Impairment of Myeloid Dendritic Differentiation and Role in the Support of Splenic Dysmegakaryopoiesis in Patients with Primary Myelofibrosis

The primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by myeloproliferation, splenomegaly with hematopoietic metaplasia and dysmegakaryopoieisis. We have previously described an increase in Flt3 ligand (FL) in plasma and spleen of patients with PMF and its role in the dysmegakaryopoiesis (Desterke and al, Cancer Res, 2011). Account the importance of FL in development of splenic myeloid dendritic cells (mDC), we studied the differentiation of mDC in patients and its potential impact on dysmegakaryopoiesis.

The mDCs were obtained from cell culture of blood and spleen mononuclear cells in presence of fetal calf serum (FCS) and bacterial lipolysaccharides (LPS). The megakaryocytes were obtained by culturing CD34+ cells from blood or spleen in specific medium serum free in presence of IL-3, IL-6, IL-11 and Tpo. Gene expression was quantified by microarray and RT-QPCR, protein analysis by immunofluorescence and flow cytometry, and migration experiments were performed in Boyden chamber.

Transcriptome of circulating CD34+ cells from PMF patients showed an increase in expression of genes encoding for integrin CD11c and also TLR4 and a decrease in the expression of gene encoding TLR9: suggesting the presence of progenitor mDCs in the blood. These results have been confirmed 1/ in cytometry by an increase in the number of CD34+ CD11c+ HLA-Dr+ cells in the blood; 2/ in cell culture by the presence of adherent cell colonies positives for TLR4+ CD11c+ HLA-Dr+ in the blood. The in vitro differentiation of mDC cells and the proportion of mature mDCs HLA-Dr+ CD11c+ cells are decreased in blood of PMF patients. Myeloid nature of circulating DCs was confirmed by the absence expression of the plasmacytoid membrane marker CD123 and by an increased of TLR4 and myeloid PU-1 (myeloid transcription factor) expression in opposite to a decreased of plasmacytoid markers: IL-23, HMGB1 and TLR9. Moreover in PMF patients, circulating adherent mDCs overexpressed CXCL12 chemokine and also FL which have abnormal chemottractant ability with respect to PMF megakaryocytic precursors still expressing flt3 receptor. Finally, in PMF patients, coculture of MK with mDC promotes their survival, differentiation and maturation (MK ploidy and transcriptional program). Primary results confirmed the presence of these mDCs precursors (CD34+ CD11c+ HLA-Dr+) in the spleen of PMF patients which harbored also an extramedullary megakaryopoiesis. These mDCs precursors are absent of the spleen from healthy subjects.

Our results show an increased presence of mDC progenitor population CD34+ HLA-Dr+ CD11c+ in the blood and the spleen of PMF patients. They also suggest that these cells are involved in migration, survival and differentiation/maturation of megakaryocytes, particulary in the spleen of patients.

No relevant conflicts of interest to declare.