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1. Synergistic effect of sildenafil combined with controlled hypothermia to alleviate microglial activation after neonatal hypoxia–ischemia in rats

Author

Pansiot Julien, Manuela Zinni, Natacha Bonnel, Marina El Kamouh, Felipe Odorcyk, Lea Peters, Emilie-Fleur Gautier, Marjorie Leduc, Cédric Broussard, and Olivier Baud

Subjects
Neurology. Diseases of the nervous system, RC346-429
Abstract

Abstract Background and purpose The only validated treatment to prevent brain damage associated with hypoxia–ischemia (HI) encephalopathy of the newborn is controlled hypothermia with limited benefits. Additional putative neuroprotective drug candidates include sildenafil citrate, a phosphodiesterase-type 5 inhibitor. The main objective of this preclinical study is to assess its ability to reduce HI-induced neuroinflammation, in particular through its potential effect on microglial activation. Methods HI was induced in P10 Sprague–Dawley rats by unilateral carotid permanent artery occlusion and hypoxia (HI) and treated by either hypothermia (HT) alone, Sildenafil (Sild) alone or combined treatment (SildHT). Lesion size and glial activation were analyzed by immunohistochemistry, qRT-PCR, and proteomic analyses performed at P13. Results None of the treatments was associated with a significant early reduction in lesion size 72h after HI, despite significant changes in tissue loss distribution. Significant reductions in both Iba1 + (within the ipsilateral hemisphere) and GFAP + cells (within the ipsilateral hippocampus) were observed in SildHT group, but not in the other treatment groups. In microglia-sorted cells, pro-inflammatory markers, i.e. Il1b, Il6, Nos2, and CD86 were significantly downregulated in SildHT treatment group only. These changes were restricted to the ipsilateral hemisphere, were not evidenced in sorted astrocytes, and were not sex dependent. Proteomic analyses in sorted microglia refined the pro-inflammatory effect of HI and confirmed a biologically relevant impact of SildHT on specific molecular pathways including genes related to neutrophilic functions. Conclusions Our findings suggest that Sildenafil combined with controlled hypothermia produces maximum effect in mitigating microglial activation induced by HI through complex proteomic regulation. The reduction of neuroinflammation induced by Sildenafil may represent an interesting therapeutic strategy for neonatal neuroprotection.

Published
2024
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2. Infected erythrocytes and plasma proteomics reveal a specific protein signature of severe malaria

Author

Jeremy Fraering, Virginie Salnot, Emilie-Fleur Gautier, Sem Ezinmegnon, Nicolas Argy, Katell Peoc’h, Hana Manceau, Jules Alao, François Guillonneau, Florence Migot-Nabias, Gwladys I Bertin, Claire Kamaliddin, and NeuroCM consortium

Subjects
Cerebral Malaria, LC-MS/MS, Plasmodium falciparum, Biomarkers, Red Blood Cells, Medicine (General), R5-920, Genetics, QH426-470
Abstract

Abstract Cerebral malaria (CM), the most lethal complication of Plasmodium falciparum severe malaria (SM), remains fatal for 15–25% of affected children despite the availability of treatment. P. falciparum infects and multiplies in erythrocytes, contributing to anemia, parasite sequestration, and inflammation. An unbiased proteomic assessment of infected erythrocytes and plasma samples from 24 Beninese children was performed to study the complex mechanisms underlying CM. A significant down-regulation of proteins from the ubiquitin–proteasome pathway and an up-regulation of the erythroid precursor marker transferrin receptor protein 1 (TFRC) were associated with infected erythrocytes from CM patients. At the plasma level, the samples clustered according to clinical presentation. Significantly, increased levels of the 20S proteasome components were associated with SM. Targeted quantification assays confirmed these findings on a larger cohort (n = 340). These findings suggest that parasites causing CM preferentially infect reticulocytes or erythroblasts and alter their maturation. Importantly, the host plasma proteome serves as a specific signature of SM and presents a remarkable opportunity for developing innovative diagnostic and prognostic biomarkers.

