| In recent years,with the increasing demand for aquatic products,the quantity and density of special economic aquatic products are increasing.How to effectively reduce the occurrence of diseases and the use of antibiotics in order to improve the quality of economic aquatic products has become an urgent problem.Chinese mitten crab(Eriocheir sinensis)as an important economic crab in China is well loved by the public.However,with the increasing expansion of breeding scale and density,it is also facing various risks of disease occurrence.Therefore,in-depth research on the basic immunology of this crab can provide important theoretical support for disease control,vaccine development,and future molecular breeding of disease resistance in this crab and other crustaceans.interleukins(ILs)are cytokines that play an important role in both innate and adaptive immunity.Vertebrate ILs have a wide variety of species and functions.The members of the interleukin(IL)superfamily are mainly cytokines produced and secreted by CD3~+and CD4~+T lymphocytes,which can promote the development and differentiation of immune-related cells,thereby participating in the regulation of the systemic immune system.However,only a very small number of ILs genes have been evolutionarily traced back to invertebrates.Interleukin-16(IL-16),as a pro-inflammatory factor,binds to the receptor CD4 in vertebrates to recruit CD4+cells to transfer to the inflammatory site and promote cell proliferation and differentiation.IL-16 is the only interleukin in Chinese mitten crab,and its immune mechanism and function are still unclear.In particular,the receptor of IL-16 in vertebrates is CD4,while invertebrates do not have this receptor.Therefore,in-depth exploration of the interaction protein of IL-16 in the Chinese mitten crab and its regulatory function can help us better understand the differences and connections between vertebrate and invertebrate immune mechanisms from the perspective of evolution.In this paper,the full-length sequence of Es IL-16 was obtained by analyzing the whole genome sequencing results and transcriptome information of Eriocheir sinensis,and the open reading frame(ORF)sequence of Es IL-16 was determined by PCR amplification and sequencing.Bioinformatics analysis showed that Es IL-16 was relatively conserved in evolution,especially that the PDZ functional domain was the same among different species.Es IL-16 was expressed in all tissues,but after stimulation by Gram-positive bacteria(represented by Staphylococcus aureus)and Gram-negative bacteria(represented by Vibrio parahemolyticus),Es IL-16 expression in hemocytes was significantly up-regulated,which initially indicated that Es IL-16 was related to immune response.Es IL-16 also exists in the form of the precursor pro-IL-16and the Caspase-3 cleavage site.Experimental verification found that under normal conditions,Caspase-3 cleavage enzyme was in a state of no activity or weak activity.However,Caspase-3 cleavage enzyme was activated after bacterial stimulation and cleaved pro-IL-16 to produce a large number of active mature IL-16.The N-terminus can enter the nucleus,and the C-terminus forms a mature peptide then is secreted out of the cell.Therefore,which receptor does the mature Chinese mitten crab(Eriocheir sinensis)bind to after secreting IL-16 from the cell?What physiological functions do they perform?In order to solve the above problems,we used T7 phage display library to select Es IL-16 as the ligand protein for receptor screening,and verified the candidate receptor by Co-IP technology,laser confocal technology and binding functional domain verification.The results showed that the receptor of Es IL-16 was Es Integrinβ1.Es IL-16 combines the INB functional domain of Es Integrinβ1,while Es Integrinβ1 combines the non-PDZ domain of Es IL-16.By Qrt-PCR detection,Es Integrinβ1 was widely expressed in various tissues of Chinese mittens crab,and the expression level of hemocytes was significantly increased after stimulation by Staphylococcus aureus and Vibrio parahemolyticus,which also suggested that Es Integrinβ1 played a positive role in antibacterial immunity.In view of the fact that both IL-16 and Integrinβ1 have the function of promoting cell proliferation in vertebrates,the EDU and CFSE cell proliferation detection techniques were used to detect the hemocytes of Chinese mitten crab.The results showed that adding recombinant Es IL-16 protein could significantly promote the proliferation of Chinese mitten crab hemocytes after 12 hours of bacterial stimulation.When the Es Integrinβ1 gene was knocked down under the same conditions,there was almost no proliferation of hemocytes compared with the control group.These results provide further evidence that Es IL-16 can promote cell proliferation only by binding to the receptor Es Integrinβ1.When Es Integrinβ1 gene was knocked down by RNAi technology and Integrinβ1 inhibitor was used,the apoptosis rate of hemocytes increased significantly.In order to further explore the mechanism of Es IL-16 and Es Integrinβ1 binding to promote the proliferation of hemocytes,this paper studied the expression of two proteins,Cyclin E and Cyclin B,which regulate cell cycle.After 12 hours of bacterial stimulation,recombinant Es IL-16 protein was added,and the expression of Cyclin E and Cyclin B was detected by Qrt-PCR.The results showed that the expression of Cyclin E was significantly increased,while Cyclin B had no significant difference compared with the control group.This also suggests that Es IL-16 may promote cell proliferation by upregulating Cyclin E expression after binding to Es Integrinβ1.In order to further verify the signal transduction mode of high expression of Cyclin E after Es IL-16 is combined with Es Integrinβ1 to promote the proliferation of hemocytes,this study used inhibitors and RNAi knockdown technology to reduce Es Integrinβ1 and related genes of its downstream signaling pathway.The protein expression of Cyclin E was detected by western blotting.The results showed that Es IL-16 combined with Es Integrinβ1 could promote the phosphorylation of adhesion spot kinase(FAK),and finally up-regulate the gene expression of Cyclin E by activating a certain signaling pathway.As a large amount of Cyclin E protein accumulated,Promote the transition from prophase G1 to late phase G1,and ultimately promote the proliferation of hemocytes.In addition,Es Integrinβ1 was found to promote the nucleation of ERK phosphate by phosphorylating FAK,and inhibit the activity of Caspase-3 cleavage enzyme,thereby inhibiting apoptosis.In conclusion,this study confirmed that the receptor of IL-16 in invertebrate Chinese mitten crab is Integrinβ1,and can promote the proliferation of hemocytes through up-regulation of Cyclin E.Integrinβ1 regulates hemocytes survival in the Chinese mitten crab through the FAK/ERK pathway.This study enriches the immune regulation mechanism of IL-16 in invertebrates and opens a new perspective on the immune system of invertebrates and vertebrates at the evolutionary level. |
PDF Full Text Request