| BackgroundCardiovascular disease (CVD) is the leading cause of death worldwide. In most cases, CVD is caused by atherosclerosis, a chronic process characterized by inflammation of blood vessels due to a variety of etiologies. More infectious agents contributing to chronic inflammatory diseases including CVD, labelled as "infectious burden", are considered to be a new and independent risk factor for atherosclerosis. Some microorganisms, such as Chlamydia pneumonia and human cytomegalovirus, may act directly on aterial wall, and others, such as influenza virus and Helicobacter pylori, may induce a systemic inflammation, which in turn damage the aterial wall. Moreover, some infectious agents, such as periodontal pathogens, may be involved in the pathogenesis of atherosclerosis via molecular mimicry. Currently, vascular inflammation and thrombosis, largely resulted from pathogens-induced platelet activation, have been demonstrated to play a vital role in the development of atherosclerosis. Staphylococcus aureus (S. aureus) is one of the most common bacteria to humans. Previous studies have reported that blood infection with S. aureus was associated with an increased risk of myocardial infarction or stroke. The precise mechanisms are still unclear but many involve platelet activation, aggravated inflammation and prothrombotic status. We previously reported that staphylococcal superantigen-like protein 5 (SSL5) is able to activate platelets and induce lung thromboembolism in mice by tail vein injection. It is now clear that in addition to thrombosis and haemostasis, platelets are also important immune and inflammatory cells. Platelets have been found to promote inflammation through shedding microparticles or interaction with leukocytes. Here I hypothesized that SSL5 may initiate proinflammatory responses by activating platelets. Thus, exploring the mechanism can provide new pieces of evidence for the role of infectious burden in atherosclerosis.Methods1. The biologic activity of recombinant SSL5 was evaluated by flow cytometry before use. Flow cytometry analysis was used to quantify the number of platelet microparticles (PMPs) in human peripheral blood and to detect surface molecule expression of SSL5-induced platelet microparticles (SSL5-PMPs). The morphological characteristics of SSL5-PMPs were observed by scanning electron microscopy. Further, SSL5-PMPs were prepared in vitro for stimulator and ADP-induced PMPs (ADP-PMPs) as well as apoptosis-induced PMPs (ap-PMPs) for controls in follow-up experiments.2. Both monocytes isolated from human peripheral blood and human acute monocytic leukaemia cell line (THP-1) were cultured for experiments. CCK-8 assay was applied to evaluate the cell viability of SSL5 on THP-1 cells. Binding of SSL5-PMPs to THP-1 cells was detected by scanning electron microscopy and flow cytometry, and flow cytometry was further used to analyse binding time-course and preference of SSL5-PMPs binding to monocyte subsets. The mRNA expression of inflammatory mediators, such as interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9), was examined by real-time quantitative PCR, and secreted IL-1β, TNFa as well as MCP-1 were quantified using a special ELISA kit. The activity of MMP-9 in supernatant of culture medium was analysed by gelatin zymography. A transwell migration assay was designed to detect the effect of SSL5-PMPs on MCP-1-induced migration of THP-1 cells. In addition, the effects of both ADP-PMPs and ap-PMPs set as control PMPs as well as SSL5 on proinflammatory responses in THP-1 cells were also investigated to explore the difference of various PMPs in their capacity of inflaming monocytes and the pathological role of SSL5 involved, respectively.3. THP-1 cells were used for mechanistic study. To examine the molecular basis of SSL5-PMP and monocyte interactions resulting in inflammatory mediator production and migration of THP-1 cells, neutralising antibodies were used to block the binding of CD40L to CD40 or of P-selectin to PSGL-1. Further, to uncover the signaling pathway involved, production of inflammatory mediators induced by SSL5-PMPs was detected after using small interfering RNA (siRNA) to suppress CD40 or TNF receptor (TNFR)-associated factor 6 (TRAF6) mRNA expression in THP-1 cells. Under the same conditions, phosphorylation and nuclear translocation of NFκB of THP-1 cells were examined using Western blot analysis and immunofluorescence assay, respectively. Finally, potential involvement of Toll-like receptor 4 (TLR4) signaling pathway in the proinflammatory response in THP-1 cells by SSL5-PMPs was also investigated by using a specific TLR4 inhibitor.ResultsThree main findings were made in the present study:1. SSL5 induces the production of PMPs.