Published
2024
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4. Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process

Author

Mélanie Robert, Bastien Laperrousaz, Diana Piedrahita, Emilie-Fleur Gautier, Travis Nemkov, Florian Dupuy, Elie Nader, Virginie Salnot, Patrick Mayeux, Angelo D'Alessandro, Catherine Lavazec, Philippe Joly, Alexander Scheer, Philippe Connes, and Agnès Cibiel

Subjects
Red blood cells, Drug carrier, Hypotonic dialysis, l-Asparaginase, Omics, Rheology, Therapeutics. Pharmacology, RM1-950
Abstract

Red blood cells (RBCs) can act as carriers for therapeutic agents and can substantially improve the safety, pharmacokinetics, and pharmacodynamics of many drugs. Maintaining RBCs integrity and lifespan is important for the efficacy of RBCs as drug carrier. We investigated the impact of drug encapsulation by hypotonic dialysis on RBCs physiology and integrity. Several parameters were compared between processed RBCs loaded with l-asparaginase (“eryaspase”), processed RBCs without drug and non-processed RBCs. Processed RBCs were less hydrated and displayed a reduction of intracellular content. We observed a change in the metabolomic but not in the proteomic profile of processed RBCs. Encapsulation process caused moderate morphological changes and was accompanied by an increase of RBCs-derived Extracellular Vesicles release. Despite a decrease in deformability, processed RBCs were not mechanically retained in a spleen-mimicking device and had increased surface-to-volume ratio and osmotic resistance. Processed RBCs half-life was not significantly affected in a mouse model and our previous phase 1 clinical study showed that encapsulation of asparaginase in RBCs prolonged its in vivo half-life compared to free forms. Our study demonstrated that encapsulation by hypotonic dialysis may affect certain characteristics of RBCs but does not significantly affect the in vivo longevity of RBCs or their drug carrier function.

Published
2022
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5. The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cell expansion

Author

Audrey Astori, Gabriel Matherat, Isabelle Munoz, Emilie-Fleur Gautier, Didier Surdez, Yaël Zermati, Frédérique Verdier, Sakina Zaidi, Vincent Feuillet, Amir Kadi, Evelyne Lauret, Olivier Delattre, Carine Lefèvre, Michaela Fontenay, Evelyne Ségal-Bendirdjian, Isabelle Dusanter-Fourt, Didier Bouscary, Olivier Hermine, Patrick Mayeux, and Frédéric Pendino

Subjects
Diseases of the blood and blood-forming organs, RC633-647.5
Abstract

The gene CXXC5, encoding a retinoid-inducible nuclear factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome and adult acute myeloid leukemia. RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknown. Here, we evaluated the consequences of RINF silencing on cytokine-induced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells, without affecting cell viability. The phenotype induced by RINF-silencing was dependent on tumor growth factor b (TGFb) and mediated by SMAD7, a TGFb-signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and myelodysplastic syndrome patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdown-dependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a TET2-anchoring platform in mouse. Collectively, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFb superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.

Published
2020
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6. Integrated analyses of translatome and proteome identify the rules of translation selectivity in RPS14-deficient cells

Author

Ismael Boussaid, Salomé Le Goff, Célia Floquet, Emilie-Fleur Gautier, Anna Raimbault, Pierre-Julien Viailly, Dina Al Dulaimi, Barbara Burroni, Isabelle Dusanter-Fourt, Isabelle Hatin, Patrick Mayeux, Bertrand Cosson, and Michaela Fontenay

Subjects
Diseases of the blood and blood-forming organs, RC633-647.5
Abstract

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content, regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequence (CDS) and 3'UTR structure are the key determinants of translation. In these cells, short transcripts with a structured 3'UTR and high CAI showed a decreased translation efficiency. Quantitative analysis of the whole proteome confirmed that the post-transcriptional changes depended on the transcript characteristics that governed the translation efficiency in conditions of low ribosome availability. In addition, proteins involved in normal erythroid differentiation share most determinants of translation selectivity. Our findings thus indicate that impaired erythroid maturation due to 5q- syndrome may proceed from a translational selectivity at the expense of the erythroid differentiation program and suggest that an interplay between the CDS and UTRs may regulate mRNA translation.

