| As important immune cell,haemocytes play important role in the immune defense of invertebrates.In the immune defense against pathogen invasion,circulating haemocytes can proliferate to rapidly increase cell numbers and immune defenses and effectively eliminate pathogens,but the potential mechanism is still unclear.Interleukin 17(IL-17)is an important pro-inflammatory cytokine that can regulate lymphocyte proliferation in the immune response of higher animals,but whether it has similar function in invertebrates is still unclear.The exploration on the regulation of interleukin-17 molecule and its receptor on the proliferation of haemocytes will provide reference for revealing the immune defense mechanism of invertebrates.In this study,an interleukin 17(CgIL17-1)and its receptor(CgIL-17R1)were identified from the oyster C.gigas by bioinformatics,molecular biology and cell biology techniques and the regulation of CgIL17-1 on proliferation of haemocytes was explored.The main results were as follows:1.The CgIL17-1 and CgIL-17R1 in C.gigas are relatively conserved in structure and constitutively expressed in different tissuesCgIL17-1 and its receptor CgIL-17R1 were screened from C.gigas genome.The open reading frame of CgIL17-1 was of 603 bp,encoding a peptide with 201 amino acid residues.The protein contains a signal peptide(1-26 aa)and a conserved IL-17 domain of the interleukin 17family(97-178 aa).The open reading frame of CgIL-17R1 was of 3141 bp,encoding 1047amino acid residues of polypeptide;The protein contains a signal peptide(1-17 aa)and two FN3domains(239-320 aa;351-435 aa)and three low-complexity regions(729-742 aa;831-842aa;984-996 aa).RT-PCR was used to detect the expression of CgIL17-1 and CgIL-17R1 m RNA in different tissues and it was found that CgIL17-1 and CgIL-17R1 were expressed in different tissues and haemocytes.The expression level of CgIL17-1 m RNA was the highest in mantle,which was5.13-fold(p<0.01)of that in hepatopancreas.The expression level of CgIL-17R1 m RNA was the highest in haemocytes which was 13.44-fold(p<0.01)of that in gills.The recombinant proteins of CgIL17-1 and CgIL-17R1 were obtained by prokaryotic expression and protein purification techniques.Mice were immunized with the recombinant proteins to obtain polyclonal antibodies.Flow cytometry was used to detect the distribution of CgIL17-1 and CgIL-17R1 in three subpopulations of oyster haemocytes,and it was found that CgIL17-1 and CgIL-17R1 were mainly distributed in granulocytes.The distribution of CgIL17-1and CgIL-17R1 in granulocytes was detected by immunofluorescence technique,and it was found that CgIL17-1 and CgIL-17R1 were mainly distributed on the cell membrane of granulocytes.2.CgIL17-1,CgIL-17R1,CgRunx-1 and cell cycle-related proteins can be induced by V.splendidus stimulation,and there is interaction between CgIL17-1 and CgIL-17R1The m RNA expression levels of CgIL17-1,CgIL-17R1,CgRunx-1 and cell cycle-related proteins(CgCDC-6,CgCDC-45 and CgCDK-2)in haemocytes of C.gigas were changed after injection of V.splendidus into adductor muscles were detected by RT-PCR.The expression levels of CgIL17-1 increased significantly at 3,6 and 48 h post V.splendidus stimulation,which were8.99-fold(p<0.01),31.36-fold(p<0.01),8.79-fold(p<0.01)of that in the control group,respectively.The expression levels of CgIL-17R1 increased significantly at 6 h post V.splendidus stimulation,which was 1.71-fold(p<0.01)of that in the control group,respectively.The expression levels of CgRunx-1 increased significantly at 6 h post V.splendidus stimulation,which was 3.69-fold(p<0.01)of that in the control group,respectively.The expression levels of CgCDC-6 increased significantly at 6,24 and 48 h post V.splendidus stimulation,which were3.15-fold(p<0.01),2.67-fold(p<0.01),2.43-fold(p<0.05)of that in the control group,respectively.The expression levels of CgCDC-45 increased significantly at 6 h post V.splendidus stimulation,which was 3.02-fold(p<0.01)of that in the control group,respectively.The expression levels of CgCDK-2 increased significantly at 6,24 and 48 h post V.splendidus stimulation,which were 2.04-fold(p<0.05),11.88-fold(p<0.01),2.18-fold(p<0.05)of that in the control group,respectively.Biofilm interference was used to analyze the binding signals of biotin-labeled r CgIL17-1with different concentrations of CgIL-17R1 protein,and it was found that there was an interaction between r CgIL17-1 and r CgIL-17R1,K_D=2.74×10-7 M.No obvious binding signal was detected between r CgIL-17R1 and the control protein r TRX,and no K_D value was found.3.CgIL17-1 and CgIL-17R1 was involved in regulating the proliferation of oyster haemocytesThe m RNA expression levels of hematopoietic related transcription factor(CgRunx-1)and cell cycle-related proteins(CgCDC-6,CgCDC-45 and CgCDK-2)in haemocytes of C.gigas after injection of r CgIL17-1 into adductor muscles were detected by RT-PCR.CgRunx-1,CgCDC-6,CgCDC-45 and CgCDK-2 were significantly increased at 6 h after treatment,which were 2.70-fold(p<0.01),1.77-fold(p<0.05),4.50-fold(p<0.01)and 24.64 fold(p<0.01)of that in the control group,respectively.The m RNA expression levels of hematopoietic related transcription factors(CgRunx-1,CgBMP-7 and CgGATA-3)and cell cycle-related proteins(CgCDC-6,CgCDC-45 and CgCDK-2)were detected by interfering CgIL-17R1 with RNAi technology.The m RNA expression levels of CgRunx-1 and CgBMP-7 were decreased,which were 0.44-fold(p>0.05)and 0.23-fold(p>0.05)of that in EGFP ds RNA-injected group,respectively.The m RNA expression levels of CgGATA-3,CgCDC-6,CgCDC-45 and CgCDK-2were decreased,which were 0.16-fold(p<0.01),0.25-fold(p<0.01),0.15-fold(p<0.05)and0.25-fold(p<0.05)of that in EGFP ds RNA-injected group,respectively.Flow cytometry was used to detect the changes of Ed U labeled new haemocytes(Ed U~+)in hemolymph after injection of r CgIL17-1 into adductor muscles.It was found that the proportion of Ed U~+cells in haemocytes increased significantly 6 h after r CgIL17-1 treatment,which was3.02-fold of that in the control group(p<0.01).CgIL-17R1 was interfered with RNAi technology to detect the changes of Ed U~+cells in hemolymph.The proportion of Ed U~+cells in the EGFP-RNAi group was 12.03%,and the proportion of Ed U~+cells in the IL-17R1-RNAi group was 5.02%.The proportion of Ed U~+cells in the IL-17R1-RNAi group was decreased,which was 0.42-fold(p<0.01)of that in EGFP-RNAi group,after CgIL-17R1 was knocked-down.In conclusion,the molecular structure of CgIL17-1 and CgIL-17R1 in C.gigas is relatively conservative,and they are expressed in different tissues and cells.CgIL17-1 and CgIL-17R1 are found on the cell membrane of granulocytes,and their expression levels are significantly increased after V.splendidus stimulation.The combination of CgIL17-1 and CgIL-17R1 induces the expression of hematopoietic related transcription factors and cell cycle-related proteins,which in turn promote the proliferation of haemocytes in C.gigas.These results provide important reference for further understanding the structure and function of interleukin-17 and its receptors in invertebrates,as well as the regulatory mechanism of interleukin-17 signaling on haemocytes proliferation. |
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