Effect Of SSL5Induced Platelets Microparticles On THP-1Cells

Background and ObjectiveAtherosclerosis (AS) is associated with a variety of cardiovascular risk factors. Inrecent years, infectious burden has been considered as a new risk factor ofatherosclerosis, which may accelerate the formation of AS lesions though the mechanismhas not been well illustrated yet.Inflammation plays a vital role in atherosclerosis. Monocytes/macrophages are veryimportant in the process of inflammation and the formation of foam cells. Plateletmicroparticles (PMPs) are released from activated platelets, the interaction ofmonocytes/macrophages and PMPs will promote vascular inflammation and foam cellformation.Staphylococcal superantigen like protein-5(SSL5) is secreted by staphylococcusaureus (S. aureus). Our previous study has demostrated that SSL5can activate plateletsby binding to GPIbα and GPVI. Here we further studied the atherosclerogenicmechanism of SSL5by observing the effect of SSL5-induced PMPs (SSL5-PMPs) onmonocytes and foam cell formation.Methods1. The expression and purification of reconstructed SSL5protein was performedaccording to the methods established in our lab.2. The preparation of PMPs released by activated plarelets1) Washed human platelets were activated by SSL5and adenosine diphosphate(ADP) respectively, the PMPs were isolated by centrifugation. PMPs were double stainedby PE labeled mouse anti-human CD62P monoclonal antibody and FITC-AnnexinV anddetected by flow cytometry (FCM) to check the activation and phosphatidylserine (PS).In addition, SSL5-PMPs were observed with scanning electron and confocal microscopy.2) The expression of CD41and CD154(CD40ligend, CD40L) on SSL5-PMPs was checked by each antibody using FCM.3) The effect of SSL5on mitochondrial membrane potential of human platelets wasassayed by JC-1kit.3. Cellular experiments1) The binding rates of SSL5-PMPs to THP-1cells were detected by FCM.Scanning electron microscopy and confocal microscopy were used to observe the bindingof SSL5-PMPs to THP-1cells.2) The activation of Mac-1(CD11b/CD18, αMβ2) on monocytes was assayed by PElabled CBRM1/5mAb using FCM.3) The mRNA expression of TNFα and IL-1β in monocytes was assayed byreal-time PCR. TNFα in the medium was assayed by ELISA.4) Lipid droplets in cytoplasm and foam cells were checked by oil red O.5) The mRNA expression of LXRα、LXRβ、ABCA1in THP-1derived foam cellswas assayed by real-time PCR.Results1. SSL5-PMPsBoth CD62P and PS were positively expressed on SSL5-PMPs. The positive rates ofCD41, CD62P, PS and CD40L on SSL5-PMPs were47%,40.6%,63%and47.6%respectively. There is not significant change on the mitochondrial membrane potentialplatelets before and after SSL5stimulation (P>0.05).2. The effect of SSL5-PMPs on THP-1cells1) The binding rates of SSL5-PMPs to THP-1cellsComparing with the control group [(16.59±2.938)%], the binding rates ofSSL5-PMPs to THP-1cells was significantly increased to（53.39±7.638）%(P <0.05).2) SSL5-PMPs induced the activation of Mac-1on THP-1cellsThe activation rates of Mac-1on THP-1cells were40.9%after co-incubation withSSL5-PMPs.3) SSL5-PMPs induced TNFα and IL-1β expression in THP-1cells.Comparing with the control group, both SSL5-PMPs and ADP-PMPs couldsignificantly increase TNFα and IL-1β mRNA expression in THP-1cells（P<0.05).Comparing with the control group （[320.3士146.5）pg/ml], TNFα protein levels in the medium of THP-1cells in SSL5-PMPs and ADP-PMPs group were significantlyincreased to (1018±246.5）pg/ml and（1041±231.7）pg/ml respectively（P<0.05).3. SSL5-PMPs promoted foam cells formationSSL5-PMPs significantly promoted the deposition of lipids in THP-1derivedmacrophages and the formation of foam cells in a concentration dependent manner.4. The mRNA expression of LXRα、LXRβ、ABCA1in THP-1derived foam cellsComparing with the control group, SSL5-PMPs didn’t affect the mRNA expressionof LXRαand LXRβ significantly in THP-1derived foam cell（P>0.05), but the mRNAexpression of ABCA1was significantly up-regulated（P <0.01).Conclusions1. SSL5-PMPs have the general characteristics of PMPs with the expression ofCD41, CD62P, CD40L and PS on the surface. The release of SSL5-PMPs was noassociated with platelet apopotosis.2. SSL5-PMPs could bind to THP-1cells, enhanced the conformation change ofMac-1and activation, up-regulated the mRNA expression of TNFα, IL-1β and increasedTNFα protein levels in the medium of SSL5-PMPs treatment THP-1cells, causedinflammation response.3. SSL5-PMPs could promote the deposition of oxLDL in THP-1derived foam cellsformation, these is likely associated with the imbalance response of ABCA1mRNA.