P49: Macrophages with JAK2V617F mutation are activated by platelets: Role in pathogenesis of myelofibrosis

Abstract

Background Myelofibrosis with myeloid metaplasia is chronic mieloproliferative disease with ineffective erythropoiesis, dysplastic-megakaryocyte hyperplasia, and an increase in the ratio of immature granulocytes to total granulocytes. The incidence rate is 3.7–5.7 per 100,000 and the median survival is estimated to be between three and six years. The pathogenesis of myelofibrosis might be explained, in part, by a somatic point mutation on exon 14 (V617F) of the JAK2 kinase gene that is located on chromosome 9p24. However, mechanisms of regulation of microenvironment in bone marrow fibrosis are not clear. Considered that main producents of pro-fibrotic factors are the megakaryocytes and the macrophages. In this study we investigated influence of platelet factors on the cellular characteristics of the macrophages with oncogenic mutation JAK2 V617F. Materials and Methods THP-1 cell line was modified by lentiviral modification. Two cell lines established: one with expression of JAK2 with oncogenic mutation JAK2 V617F, another wild type JAK2. Cells were cultured in RPMI-1640 containing 0.1% gentamicin, 1% l-glutamine and 10% fetal bovine serum (FBS) or 5%, 10% and 15% platelet lysate (PL). Cell lines were differentiated into the macrophages using 50 ng/ml phorbolmyristate acetate (PMA). Level of expression transforming growth factor β (TGF β ), galectin-3, matrix metalloproteinases (MMP) 2, 9, 12, 13 and tissue inhibitors of matrix metalloproteinase (TIMP) accessed by specific RT-qPCR. Results We observed increased expression level of galectin-3, MMP-2, MMP-13 and TIMP-3 in JAK2 V617F expressing macrophages cultured with 10% FBS compare to ones with WT JAK2. Also macrophages containing mutation JAK2 V617F has increased level of expression of MMP-2, TIMP-3 and TIMP-4 when cultured with 10% platelet lysate. Also, cell line containing mutation JAK2 V617F has increased levels of expression of MMP-12 for cells cultured with 15% PL. We did not observe any significant difference in expression of TGF β , MMP-9, TIMP-1 and -2. Conclusion Increased levels of expression of galectin-3, MMP-12 and -13 suggest that platelet factors induce macrophages with JAK2 V617F mutation to release of profibrotic factors. On the other hand, increase of antifibrotic MMP-2 and TIMP-3 revealing complex nature of melofibrosis development and need to be analyzed further. This study confirms the idea of interaction between macrophages and platelets in pathogenesis of primary myelofibrosis.