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

D'ATRI, LINA PAOLA; POZNER, ROBERTO GABRIEL; CARRERA-SILVA, EUGENIO ANTONIO; MIGUEL, CAROLINA PAULA; NEGROTTO, SOLEDAD; SCHATTNER, MIRTA; RODRÍGUEZ, CAMILA SOFÍA; ORTIZ WILCZYÑSKI, JUAN MANUEL; HELLER, PAULA GRACIELA

JOURNAL OF THROMBOSIS AND HAEMOSTASIS

WILEY-BLACKWELL PUBLISHING, INC

Año: 2019 vol. 17 p. 2196 - 2210

1538-7933

Background: Platelet toll-like receptors (TLR) 2 and 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 LPS or Pam3CSK4 were added 7 days after TPO addition. 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 NF-B inhibitors. Additionally, increased proplatelet and platelet production were associated with enhanced nuclear translocation of NF-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-B axes.