Expression of t ype I interferon receptor in megakaryopoiesis

S. NEGROTTO; C. JAQUENOD DE GIUSTI; M.J. LAPPONI; J. ETULAIN; A. CARESTIA; R.M. GOMEZ; M. SCHATTNER

Kyoto

Congreso; XXIII Congress of the International Society on Thrombosis and Haemostasis 57th Annual SSC Meeting; 2011

The International Society on Thrombosis and Haemostasis

We have recently shown evidences that link viral infection of human hematopoietic progenitors with selective inhibition of thrombopoiesis through the type I IFN pathway, providing a mechanistic explanation for the thrombocytopenia and bleeding observed in Argentine Hemorrhagic Fever and other viral diseases. Since very little is known about the expression of type I IFN receptor (IFNR) in the megakaryocytic lineage, we characterized the expression, regulation and functionality of the IFNR in CD34+ cells, early megakaryocytic progenitor cell line MEG01, megakaryocytes and platelets (n=6-7).We found that the mRNA encoding the INFR subunits 1 and 2 are expressed throughout all stages of megakaryopoiesis, from early hematopoietic progenitors (CD34+ cells and MEG01) to mature megakaryocytes and platelets (RT-PCR). To verify that the mRNA is ultimately translated into protein, we studied the IFNR1 expression using two different antibodies (Abcam #ab45172 and Santa Cruz #sc-845). Immunofluorescence and flow cytometry analysis revealed that CD34+ cells, megakaryocytes, and MEG01, but not platelets, express IFNR1. Immunoblotting studies showed the typical 90kD band of IFNR1 (heavily glycosylated isoform) is present in CD34+, MEG01, and megakaryocytes, but not in platelets. An additional weak band of 60kD (non glycosylated isoform) was observed in megakaryocytes and platelets. Kinetic studies in MEG01 cells demonstrated that IFNR expression was upregulated in a time- and concentration-dependent manner by either dsRNA-mimetic PolyI:C (peak at 48 h, EC50: 20 μg/ml) or Junin virus infection (peak at 48 h, 50% of maximal response at MOI 0.5). IFNR activation in megakaryocytes with IFN-β (500 U/ml) resulted in the expression of significant amounts of IRF7 transcript (IFNR downstream protein), which was undetectable in unstimulated controls (RQ-PCR).In conclusion, early progenitors and mature megakaryocytes, but not platelets, express functional type I INFR.