| Immune inhibitory receptors are an important regulatory factor of the immune system,which can inhibit the overactivation of immune system and play a vital role in maintaining immune homeostasis.At present,the research on inhibitory receptors and their negative regulation has become a hot spot in the field of immunology and this mechanism is usually caused by inhibitory receptors.It has been found that inhibitory receptors are mainly used in cancer and tumor therapy in vertebrates,but there are few studies in invertebrates.In the present study,the inhibitory receptors Cg Ig IT2 and Cg PD-1-like were isolated and identified from Crassostrea gigas by bioinformatics,cell biology and molecular immunology.The study not only verified the binding activity of Cg Ig IT2 and Cg PD-1-like to microbes and pathogen-associated molecular patterns(PAMPs),but also verified the regulatory functions of autophagy and inflammation in oysters.The results obtained are as follows:1.Molecular structure and expression characteristics of Cg Ig IT2 and Cg PD-1-likeIn the present study,two novel inhibitory receptors Cg Ig IT2 and Cg PD-1-like was identified from the Pacific oyster,C.gigas.The open reading frame(ORF)of Cg Ig IT2 was of2649 bp,which encoded 882 amino acids.The protein sequence of Cg Ig IT2 contained one signal peptide,four Ig domains,one fibronectin type III domain,one transmembrane domain,and a cytoplasmic tail with two intracellular immunoreceptor tyrosine-based inhibitory motifs(ITIMs)and one immunoreceptor tyrosine-based switch motif(ITSM).The full-length c DNA sequence of Cg PD-1-like was of 1499 bp and its ORF was of 1473 bp,which encoded 490 amino acids.The protein sequence of Cg PD-1-like included a signal peptide(1-16 aa),two Ig domains(24-139 aa;268-333 aa),a TM domain(389-411 aa),two ITIMs(442 aa;452-457 aa)and two ITSMs(468-473 aa;478-483 aa).The tissue distribution,developmental stages and expression pattern of Cg Ig IT2 and Cg PD-1-like in haemocytes after Vibrio splendidus stimulation were examined by quantitative reverse transcriptase PCR(q RT-PCR).It was found that the m RNA transcripts of Cg Ig IT2 and Cg PD-1-like were widely expressed in all the tested tissues,with Cg Ig IT2 was expressed the highest in haemocytes,and Cg PD-1-like was expressed the highest in mantle,followed by gill and haemocytes.The m RNA transcripts of Cg Ig IT2 were expressed in all developmental stages,and the m RNA expression of Cg Ig IT2 was the highest in 8 cell stage.The m RNA expressions of Cg Ig IT2 in haemocytes increased significantly at 24,48 and 72 h after V.splendidus stimulation.The recombinant proteins Cg Ig IT2-4 × Ig,Cg Ig IT2-FN3 and Cg PD-1-like were obtained by prokaryotic expression and protein purification in vitro,and the specific polyclonal antibodies were obtained by immunizing mice with the recombinant protein.Flow cytometry analysis showed that the positive signals of Cg Ig IT2 protein were mainly detected in granulocytes of haemocytes,which were 1.27-fold and 2.15-fold(p < 0.05)higher than that of semi-granulocytes and agranulocytes,respectively.The immunocytochemistry suggested that Cg Ig IT2 and Cg PD-1-like was mainly located in the membrane and cytoplasm of haemocytes.2.Binding activity of r Cg Ig IT2-4 × Ig and r Cg PD-1-like to different microbes and PAMPsWestern blot analysis suggested that the recombinant protein of Cg Ig IT2-4 × Ig(r Cg Ig IT2-4× Ig)and Cg PD-1-like(r Cg PD-1-like)could bind Gram-negative bacteria(V.splendidus,V.anguillarum,Escherichia coli),Gram-positive bacteria(Staphylococcus aureus,Bacillus subtilis),and fungi(Pichia pastoris),in which r Cg Ig IT2-4 × Ig V.splendidus,E.coli and P.pastoris had strong binding activity,and r Cg PD-1-like had strong binding ability with V.anguillarum,Escherichia coli and Staphylococcus aureus.The Enzyme-linked Immunosorbent assay suggested that r Cg Ig IT2-4 × Ig exhibited binding activity towards multiple pathogen-associated molecular patterns(PAMPs),including lipopolysaccharides(LPS),peptidoglycan(PGN),mannose(MAN)and polyinosinic-polycytidylic acid(Poly(I: C)),with r Cg Ig IT2-4 × Ig had the highest affinity for LPS.r Cg PD-1-like could bind to LPS,PGN,MAN,Lipoteichoic acid(LTA)and Poly(I: C),which had the strongest binding activity to Poly(I: C).3.The regulation of Cg Ig IT2 and Cg PD-1-like autophagy and inflammation in haemocytesThe expression of autophagy-related genes and cytokines after blocking assay with anti-Cg Ig IT2 antibody were detected by q RT-PCR and Western blot.It was found that compared with the control group(negative serum group),the expression of SHP2 phosphorylation decreased.Compared with the control group,the m RNA expressions of transcription-related factors(Cg AP-1 and Cg Rel)were increased significantly,which were 2.75-fold(p < 0.001)and2.56-fold(p < 0.01),respectively.The m RNA expression levels of autophagy-related genes autophagy-related genes(Cg ATG5,Cg ATG16 L,Cg LC3 and Cg OPTN)were increased,which were 3.82-fold(p < 0.001),3.35-fold(p < 0.05),4.81-fold(p < 0.01),2.94-fold(p < 0.001),respectively.The m RNA expression levels Cg IL-17-1,Cg IL-17-3,Cg IL-17-6,Cg TNF-1,Cg TNF-2 and Cg Defh-2 in haemocytes increased significantly at 12 h after V.splendidus stimulation,which were 6.05-fold(p < 0.01),2.46-fold(p < 0.05),7.44-fold(p < 0.05),1.81-fold(p < 0.05),4.45-fold(p < 0.05),3.16-fold(p < 0.01),respectively.At the same time,tissue sections showed that the gill swelling,cilia shedding,gill adhesion,vacuoles and gill flap enlargement were obvious in the antibody blocking group compared with the control group.The expression of autophagy-related genes and cytokines were detected after Cg PD-1-like genes was interfered by RNAi technology.The results showed that the m RNA expression of autophagy-related genes(Cg Beclin1,Cg OPTN,Cg P62,Cg ATG10 and Cg ATG16L)decreased significantly at 24 h after V.splendidus stimulation than that control group,which were 0.31-fold(p < 0.05),0.23-fold(p < 0.01),0.48-fold(p < 0.05),0.24-fold(p < 0.05)and 0.08-fold(p <0.05),respectively.Western blot showed that the expression of LC3 Ⅱ was decreased by RNAi.Compared with the control group,the m RNA expression of cytokines(Cg IL-17-1 and Cg IL-17-2)were significantly increased and Cg IFNLP was decreased significantly,which were 6.75-fold(p< 0.05),3.76-fold(p < 0.01)and 0.35-fold(p < 0.05),respectively.In conclusion,two inhibitory receptors Cg Ig IT2 and Cg PD-1-like were isolated and identified from oyster C.gigas.Both of them could bind to different microbes and PAMPs,and regulated the expression of autophagy-related genes and cytokines in oyster.These results would provide a reference for the further study of inhibitory receptors and their functions in invertebrates. |
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