Published
2020
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8. Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

Author

Ralfs Buks, Tracy Dagher, Maria Giustina Rotordam, David Monedero Alonso, Sylvie Cochet, Emilie-Fleur Gautier, Philippe Chafey, Bruno Cassinat, Jean-Jacques Kiladjian, Nadine Becker, Isabelle Plo, Stéphane Egée, and Wassim El Nemer

Subjects
polycythemia vera, JAK2V617F, red blood cells, Ca2+, organelle sorting, enucleation, Cytology, QH573-671
Abstract

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

Published
2021
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9. Comprehensive Proteomic Analysis of Human Erythropoiesis

Author

Emilie-Fleur Gautier, Sarah Ducamp, Marjorie Leduc, Virginie Salnot, François Guillonneau, Michael Dussiot, John Hale, Marie-Catherine Giarratana, Anna Raimbault, Luc Douay, Catherine Lacombe, Narla Mohandas, Frédérique Verdier, Yael Zermati, and Patrick Mayeux

Subjects
Biology (General), QH301-705.5
Abstract

Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis.

Published
2016
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10. Red blood cell proteomics reveal remnant protein biosynthesis and folding pathways in PIEZO1-related hereditary xerocytosis

Author

Alexis Caulier, Nicolas Jankovsky, Emilie Fleur Gautier, Wassim El Nemer, Corinne Guitton, Hakim Ouled-Haddou, François Guillonneau, Patrick Mayeux, Virginie Salnot, Johanna Bruce, Véronique Picard, Loïc Garçon, CHU Amiens-Picardie, HEMATIM - Hématopoïèse et immunologie - UR UPJV 4666 (HEMATIM), Université de Picardie Jules Verne (UPJV)-CHU Amiens-Picardie-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Biologie Intégrée du Globule Rouge (BIGR (UMR_S_1134 / U1134)), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pointe-à-Pitre/Abymes [Guadeloupe] -Université des Antilles (UA)-Université Paris Cité (UPCité), AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Plateforme protéomique 3P5 [Institut Cochin] (3P5), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hôpital Bicêtre, Université Paris-Saclay, and DESSAIVRE, Louise

Subjects
piezo1 channel, proteomics, xeroxytosis, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Physiology, Physiology (medical), ubiquitin, red blood cell, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

Hereditary xerocytosis is a dominant red cell membrane disorder characterized by an increased leak of potassium from the inside to outside the red blood cell membrane, associated with loss of water leading to red cell dehydration and chronic hemolysis. 90% of cases are related to heterozygous gain of function mutations in PIEZO1, encoding a mechanotransductor that translates a mechanical stimulus into a biological signaling. Data are still required to understand better PIEZO1-HX pathophysiology. Recent studies identified proteomics as an accurate and high-input tool to study erythroid progenitors and circulating red cell physiology. Here, we isolated red blood cells from 5 controls and 5 HX patients carrying an identified and pathogenic PIEZO1 mutation and performed a comparative deep proteomic analysis. A total of 603 proteins were identified among which 56 were differentially expressed (40 over expressed and 16 under expressed) between controls and HX with a homogenous expression profile within each group. We observed relevant modifications in the protein expression profile related to PIEZO1 mutations, identifying two main “knots”. The first contained both proteins of the chaperonin containing TCP1 complex involved in the assembly of unfolded proteins, and proteins involved in translation. The second contained proteins involved in ubiquitination. Deregulation of proteins involved in protein biosynthesis was also observed in in vitro-produced reticulocytes after Yoda1 exposure. Thus, our work identifies significant changes in the protein content of PIEZO1-HX erythrocytes, revealing a “PIEZO1 signature” and identifying potentially targetable pathways in this disease characterized by a heterogeneous clinical expression and contra-indication of splenectomy.

