Immunophenotypic and molecular characterization of monocyte subpopulations in myeloproliferative neoplasms

Abstract

Myeloproliferative Neoplasms (MPNs) are clonal hematological disorders resulting from genetic mutations in the hematopoietic stem cell that promotes cellular hyperproliferation and accumulation of precursor and mature myeloid cells in the bone marrow and peripheral blood. The classic negative BCR-ABL1 MPNs, in this work, are represented by the neoplasms Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), whose pathophysiology is associated with alterations involving Janus Kinases (JAK), CALR, MPL proteins and cell proliferation regulating epigenetic complexes such as TET2 enzymes. Among these changes, the JAK2V617F mutation is the most frequent, giving to the stem cell the ability to proliferate autonomously and exacerbated, resulting in the excessive production of one of the myeloid hematopoietic lineages. Although unregulated hematopoiesis is mainly attributed to genetic mutations in stem cell, abnormalities in the immune system and in the cells that are present in the medullary microenvironment seem to contribute to the pathophysiology of these diseases. MPNs have recently been considered as pre-leukemic oncoinflammatory diseases, endowed with elevated serum cytokine levels and exacerbated inflammatory activity, which appear to contribute to genetic instability and increased risk of thrombosis, hemorrhage and other cardiovascular changes. Human monocyte populations, subdivided into classical, intermediate and non-classical, have been involved in the genesis and modulation of various chronic, autoimmune and neoplastic inflammatory conditions. Although studies show the presence of monocytosis in patients with MPNs, the phenotypic and molecular profile of these cells during these conditions has not been elucidated. Thus, the aim of this study is to characterize the immunophenotypic, secretory and molecular profile of peripheral blood monocytes of patients with PV, ET, PMF and healthy individuals. Monocytes will be subjected to immunophenotyping to determine circulating populations, as well as quantification of cytokine expression related to different monocyte acquired phenotypes (IL-1beta, IL-6, IL-8, IL-12, TNF-alpha, TGF-beta) and epigenetic regulation enzymes (DNMT3A, DNMT3B, HDAC1, HDAC2) by Real Time PCR. In addition, the phagocytosis assay and treatment of monocytes with hypomethylating agent and JAK2 inhibitor will be performed in order to clarify their influence on the effector activity of monocytes. The results will help to understand the participation of immune cells in the pathophysiology of MPNs and to elucidate the contribution of monocytes to the oncoinflammatory profile of these diseases. (AU)