Activation of toll‐like receptors 2 and 4 on CD 34 + cells increases human megakaryo/thrombopoiesis induced by thrombopoietin

Background Platelet Toll‐like receptor (TLR) 2/4 are key players in amplifying the host immune response; however, their role in human megakaryo/thrombopoiesis has not yet been defined. Objectives We evaluated whether Pam3 CSK 4 or lipopolysaccharide ( LPS ), TLR 2/4 ligands respectively, modulate human megakaryocyte development and platelet production. Methods CD 34 + cells from human umbilical cord were stimulated with LPS or Pam3 CSK 4 with or without thrombopoietin ( TPO ). Results CD 34 + cells and megakaryocytes express TLR 2 and TLR 4 at both RNA and protein level; however, direct stimulation of CD 34 + cells with LPS or Pam3 CSK 4 had no effect on cell growth. Interestingly, both TLR ligands markedly increased TPO ‐induced CD 34 + cell proliferation, megakaryocyte number and maturity, proplatelet and platelet production when added at day 0. In contrast, this synergism was not observed when TLR agonists were added 7 days after TPO addition. Interleukin‐6 ( IL ‐6) release was observed upon CD 34 + or megakaryocyte stimulation with LPS or Pam3 CSK 4 but not with TPO and this effect was potentiated in combination with TPO . The increased proliferation and IL ‐6 production induced by TPO  +  LPS or Pam3 CSK 4 were suppressed by TLR 2/4 or IL ‐6 neutralizing antibodies, as well as by PI 3K/ AKT and nuclear factor ‐κB inhibitors. Additionally, increased proplatelet and platelet production were associated with enhanced nuclear translocation of nuclear factor ‐E2. Finally, the supernatants of CD 34 + cells stimulated with TPO + LPS ‐induced CFU ‐M colonies. Conclusions Our data suggest that the activation of TLR 2 and TLR 4 in CD 34 + cells and megakaryocytes in the presence of TPO may contribute to warrant platelet provision during infection episodes by an autocrine IL ‐6 loop triggered by PI 3K/ NF ‐κB axes.