Published
2022
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11. Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process

Author

Angelo D'Alessandro, Travis Nemkov, Virginie Salnot, Alexander Scheer, Philippe Connes, Philippe Joly, Florian Dupuy, Bastien Laperrousaz, Catherine Lavazec, Emilie-Fleur Gautier, Elie Nader, Diana Piedrahita, Agnès Cibiel, Patrick Mayeux, Mélanie Robert, ERYTECH Pharma, Laboratoire Interuniversitaire de Biologie de la Motricité (LIBM ), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Université Savoie Mont Blanc (USMB [Université de Savoie] [Université de Chambéry]), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Plateforme protéomique 3P5 [Institut Cochin] (3P5), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), University of Colorado Anschutz [Aurora], Centre de Biologie et Pathologie Est (CBPE), Hospices Civils de Lyon (HCL)-Centre National de Référence des Légionelles, and Lavazec, Catherine

Subjects
Drug, Morphology, [SDV.MHEP.HEM] Life Sciences [q-bio]/Human health and pathology/Hematology, Drug carrier, media_common.quotation_subject, Physiology, Omics, Red blood cells, Pharmacokinetics, In vivo, hemic and lymphatic diseases, medicine, General Pharmacology, Toxicology and Pharmaceutics, media_common, Senescence markers, Chemistry, hemic and immune systems, [SDV.MHEP.HEM]Life Sciences [q-bio]/Human health and pathology/Hematology, Red blood cell, l-Asparaginase, medicine.anatomical_structure, Tonicity, Hypotonic dialysis, Dialysis (biochemistry), Rheology, Intracellular, circulatory and respiratory physiology
Abstract

International audience; Red blood cells (RBCs) can act as carriers for therapeutic agents and can substantially improve the safety, pharmacokinetics, and pharmacodynamics of many drugs. Maintaining RBCs integrity and lifespan is important for the efficacy of RBCs as drug carrier. We investigated the impact of drug encapsulation by hypotonic dialysis on RBCs physiology and integrity. Several parameters were compared between processed RBCs loaded with l-asparaginase (“eryaspase”), processed RBCs without drug and non-processed RBCs. Processed RBCs were less hydrated and displayed a reduction of intracellular content. We observed a change in the metabolomic but not in the proteomic profile of processed RBCs. Encapsulation process caused moderate morphological changes and was accompanied by an increase of RBCs-derived Extracellular Vesicles release. Despite a decrease in deformability, processed RBCs were not mechanically retained in a spleen-mimicking device and had increased surface-to-volume ratio and osmotic resistance. Processed RBCs half-life was not significantly affected in a mouse model and our previous phase 1 clinical study showed that encapsulation of asparaginase in RBCs prolonged its in vivo half-life compared to free forms. Our study demonstrated that encapsulation by hypotonic dialysis may affect certain characteristics of RBCs but does not significantly affect the in vivo longevity of RBCs or their drug carrier function.

Published
2022
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12. Integrated analyses of translatome and proteome identify the rules of translation selectivity in RPS14-deficient cells

Author

Michaela Fontenay, Pierre-Julien Viailly, Bertrand Cosson, Patrick Mayeux, Anna Raimbault, Dina Al Dulaimi, Emilie-Fleur Gautier, Barbara Burroni, Isabelle Dusanter-Fourt, Ismael Boussaid, Salomé Le Goff, Célia Floquet, Isabelle Hatin, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Génomique, Structure et Traduction (GST), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
Ribosomal Proteins, 5q- syndrome, Proteome, [SDV]Life Sciences [q-bio], Red Cells, Biology, Ribosomopathies, medicine.disease_cause, Ribosome, Article, 03 medical and health sciences, 0302 clinical medicine, Ribosomal protein, hemic and lymphatic diseases, medicine, Coding region, Humans, Diamond-Blackman, Anemia, Macrocytic, Gene, 030304 developmental biology, Anemia, Diamond-Blackfan, 0303 health sciences, Mutation, Translation (biology), GATA1, Hematology, Cell biology, 030220 oncology & carcinogenesis, Codon usage bias, Myelodysplastic Syndromes, Ribosomes
Abstract

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequence (CDS) and 3’UTR (untranslated region) structure are the key determinants of translation. In these cells, short transcripts with a structured 3’UTR and high codon adaptation index (CAI) showed a decreased translation efficiency. Quantitative analysis of the whole proteome confirmed that the post-transcriptional changes depended on the transcript characteristics that governed the translation efficiency in conditions of low ribosome availability. In addition, proteins involved in normal erythroid differentiation share most determinants of translation selectivity. Our findings thus indicate that impaired erythroid maturation due to 5q- syndrome may proceed from a translational selectivity at the expense of the erythroid differentiation program, and suggest that an interplay between the CDS and UTR may regulate mRNA translation.

