Abnormal retention of mitochondria in mature red blood cells (RBC) has been recently reported in sickle cell anemia (SCA) but their functionality and their role in the pathophysiology of SCA remain unknown. The presence of mitochondria within RBC was determined by flow cytometry in 61 SCA patients and ten healthy donors. Patients were classified according to the percentage of mature RBC with mitochondria contained in the whole RBC population: low (0-4%), moderate (>4% and <8%), or high level (>8%). RBC rheological, hematological, senescence and oxidative stress markers were compared between the three groups. RBC senescence and oxidative stress markers were also compared between mature RBC containing mitochondria and those without. The functionality of residual mitochondria in sickle RBC was measured by high-resolution respirometry assay and showed detectable mitochondrial oxygen consumption in sickle mature RBC but not in healthy RBC. Increased levels of mitochondrial reactive oxygen species were observed in mature sickle RBC when incubated with Antimycin A versus without. In addition, mature RBC retaining mitochondria exhibited greater levels of reactive oxygen species compared to RBC without mitochondria, as well as greater Ca2+, lower CD47 and greater phosphatidylserine exposure. Hematocrit and RBC deformability were lower, and the propensity of RBC to sickle under deoxygenation was higher, in the SCA group with a high percentage of mitochondria retention in mature RBC. This study showed the presence of functional mitochondria in mature sickle RBC, which could favor RBC sickling and accelerate RBC senescence, leading to increased cellular fragility and hemolysis.

Summary Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca 2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1–10 μM). Oxygen‐gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca 2+ influx. Yoda1 induced Ca 2+ ‐dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo−/heterozygous for the rs59446030 gain‐of‐function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 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.

Sickle cell disease (SCD) defines a group of hemoglobinopathies affecting the β-globin gene. Sickle cell SC disease (HbSC) is a SCD syndrome characterized by the presence of both haemoglobin S (HbS) and haemoglobin C (HbC) in red blood cells (RBCs).1 HbS is caused by a single point mutation in the β-globin gene, responsible for the substitution of valine for glutamic acid. HbS may polymerize upon deoxygenation.2 HbC is another abnormal haemoglobin and is caused by a single point mutation in the same position of the β-globin gene as for HbS, but with a lysine replacing glutamic acid.3 HbC has the tendency to crystallize in both oxygenated and deoxygenated conditions and promotes RBC dehydration.4, 5 HbSC patients are usually characterized by milder anaemia than the homozygous form of SCD (i.e. HbSS), low RBC deformability and very high blood viscosity.6 Recently, abnormal mitochondria retention has been reported in mature RBCs from HbSS patients, which correlated with accelerated RBC senescence and hemolysis.7 In another recent study, Esperti et al.8 reported an association between mitochondria retention in mature RBCs, RBC senescence and RBC sickling in HbSS individuals. However, no data about the presence of mitochondria in mature RBCs from HbSC patients have been published so far. In this study, we tested for the presence of mitochondria-positive RBCs in patients with HbSC disease and investigated the association between mitochondria retention into mature RBCs and RBC deformability, senescence, oxidative stress, haemolytic markers and clinical manifestations. Fresh blood samples from 16 healthy donors and 16 HbSC individuals (adults and children at steady state, non-transfused, without clinical complications in the last 3 months before this study) were collected in EDTA tubes and analysed by Flow cytometry less than 5 h after sampling. A group of 16 HbSS patients was also included for the comparisons of selected parameters. All patients were regularly followed at the Hospitals of Lyon (France). The study was conducted in accordance with the guidelines set by the Declaration of Helsinki and approved by the Regional Ethics Committees (L14-127, Université de Lyon). Blood samples