Adenosine signaling inhibits erythropoiesis and promotes myeloid differentiation

Intracellular uptake of adenosine is essential for optimal erythroid commitment and differentiation of hematopoietic progenitor cells. The role of adenosine signaling is well documented in the regulation of blood flow, cell proliferation, apoptosis, and stem cell regeneration. However, the role of adenosine signaling in hematopoiesis remains unclear. In this study, we show that adenosine signaling inhibits the proliferation of erythroid precursors by activating the p53 pathway and hampers the terminal erythroid maturation. Furthermore, we demonstrate that the activation of specific adenosine receptors promotes myelopoiesis. Overall, our findings indicate that extracellular adenosine could be a new player in the regulation of hematopoiesis

The endothelin B receptor plays a crucial role in the adhesion of neutrophils to the endothelium in sickle cell disease

Although the primary origin of sickle cell disease is a hemoglobin disorder, many types of cells contribute considerably to the pathophysiology of the disease. The adhesion of neutrophils to activated endothelium is critical in the pathophysiology of sickle cell disease and targeting neutrophils and their interactions with endothelium represents an important opportunity for the development of new therapeutics. We focused on endothelin-1, a mediator involved in neutrophil activation and recruitment in tissues, and investigated the involvement of the endothelin receptors in the interaction of neutrophils with endothelial cells. We used fluorescence intravital microscopy analyses of the microcirculation in sickle mice and quantitative microfluidic fluorescence microscopy of human blood. Both experiments on the mouse model and patients indicate that blocking endothelin receptors, particularly ETB receptor, strongly influences neutrophil recruitment under inflammatory conditions in sickle cell disease. We show that human neutrophils have functional ETB receptors with calcium signaling capability, leading to increased adhesion to the endothelium through effects on both endothelial cells and neutrophils. Intact ETB function was found to be required for tumor necrosis factor α-dependent upregulation of CD11b on neutrophils. Furthermore, we confirmed that human neutrophils synthesize endothelin-1, which may be involved in autocrine and paracrine pathophysiological actions. Thus, the endothelin-ETB axis should be considered as a cytokine-like potent pro-inflammatory pathway in sickle cell disease. Blockade of endothelin receptors, including ETB, may provide major benefits for preventing or treating vaso-occlusive crises in sickle cell patients

Role of Endothelin-1 in neutrophils activation and adhesion in sickle cell disease