Published
2020
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13. Comprehensive proteomic analysis of murine terminal erythroid differentiation

Author

Narla Mohandas, Frédérique Verdier, Patrick G. Gallagher, Michaela Fontenay, Vincent P. Schulz, Christopher D. Hillyer, C. Lacombe, Patrick Mayeux, Emilie-Fleur Gautier, Ismael Boussaid, Marjorie Leduc, Carine Lefevre, François Guillonneau, Meriem Ladli, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Plateforme protéomique 3P5 [Institut Cochin] (3P5), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Yale University [New Haven], New York Blood Center, Mayeux, Patrick, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)

Subjects
Proteomics, Erythroblasts, Cell, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Transcriptome, 03 medical and health sciences, Mice, 0302 clinical medicine, Red Cells, Iron, and Erythropoiesis, Erythroid Cells, hemic and lymphatic diseases, medicine, Animals, Humans, Erythropoiesis, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, 030304 developmental biology, 0303 health sciences, biology, GATA1, Hematology, Embryonic stem cell, Cell biology, Chromatin, Histone, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Proteome, biology.protein, Leukemia, Erythroblastic, Acute
Abstract

Murine-based cellular models have provided and continue to provide many useful insights into the fundamental mechanisms of erythropoiesis, as well as insights into the pathophysiology of inherited and acquired red cell disorders. Although detailed information on many aspects of these cell models is available, comprehensive proteomic data are lacking. This is a critical knowledge gap, as proteins are effectors of most biologic processes. To address this critical unmet need, proteomes of the murine cell lines Friend erythroleukemia (MEL), GATA1 erythroid (G1ER), and embryonic stem cell–derived erythroid progenitor (MEDEP) and proteomes of cultured murine marrow–derived erythroblasts at different stages of terminal erythroid differentiation were analyzed. The proteomes of MEDEP cells and primary murine erythroid cells were most similar, whereas those of MEL and G1ER cells were more distantly related. We demonstrated that the overall cellular content of histones does not decrease during terminal differentiation, despite strong chromatin condensation. Comparison of murine and human proteomes throughout terminal erythroid differentiation revealed that many noted transcriptomic changes were significantly dampened at the proteome level, especially at the end of the terminal differentiation process. Analysis of the early events associated with induction of terminal differentiation in MEDEP cells revealed divergent alterations in associated transcriptomes and proteomes. These proteomic data are powerful and valuable tools for the study of fundamental mechanisms of normal and disordered erythropoiesis and will be of broad interest to a wide range of investigators for making the appropriate choice of various cell lines to study inherited and acquired diseases of the erythrocyte.

Published
2020
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14. The proteome of neutrophils in sickle cell disease reveals an unexpected activation of interferon alpha signaling pathway

Author

Darragh Duffy, Pierre-Louis Tharaux, Patrick Mayeux, Patricia Hermand, Slim Azouzi, Vincent Bondet, Bérengère Koehl, Emilie-Fleur Gautier, François Guillonneau, Caroline Le Van Kim, Sébastien Dechavanne, Institut National de la Transfusion Sanguine [Paris] (INTS), Université de Paris (UP), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Immunobiologie des Cellules dendritiques, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Paris-Centre de Recherche Cardiovasculaire (PARCC (UMR_S 970/ U970)), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Service d'Hématologie Biologique [Hôpital Robert Debré, Paris], AP-HP Hôpital universitaire Robert-Debré [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), This work was supported by a grant from l’Association Recherche et Transfusion, the Institut National de la Transfusion Sanguine, and the Laboratory of Excellence GR-Ex, reference ANR-11-LABX-0051, GR-Ex is funded by the program 'Investissements d’avenir' of the French National Research Agency, reference ANR-11-IDEX-0005-02. The Orbitrap Fusion mass spectrometer was acquired with funds from the FEDER through the 'Operational Programme for Competitiveness and Employment 2007-2013' and from the 'Canceropole Ile de France'., ANR-11-IDEX-0005,USPC,Université Sorbonne Paris Cité(2011), Université Paris Cité (UPCité), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Vougny, Marie-Christine, and Université Sorbonne Paris Cité - - USPC2011 - ANR-11-IDEX-0005 - IDEX - VALID