were washed twice in PBS 1X (1:1) at 1000 g for 5 min at room temperature (RT). RBCs pellets were resuspended at 0.2% Ht in PBS 1X (1:1) or in the appropriate staining buffer according to manufacturer instructions. The percentage of mature RBCs retaining mitochondria was assessed by flow cytometry performing a double staining using MitoTracker(R) Deep Red probe (MTKdr; Sigma-Aldrich; 5 nM final concentration) and an anti-CD71 antibody (1:50 dilution, Miltenyi 130-115-070), to discriminate the percentages of reticulocytes and mature RBCs retaining mitochondria. The percentage of positive events was recorded to quantify mitochondria+ and CD71− RBCs. Phosphatidylserine (PS) exposure on the outer membrane of RBCs was assessed incubating RBCs with Annexin V-PE (1:11 dilution, Miltenyi 130-118-363) and intracellular reactive oxygen species (ROS) were determined using 2′,7′–dichlorofluorescin diacetate (DCFDA, 20 μM final concentration, Sigma-Aldrich). After incubation (20 min in the dark at RT), the samples were washed, resuspended in their respective staining buffer and analysed by flow cytometry (MACSQuant 16, Miltenyi). Ektacytometry was performed with the Laser-assisted optical rotational red cell analyser (LORRCA, Mechatronics) to evaluate RBC deformability (Elongation Index, EI) in isotonic medium (Polyvinylpyrrolidone, PVP) over a shear stress gradient (0.3–30 Pa). Lactate dehydrogenase levels were determined by the standard biochemical method. The results showed no or very low levels of mitochondria-positive mature RBCs in healthy donors, compared to RBCs from HbSC patients (Figure 1A). The level of mitochondria-positive mature RBCs was lower in HbSC compared to HbSS patients (Figure 1A). RBC deformability was lower in both HbSC (0.41 ± 0.07) and HbSS (0.36 ± 0.06) patients compared to healthy individuals (0.64 ± 0.005; p < 0.0001). The percentages of mature RBCs with mitochondria negatively correlated with RBC deformability in HbSC patients (Figure 1B). The level of RBCs exposing PS was different between the three groups (HbSS = 3.03 ± 1.86; HbSC = 1.4 ± 1.1%, healthy individuals = 0.2 ± 0.08%; p < 0.0001). Several associations were tested in HbSC patients. A positive correlation between the percentage of mature RBCs retaining mitochondria and PS exposure was observed (Figure 1C). No significant correlation was observed between the percentage of mature RBCs retaining mitochondria and intracellular ROS levels (r = −0.17; p > 0.05), LDH (r = −0.01; p > 0.05), percentages of reticulocytes (r = −0.03; p > 0.05) or haematocrit (r = 0.20; p > 0.05). No association between mature sickle RBC mitochondria retention and previous history of clinical manifestations (i.e. vaso-occlusive crises and acute chest syndrome rates in the three preceding years, glomerulopathy, priapism, leg ulcers, osteonecrosis, stroke or pulmonary hypertension) was observed. During the terminal erythroid differentiation the removal of mitochondria is ensured by a specific autophagy pathway, called mitophagy. The lack of mitochondria-positive RBCs in healthy controls is in agreement with previous studies.7, 8 However, recent studies reported mitochondria retention in mature RBCs in HbSS patients, probably as a consequence of deficient mitophagy.7, 9, 10 Mitochondria are a source of reactive oxygen species (ROS), which could contribute to increase the intracellular oxidative environment of RBCs, which in SCD is already increased.11-13 In a sickle mice model, the pharmacological inhibition of mitochondria retention decreased intracellular ROS levels and improved RBCs survival.14 Our results show for the first time abnormal mitochondria retention in RBCs from HbSC patients, although the extent of mitochondrial retention is lower compared to HbSS individuals. The reasons for such retention are unknown and whether it is due, as for HbSS patients, to deficienct mitophagy remains uknown. Although we did not find any association between mitochondria retention into mature RBCs and hemolytic markers or the level of anaemia, our findings suggest that the retention of mitochondria into mature RBCs could promote senescence, as demonstrated by the associations between the percentages of matures RBCs with mitochondria and RBC deformability and PS externalization. The accumulation of intracellular ROS, as well as