Role And Mechanism Of Staphylococcal Enterotoxin B-induced THP-1 Cell Apoptosis

Apoptosis is an important biological process in multicellular organisms,which plays a regulatory role in tissue differentiation,metabolism and the immune regulation.In bacterial-caused infectious diseases,certain components of bacteria can regulate host cell apoptosis through a variety of ways,and this has also been shown to ha ve a crucial impact on the progression of the disease.As an important human pathogen,Staphylococcus aureus(S.aureus)can cause host cell apoptosis in its infection process.In many diseases closely related to S.aureus,such as atopic dermatitis(AD)and sepsis,abnormal host cell apoptosis has significantly affected the course,severity,and prognosis of the disease.An important feature of S.aureus is its secretion of a variety of toxins,including hemolysins,leukocidins,enterotoxins,secreted enzymes etc.Among these toxins,enterotoxins as important S.aureus superantigens,play an important role in many diseases closely related to S.aureus.The natural receptors of enterotoxins include T cell receptor(TCR)and major histocompatibility complex II(MHCII).Staphylococcal enterotoxins(SEs)can not only bind to TCR to continuously activate T cells,causing hyper-inflammatory responses;but also bind to MHCII to induce cell apoptosis.As a widely distributed and deeply studied enterotoxin,staphylococcal enterotoxin B(SEB)has been shown to cause apoptosis in T cells and peripheral blood mononuclear cells(PBMCs),and this apoptosis is closely relat ed to S.aureus-induced diseases such as AD.But whether SEB can cause monocytes apoptosis,and the mechanisms underlying are still unclear.In this study,we cloned,expressed and purified the recombinant SEB of the S.aureus strain XQ,which was isolated and sequenced by our research group,and explored the role and mechanism of SEB-induced apoptosis in THP-1 monocytic cell line.We demonstrated that a positive feedback cycle of TNF? played a very important role in this process.At the same time,we found that metallothionein 2A(MT2A)could affect the positive feedback cycle by promoting the SEB-induced up-regulation of TNF? and HLA-DRa,therefore take part in SEB-induced THP-1 cell apoptosis.The results of this study shed lights on further researches on the mechanism of SEs and the response mechanism of host cell to bacterial toxin.The main methods and results are as followings:1.Construction,expression and purification of the recombinant SEBWe obtained the SEB coding sequence from the whole genome of S.aureus strain XQ by PCR,and successfully constructed the expression plasmid p ET30a-SEB by Bam HI+Xho I double enzymes digestion of the coding sequence and the plasmid,and subsequently ligation with T4 ligase.The recombinant SEB was finally gained from p ET30a-SEB transformed Escherichia coli(E.coli)strain C43.The recombinant SEB was purified by nickel affinity chromatography,and the endotoxins were removed by endotoxin-removing gel,the purified recombinant SEB was concentrated and the buffer was replaced by PBS using Amicon Centrifugal Filter Units.The purity,concentration and residual LPS of the recombinant SEB were determined by SDS-PAGE,Bradford assay kit and tachypleus amebocyte lysate test.The purity was abov e 95%,the concentration was 539.2?g/ml and the concentration of the residual LPS was less than 2EU/ml,which all satisfied the further cell experimental requirements.2.The recombinant SEB-induced THP-1 cell apoptosisThe THP-1 cell line was subjected to short tandem repeat(STR)testing,and no cell contamination was observed.The THP-1 cell line had a similarity of 93.33% compared with that retained in American type culture collection(ATCC),confirming that the cells used in this study is a derivative cell line of THP-1.Using Annexin V + PI double staining and flow cytometry,we determined the recombinant SEB induced THP-1 cell apoptosis with a proportion of about 3%,in a timeand dose-dependent manner.Using broad-spectrum caspase inhibitor Z-VAD-FMK,we verified the recombinant SEB-induced THP-1 cell apoptosis is caspase-dependent.In the IFN-? activated or PMA differentiated THP-1 cells,the recombinant SEB induced a higher proportion of apoptosis with a proportion of 7.5% and 15%,respectiv ely,determined