Gender effect on production and enrichment of F cell numbers in sickle cell disease patients in Tanzania

In sickle cell disease (SCD), the tendency of the pathological hemoglobin (HbS) to polymerize when deoxygenated is the driver of subsequent pathology, rigid and abnormally shaped red blood cells (RBC) causing vascular damage and inflammation and a short erythrocyte life span causing hemolytic anemia. A phenomenon occurring in the majority of patients is a substantially increased level of fetal hemoglobin (HbF), due to the increased prevalence (>25%, on average) of the fraction of erythrocytes carrying HbF, so-called ‘F cells’.1 While F-cell prevalence in non-anemic individuals (usually under 10%) is mostly determined by genetic factors,2 their accumulation in SCD patients is thought to result from F cells surviving longer in circulation than other RBC3: HbF is an effective inhibitor of HbS polymerization and RBC sickling. Patients with higher blood HbF levels live longer and present generally milder.4 Raising HbF levels is a major target of established and new SCD therapies. The dissection of the genetic, pathological, and compensatory mechanisms influencing them is therefore of fundamental interest. To shape a patient's specific HbF level, several distinct processes and variables come into play: (i) the proportion of F cell precursors starting terminal differentiation in the bone marrow, which is likely variable and under strong genetic control2 (ii) the amount of F cell precursor expansion and survival under the influence of erythropoietin/anemia under conditions of ineffective erythropoiesis,5 (iii) the amount of HbF expressed per F cell and (iv) the differential survival of F cells and non-F RBC in circulation. Some processes are linked, for example, the amount of HbF present in individual RBC affects their survival in sickle cell patients and influences clinical outcomes.4 To dissect these processes and identify influencing factors will help to understand pathogenesis and disease progression and to develop and evaluate new therapeutic approaches. One fundamental biological factor promoting HbF and F-cell levels is the female sex. In female SCD patients, HbF levels are generally higher driven by higher levels of circulating F cells (31% in females versus 22% in males6), following increased F-cell production (F-reticulocytes).3 It is unclear whether other HbF-shaping processes, such as RBC survival in circulation, contribute to the observed HbF sex difference in SCD. Here, we set out to compare F cell production and changes in F cell abundance in circulation in male and female SCD patients. From sickle cell anemia patients enrolled in the Sickle Cell Programme at the Muhimbili University of Health and Allied Sciences (MUHAS), 101 males and 126 females, aged 5–60 years, were studied under steady-state conditions, excluding hydroxyurea therapy and recent transfusion. To estimate the proportion of RBC released as F reticulocytes from the bone marrow (‘F cell production’), we used flow cytometry of peripheral blood cells (Supplementary Methods, Data S1) after anti-gamma globin (labeling HbF) and anti-CD71 staining (labeling reticulocytes) to enumerate HbF+/CD71+ cells (F reticulocytes) and calculated the ‘F-retics%’ parameter, as a proportion of all reticulocytes (for distribution of F retics, F cells, MCH and MCV values see Figures S2–S5). Analogously, we identified mature F cells (HbF+/CD71-) and calculated the ‘F-RBC%’ parameter, as a proportion of all mature RBC. Finally, to evaluate changes in F cell abundance during circulation, we calculated an ‘F cell enrichment ratio’ (FER: F-RBC%/F retics%) for each sample analyzed (Figure S1). We regarded patients with a FER between 0.9 and 1.1 as maintaining the same F cell percentage, those with FER <0.9 as having lost F cells proportionally, and those with FER >1.1 as having accumulated F cells in circulation. Also, automated full blood counts and HbF% were measured in a Hematology clinical and research laboratory. As we and others3, 6 observed previously, F-RBC% was significantly higher in our female patients, as were HbF% levels, MCV and MCH, whereas total hemoglobin was not significantly different between males and females. Our median estimate for F cell production (F-retics%) was 18.9%. It is quite revealing to compare this value to the 3% average F cell levels observed2 in non-anemic individuals, where the proportion of F cells circulating can be assumed to be equivalent to the proportion of F cells produced. Thus, the bulk of F cell accumulation in SCD, that is, the depletion of non-HbF erythrocytes, is already evident at the reticulocyte stage. Much of it is likely to have occurred during terminal erythroid differentiation under conditions of ineffective erythropoiesis. The enhanced entry of early F-like precursors into terminal differentiation stimulated by erythropoietin and hypoxia may also contribute to the increased levels of F cell abundance in circulation. As previously described,3 F-retics% was higher in females, but the difference was small (19.6% compared to 17.4% in males, p = 0.05) (Table S1). This nevertheless suggests that part of the eventually considerable difference in F-cell levels between the sexes is already present when cells leave the bone marrow. Evaluating the change in F cell prevalence while in circulation (FER), we found that a majority of the patients (n = 116, 51%) increased F cell abundance (F-RBC%) compared to the reticulocyte stage (FER >1.1) (Figure 1A). This indicates that in most patients indeed F-cells survive longer in circulation than non-F erythrocytes. However, in a substantial number of patients, F-cell abundance did not change (FER 0.9–1.1, n = 34, 15%) or even