Immunopheonotypic and molecular characterization of monocyte subpopulations in Myeloproliferative Neoplasms

Author(s)

Vitor Leonardo Bassan

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-03-07

Examining board members

Fabíola Attié de Castro; Tiago da Silva Medina; Raquel Tognon Ribeiro

• Advisor: Fabíola Attié de Castro; Fabiani Gai Frantz

Abstract

Myeloproliferative Neoplasms (MPN) are clonal hematological disorders characterized by the hyperproliferation and accumulation of precursor and mature myeloid cells in the bone marrow and peripheral blood. The BCR-ABL1-negative MPN evaluated in this study were Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (MF). The pathophysiology of these diseases is associated with the presence of mutations involving the Janus Kinase 2 (JAK2), Calreticulin (CALR), thrombopoietin receptor (MPL) genes and changes in epigenetic complexes. Although dysregulated hematopoiesis in MPN is mainly attributed to genetic mutations in the hematopoietic stem cell, abnormalities in the immune system and in the cells that compose the bone marrow microenvironment seem to contribute to the pathophysiology of these diseases. Recently, MPN have been considered as pre-leukemic oncoinflammatory diseases, once the patients have elevated levels of cytokines, chemokines and inflammatory markers in plasma and bone marrow. The MPN present a state of exacerbated inflammatory activity, which contributes to genetic instatility and cardiovascular damages in patients. Human monocyte subpopulations, subdivided into classical, intermediate and non classical, have been involved in the genesis and modulation of chronic inflammatory, autoimmune and neoplastic conditions. Although studies show the presence of monocytosis in MPN patients, the immunophenotypic profile of monocytes in these diseases has not been elucidated yet. In this context, the present study characterized the immunophenotypic and molecular profile of peripheral blood monocytes from patients with PV, ET, MF and healthy subjects. Peripheral blood monocytes were isolated and submitted to immunophenotyping to determine the frequency of circulating subpopulations and the expression of CD56, CD64, CD80/86 and HLA-DR markers. The plasma concentration of the soluble CD163 marker was quantified by ELISA and the monocytes transcriptome was analyzed by the in silico methodology. Compared to controls, PV, ET and MF patients had increased frequency of intermediate and non classical monocytes and lower frequency of classical monocytes. Higher expression of HLA-DR was identified in monocytes in the three disease categories, indicating cell activation. The expression of CD80/86 was lower in monocytes from patients with PV and ET than in patients with MF and controls, indicating that, in these diseases, there seem to be more monocytes with less costimulatory activity. The frequency of CD56-positive monocytes was increased in the three MPN, mainly in MF. The plasma concentration of CD163, an indicator of monocyte activation, is elevated in PV, ET and MF patients. The MPN patients transcriptome revealed distint immunological signatures among patients with PV, ET and MF, and indentified the activation of inflammatory pathways in monocytes, possible alterations in cellular metabolism, in the regulation of apoptosis, in activities associated with coagulation and prothrombotic state, interaction with neutrophils and platelets, and the differential expression of genes associated with the type I and type II interferon pathways. Taken together, the results suggested that the increased frequency of intermediate and non classical monocytes contributes to the oncoinflammation process in MPN. PV and ET shared monocytes with similar immunophenotypic profiles, marked by high activation and lower costimulatory potential. The increase of the frequency of CD56-positive monocytes suggests the existence of an immunosurveillance process mediated by monocytes in MPN, since these cells are associated with the induction of cytotoxic activity and antitumor immune response. In conclusion, monocytes seem to be influenced by the pathophysiological alterations triggered by the neoplastic process in MPN and should be considered as a potential target for pharmacological interventions in these diseases. (AU)

• FAPESP's process: 19/18013-8 - Immunophenotypic and molecular characterization of monocyte subpopulations in myeloproliferative neoplasms
• Grantee: Vitor Leonardo Bassan
• Support Opportunities: Scholarships in Brazil - Master