How soluble erythroid factors impact on iron homeostasis locally, maintaining the physiological integrity of the erythroblastic islands, and systematically, through regulation of hepcidin synthesis, has not yet been elucidated. During terminal differentiation, erythroblasts produce cytokines whose role and cell targets remain largely unknown. Several of these cytokines such as GDF15, TWSG1 or OSM, could be one way by which erythroid precursors communicate their iron needs to the liver to influence the production of hepcidin and thus the amount of iron available for use. Whether these cytokines are also involved in a crosstalk between erythroblasts and macrophages within the erythroblastic island will be tested. Tissue macrophages play a predominant role in providing iron to the developing erythroblasts, by recycling heme iron following degradation of senescent RBC. Perturbations in erythrophagocytosis play a role in the pathophysiology of several diseases, including hemochromatosis, anemia of chronic disorders and thalassemia. Recently P1 has shown that serotonin is produced by erythoblasts in response to Epo and that mice devoid of serotonin synthesis exhibit iron metabolism disturbances.

The roles of the HIF (Hypoxia-inducible transcription Factor) proteins in erythropoiesis is currently studied by several partners of the GREx (P1, P5, P6, P7, P18) and animal models required for these studies, including mice with tissue-specific inactivation of HIF-1, HIF-2 and vHL (the “negative regulator” of HIFs) genes, are already available in these teams. Apart from hypoxia, the kidney is also able to sense iron deficiency although not much is known on the molecular mechanisms. There is also some evidence that the kidney can contribute to trans-epithelial iron reabsorption. Hepcidin levels in the urine, or local production of hepcidin by kidney cells, could play a role in controlling the amount of iron present in the interstitium. The first objective of this project is to define the role of hepcidin and kidney iron deficiency in controlling Epo production in interstitial medulla. Hepcidin deficient mice, with a complete or kidney-specific deficiency will be provided by P7.

This project will exploit the unique expertise of several partners in various aspects of iron and erythropoiesis to decipher the underlying mechanisms of anemia (anemia of inflammation, intensive care unit) and evaluation of new therapeutics.

The main goal is to decrypt the molecular events leading to SCD vaso-occlusion in order to develop new therapeutics. The SCD is the major hemoglobinopathy resulting from a unique mutation of b Hemoglobin (HbS) that polymerizes in hypoxic conditions leading to less deformable sickle-shaped RBC. The main complications arise from the tendency of sickle RBCs to block capillaries at low oxygen tension leading to chronic anemia, recurrent and unpredictable severe painful vaso-occlusive crisis (VOC), as well as “acute chest syndrome” (pneumonia or pulmonary infarction), bone or joint necrosis, priapism or renal failure. SCD has been designated as a public health priority by UNESCO in 2003, the French Ministry of Health in 2004 and WHO in 2006. Substantial evidence supports the hypothesis that VOC could be initiated by abnormal RBC adhesion to the vascular endothelium. Despite considerable efforts in the search of drugs for the treatment of SCD, to date only one, Hydroxycarbamide (HC) has demonstrated benefit for SCD patients, with fewer VOC and lower mortality and morbidity.

We aim to:

• Decipher the mechanisms of RBC interactions and signaling that trigger VOC
• Analyze the effect of secreted vascular mediators (endothelin, sympathetic, serotonin systems) on RBC deformability and blood viscosity
• Define molecular targets of HC and HC-induced signaling pathways in endothelial and circulating cells
• Identify small molecules that prevent and/or reverse SS RBC/endothelium interactions
• Characterize the metabolome of sickle cell RBCs before, during and after VOC to define potential metabolic bio-markers that can be used for prognosis
• Explore the splenic retention of hyperdense RBCs, their role in hyposplenism and their abundance as a prognosis factor for VOC.

If a role is confirmed we will develop a point-of-care prognostic test. In parallel, a transfusion strategy specifically tailored for the management of SCD patients will be devised using ex vivo generated RBCs containing a high level of fetal hemoglobin (HbF), derived from hematopoietic stem cells (HSC) of SCD patients (WP4). This consortium will play a critical role in facilitating a standardized follow-up of a very large cohort of patients (University hospitals of Pointe-à-Pitre in Guadeloupe, Henri Mondor in Créteil, Robert Debré and Necker hospitals in Paris), thereby enhancing the power of epidemiological and clinical analyses. 

Each year, 250 million clinical cases of malaria are reported, causing approximately 850,000 deaths. A key feature of the biology of Plasmodium falciparum is its ability to modify the surface and the deformability of parasitized erythrocytes (PEs) so that parasites either adhere to host cells or are trapped in the spleen. PE sequestration via PfEMP1 is the parasite’s mode of defense against destruction during passage through the spleen. Such sequestered PEs cause considerable obstruction to tissue perfusion contributing to severe clinical manifestations of malaria. PE adhesion chondroitin-4-sulfate (CSA) is linked to the severe disease outcome of pregnancy-associated malaria. Evidence strongly supports var2CSA as the leading candidate for a pregnancy malaria vaccine.

This aim will target key knowledge gaps in our understanding of the molecular basis for PE binding to endothelial cells and placenta in order to develop vaccine and therapeutic strategies that prevent or reverse PE sequestration. We will also validate our microfiltration system to determine the pitting rate threshold that differentiates PEs from patients with slow or rapid parasite clearance in areas where artemisinin resistance is spreading; develop a test to evaluate resistance to artemisinin and set-up a medium through-put screening system for compounds reducing the deformability of P. falciparum gametocytes (collaboration with Sanofi). We’ll evaluate the hypothesis that placental PE sequestration is reduced in SCD patients, conferring a protective trait to SCD pregnant women and their newborns, which could account for SCD expansion in Africa.

