The Functions Of Histone Deacetylases 3 In Innate And Trained Immunity Of Marsupenaeus Japonicus

Shrimp farming is an important marine aquaculture industry and creates huge economic benefits,but frequently outbreaks of shrimp diseases have caused significant losses in the farming industry.In order to effectively control the diseases,the innate immune mechanism of shrimp needs to be further explored Acetylation modification is one of the important epigenetic modifications in organisms,which is related to the regulation of innate immune response and innate immune memory(trained immunity).Several histone acetyltransferase(HATs)and histone deacetylase(HDACs)involve in acetylation modification in shrimp,but there are few reports on the study of acetylation modification in shrimp immunty.In this thesis,we studied the function of the deacetylases in the shrimp innate immunity and memory of innate immunity.1.Histone deacetylase 3 regulates histone H3K27 acetylation modification to promote shrimp innate immunity against bacteriaAcetylation modification is a kind of reversible dynamic modification performed by histone acetyltransferase(HATs)and histone deacetylase(HDACs)in organisms.Studies have found that acetylation modification not only occurs on histones,affects the assembly and construction of chromatins,and regulation of gene expression.Acetylation modifications also exist in non-histones,affecting many important signal pathways in the growth and development,metabolism,and disease occurrence of organisms.HDACs are a class of zinc-dependent metalloproteinases that can promote protein deacetylation,regulate gene transcription,protein activity,and cytoplasmic and nuclear localization.At present,the functions of HDACs have been thoroughly researched in mammals,but few studies are reported in invertebrates.In order to study the function of histone deacetylase in the innate immunity of shrimp,we screened the Marsupenaeus japonicus transcriptome data and identified five histone deacetylases(HDACs)in the shrimp,including class ? HDACs:HDAC1,HDAC3,HDAC8,and class ? HDACs:Hdac4,Hdac6.Expression pattern analysis of the Hdacs was carried out,and the results showed that their mRNA expression was upregulated in shrimp infected by Vibrio anguillarum.Histone deacetylase 3 plays an important role in life processes such as pro-inflammatory factor expression,cancer development,biological development and cell differentiation in mammals.There are few reports on the functions of the enzyme in invertebrate innate immunity.Therefore,we selected histone deacetylase 3(HDAC3)for functional and mechanism studies.we firstly used RNA interference to knock down Hdac3 expression,and the bacterial loads,and survival rate of the shrimp challenged with Vibrio anguillarum were analyzed.The results showed that the number of bacteria in the hemolymph and gastrointestinal tract of shrimp increased significantly,the survival rate of shrimp was declined significantly.The possible mechanism of HDAC3 against bacteria in shrimp was further studied.After knockdown of Hdac3 in shrimp following bacterial infection,the expression of antimicrobial peptide(Amps)genes such as anti-lipopolysaccharide factor(Alf)were significantly declined.To determine the function of HDAC3,we used RGFP966,a selective inhibitor of HDAC3,to inhibit the activity of HDAC3 in shrimp,and similarly found that the gene expression of the antimicrobial peptides Alf-b1,Alf-c1,Alf-c2,and Alf-dl was significantly reduced and the bacterial number of hemolymph and gastrointestinal tract was significantly increased.In further mechanism study,we found that HDAC3 translocated from nucleus to the cytoplasm in hemocytes of shrimp infected with V anguillarum.The cytoplasmic translocation of HDAC3 enhanced acetylation modification of chromatin H3K27 in the nucleus and may promote the transcription factor STAT and Dorsal translocated into the nucleus,eventually regulate downstream antimicrobial peptide gene expression.Therefore,histone deacetylase 3 regulates the innate immune process of shrimp by regulating histone acetylation modification and nuclear translocation of transcription factors of STAT and Dorsal.2.The function of Histone deacetylase 3 in antimicrobial training immunity of shrimpThe discovery of trained immunity has subverted the theory of classical innate immunity having no memory.Current studies show that there is trained immunity in shrimp,but the mechanism of the trained immunity is not clear.To investigate the mechanism of shrimp trained immunity,we constructed a training model using inactivated V.anguillarum to induce trained immunity in M.japonicus.Through this model,we found that oral feeding inactivated V.anguillarum can promote the non-specific trained immunity in the shrimp.The shrimp were fed with inactivated V.anguillarum for immune priming,and then infected with V.anguillarum or white spot syndrome virus(WSSV)after 7 day-resting time.The loads of bacteria or viruses were analyzed.The results showed that the bacteria or viral numbers were significantly declined,and survival rate obviously increased comparing with the control.We also found the expression of immune related genes,such as Alf-c2?Crus I-2?ProPO and Toll1,was faster and stronger than the control.To explore the possible mechanisms of the trained immunity induced by the bacteria,the RGFP966,an inhibitor of histone deacetylase 3,was injected into inactivated Vibio primed shrimp,and bacterial number and survival rate of the shrimp were analyzed.The results showed that the HDAC3 inhibitor blocked the process of inactivated V.anguillarum induced trained immunity and reduced the survival rate of the shrimp.The result suggest that Acetylation modifications associated with HDAC3 play an important role in the antimicrobial trained immunity of shrimp.The mechanism in detail needs further study.