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Team: Heme, Iron and Inflammatory diseases


Our group works in strong interaction with several referent hospital structures and network that provide national and European readability, expertise, technologies and unique ability to recruit patients and biological samples. It includes: the national reference center for rare diseases Porphyrias (Director J.C. Deybach and H. Puy) and the European network EPNET (European Porphyria Network, President J.C;Deybach), the reference laboratory for diagnosis of rare microcytic anemia (B. Grandchamp and C. Kannengiesser) and the reference laboratory for the management of the Diamond-Blackfan anemia (L. Da Costa).

Heme synthesis requires the fine-tuning of protoporphyrin production and iron bioavailability. This pathway is highly active in bone marrow erythroblasts, and to a lesser extent in liver and kidney. Besides being the prosthetic group of hemoproteins, heme is a transcriptional as well as post-transciptional regulator and can exert pro- or anti-inflammatory effects, according to circumstances.  Mutations in enzymes of heme biosynthetic pathway lead to porphyrias, symptoms resulting mostly from accumulation of heme precursors, in hepatocytes or erythroblasts according to the defect. Reduced heme synthesis may underlie the erythroid-specificity of Diamond-Blackfan anemia, a ribosomopathy. Defective iron bioavailability leads to microcytic anemia, or can contribute to the anemia of inflammation, mostly because of upregulation of hepcidin, the negative regulator of iron homeostasis. Both liver and kidney contribute to a tight control of serum hepcidin levels.

Two members of our group have launched a start-up called RedoxyLab to explore markers of oxidative stress and the levels of anti-oxidant defence in normal and pathological conditions.

Identification of candidate gene in rare microcytic anemia and in DBA 

In recent years, our team has contributed to the identification of new genes involved in rare anemia, in non-classical EPP and in Diamond-Blackfan anemia (DBA). However, for up to 30 % of these patients, the causative gene remains to be identified.

The specific objectives of this project are to identify known or unknown genes involved in the disorder of iron acquisition and heme synthesis by erythroid cells, and/or in the regulation of hepcidin. We will then perform functional studies of the newly identified causative genes. We have developed cultures of erythroid progenitors from CD34+ cells isolated from circulating blood of patients and are now able to characterize heme synthesis, iron uptake, as well as erythroid cell proliferation and differentiation.

Reappraisal of the role of kidney in iron homeostasis, hepcidin synthesis and control of erythropoietin (EPO) production in the anemia of inflammation, in hemolysis and in ineffective erythropoiesis

 As a prerequisite to these studies, we are developing a mass spectrometry-based assay for mouse serum and urine hepcidin

  1. 1.    Anemia of inflammation

Anemia of inflammation (AI) is characterized by functional iron deficiency due to increased serum hepcidin level, ineffective bone marrow erythropoiesis and impaired production of EPO by the kidney. AI is partially resistant to EPO therapy. The interactions between kidney hepcidin production and EPO regulation in inflammation are not known. Iron deficiency has been shown to stimulate EPO synthesis and since, the medullar interstitial fibroblasts that produce EPO are immersed in an environment faintly vascularized, we presume that their iron content may be modulated by the degree of iron reabsorption in the tubular cells. We have shown that hepcidin tightly control renal handling of iron but the possibility that this hormone exerts a feedback control on Epo production during AI remains to be determined. We plan to explore physiological relationship between hepcidin, Epo and iron metabolism during anemia of inflammation, using several mouse models lacking normal expression of hepcidin (constitutive and renal Hamp-/-, Hjv-/-, Hfe-/-) in order to study the effect of inflammation and hepcidin on EPO synthesis and on HIF1α /Hif2α pathways in kidney.

