Lipopolysaccharide Signaling without a Nucleus: Kinase Cascades Stimulate Platelet Shedding of Proinflammatory IL-1β-Rich Microparticles

Platelets contain unspliced heteronuclear IL-1 beta RNA, which is rapidly spliced and translated upon activation. LPS is a superior agonist for this atypical platelet response, but how LPS induces proinflammatory cytokine production in anucleate cells lacking NF-kappa B is unknown. Platelets express functional TLR4, and stimulation by LPS induced rapid splicing, translation, and secretion of mature IL-1 beta after caspase-1 processing. LPS stimulated microparticle shedding, and secreted IL-1 beta was exclusively present in these particles. Microparticles from LPS-stimulated platelets induced VCAM-1 production by cultured human endothelial cells, and blockade of endothelial IL-1 beta receptor with IL-1 receptor antagonist completely suppressed endothelial activation. Splicing was posttranscriptional as the SR kinase inhibitor TG003 blocked IL-1 beta RNA production by platelets, but not by monocytes, and was dependent on exogenous CD14-a property of platelets. We used a combination of small-molecule inhibitors, cell-penetrating chimeric peptide inhibitors, and gene-targeted animals to show splicing required MyD88 and TIRAP, and IRAK1/4, Akt, and JNK phosphorylation and activation. Traf6 couples MyD88 to the Akt pathway and, remarkably, a Traf6 interacting peptide-antennapedia chimera was more effective than LPS in stimulating IL-1 beta splicing. The Traf6 chimera did not, however, stimulate microparticle shedding, nor was IL-1 beta released. We conclude LPS-induced kinase cascades are sufficient to alter cellular responses, that three signals emanate from platelet TLR4, and that Akt and JNK activation are sufficient to initiate posttranscriptional splicing while another event couples microparticle shedding to TLR4 activation. Platelets contribute to the inflammatory response to LPS through production of microparticles that promote endothelial cell activation. The Journal of Immunology, 2011, 186: 5489-5496.