Ca2+ released by mitochondria,15 into the cytosol of RBCs could accelerate senescence. These findings reinforce the role that abnormal mitochondrial retention could play in modulating RBC properties and sickling in SCD, as recently shown in HbSS patients.8 Futher investigations about the functionality of these residual mitochondria in HbSC RBCs, as well as the clinical relevance of such findings, are needed. Sofia Esperti, Elie Nader, Camille Boisson and Philippe Connes designed the research. Sofia Esperti, Elie Nader, Camille Boisson, Romain Carin, Céline Renoux and Philippe Joly performed the biological analyses. Alexandra Gauthier and Solène Poutrel included patients. Sofia Esperti, Elie Nader and Philippe Connes performed the statistical analyses. Sofia Esperti, Elie Nader and Philippe Connes wrote the first version of the paper. Camille Boisson, Romain Carin, Françoise Horrand, Diana Piedrahita, Céline Renoux, Philippe Joly, Alexandra Gauthier and Solène Poutrel read and approved the final version of the manuscript. This study was supported by the European Framework Horizon 2020 under grant agreement number 860436 (EVIDENCE). No conflict of interest to declare. The study was conducted in accordance with the guidelines set by the Declaration of Helsinki and approved by the Regional Ethics Committees (L14-127, Université de Lyon). Data are available upon reasonable request to the corresponding author.

THC triggers a pronounced entry of Ca2+ , which may be deleterious, into sickle cell red blood cells via activation of the TRPV2 channel.

Glucose-6-dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in the world,1 which leads to a lower level of reduced glutathione, an antioxidant, in red blood cells (RBCs). Most of the time, those who are affected have no symptoms. However, they should avoid specific triggers that may promote oxidative stress such as fava beans, that may fragilize RBCs and cause haemolysis. G6PD deficiency has been proposed to be a modulator of certain complications in sickle cell anaemia (SCA), such as stroke2 and vaso-occlusive crises,3 but others found no major role of G6PD deficiency in the clinical manifestations and haematological phenotype of SCA.4-6 The present study investigated the effects of G6PD deficiency on RBC deformability, RBC senescence markers, haematological parameters and clinical severity in SCA. We hypothesized that RBC oxidative stress would be greater in SCA patients with G6PD deficiency than those without, and this would be accompanied by greater alterations in RBC rheology. Seventy-one patients with homozygous sickle cell disease (SCA, HbSS), no recent blood transfusion or acute complication participated in the present study after giving written informed consent. Medical records were reviewed by two physicians to identify patients who had acute chest syndrome (ACS) or vaso-occlusive crises (VOC) in the four years preceding the study, as well as a diagnosis of osteonecrosis, glomerulopathy, retinopathy and leg ulcers. A patient was considered as having frequent VOC when the number of VOC per year was greater or equal to two. This study was performed in accordance with the Declaration of Helsinki and approved by the HCL Ethics committee (L14-127). Screening for G6PD deficiency in SCA patients was first done at the enzyme level in all patients. Both G6PD and pyruvate kinase (PK) activities were measured and the ratio G6PD/PK calculated. A ratio comprised between 0.5 and 1.5 indicates no deficiency. For hemizygotes or homozygotes, the ratio is very low (<0.10). When the ratio was lower than 0.5, high resolution melting (HRM) analysis was performed focusing on the Med and A(−) variants7 to detect a possible heterozygous carrier state. Haematological parameters were determined with a haematology analyser (Advia, Siemens, Rungis, France). Ektacytometry was carried out with the Laser-assisted optical rotational red cell analyser (Lorrca, RR-Mechatronics) with the oxygenscan module to measure RBC deformability (Elongation Index; EI) over an oxygen gradient and at 30 Pa, as previously described.8 Several parameters were derived: (1) EImax (normoxic RBC deformability), (2) EImin (the lowest RBC deformability reached during deoxygenation) and (3) PoS (the pO2 at which RBC deformability decreases below 95% of EImax during deoxygenation), which