Monocytes from Patients with Polycythemia Vera Express Molecules Related to Stress Erythropoiesis and Have Increased Erythrocyte Phagocytosis
Authors: Borges Marina Dorigatti , Sesti-Costa Renata , Albuquerque Dulcinéia Martins de , Lanaro Carolina , Fertrin Kleber Yotsumoto , Costa Fernando Ferreira 
Published: November 2021

Abstract
Stress erythropoiesis (SE) is characterized by an increase in erythropoietic activity in the bone marrow and in extramedullary sites. The central macrophage present in the erythroblastic island (EBI) plays a key role in regulating SE through the expression of molecules mediating cell adhesion, iron metabolism, and those capable of identifying and engulfing damaged and senescent erythrocytes. Those receptors are also expressed in monocytes (MC), suggesting that their monocytic expression could be involved in erythropoiesis and erythrophagocytosis. CD14 +CD16 + intermediate MC (I-MC) express markers that are typically found in EBI macrophages and, together with CD14 +CD16 - classic MC (C-MC), are able to remove circulating iron. Polycythemia vera (PV) is characterized by autonomous overproduction of red blood cells (RBCs) with extramedullary hematopoiesis most often caused by an acquired JAK2 V617F mutation, resulting in a state of chronic SE. The depletion of macrophages from EBIs in animal model of PV reverses splenomegaly and erythrocytosis indicating that they are essential for the development of chronic SE. It is unknown if MC play the same role in humans or if they have different expressions of those key molecules, which could contribute to the severity of the disease.
We aimed to investigate the role of MCs in SE present in PV by characterizing the expression of molecules relevant to RBC adhesion, anti-inflammation, erythrophagocytosis, and iron metabolism in MCs from PV patients and from healthy controls (HC).
Peripheral blood MCs were isolated from HC (n=21) and PV patients (n=17) and phenotyped by flow cytometry (FC) for sialoadhesin (CD169), VCAM1 (CD106), the receptor for the hemoglobin-haptoglobin complex (CD163), mannose receptor (CD206), SIRPα (CD172), ferroportin (Fpn), and separated into subtypes according to expression of CD14 and CD16. We also evaluated MC erythrophagocytosis by determining positivity for intracellular glycophorin (CD235a) (nHC=13 and nPV=17).
In PV, we observed significantly higher expression of CD169 and CD106, and lower expression of CD172 in C-MC (1,167±216.6 vs 1,834±241.9, p=0.009; 1,427±217.1 vs 2,849±182.3, p=0.0004; 104,707±9,546 vs 77,070±8,756, p=0.0428, respectively); higher CD169 and CD106 in I-MC (2,221±322.4 vs 3,150±321.8, p=0.0371; 2,186±201.7 vs 2,721±153.5, p=0.0238, respectively); and higher CD206, CD163, CD172, and CD106 in CD14 lowCD16 + non-classical MCs (NC-MC) (181.2±8.5 vs 268.6±13.4, p&lt;0.0001; 312.3±15.1 vs 368.8±12.1, p=0.0174; 13,923±2256 vs 22,792±3211, p=0.0161; 1,234±96 vs 1,498±58.9 p=0.004, respectively). Fpn expression was not significantly different. A lower expression of CD172 in the C-MC suggests less inhibitory signaling for erythrophagocytosis in those cells. Although C-MC and I-MC have been previously linked to erythropoiesis, we saw a larger number of the investigated molecules being more expressed in NC-MC, supporting their possible involvement in regulating erythropoiesis. Our results suggest that MCs in PV could be more likely to attach erythroid cells and could therefore contribute to form EBIs if differentiated to macrophages. Higher molecule expression was associated to a higher percentage of MCs containing intracellular CD235a (0.35±0.07 vs 4.48±1.08, p&lt;0.0001). This is evidence of circulating PV MCs performing erythrophagocytosis and supports a role for them in RBC clearance.
Our findings reveal an increase in the expression of markers relevant to the adhesion of erythrocytes to MCs in all MC subsets from PV patients along with more frequent erythrocyte phagocytosis in circulating cells. Further studies should yield better understanding of the role of MCs in SE and in the formation of EBIs, providing future targets for the treatment of chronic SE in PV patients.

Disclosures
Fertrin: Sanofi Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Costa: Novartis: Consultancy.