La drépanocytose est une maladie génétique du globule rouge, due à une mutation ponctuelle du gène β de la chaine de l’hémoglobine. Néanmoins, la physiopathologie de la maladie va bien au-delà des anomalies érythrocytaires, avec notamment un dysfonctionnement vasculaire et leucocytaire qui font toute la complexité de la maladie. Au cours de ce travail, nous avons étudié le rôle de l’endothéline-1, peptide vaso-constricteur impliqué de nombreuses pathologies notamment vasculaires, sur l’activation et l’adhérence des polynucléaires neutrophiles dans la drépanocytose. Nous avons travaillé à la fois sur un modèle murin de drépanocytose (les souris SAD) et sur des échantillons sanguins de patients. Sur le modèle animal, nous avons réalisé des expériences de microscopie intravitale, permettant de tester in vivo l’effet des antagonistes des récepteurs ETA et ETB à l’endothéline sur le recrutement des polynucléaires neutrophiles. Sur les échantillons de sang de patients drépanocytaires, nous avons testé in vitro l’adhérence en flux des neutrophiles à l’endothélium vasculaire en réponse au blocage des récepteurs ETA et ETB. Nous avons enfin étudié l’expression des récepteurs ETA et ETB à la surface des neutrophiles et les voies de signalisations découlant de leur activation.Sur le modèle de souris drépanocytaire, l’inhibition des récepteurs ETA, mais surtout ETB permet de limiter le recrutement leucocytaire important provoqué par un stimulus inflammatoire. Ces résultats confirment le rôle d’ETA et celui, plus inattendu, d’ETB dans toutes les étapes d’adhérence des polynucléaires neutrophiles et dans leur transmigration tissulaire en contexte drépanocytaire.Sur des échantillons humains, nous avons confirmé le rôle crucial d’ETB dans l’adhérence des polynucléaires neutrophiles. Nous avons également confirmé la présence de récepteurs ETA et ETB à la surface des neutrophiles. Le récepteur ETB active une voie de signalisation responsable d’une mobilisation du calcium intra cytoplasmique, mais indépendante de l’activation de la phospho-inositide 3-kinase. Enfin, nous avons montré la capacité de ces cellules à synthétiser et excréter elles-mêmes de l’endothéline-1, pérennisant ainsi probablement la réponse inflammatoire et le recrutement leucocytaire. En conclusion, notre travail a permis de mettre en évidence le rôle important du récepteur ETB dans le recrutement des polynucléaires neutrophiles dans la drépanocytose. Ces données suggèrent que les antagonistes des récepteurs à l’endothéline pourraient être bénéfiques en prévention des phénomènes vaso-occlusifs chez les patients drépanocytaires.Sickle cell disease is a genetic disorder affecting red blood cells, due to a point mutation in the β chain of the hemoglobin gene. However, the pathophysiology of the disease goes well beyond the erythrocyte abnormalities, including vascular and white blood cell dysfunctions that contribute to the complexity of the disease. In this project, we investigated the role of endothelin-1, a powerful vasoconstrictor peptide involved in many vascular diseases, on activation and adhesion of neutrophil in sickle cell disease.We worked on both a mouse model of sickle cell disease (SAD mice) and blood samples from patients. In mice, we performed intravital microscopy experiments, to test the in vivo effect of endothelin receptor antagonists ETA and ETB on neutrophils recruitment. On blood samples from patients with sickle cell disease, we tested in vitro adhesion of neutrophils to vascular endothelium in response to the blocking of ETA and ETB receptors. Finally, we studied the expression of ETA and ETB receptors on neutrophils and the signaling pathways resulting in their activation.In our mouse model of sickle cell disease, the inhibition of both endothelin receptors ETA and ETB limits the major leukocyte recruitment caused by an inflammatory stimulus. These results confirm the role of ETA and the more unexpected important role of ETB in all stages of neutrophil adhesion and transmigration in sickle cell context.On human samples, we demonstrated the crucial role of ETB in neutrophils adhesion. We also confirmed the expression of ETA and ETB receptors on neutrophils. ETB receptor activates a signaling pathway responsible for intracytoplasmic calcium mobilization, but not involving the activation of phosphoinositide 3-kinase. Finally, we have shown the ability of neutrophils to synthesize and secrete endothelin-1, which can contribute to sustain the inflammatory stimulation and increased leukocyte recruitment.In conclusion, our work has highlighted the important role of ETB receptor in the recruitment of neutrophils in sickle cell disease. These data suggest that the antagonists of endothelin could be beneficial in prevention of vaso-occlusive events in sickle cell patients

Thérapeutiques transfusionnelles dans la prise en charge de la vasculopathie cérébrale de l enfant drépanocytaire : échanges transfusionnels versus saignées-transfusions, bénéfice/risque des deux méthodes

La drépanocytose est une maladie génétique de l hémoglobine, exposant notamment les patients à un risque d accident vasculaire cérébral, qui peut être prévenu par la réalisation de transfusions sanguines ou d échanges transfusionnels itératifs. Nous avons décrit et comparé, en termes d efficacité et de tolérance, les protocoles transfusionnels proposés aux enfants drépanocytaires ayant une vasculopathie cérébrale dans 2 centres hospitaliers : dans l un, un programme d échanges transfusionnels manuels ou automatisés, et dans l autre un programme de transfusions itératives et de saignées-transfusions. L échange transfusionnel automatisé est la méthode qui permet la meilleure diminution du taux d HbS par séance en comparaison avec l échange transfusionnel manuel et la transfusion simple. En ce qui concerne la normalisation de la vasculopathie cérébrale, il n existait pas de supériorité des échanges transfusionnel, manuels et automatisés, par rapport aux transfusions simples. Le taux d allo-immunisation était plus important dans le groupe de transfusions itératives que dans le groupe d échanges transfusionnels. Les patients sous transfusions itératives présentaient une surcharge martiale plus fréquente et plus importante que les patients sous échanges transfusionnels. En conclusion, la réalisation de transfusions itératives chez les enfants drépanocytaires permet d améliorer la vasculopathie cérébrale de manière aussi efficace que les échanges transfusionnels manuels et automatisés. En revanche, cette méthode comporte d avantages de risques en termes d allo-immunisation, et surtout de surcharge martiale, difficilement accessible ensuite au traitement médicamenteux chélateurPARIS6-Bibl.Pitié-Salpêtrie (751132101) / SudocSudocFranceF

Variability of Prognostic Results Based on Biological Parameters in Sickle Cell Disease Cohort Studies in Children: What Should Clinicians Know?