Subjects
[SDV.IMM] Life Sciences [q-bio]/Immunology, Proteome, Neutrophils, Cell, Red Cells, Alpha interferon, Inflammation, Disease, Anemia, Sickle Cell, 03 medical and health sciences, 0302 clinical medicine, Text mining, Medicine, Humans, Letters to the Editor, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, business.industry, Interferon-alpha, Hematology, 3. Good health, Hemoglobinopathies, medicine.anatomical_structure, Cancer research, [SDV.IMM]Life Sciences [q-bio]/Immunology, medicine.symptom, Signal transduction, business, 030215 immunology, Signal Transduction
Abstract

International audience

Published
2020
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15. Proteomic Landscape of Neutrophils in Sickle Cell Anemia: An Unexpected Autoimmune Profile

Author

Emilie Fleur Gautier, Darragh Duffy, Yves Colin, Patrick Mayeux, Virginie Salnot, Caroline Le Va Kim, Slim Azouzi, Bérengère Koehl, Pierre-Louis Tharaux, Patricia Hermand-Tournamille, Vincent Bondet, Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Biologie Intégrée du Globule Rouge (BIGR (UMR_S_1134 / U1134)), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université des Antilles (UA), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Immunobiologie des Cellules dendritiques, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Paris-Centre de Recherche Cardiovasculaire (PARCC - UMR-S U970), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Robert Debré, Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Toll-like receptor, Innate immune system, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Immunology, Cell Biology, Hematology, Biology, Biochemistry, 03 medical and health sciences, 030104 developmental biology, Cytokine, Interferon, medicine, biology.protein, L-selectin, Signal transduction, Receptor, Interferon type I, medicine.drug
Abstract

Although sickle cell disease (SCD) is a red cell disorder, many cell types, including endothelial cells and polymorphonuclear neutrophils (PMNs), contribute to its pathophysiology. In particular, activated PMNs have been implicated to play an important role in the initiation and propagation of vaso-occlusive events in SCD. Activated PMNs engage in a complex process of abnormal interactions with activated endothelial cells, platelets and circulating erythrocytes contributing to endothelial injury and decreased blood flow. In the present study, global proteomic analysis was performed using label-free mass spectrometry of PMNs from 4 SCD patients (SS) in steady state and from 4 control subjects (AA). We identified a total of 4,534 proteins both in AA and SS PMNs with 3,080 of these proteins identified in at least three samples for each condition. 50 proteins were significantly over-expressed in SS PMNs compared to AA PMNs (ratio > 1.4). STRING employed to monitor potential interaction between the overexpressed proteins showed that the main interactive clusters consist of STAT1 and STAT2, OAS 1, 2 and 3, and many Interferon Signaling Proteins i.e. IFIT1, IFIT2, IFIT 3, ISG15, ISG20, GBP2, IFI35, MX1 and MX2, TLR8 proteins (Fig. 1). This finding implies a strong activation of the type I interferon (IFN) signaling pathway in the SS PMNs (between 10 and 100-fold increase in SS vs AA). In addition, 33 proteins showed significantly lower expression in SS PMNs compared to AA PMNs. Among these were L-selectin (CD62L) and IL-17 receptor A (IL17RA) (p = 0.01). These findings are consistent with previously described phenotypes of aged neutrophils and acute inflammatory responses in SCD. Similar proteomic analysis performed on PMNs from SS patients treated with hydroxycarbamide (HC, n=4) showed that 14 proteins had significantly lower expression compared to untreated-SS patients (ratio Type I IFNs represent the major effector cytokines of the host immune response against viruses and other intracellular pathogens. These cytokines are produced via activation of STAT1 and of pattern recognition receptors, including the Toll-like receptor signaling network. To determine if type I IFN-α could be detected at the protein level in the plasma of SS patients, we used the novel digital-ELISA technology (SIMOA, Quanterix) developed by Wilson et al (J Lab Autom, 2016). Interestingly, we found an increased level of INFα in plasma from SS patients compared to AA (n=32) (p In summary, our novel proteomic analysis documents a high level expression of interferon signaling proteins, STAT1 and TLR8 in the proteome of neutrophils from SS patients and strongly suggests autoimmune or auto-inflammatory phenomena at basal state in SCD. Our results provide strong support for an important role for the innate immune system in the pathophysiology of SCD. Future studies will help determine the relationship between the plasmatic level of IFN-α and clinical complications and will establish if interferon signaling proteins and IFN-α could represent new therapeutic targets in SCD. Disclosures Hermand-Tournamille: Imara: Research Funding. Le Va Kim:Imara: Research Funding. Koehl:Imara: Research Funding.