by Annexin V + PI double staining and flow cytometry.Whereas the enhanced inducing effect on apoptosis was not observed in endotoxin(LPS)activated THP-1 cells.However,in the IFN-? activated or PMA differentiated THP-1 cells,the percentage of apoptosis cells was also increased in the control group treated with PBS.Therefore,treating THP-1 cells directly with the recombinant SEB was preferred in our further experiments to exclude the confusion on the actual effect of SEB with additional cytokines.3.The recombinant SEB-induced THP-1 cell apoptosis depended on a positive feedback cycle of TNF?There are two classical apoptosis pathways: the extrinsic pathway(initiated by caspase-8)and the intrinsic pathway(initiated by caspase-9).Using caspase-3,-8,-9 activity assay kits,we found that the recombinant SEB-induced THP-1 cell apoptosis depended on the cascade activation of caspase-3 and-8,but not caspase-9,which indicated the extrinsic pathway played an important role in the recombinant SEB induced THP-1 cell apoptosis.Using RT-q PCR,we next detected the transcriptional level of extrinsic apoptosis pathway related molecules in THP-1 cells treated with the recombinant SEB.We found that the transcriptional level of TNF? and HLA-DRa were up-regulated,which were further confirmed by ELISA and Western blot.Using anti-TNF? monoclonal antibody to neutralize TNF?,we found the recombinant SEB-induced apoptosis was significantly decreased to about 1%,whereas the proportion of apoptosis cells was still about 3.5% treated directly with the recombinant SEB or in the control group using an isotype control IgG.These results indicated TNF? played a central role in the process of SEB-induced THP-1 cell apoptosis.Using siRNA to knock down HLA-DRa and TNFR1,and a non-target siRNA as control,we demonstrated the recombinant SEB-induced THP-1 cell apoptosis was much lower in the knock down cells(the proportion was about 2.5%)than that in the control cells(the proportion was about 9%).Meanwhile,the level of TNF? secretion was also significantly lower in the knock down cells than that in the control cells.In consideration of TNF? is a key molecule to up-regulate HLA-DRa expression,augmenting the ligation of recombinant SEB to its receptor HLA-DRa,so that the secretion of TNF? further increased,thus,a positive feedback cycle of TNF? formatted.The recombinant SEB-induced THP-1 cell apoptosis depended on this positive feedback cycle of TNF?.4.MT2 A affected the recombinant SEB-induced THP-1 cell apoptosis by affecting the positive feedback cycle of TNF?As the receptor of SEB,HLA-DRa contains a relatively short cytoplasmic tail without a predicted motif,its signal transduction function was presumed to be achieved by other membrane proteins that bind to it.Although the recombinant SEB-induced THP-1 cell apoptosis depended on the positive feedback cycle of TNF?,but the mechanisms by which TNF? secretion was up-regulated after the recombinant SEB binding with HLA-DRa was still unclear.Therefore we further used immunoprecipitation techniques to screen the proteins interacting with HLA-DRa by an anti-HLA-DRa monoclonal antibody and found that metallothionein 2A(MT2A)may play a key role.We determined the expression profile of metallothioneins(MTs)in THP-1 cells and their changes in transcription level after treatment with recombinant SEB by RT-q PCR.The result showed the MTs expressed in THP-1 cells include MT1 E,F,G,X and MT2 A,and only the expression of MT2 A increased after treatment of the recombinant SEB.The up-regulation of MT2 A in recombinant SEB treated THP-1 cells was further confirmed by Western blot.Using si RNA to knock down MT2 A expression and a non-target si RNA as control,we found that the recombinant SEB-induced apoptosis was much lower in the MT2 A si RNA knocked down cells(the proportion was about 4%)than that in the control cells(the proportion was about 8%),determined by Annexin V + PI double staining and flow cytometry.Using the ELISA and Western blot,we further detected that,the expression of TNF? and HLA-DRa were both decreased in the MT2 A si RNA knock down THP-1 cells,indicating that MT2 A played an important role in the positive feedback cycle of TNF?,and thus promoting the recombinant SEB-induced THP-1 cell apoptosis.