Expression and functionality of Toll‐like receptor 3 in the megakaryocytic lineage

Summary Background In addition to their key role in hemostasis, platelets and megakaryocytes regulate immune and inflammatory responses, in part through their expression of Toll‐like receptors ( TLR s). Among the TLR s, TLR 3 recognizes ds RNA associated with viral infection. Thrombocytopenia is a frequent complication of viral infection. However, the expression and functionality of TLR 3 in megakaryocytes and platelets is not yet well understood. Objective To study the expression and functionality of TLR 3 in the megakaryocytic lineage. Methods and results RT ‐ PCR , flow cytometric and immunofluorescence assays showed that TLR 3 is expressed in CD 34 + cells, megakaryocytes, and platelets. Immunoblotting assays showed that stimulation of megakaryocytes with two synthetic agonists of TLR 3, Poly(I:C) and Poly(A:U), activated the nuclear factor‐κB ( NF ‐κB), phosphoinositide 3‐kinase ( PI 3K)/Akt, extracellular signal‐related kinase ( ERK )1/2 and p38 pathways. TLR 3–megakaryocyte activation resulted in reduced platelet production in vitro and interferon‐β release through the PI 3K–Akt and NF ‐κB signaling pathways. TLR 3 ligands potentiated the aggregation mediated by classic platelet agonists. This effect was also observed for ATP release, but not for P‐selectin or CD 40L membrane exposure, indicating that TLR 3 activation was not involved in α‐granule release. In addition, TLR 3 agonists induced activation of the NF ‐κB, PI 3K–Akt and ERK 1/2 pathways in platelets. Reductions in platelet production and platelet fibrinogen binding mediated by Poly(I:C) or Poly(A:U) were prevented by the presence of an inhibitor of the TLR 3–ds RNA complex. Conclusions Our findings indicate that functional TLR 3 is expressed in CD 34 + cells, megakaryocytes, and platelets, and suggest a potential role for this receptor in the megakaryopoiesis/thrombopoiesis alterations that occur in viral infections.