Activation of toll-like receptors 2 and 4 on CD34 + 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 Pam3CSK4 or lipopolysaccharide (LPS), TLR2/4 ligands respectively, modulate human megakaryocyte development and platelet production.
Methods: CD34 ⁺ cells from human umbilical cord were stimulated with LPS or Pam3CSK4 with or without thrombopoietin (TPO).
Results: CD34 ⁺ cells and megakaryocytes express TLR2 and TLR4 at both RNA and protein level; however, direct stimulation of CD34 ⁺ cells with LPS or Pam3CSK4 had no effect on cell growth. Interestingly, both TLR ligands markedly increased TPO-induced CD34 ⁺ 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 CD34 ⁺ or megakaryocyte stimulation with LPS or Pam3CSK4 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 Pam3CSK4 were suppressed by TLR2/4 or IL-6 neutralizing antibodies, as well as by PI3K/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 CD34 ⁺ cells stimulated with TPO+LPS-induced CFU-M colonies.
Conclusions: Our data suggest that the activation of TLR2 and TLR4 in CD34 ⁺ 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 PI3K/NF-κB axes.
(© 2019 International Society on Thrombosis and Haemostasis.)