This project is led by Maria De Grandis (PhD, CR EFS), Sylvie Fernandes (PhD, DR EFS) and Stéphanie Morin (PhD, CR EFS), with the technical support of Alexandra Grimaldi (Research Assistant EFS), Cécile Durousseau (Research Assistant EFS) and Thomas Granier (Research Assistant EFS); it is structured in 3 parts:
The microenvironment of the bone marrow: erythroid niches and erythroblast islets "
Erythroid differentiation takes place in specific microenvironments of the bone marrow called erythroid niches. It is regulated by soluble factors and cellular interactions that remain largely unknown due to the lack of tools integrating the complexity of this medullary environment and to the difficulties of access to biological material in humans. These factors and molecules play an important role during erythropoiesis, particularly in the erythroblast islet (EI), which is the manufacturing unit of the RBC. The EI is composed of a central macrophage in close interaction with differentiating erythroblasts.
We will explore the role of the microenvironment and cell interactions through an in vitro experimental approach in which we develop a 3D cell culture system of organoid type and within a scaffold (collaboration with Nicolas Espagnolle, EFS Toulouse) to include several cell types and mimic the medullary environment of erythroid niches. Erythroid differentiation will be followed and studied in this system with molecular and biochemical approaches to determine the effect of the different components.
We will also develop a large scale in silico screening approach to identify the interactome of macrophage and erythroblasts within the EI using already available public data and data that we will generate with our experiments (partnership with Xegen, Marseille) In a second step, we will develop functional studies to investigate the role of candidate genes identified by this screening with adapted molecular, cellular and biochemical approaches, and a possible use of mouse models. Then we will use the same pipeline to identify the interactions between erythroid progenitors, erythroblasts and other cells of the marrow microenvironment (stromal cells, endothelial cells... etc).
This component will benefit from collaborations with Dr. Mohandas Narla (New York Blood Center), Dr. Emile van den Akker (Sanquin, Amsterdam) and Dr. Ashley Toye (University of Bristol, UK).
Component "The role of surface proteins "
This component is related to our previous five-year project on erythroid blood group carrier molecules. We are interested in the role that some of these molecules may have during erythroid differentiation, focusing on two families: adhesion molecules and channels and transporters. Although the RBC is a non-adherent cell, it expresses adhesion molecules on its surface, such as ERMAP, CD47, LW/ICAM4 and Lu/BCAM, whose function in the circulation is not clearly defined, and which are thought to play an important role in cellular interactions with the marrow microenvironment. Conversely, channels and transporters, such as the mechanosensor PIEZO1 and the Gardos channel (KCNN4), are known to play an important role in the regulation of the red blood cell volume, in order to maintain it constant during the 120 days of life in the circulation, but their function during erythropoiesis is very little studied.
We will study (1) the role of these molecules during normal and pathological erythropoiesis (sickle cell disease, project 3.2), with gene inactivation (CRISPR/Cas9, shRNA) and overexpression (recombinant protein expression) approaches (Collaborations: Dr Stéphane Egée, Roscoff; Pr Loïc Garçon, Amiens; Dr Emile van den Akker, Amsterdam).
Cellular signaling and metabolism component "
Signaling during erythropoiesis is well described for the axes involved in cell proliferation and survival such as those of interleukins, M-CSF and especially the EPO/JAK2/STAT5 axis for the terminal phase of differentiation. Kinases play a central role in these signaling axes during differentiation, however their role in the circulating RBC is relatively poorly described. We recently identified several kinases in the mature RBC using a proteomic approach (Gautier et al, 2018), among which is OXSR1 (oxidative stress-responsive kinase 1) whose function during erythropoiesis is unknown. In the mature RBC, OXSR1 has been described in a single paper as regulating Na+/K+/2Cl- cotransport (catalyzed by the cotransporter NKCC1). We will explore the role of this kinase during erythropoiesis by inactivation, overexpression and inhibition approaches (Collaboration with Dr Stéphane Egée, Roscoff).
Signaling and cellular metabolism are often linked, one regulating the other and vice versa. We will explore cell metabolism during erythroid differentiation by profiling each stage using the SCENITH method, which we will adapt to the study of erythropoiesis in collaboration with its inventor, Dr Rafael Argüello (CIML, Marseille).
The 3 parts of this component will allow an integrated approach of erythropoiesis, taking into account the microenvironment, the cellular interactions, as well as the intracellular signaling and metabolism that regulate it. A very important parameter that will be taken into account and explored for the first time in our experiments is the application of partial hypoxia in order to mimic the medullary oxygenation conditions, thanks to a hypoxia chamber that we recently acquired. Indeed, the medullary environment is a hypoxic environment, with an oxygen percentage around 1 to 7%, but the vast majority of data published to date have been obtained from experiments performed under normoxic conditions. We believe that it is essential to revisit these data with the angle of medullary hypoxia in order to integrate this important dimension and get closer to physiological conditions.