Published
2018
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16. Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes

Author

Karine Bailly, Patrick Mayeux, Emilie-Fleur Gautier, Wassim El Nemer, Marjorie Leduc, Sylvie Cochet, François Guillonneau, Narla Mohandas, Catherine Lacombe, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Dynamique des Structures et Interactions des Macromolécules Biologiques (DSIMB), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Red Cell Physiology Laboratory [New York, USA], New York Blood Center, Plateforme protéomique 3P5 [Institut Cochin] (3P5), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Transfusion Sanguine [Paris] (INTS), and CHU Pitié-Salpêtrière [APHP]

Subjects
0301 basic medicine, Erythrocytes, Reticulocytes, Proteome, [SDV]Life Sciences [q-bio], Population, 03 medical and health sciences, chemistry.chemical_compound, Red Cells, Iron, and Erythropoiesis, 0302 clinical medicine, Biotin, Reticulocyte, medicine, Humans, education, ComputingMilieux_MISCELLANEOUS, education.field_of_study, Kinase, Hematology, Cell sorting, Adenosine diphosphate, 030104 developmental biology, medicine.anatomical_structure, chemistry, Biochemistry, 030220 oncology & carcinogenesis, Adenosine triphosphate
Abstract

Reticulocytes produced in the bone marrow undergo maturation in the bloodstream to give rise to erythrocytes. Although the proteome of circulating red cells has been the subject of several reports, the cellular populations used for these studies were never completely devoid of reticulocytes. In our current study, we used highly purified erythrocyte and reticulocyte populations to quantify the absolute expression levels of the proteins in each cell population. Erythrocytes and reticulocytes were purified in a multistep process involving cellulose chromatography, Percoll gradient centrifugation, and fluorescence cell sorting after thiazole orange labeling. Proteins were analyzed by mass spectrometry from whole cells and erythrocyte plasma membrane (ghosts), leading to the identification and quantification of 2077 proteins, including 654 that were reticulocyte-specific. Absolute quantifications of these proteins were made using the mean corpuscular hemoglobin content of the cells as a standard. For each protein, we calculated the percentage loss during the terminal stages of reticulocyte maturation and the percentage of association with the plasma membrane. In addition, we used modified adenosine triphosphate and adenosine diphosphate molecules that enable the transfer of a biotin molecule to the catalytic sites of kinases to isolate active kinases in the erythrocytes and determined the absolute expression of 75 protein kinases and the modification of their expression during reticulocyte maturation. Our findings represent the first absolute quantification of proteins that are specifically expressed in normal erythrocytes with no detectable contamination by reticulocytes. Our findings thus represent a reference database for the future proteomic analysis of pathological erythrocytes.

Published
2018
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17. XPO1 regulates erythroid differentiation and is a new target for the treatment of β-thalassemia

Author

Flavia Guillem, Michaël Dussiot, Elia Colin, Thunwarat Suriyun, Jean Benoit Arlet, Nicolas Goudin, Guillaume Marcion, Renaud Seigneuric, Sebastien Causse, Patrick Gonin, Marc Gastou, Marc Deloger, Julien Rossignol, Mathilde Lamarque, Zakia Belaid Choucair, Emilie Fleur Gautier, Sarah Ducamp, Julie Vandekerckhove, Ivan C. Moura, Thiago Trovati Maciel, Carmen Garrido, Xiuli An, Patrick Mayeux, Narla Mohandas, Geneviève Courtois, and Olivier Hermine

Subjects
Diseases of the blood and blood-forming organs, RC633-647.5
Abstract

β-thalassemia major (β-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human β-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of β-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of β-TM.

Published
2019
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