In this project we are interested in red blood cell diseases and the vascular abnormalities in which they may be involved, with a particular focus on dyserythropoiesis and blood cell interactions in the circulation. Initially we will concentrate our efforts on sickle cell disease as a pathological model, and then we will apply our methodology to explore other diseases, notably membrane diseases, in which potential defects in erythropoiesis have never been examined. This component is led by Wassim El Nemer (PhD, HDR, DR EFS), Soraya Mezouar (PhD, HDR, MCU AMU), Maria De Grandis (PhD, CR EFS) and François Jordier (PhD, CR EFS) with the technical support of Alexandra Grimaldi (Research Assistant EFS), Catherine Movia (Research Technician EFS), Audrey Charlier Tous (Research Technician EFS) and Marie-Laurence Deligny (Lab Manager)
Hemolytic anemia is a clinical manifestation of red blood cell diseases. This anemia is due to the destruction of RBCs in the circulation, most often caused by intrinsic defects affecting its ability to deform, as is the case in sickle cell disease. The cause of anemia can also be central, as a result of defects in the manufacture of RBCs in the bone marrow, as in β thalassemia major. We have recently demonstrated the existence of dyserythropoiesis in sickle cell disease, which would contribute to the hemolytic anemia in this condition.
Sickle cell disease is a recessive genetic disorder, the most common in France, characterized by hemolytic anemia and vaso-occlusive crises. It is caused by a point mutation in the β-globin gene leading to the production of an abnormal hemoglobin (HbS) which, under deoxygenated conditions, polymerizes and causes sickle cell disease. Sickle cell disease, in its homozygous SS form, presents a wide spectrum of clinical manifestations, with varying degrees of severity. This is due to modulating factors such as the level of fetal hemoglobin (HbF) expression, which is influenced by nucleotide polymorphisms (SNPs) whose combination defines 5 haplotypes. The relationship between haplotypes and disease severity is poorly documented at the mechanistic and cellular level.
We have shown that HbF protects erythroblasts from apoptosis during terminal differentiation. In this component, we will extend the exploration of this dyserythropoiesis as a factor in anemia, and study it in relation to the genetic variability of patients, including the two genotypes SS (HbS) and SC (heterozygosity of HbS and HbC, which has a Glu6Lys substitution) and the different haplotypes.
We will determine the metabolic profile of the differentiation stages with the SCENITH method, as described above, and explore the metabolic pathways, including glycolysis and associated metabolic pathways involved in the maintenance of redox homeostasis (collaboration with Dr Sandrina Kinet, IGMM, Montpellier). These experiments will be conducted with primary CD34+ cells from patients included in the clinical research protocol of the Labex GR-Ex in which we are partners, and from APHM (Timone and Conception) and APHP (Necker) in collaboration with the clinical teams of the hospitals in question. In addition, we will develop a cell immortalization program to establish proerythroblastic lines from primary cells of patients with interesting genetic profiles (collaboration with Dr Laurence Guyonneau Harmand, Paris) to overcome the lack of biological material.
In our ANR project IRIS (2022-2024), coordinated by our team in collaboration with Dr. Annarita Miccio (Imagine Institute, Paris) and Dr. Mario Amendola (Genethon, Paris), we are exploring the effect of dyserythropoiesis on the EI. Our preliminary results obtained in vitro show a cell death of the central macrophage by ferroptosis. We will continue this project by developing a multicellular 3D cell culture system (sickle cell bone marrow microenvironment, project 3.1.1) to mimic the complexity of the bone marrow environment. This cellular model will be completed by an in vivo approach using the Townes mouse model that we recently acquired and that is hosted and followed at CEFOS (Centre d'Exploration Fonctionnelle Scientifique, UAR 2020).
Orientation "Effects of therapeutic molecules "
On the translational side, we will determine the effect of therapeutic molecules on this dyserythropoiesis, which can interfere with the polymerization of HbS (GBT440, GBT/pfizer) and / or induce HbF (HU). In this context, we will reinforce our interactions with the pharmaceutical industry by evaluating molecules in clinical trials (LGD, GBT/pfizer, Agios, Forma Therapeutics...). In addition, we will seek to identify new markers that could reflect the degree of dyserythropoiesis by exploring the potential of circulating DNA (cell free DNA: cfDNA) of erythroid origin. Indeed, the level of cfDNA of erythroid origin has been shown to be related to types of anemia (Lam et al, 2014) and to be a marker of dyserythropoiesis in β thalassemia (Tavazzi et al, 2018). We will develop this cross-cutting project with Axis 2 of our team (Pascal Pedini) on the aforementioned cohorts of sickle cell patients and that of our collaborators from the Hemocentro de Ribeirao Preto in Brazil (Dr Dimas Covas, Dr Simone Kashima and Dr Ana Cristina Silva Pinto, University of Sao Paulo).
Track "Cellular interactions of RBC in the circulation "
Orientation "Red cell and thrombus "
ABO blood type is one of the primary risk factors for venous thrombosis (VT) and is associated with the risk of stroke and myocardial infarction (Stowell et al., Vox Sang 2019). Currently, the biological mechanisms by which ABO influences thrombosis remain unclear. Recently, our team reported 4 specific ABO "single nucleotide polymorphism" (SNP) whose thrombotic effects were considered to be mediated by ABO(H) structures, which are expressed by factor VIII (FVIII) and von Willebrand (vWF) (Goumidi et al., Blood 2021). Among them, blood groups A1 and B have also been associated with VT risk. However, the role of ABO groups on the endothelial side in the interaction between RBCs and endothelial cells remains unexplored. On the other hand, interestingly, the platelet-RG relationship has been previously reported (Klatt et al, JCI 2018). This study highlighted the key role of FasL/FasR in the induction of procoagulant activity in thrombosis. In this context, we propose to sequentially evaluate the role of these vascular bed actors in thrombosis by generating endothelial cell models specifically presenting each ABO group in partnership with Pr.P.E. Morange and Dr. F. Peiretti (C2VN, Marseille). We will decipher the molecular mechanisms involved in the interaction of these cellular actors (i) in vitro via the use of flow chamber; and (ii) in vivo via the use of mouse models coupled with intravital microscopy in partnership with Pr. C. Dubois (C2VN). This project, which will be led by Dr. S. Mezouar with expertise in the study of cellular actors of thrombosis and associated models (vitro/vivo) will allow to dissect the mechanisms of the cellular trio GR - platelets - endothelium on the association of ABO blood groups in thrombosis in order to identify biomarkers as well as new therapeutic targets in pathologies characterized by thrombosis such as myeloproliferative neoplasias.
Orientation "Red blood cell and vaso-occlusive crises in sickle cell disease "
Vaso-occlusive crises in sickle cell disease are a major clinical complication generated by the formation of micro "thrombi" in the microcirculation. Previous studies conducted by S. Mezouar were able to highlight the key role of the cooperation of leukocytes, RBCs and platelets in thrombus formation by intravital microscopy (Blood, 2012; Blood, 2013). In partnership with Pr. C. Dubois (C2VN, Marseille) we propose to dissect the molecular mechanisms and the interactions of RBCs-Leukocytes-Platelets in in vivo animal models in order to identify biomarkers as well as new therapeutic targets allowing to improve the management of patients who remain to date deprived of adapted treatment.
We will also explore the contribution of physical flow parameters to these seizures through microfluidic approaches, in collaboration with Annie Viallat's team (CINaM, Mareille), and acoustic approaches, in collaboration with Emilie Franceschini's team (LMA, Marseille).