decreased (FER <0.9, n = 77, 34%, Figure 1A). Across all patients, there was only a small increase in F cell prevalence (median FER 1.13, p = 0.02). However, there was a fundamental difference between the sexes, with female patients alone showing a significant gain (FER 1.26, p = 0.002) and males showing overall no change (1.01, n.s.) (Table 1). Our results presently do not offer an explanation for why a loss of F cells in circulation should occur in some patients. Possibly not all F cells, but only those with the highest HbF content, are protected from HbS polymerization and sickling,4 and other features of F cells, such as certain surface adhesion molecules, might promote the elimination or sequestration of those with little HbF. The difference between male and female patients' FER was significant (p = 0.01) and patient-for-patient, there appeared to be a systematic higher F cell enrichment in females (Figure 1B). Again, it is unclear what the cause for this is, but it appears that more stringent F cell enrichment, that is, loss of HbF-free erythrocytes, in females affects both, erythroid precursors and circulating erythrocytes. Other female-specific biological factors might play a role, since erythropoiesis in general is regulated differently to the male environment, with lower hemoglobin and hematocrit values, but higher erythropoietin levels. In addition, vascular biology and its regulation by nitric oxide show significant sex differences in SCD. We have recently re-investigated a long-sought X-linked genetic determinant of F cells and found a genetic association of F-cell abundance with the FRMPD4 locus at chromosome Xp22.2.6 It is yet to be determined which of the biological processes shaping F-cell prevalence are affected by this locus. In our patients, F-retics% (F cell production) was positively associated with hemoglobin and negatively with the percentage of reticulocytes, implying that the preferential survival of F cell precursors during terminal erythroid differentiation, along with a genetic predisposition favoring F cell production, might be a mechanism alleviating hemolysis and anemia in certain patients. We found FER to be positively associated with MCH and MCV (Table S2), two parameters that are generally correlated with circulating F cells, probably due to them being marginally larger than other RBC.2 More revealing is the inverse correlation of FER with RBC count (p < 0.001) (Table S2), and its direct correlation with reticulocyte count (p < 0.001) (Table S2), both suggesting that hemolysis is indeed a key driver of F cell accumulation in the peripheral blood of SCD patients. While our flow-cytometry experimental approach has been informative, we feel that there is significant measurement-related variability, especially in the quantification of F reticulocytes. For instance, the specifics of patient recruitment and sample collection make it sometimes difficult to have all samples collected and analyzed on the same day, which would be an ideal scenario. Thus, some patients might appear to have a loss of F cells, while in reality there is none or a very mild enrichment. Such experimental variability would reduce our power to detect additional, weaker relationships between parameters. For instance, the enrichment in males is clearly less pronounced than in females, but possibly any mild enrichment that might be present also in this group was not detected due to power/variability issues. In the continuation of our research, the reduction of experimental variability will be a major focus, along with the inclusion of genetic data, such as alpha thalassemia, BCL11A, or Xp22.2, as well as the evaluation of iron deficiency. In conclusion, while most F cell accumulation appears to occur during terminal erythroid differentiation prior to release as F cells, further F cell enrichment occurs in some, but not all patients, and is significantly more prominent in females. F cell enrichment at the precursor stage appears to be associated with a mitigation of hemolysis and anemia, whereas when it occurs in circulation it is not. The stimulation of HbF expression is a major goal of novel therapeutic approaches, and a better understanding of the causes of any sex differences will be important. We would like to thank our patients without whom this study would not have been possible. Our thanks go to staff of the Sickle Cell Programme, Departments of Hematology and Blood Transfusion, Biochemistry, Microbiology, and Immunology for their support. We thank Prof. Lucio Luzzatto for his great contribution to interpreting the data and reviewing the manuscript. We would also like to thank MUHAS and Kings College London staff who supported this study and made it possible to happen. This work was supported by a grant from MUHAS-SIDA (Grant to Florence Urio; 3177/03030300) and The Commonwealth Split-Site Scholarship Commission awarded to Florence Urio; TZCN-2018-370. Stephan Menzel and Helen Rooks are supported by the Research Councils UK MRC project grant MR/T013389/1 to Stephan Menzel. Sara El Hoss has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 101024970. The authors declare no competing financial or other interests. Data S1. Supporting Information. Figure S1. Dot-plot diagram showing F-RBC and F-retics in quadrant 1 and 2, respectively. The proportion of F retics (F-retics%) was calculated as Q2/Q2 + Q3. The F-retics% value from above figure is 32.9%, with F-RBCs% being 56.1% (F cell enrichment ratio 1.71). Figure S2. Distribution of F-retics in SCD patients. Figure S3. Distribution of F cells in SCD patients. Figure S4. Distribution of MCH in SCD patients. Figure S5. Distribution of MCV in SCD patients. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.