①Recombinant SSL5 was identified with biological activity, which could be used for subsequent experiments.② SSL5 treatment resulted in a significant increase of PMPs in human blood ex vivo.③ SSL5-activated PMPs were identified by flow cytometry, according to their expression of glycoprotein (GP) Ⅱb as a platelet-specific marker and exposure of phosphatidylserine (PS). Signalling molecules involved in cellular interactions, such as P-selectin and CD40L, were also highly expressed on the SSL5-PMPs’ surface. In addition, scanning electron microscopy showed that SSL5-PMPs were heterogeneous in size ranging from 0.1 to 1 μm.2. SSL5-PMPs provoke proinflammatory responses in monocytes.①THP-1 cell viability was not influenced by SSL5, ruling out proinflammatory responses caused by cytotoxicity of SSL5 in monocytes.② Flow cytometry analysis revealed that the number of SSL5-PMPs binding to THP-1 cells increased with the prolongation of coincubation time and that SSL5-PMPs predominantly bound to CD14++CD16+(intermediate) monocyte subset. In addition, the electro-micrograph also verified SSL5-PMPs adhering to the surface of THP-1 cells after coincubation.③ SSL5-PMPs time- and dose-dependently upregulated mRNA expression of inflammatory mediators in THP-1 cells, including IL-1β, TNFα, MCP-1 and MMP-9. Moreover, SSL5-PMPs treatment evoked a dose-dependent secretion of IL-1β, TNFα and MCP-1 by both THP-1 cells and monocytes. SSL5-PMPs promoted THP-1 cells and monocytes to secret active MMP-9 and the increase of MMP-9 activity quantified by zymograms was SSL5-PMPs dose-dependent.④ ADP-PMPs or ap-PMPs also led to increased mRNA expression of all four inflammatory mediators in THP-1 cells, the level of which is lower than that caused by SSL5-PMPs stimulation, while SSL5 had no effect on mRNA expression of inflammatory mediators.⑤ SSL5-PMPs enhanced MCP-1-induced migration of THP-1 cells, whereas SSL5 itself potently inhibited MCP-1 chemotaxis.3. The mechamism by which SSL5-PMPs provoke proinflammatory responses in monocytes.①Neutralising antibody based blockade of CD40L-CD40 interactions significantly decreased IL-1β mRNA expression and secretion in THP-1 cells. In contrast, interruption of P-selectin binding to PSGL-1 using PSGL-1 mAb did not have such an effect.② In the presence of PSGL-1 mAb, SSL5-PMPs-mediated increase of THP-1 cell migration was largely abolished. Similarly, blockade of CD40L-CD40 interactions also reduced the percent of THP-1 cell migration in response to SSL5-PMPs.③ SSL5-PMP-induced IL-1β production was inhibited by downregulation of CD40 or TRAF6 gene expression in THP-1 cells by siRNA. On the other hand, siRNA-mediated silencing of CD40 or TRAF6 gene largely abrogated phosphorylation and nuclear translocation of NFκB (p65) in THP-1 cells.④ In THP-1 cells, blockade of TLR4-mediated signalling did not significantly decrease IL-1βrelease stimulated by SSL5-PMPs.ConclusionsIn the present study, we demonstrated that SSL5, a S. aureus secreted toxin, activates generation of microparticles by platelets (SSL5-PMPs), thereby leading to a significant increase of PMPs in human blood ex vivo. SSL5-PMPs are able to bind predominantly to proinflammatory intermediate monocytes. As a result, SSL5-PMPs provoke extensive proinflammatory responses in monocytes, including release of cytokines and chemokines, activation of matrix metalloproteinases and monocyte migration. The possible mechanism is that SSL5-PMPs trigger proinflammatory responses via PMPs-derived CD40L directly interacting with monocyte CD40 and further activation of CD40-TRAF6-NFκB signaling pathway. This occurs despite the fact that SSL5 itself normally inhibits leukocyte function. Our findings reveal a novel mechanism by which S. aureus induces inflammation, which facilitates to clarify the mechanism underlying infectious burden related to atherosclerosis in a new perspective. |
