The Regulatory Effect Of CgTLR2 Signaling On Inflammatory Factors In Crassostrea Gigas

Toll like receptors（TLRs）are evolutionarily ubiquitous recognition molecules in the animaland plant kingdoms,which play an important role in immune defense and homeostasis maintenance.After recognizing ligands,TLR transmits signals downstream through My D88（Myeloid differentiation primary response 88）-dependent and My D88-independent signaling pathways,inducing the production of pro-inflammatory cytokines and type I interferon.At present,10 and 12 TLR have been identified in humans and mice,respectively.They are distributed in different parts of cells,have significantly different ligand recognition characteristics and cause different immune responses.In contrast,significantly expanded TLR family members were found in invertebrate genomes,for example,83 TLRs and 10 My D88s were found in Crassostrea gigas,which indicated that the innate immune defense mechanism of invertebrates was more complex,but their specific roles in immune response were not fully understood.C.gigas,a mollusk,is a typical representative of intertidal organisms and the main species of shellfish culture in the world,which has important ecological and economic value.With the analysis of genome,C.gigas has gradually become an excellent model organism for studying pathogen-host interaction.In this study,three key components of TLR signal（CgTLR2,CgMyD88-2 and CgMyD88s）were identified by bioinformatics analysis,ELISA and Bio-Layer Interferometry（BLI）,and the molecular structure,binding activity,expression characteristics and immune function of TLR signal in C.gigas were preliminarily explored,in order to analyze the regulation of TLR signal on inflammatory factors in C.gigas.The main research results are as follows:1.The molecular structure characteristics of CgTLR2,CgMyD88-2 and CgMyD88s in C.gigasOne TLR（named CgTLR2）and two connector molecules My D88（named CgMyD88-2 and CgMyD88s,respectively）were screened from the genome of C.gigas.The coding frame length of CgTLR2 gene is 2289 bp,which can encode a peptide consisting of 762 amino acid residues,and the predicted molecular weight of CgTLR2 protein is 88.9 k Da.CgTLR2 contains conserved domains of the TLR family,including 8 extracellular LRR domains,1 transmembrane domain and 1 intracellular TIR domain.The coding frame length of CgMyD88-2 gene is 1845 bp,which can encode a peptide consisting of 457 amino acid residues,and the molecular weight of CgMyD88-2 gene is predicted to be 50.9 k Da.CgMyD88-2 protein contains a N-terminal Death domain and a C-terminal TIR domain,and the structure is complete.The length of the coding frame of CgMyD88s gene is 1107 bp,and it can encode a polypeptide consisting of 178 amino acid residues,with a predicted molecular weight of 20.7 k Da.CgMyD88s protein contains only one C-terminal TIR domain,and the N-terminal Death domain is missing.The phylogenetic trees of CgTLR2,CgMyD88-2 and CgMyD88s were constructed by MEGAX software,and the results showed that the three genes were relatively conserved in evolution.2.The binding activity of CgTLR2 to ligand and intracellular linker molecules in C.gigasRecombinant proteins of CgTLR2,CgMyD88-2 and CgMyD88s（r CgTLR2-LRR,r CgTLR2-TIR,r CgMyD88-2,r CgMyD88s）were obtained by prokaryotic expression and affinity purification techniques,respectively,and highly specific polyclonal antibodies were prepared by using the recombinant proteins.The binding activity of r CgTLR2-LRR to different PAMPs ligands was detected by ELISA.CgTLR2 bound to LPS,MAN and poly（I:C）in a dose-dependent manner,and showed the highest affinity for LPS,a relative low affinity for MAN and poly（I:C）.The binding activity between r CgTLR2-TIR and r CgMyD88-2 and r CgMyD88s was detected by BLI method.The binding constants（K_D）of CgTLR2 to CgMyD88-2 and CgTLR2 to CgMyD88s were 1.96 x10^-9 M and 4.84 x10^-8 M,respectively,which indicated that the binding ability of CgTLR2 to CgMyD88-2 was stronger than that of CgTLR2 to CgMyD88s.ELISA further proved that the affinity of CgTLR2 with CgMyD88-2was higher than that with CgMyD88s.3.The expression characteristics of CgTLR2,CgMyD88-2 and CgMyD88s in C.gigasImmunohistochemical method was used to detect the localization of CgTLR2 in haemocytes of C.gigas,and it was found that CgTLR2 was distributed on the cell membrane of haemocytes.The expression characteristics of three TLR signal elements were detected by fluorescence