This aim is a first step in building population based approaches in haematology to study specific disease entities. We will build dedicated human resources in order to have in France, haemato-epidemiologists who do not currently exist. We will build a large registry of SCD patients that will encompass more than 2 500 adults and 1500 children from 5 African countries: Senegal, Ivory Coast, Mal, Gabon and Cameroon. Based on our expertise in population-based epidemiological studies, we will build this registry with the help of the medical teams within each country. The study will be conducted in close collaboration with the newly established National Reference Center for SCD in Yaoundé, the International Center for medical research in Libreville and the Centre de Recherche et de lutte contre la drépanocytose in Bamako. The following data will be collected and computerized: administrative data, medical history, clinical examination, biological examination including sickle cell genotype, hematological parameters and renal function, as well as two additional examinations: cardiac echocardiography and PWV (pulse wave velocity). The first aim is to assess the correlation between the severity of the SCD, assessed by the frequency of VOC and hospitalizations, the chronic microvascular complications (osteonecrosis, stroke, leg ulcers, retinopathy, renal insufficiency…) and the endothelial dysfunction assessed by arterial stiffness and wave reflexion. This large cohort will be also used to test other potential biological prognostic markers and pathophysiological hypotheses emerging from other findings at the epidemiological level. For example, it has been established that SCD protects against malaria and that malaria is associated with the development of Burkitt lymphoma. We will test at the epidemiological level the hypothesis that SCD may protect against the occurrence of Burkitt lymphoma and may worsen the outcome of other lymphoma subtypes. 

Prolonged storage reduces the survival of RBCs after transfusion and contributes to transfusion-related side effects, including respiratory distress and systemic sepsis. It has been shown that in SCD, stored RBCs incubated in the patient plasma can suffer from the inflammatory environment of the patient as compared to healthy individuals (F Noizat-Pirenne, EFS, Créteil). Therefore, we hypothesize that a patient effect has to be taken into account in the evaluation of storage lesions and harmful effects of “old” RBCs. The objectives are to optimize RBC concentrates, improve transfusion yields and decrease risk of post-transfusion severe adverse events. To achieve these objectives we aim to:

• Evaluate if plasma from inflammatory patients induce toxic effects on RBCs, depending of the age of the RBCs. A predictive test could be also implemented.
• Restore the deformability of RBC populations stored for more than 3 weeks under blood bank storage conditions. Microfiltration, a process that mimics the mechanical sensing of RBC by the human spleen can separate poorly deformable RBCs from RBCs with a normal deformability. Scaling-up of the microfiltration method may help spare rare blood resources and improve prognosis when transfusing critically ill patients. This approach will also be useful for improving the quality of red blood cells frozen before transfusion (this is the case for very rare phenotypes), and to prevent blockade of leukodepletion filters when the donor of red blood cells carries one HbS allele.
• Evaluate the potential of serotonin (5-HT) in the enhancement of the life span of RBC during storage and after transfusion. Indeed, RBCs from 5-HT (serotonine)–deficient mice are more sensitive to macrophage phagocytosis and have a shortened half-life.

The consortium aims to develop blood substitutes following two approaches: first is developing cultured RBC (cRBC) from stem cells, second is developing the production of recombinant Hb (in micro-organisms, animals, or plants).  Independent of the final choice of molecules, an extremely large quantity is required to satisfy the demands of blood transfusion. The objectives of cRBC production is to:

• Obtain complete terminal maturation to the stage of mature functional RBCs.
• Reach quantities equal to those provided by a standard RBC concentrate.
• Establish industrial production conditions compatible with transfusion requirements and Good Manufactory Practice (GMP) standards.
• Have at one’s disposal an unlimited source of stem cells of specific blood group phenotypes.

RBCs can be now cultured in vitro from human hematopoietic stem cells (hHSCs), human embryonic stems cells (hESCs), or human induced pluripotent stem cells (hiPSCs). The highly promising hiPSC technology represents a potentially unlimited source of RBCs and opens the door to the revolutionary development of a new generation of allogeneic transfusion products. Partner 13 originated the concept of cRBC generation from stem cells for transfusion purposes. An in vitro protocol has been published, which allows the proliferation of HSC and the induction of their erythroid progeny and the final differentiation into RBCs. The functionality of these RBCs has been demonstrated in vitro and in vivo in the murine model. Starting from cord blood HSC it permits a 30 million-fold expansion and differentiation into functional RBC. The team has recently demonstrated the proof of principle for transfusion of cRBCs in humans. RBCs generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients. Indeed, only 3 hiPSC clones would have been sufficient to match more than 99% of the patients in need of RBC transfusions⁵¹.This project is funded by OSEO, and is one of the purposes of the STEMRED project that brought together academic and industrial partners (University Paris 6, Etablissement Français du Sang, Cellectis, Bertin Technologies).

Currently the production of Hb based blood substitutes are based on recycled human or bovine Hb. Clinical testing of Hb based blood substitutes has revealed two critical problems: the size of the transporter, and the oxidation of the iron atom that binds the oxygen. Two technologies are available to assist in the development of an Hb-based oxygen delivery system. The genetic engineering methods combined with co-expression systems are available to produce novel Hb molecules. In addition drug delivery systems based on nanoparticles could be adapted to the blood substitute application. One strategy of producing a blood substitute of appropriate size is the use of genetically engineered Hbs; therefore, we have undertaken the development of a stable octameric Hb based on surface cysteines to increase the size of the transporter. In parallel we will consider vectors originally developed for drug delivery; a new generation of core-crown nanoparticles coupled to Hb may allow a more robust delivery.  In addition the use of a nanoparticle would also allow the addition of other molecule to help reverse the oxidation. In both cases a large quantity is required for use as a blood substitute, and we will consider a scalingup of the methods for producing recombinant proteins.