PIEZO1 ACTIVATION INCREASES NEUTROPHIL ADHESIVE PROPERTIES IN SICKLE CELL ANEMIA

Abstract

Sickle cell anemia (SCA) is an inherited disorder caused by a mutation in the gene encoding β-globin. This mutation brings about alterations in the hemoglobin molecule, resulting in abnormal red blood cell (RBC) properties, shape, and subsequent destruction in the circulation, leading to anemia and various complications. Genetic mutations that increase the activation of Piezo1, a mechanosensitive protein involved in calcium influx and Gardos channel activation, have been shown to be responsible for hereditary xerocytosis, a disorder in which RBCs are severely dehydrated. We recently showed that Piezo1 plays a role in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease (Nader et al.; PMID: 37011913). The aim of the current study was to explore the role of Piezo1 in neutrophil adhesive properties in SCA. This investigation was conducted collaboratively between Lyon and UNICAMP (Fapesp grant 2020/06133-6). Neutrophils were isolated from the peripheral blood of healthy control individuals (HbAA; N = 7) and individuals with SCA (HbSS; N = 7), by density separation. Neutrophils were incubated with Yoda1 (0.1-10 μM), a Piezo1 channel agonist that does not require mechanical activation, or DMSO vehicle, for 30 min (37ºC), and then perfused over fibronectin-coated microchannels (400 μm-wide) using the Venaflux microfluidic platform, under 0.5 dyn/cm2 shear stress, 37°C, 3 min for monitoring adhesion. Flow cytometry analysis was performed with fluorescent antibodies to detect activated CD11a (LFA-1 integrin subunit) and CD11b (Mac-1 integrin subunit) epitopes. Yoda1-treated neutrophils (0.1-10 μM) were co-incubated with antibodies that bind to either the activated epitopes of CD11a or CD11b when in their activated states (MEM-83 antibody/anti-mouse IgG-FITC, and CBRM1/5-PE; Invitrogen), and cells were analyzed by flow cytometry. Interestingly, in the microfluidic adhesion assays we found that incubation of SCA neutrophils, but not neutrophils from control individuals, with 1 μM Yoda1 significantly increased the adhesion of the cells to fibronectin ligand (p = 0.001). Yoda1-induced adhesion of the SCA neutrophils was not associated with any increase in the activation of either the MAC-1 or LFA-1 integrin subunits, as demonstrated by flow cytometry assays (p > 0.05). These results suggest that Piezo1 may influence the adhesive properties of neutrophils in SCA, implying that changes in flow and shear stress could also modulate this mechanosensor on neutrophils. This study highlights the significance of Piezo1 in the pathophysiology of SCA and could offer new insights for therapeutic approaches for treating this condition.