed that Cg TLR3 has a regulatory effect on the proliferation and phagocytosis of haemocytesBy injecting the si RNA interference fragment of Cg TLR3,the expression level of Cg TLR3gene in haemocytes was knocked down（0.41-fold that of control group,p<0.01）.The expression changes of genes related to cell cycle,inflammatory factors,hematopoietic transcription factors and different types of haemocytes marker molecules were detected after knockdown Cg TLR3 gene expression.The results showed that the m RNA expressions of cell cycle related genes Cg CDC-45,Cg CDK-2 and Cg PCNA were significantly decreased,which were 0.44-fold of that of the control group（p<0.01）,0.53-fold（p<0.01）and 0.48-fold（p<0.01）.The m RNA expression of inflammatory cytokine Cg IL17-1 decreased significantly,which was 0.47-fold that of the control group（p<0.01）.The m RNA expression of hematopoietic transcription factors Cg GATA-3 and Cg Runx decreased significantly,which was 0.43-fold that of control group（p<0.01）and 0.83-fold（p<0.05）.The m RNA expressions of lymphocyte marker Cg CD-9 and Cg AATase were significantly decreased,which were 0.70-fold that of control group（p<0.05）and 0.62-fold（p<0.01）.Flow cytometry was used to detect the effect of knockdown Cg TLR3 on the proliferation of haemocytes.The result showed that the proportion of Ed U⁺haemocytes cells in SW group was0.7%.At 6 h after V.splendidus stimulation,the proportion of Ed U⁺cells in haemocytes of si-NC+V.s group was 2.7%;The proportion of Edu⁺cells in haemocytes of si-Cg TLR3+V.s group was 1.47%,which was significantly lower than that of si-NC+V.s group（0.54-fold of that of si-NC+V.s group,p<0.05）.Cg TLR3 on haemocytes surface was blocked by Cg TLR3 antibody to detect its effect on haemocytes cell phagocytosis.Flow cytometry showed that the phagocytic rate of V.splendidus in the Anti-Cg TLR3 blocking group was 13.43%,which was significantly lower than that of the control group（28.53%）（p<0.01）.Microscope observation showed that the fluorescence content of haemocytes cells in Anti-Cg TLR3 blocking group was significantly lower than that in C.gigas in control group.The results showed that the phagocytic effect of haemocytes of C.gigas on V.splendidus was significantly reduced after antibody blocking Cg TLR3.In summary,Cg TLR3 of C.gigas has a relatively conserved molecular structure.The extracellular ligand-binding domain of Cg TLR3（r Cg TLR3-LRR）recognizes a variety of PAMPs,while the downstream adaptor protein-binding intracellular domain（r Cg TLR3-TIR）interacts with Cg My D88-2.Cg TLR3 is mainly expressed in granulosa cells of C.gigas,and its expression level is significantly increased after pathogen stimulation,and Cg TLR3 has regulatory effects on the proliferation and phagocytosis of haemocytes.The results of this study provide a reference for the analysis of the mechanism of TLR signaling pathway in the proliferation of haemocytes of C.gigas,and for further understanding of the immune response and defense mechanism of invertebrates.