Staphylococcal SSL5-induced platelet microparticles provoke proinflammatory responses via the CD40/TRAF6/NFKB signalling pathway in monocytes

Abstract

Pathogens-induced platelet activation contributes to inflammation in cardiovascular diseases, but underlying mechanisms remain elusive. Staphylococcal superantigen-like protein 5 (SSL5) is a known activator of platelets. Here we examined whether SSL5 is implicated in Staphylococcus aureus (S. aureus)-induced inflammation and potential mechanisms involved. As expected, we show that SSL5 activates human platelets and induces generation of platelet microparticles (PMPs). Flow cytometry and scanning electron microscopy studies demonstrate that SSL5-induced PMPs (SSL5-PMPs) bind to monocytes, causing aggregate formation. In addition, SSL5-PMPs provoke monocyte expression and release of inflammatory mediators, including interleukin-1β (IL-1β), tumour necrosis factor-α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) in a dose- and time-dependent manner. SSL5-PMPs also enhance MCP-1-induced monocyte migration. Blockade of CD40 and CD40 ligand (CD40L) interactions with neutralising antibodies significantly reduce monocyte release of inflammatory mediators and migration induced by SSL5-PMPs. SiRNA-mediated silencing of CD40 or TNF receptor (TNFR)-associated factor 6 (TRAF6) gene largely abrogates phosphorylation and nuclear translocation of NFκB (p65). In conclusion, SSL5 provokes the release of inflammatory mediators in monocytes, at least in part, via PMPs-mediated activation of the CD40/TRAF6/NFκB signalling pathway, though it normally inhibits leukocyte function. Our findings thus reveal a novel mechanism by which S. aureus induces inflammation.

Up-Regulated Expression of Matrix Metalloproteinases in Endothelial Cells Mediates Platelet Microvesicle-Induced Angiogenesis

“…Of the microvesicles circulating in blood, 70% to 90% are derived from platelets [15, 26] in response to various stimuli, such as ADP, collagen, thrombin, or following complement activation [27]. Since platelet activation, characteristically occurs at the sites of blood flow disturbances or endothelial injury, where angiogenesis takes place (e.g., in tumor vasculature [28], or in proximity to thickened tunica intima [29]), the possible impact of PMVs on blood vessel development is an important issue.…”

“…Washed platelets were prepared as previously described [15]. Briefly, Platelet rich plasma (PRP) was obtained by centrifugation of the above sample at 110×g for 15 min to remove RBCs and leukocytes.…”

“…Finally, the pellets containing microvesicles were resuspended in Tyrode’s buffer, which were directly used, or snap-frozen at -80°C to avoid repeated freezing and thawing. The amount of PMVs was quantified by both bicinchoninic acid (BCA) protein assay (Beyotime Biotechnology) and flow cytometry as we described previously [15]. We found that 50 µg/ml of protein concentration was equivalent to 6 × 10 6 /ml PMVs.…”

“…MMP-2/MMP-9 protein abundance and activation in conditioned medium were analyzed by gelatin zymography as described previously [15, 16]. Briefly, HUVECs were incubated in the presence of Tyrode’s buffer, supernatants, or different concentrations (10, 20, or 50 µg/ml) of PMVs in serum-free medium for 24 hours.…”

“…Mounting evidences indicate that PMVs not only passively transport various proteins and receptors from platelets or megakaryocytes to recipient cells, but also act as circulating signaling modules by inducing specific responses in target cells. We previously demonstrated that SSL5-PMPs, staphylococcal superantigen-like protein 5 (SSL5)-induced PMPs, provoke inflammatory mediator release in monocytes [15]. Besides, PMVs induce changes in early outgrowth cells (EOCs) secretome towards a more pro-angiogenic profile [10], which plays a pivotal role in vasculogenesis.…”