quantitative PCR.It was found that the m RNA transcriptions of CgTLR2 were detected in haemocyte,mantle,gill,adductor muscle,hepatopancrease and labial palp,and the highest expression was in haemocytes,which was 30.35-fold of that in gill.CgMyD88-2 and CgMyD88s expressed constitutively in all tissues and were highest in gill.The expression of CgMyD88-2 in gill was 3.17-fold of that in hepatopancrease,and that of CgMyD88s in gill was45.51-fold of that in labial palp.Further analysis of the expression of CgTLR2,CgMyD88-2 and CgMyD88s m RNA in different types of lymphocytes showed that CgTLR2,CgMyD88-2 and CgMyD88s m RNA were expressed in granulocytes,semi-granulocytes and agranulocytes,but the expression level was the highest in g granulocytes and the lowest in agranulocytes.The expression levels in granulocytes were 8.25-fold,13.76-fold and 43.27-fold in agranulocytes,respectively.Then they examined temporal expression after stimulation of Vibrio splendidus and found that the m RNA expression level of CgTLR2 increased gradually at 3 h after stimulation by V.splendidus,and reached the highest level at 6 h,which was 2.80-fold of that of the control group（p<0.001）.The m RNA expression level of Cg IL17-1 increased gradually at 3 h after stimulation by V.splendidus,and reached the highest level at 6 h,which was 12.55-fold（p<0.001）and 21.42-fold（p<0.001）of the control group,respectively.The m RNA expression level of CgMyD88-2 was also significantly increased at 3 h and 6 h after stimulation by V.splendidus,which was 5.18-fold（p<0.001）and 7.55-fold（p<0.001）of the control group,respectively.The m RNA expression level of CgMyD88s increased until 24 h after stimulation by V.splendidus,and reached the highest level at 48 h,which was 35.99-fold that of the control group（p<0.001）.CgTLR2,CgMyD88-2 and CgMyD88s were highly expressed after secondary stimulation by V.splendidus,and their expression levels were 1.50-fold（p<0.05）,1.68-fold（p>0.05）and1.95-fold（p<0.01）of that after primary stimulation by V.splendidus,respectively.4.The regulation of CgTLR2 signal on the expression of downstream genes and inflammatory factors in C.gigasThe m RNA expression of CgTLR2 in i CgTLR2+VS group（experimental group）was significantly decreased by siRNA interference,which was 0.27-fold of that in NC+VS group（control group）（p<0.001）.At the same time,the m RNA expression of CgMyD88-2,CgMyD88s and Cg IL17-1 in the middle and lower reaches of the experimental group also decreased significantly,which were 0.32-fold（p<0.001）,0.23-fold（p<0.001）and 0.26-fold（p<0.001）times of those in the control group,respectively.The m RNA expression of CgMyD88-2in i CgMyD88-2+VS group（experimental group）was significantly decreased by siRNA interference,which was 0.20-fold of that in NC+VS group（control group）（p<0.001）.At the same time,the m RNA expression of CgMyD88s and Cg IL17-1 in the middle and lower reaches of the experimental group also decreased significantly,which were 0.32-fold（p<0.001）and0.43-fold（p<0.01）of those in the control group,respectively.The m RNA expression of CgMyD88s in i CgMyD88s+VS group（experimental group）was significantly decreased by siRNA interference,which was 0.37-fold of that in NC+VS group（control group）（p<0.001）.At the same time,the m RNA expression of CgMyD88-2 and Cg IL17-1 in the middle and lower reaches of the experimental group also increased significantly,which were 3.98-fold（p<0.001）and 2.48-fold（p<0.01）of those in the control group,respectively.In conclusion,the molecular structures of CgTLR2,CgMyD88-2 and CgMyD88s were relatively conservative.The extracellular r CgTLR2-LRR of CgTLR2 could recognize many kinds of PAMPs,and the intracellular r CgTLR2-TIR could interact with CgMyD88-2 and CgMyD88s,with stronger binding ability with CgMyD88-2.CgTLR2,CgMyD88-2 and CgMyD88s were expressed constitutively in various tissues,with the highest expression in granulocytes,and all of them could respond to the immune stimulation of V.splendidus.CgTLR2-CgMyD88-2 signal was activated in the early stage of immune response and promoted the production of Cg IL17-1,but this signal was inhibited by CgMyD88s in the later stage of immune response to avoid excessive production of Cg IL17-1.The results preliminarily clarified the regulatory role of TLR signal on inflammatory factors in the immune defense of C.gigas,which provided an important reference for further understanding the immune defense mechanism of invertebrates.