  1. 2.    Intravascular hemolysis and ineffective erythropoiesis

Both intravascular hemolysis and ineffective erythropoiesis are known to suppress liver hepcidin expression and increase intestinal iron absorption. In the former situation, this is barely sufficient to compensate urinary iron losses whereas in the latter situation it leads to hepatocyte iron overload. Using mouse models of regenerative anemia following hemolysis, of intermediate or major thalassemia, and of sickle cell disease with or without intravascular hemolysis, we plan to i) evaluate red cell half-life, bone marrow and spleen erythropoiesis and the degree of apoptosis of erythroid progenitors, ii) evaluate the iron balance between intestinal absorption, tissue iron distribution between hepatocyte, macrophage and kidney epithelial cells, and finally urinary iron losses, iii) assess hepcidin synthesis in liver and kidney, in response to ineffective or regenerative erythropoiesis in the presence of iron overload or not.

New therapeutic approaches of erythropoietic protoporphyrias (EPP)

a. Antisense oligonucleotide therapy

Deleterious accumulation of protoporphyrin IX (PPIX) in erythroid cells can result from a partial deficiency in ferrochelatase (FECH) or from induction of ALAS2. Most EPP patients present in trans to a FECH gene mutation an IVS3-48C hypomorphic allele due to a splice mutation. The aberrantly spliced mRNA is degraded and contributes to a low FECH enzyme activity allowing EPP phenotypic expression. We have identified an antisense oligonucleotide (V1-ASO) which allows redirecting splicing from the cryptic to the physiological site. This V1-ASO-mediated redirection allows reducing by 60% the PPIX overproduction in primary cultures of EPP erythroid progenitors. We plan to improve the targeting of the V1-ASO to erythroid progenitors, either using lentiviral particles in cultured erythroid progenitors, in collaboration with Hubert de Verneuil (partner 18) or by linking the V1-ASO to peptides with high affinity to transferrin receptor 1, highly expressed in erythroid cells.

b. Inhibitors of heme synthesis

Inhibiting ALAS2, the first enzyme of the pathway, would be another therapeutic approach to reduce PPIX accumulation, which could also be beneficial in thalassemia where reduced heme biosynthesis is known to improve the clinical phenotype. We propose to screen a chemical databank using a fluorescent test at the “Plateforme de Chimie Biologie Intégrative” in Strasbourg. The fluorescent test is based on the induction of ALAS2, the erythroid isoform of the first enzyme of the heme biosynthetic pathway and the accumulation of easily detectable porphyrins in erythroid cell lines, MEL or K562.

Diamond-Blackfan anemia (DBA), a ribosomopathy with an erythroid tropisms.

 Pathophysiology of DBA remains poorly understood. Mutations of ribosomal protein (RP) genes induce a maturation defect in ribosomal RNA followed by defective assembly of ribosomal subunit.   Animal models and cultures of primary CD34+ cells from patients or infected with RP-sh RNAs have shown that activation of the p53 pathway is responsible for apoptosis and cycle cell arrest at the G0/G1 stage. In addition, an alternate splicing of the FLVCR mRNA, coding for a heme exporter, has been observed in DBA patients and in K562 cells infected with RPS19-shRNA leading to reduced expression of this transporter at the cell membrane. FLVCR1-deficient mice develop a DBA-like phenotype; however, no mutation in this gene has been reported so far in DBA patients.

Aim: Test the hypothesis that the heme exporter FLVCR could act as a modifier gene, responsible in part for the phenotypic variability of the DBA patients and/or that defective heme export is an aggravating factor by inhibiting globin transcription or mRNA translation.

1-  We will study heme and iron metabolism as well as oxidative stress (in collaboration with RedoxyLab) in cord blood CD34+ cells infected with RPS19-, RPL5- or RPL11-sh RNAs. We will also try to reverse the phenotype by overexpressing FLVCR in these cells (in collaboration with Janis Abkowitz, Seattle, USA).

2- We have developed FLVCR shRNA to see the consequences of FLVCR inhibition on cell proliferation, differentiation and apoptosis as well as on the p53 pathway. The results will be confirmed on primary cultures of erythroid progenitors from DBA patients.

3- At odds with the above-mentioned hypothesis, it has been recently suggested, using cultured erythroid progenitors from patients, that exogenous heme can revert the DBA phenotype. We will test whether we can confirm these data and if this is so, we will test the possibility to treat the anemia of DBA patients using Normosang® .