Activation of Toll like receptors 2 and 4 on CD34+cells increases human megakaryothrombopoiesis induced by thrombopoietin

RODRÍGUEZ, CAMILA SOFÍA; HELLER, PAULA; SCHATTNER, MIRTA; NEGROTTO, SOLEDAD; POZNER, ROBERTO GABRIEL; D'ATRI, LINA PAOLA

Melbourne

Congreso; XXVII Congress of the International Society of Thrombosis and Haemostasis; 2019

ISTH

Background: Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of immune responses. Although it is known that activation of platelet ´s TLR2/TLR4 amplifies the host immune response against infections, its role on human megakaryopoiesis has not yet been investigated. Aims: Since thrombocytosis is usually observed during bacterial infections we evaluated whether Pam3CSK4 or lipopolysaccharide (LPS) (components of Gram positive and negative bacteria and TLR2/TLR4 ligands, respectively) modulate human megakaryocyte development and platelet production. Methods: CD34+ cells derived from human umbilical cords were cultured with TPO; LPS or Pam3CSK4; TPO+ LPS or Pam3CSK4 during 11-16 days. Results: CD34+ cells and megakaryocytes express TLR2/TLR4 at both, RNA and protein level. Incubation of CD34+ cells with Pam3CSK4 or LPS showed no differences in cell growth vs control. However, both TLR ligands markedly increased TPO-induced CD34+ cell proliferation (Fig.1). Although there was a slight but significant decrease in megakaryocytes differentiation (CD61+), megakaryocyte number, maturation degree (CD42b+), proplatelet and platelet production were higher in cultures treated with TPO + LPS or Pam3CSK4 vs TPO alone (Fig.1). Stimulation of CD34+ cells with TPO and TLR2/4 ligands but not with TPO alone triggered IL-6 release (Fig.2). The increased proliferation and IL-6 production induced by TPO + LPS or Pam3CSK4 were suppressed by TLR2/4 or IL-6 neutralizing antibodies or PI3K/AKT or NF-kB inhibitors and. The increment in proplatelet and platelet production was associated with an increased nuclear translocation of NF- E2 (Fig.2). The synergistic effects of TPO- induced megakaryo/thrombopoiesis mediated by TLR2/4 ligands were not observed when Pam 3 CSK 4 or LPS were added at day 7 of TPO- treated cultures. Supernatants from CD34+ cells stimulated with TPO + LPS induced CFU- M colonies (cytokine- free methocult). Conclusions : Our data suggest that recognition of bacteria by CD34+ cell TLR2/4 in the presence of TPO might contribute to thrombocy-tosis by an autocrine IL- 6 loop.