reflects the pO2 at which RBCs start to sickle during deoxygenation. Phosphatidylserine (PS) exposure at the outer membrane leaflet of RBCs and CD47 were assessed using Annexin V-PE (Miltenyi, 130-118-363) and anti-CD47-PE antibody (Miltenyi 130-101-348), respectively.9 Intracellular RBC reactive oxygen species (ROS) were determined using 2′,7′–dichlorofluorescin diacetate (DCFDA, Sigma-Aldrich, Saint-Quentin-Fallavier, France).9 DCFDA is a non-specific probe that allows the determination of the degree of overall oxidative stress in the cells by reacting with various reactive oxygen species. Despite the fact that nitric oxide may oxidize DCFDA, this probe seems sensitive enough to detect intracellular oxidative stress levels in RBCs in a pathophysiological context and for in vitro pharmacological modulation assessments.9 Ten patients had G6PD deficiency (six homozygotes, four hemizygotes), all A(−) variants. Age was not different between the two groups (23.7 ± 16.8 vs 24.9 ± 13.8 years in patients with and without G6PD deficiency, respectively). Biological results (Table 1) showed greater intracellular ROS content and higher percentage of positive RBCs for ROS in SCA patients with G6PD deficiency. RBC ROS level was also analysed in a group of healthy subjects (36.9 ± 7.8 years): positive RBCs = 55.0% ± 24.3%; median fluorescence intensity (MFI) = 0.57 ± 0.14. Both parameters were below the values found in the two SCA groups. The other markers were not different between the two groups. Table 2 shows no difference in the clinical parameters between the two groups. Bouguerra et al.10 previously showed higher numbers of PS-exposing RBCs and higher ROS level in the RBCs from non-SCA G6PD-deficient patients than from healthy volunteers. Indeed, it is not surprising that greater intracellular ROS levels were observed in SCA patients with G6PD deficiency than in those without in the present study. However, no difference was observed between the two groups for RBC senescence markers (percentage of RBCs exposing PS and CD47 expression), RBC deformability in both normoxia and hypoxia, the propensity of sickling (i.e., PoS), the level of anaemia (Hct and Hb) and haemolytic markers. Although some studies reported lower Hb concentrationds in SCA patients with G6PD deficiency compared to those without,11, 12 the majority of previous studies showed no difference in haematological parameters between the two populations.13, 14 Nevertheless, these results are quite surprising since oxidative stress is known to further reduce RBC deformability in SCA and fragilize RBC,9 as well as promote RBC senescence. The exact reasons for these discrepancies are not clearly understood. Although our study did not show further RBC alterations in G6PD-deficient patients, the higher baseline RBC oxidative stress found in this group justifies patients becoming acquainted with their G6PD genotype in order to avoid consuming oxidative substances that would further enhance oxidative stress and worsen their haematological phenotype. The clinical impact of G6PD deficiency in SCA is also debated in the literature. Some studies reported an association between cerebral vasculopathy and G6PD deficiency2, 15 while others did not find such an association.5, 6 Patients with a chronic transfusion programme were not included in this study and it was thus not possible to test whether G6PD deficiency was associated with brain infarcts or abnormal transcranial Doppler velocity since these patients usually have transfusion therapy. Instead, we tested the associations between G6PD deficiency and other acute and chronic complications. Although the sample size of our G6PD-deficient group was small, we did not detect any association which confirms previous studies.5, 13, 14 In conclusion, our study did not show any difference in the RBC rheological, haematological and clinical profiles between SCA patients with and without G6PD deficiency despite higher RBC oxidative stress in the former population. All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. The authors thank all the participants. This study was supported by the European Framework Horizon 2020 under grant agreement number 860436 (EVIDENCE). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data are available upon requests to the corresponding author.