Background: Many pediatric studies describe the association between biological parameters (BP) and severity of sickle cell disease (SCD) using different methods to collect or to analyze BP. This article assesses the methods used for collection and subsequent statistical analysis of BP, and how these impact prognostic results in SCD children cohort studies. Methods: Firstly, we identified the collection and statistical methods used in published SCD cohort studies. Secondly, these methods were applied to our cohort of 375 SCD children, to evaluate the association of BP with cerebral vasculopathy (CV). Results: In 16 cohort studies, BP were collected either once or several times during follow-up. The identified methods in the statistical analysis were: (1) one baseline value per patient (2) last known value; (3) mean of all values; (4) modelling of all values in a two-stage approach. Applying these four different statistical methods to our cohort, the results and interpretation of the association between BP and CV were different depending on the method used. Conclusion: The BP prognostic value depends on the chosen statistical analysis method. Appropriate statistical analyses of prognostic factors in cohort studies should be considered and should enable valuable and reproducible conclusions

Alterations of red blood cell metabolome in overhydrated hereditary stomatocytosis

Overhydrated hereditary stomatocytosis, clinically characterized by hemolytic anemia, is a rare disorder of the erythrocyte membrane permeability to monovalent cations, associated with mutations in the Rh-associated glycoprotein gene. We assessed the red blood cell metabolome of 4 patients with this disorder and showed recurrent metabolic abnormalities associated with this disease but not due to the diminished half-life of their erythrocytes. Glycolysis is exhausted with accumulation of ADP, pyruvate, lactate, and malate. Ascorbate metabolic pathway is altered probably due to a limited entry of dehydroascorbate. Although no major oxydative stress has been reported in patients with overhydrated hereditary stomatocytosis, we found decreased amounts of oxydized glutathione, creatine and ergothioneine, suggesting transporter abnormalities and/or uncharacterized oxydative stress. These results pinpoint major metabolic defects of overhydrated hereditary stomatocytosis erythrocytes and emphasize the relevance of red blood cell metabolomics for a better understanding of the pathophysiological bases of hemolytic anemia associated with erythrocyte abnormalities

Factors associated with adverse outcome among children with sickle cell disease admitted to the pediatric intensive care unit: an observational cohort

International audienceAbstract Background Sickle cell disease (SCD) is one of the most frequent inherited diseases in the world. Over the last decades, in high-income countries, an important decrease in mortality have been observed due to the improvement of care. However, children with SCD can become critically ill and require admission in Pediatric Intensive Care Units (PICU). The purpose of this study was to describe the epidemiology of children with SCD admitted to PICU for acute crisis and to identify factors associated with adverse outcome (AO). Methods We conducted a retrospective study in a Tertiary Hospital in France including all consecutive children with SCD admitted to PICU between January 1st, 2009 and December 31, 2019. We collected baseline patient’s characteristics, clinical and biological data as well as treatments and life sustaining therapies used in the PICU. Patients were defined as experiencing AO in case of death during stay and/or need for invasive mechanical ventilation (MV) and/or for non-invasive ventilation (NIV) for more than 3 days and/or need for vasopressors and/or need for renal replacement therapy. Results We included 579 admissions in 395 patients, mainly of SS genotype (90%) with a median age of 9.2 years [5.5–13.4] and a median baseline hemoglobin of 8.0 g/dl (7.5–8.8). The two main reasons for admission were acute chest syndrome (ACS) ( n = 331, 57%) and vaso-occlusive crisis refractory to first line therapy ( n = 99, 17%). Half of patients required NIV and 47 (8%) required MV. The overall length of stay was 3 days [1–4] and seven (1%) patients died during PICU stay.There was a total of 113 (20%) admissions with AO and on multivariable analysis, baseline hemoglobin < 8 g/dL, history of bronchial obstruction and admission for ACS were associated with AO. There was no difference in the proportion of hydroxyurea treatment or exchange transfusion program between patients with AO and the other patients. Conclusions Baseline hemoglobin < 8 g/dL, history of bronchial obstruction and admission for ACS were the strongest risk factors for severe evolution in SCD children admitted to PICU. These factors could be taken into consideration when choosing the adequate therapeutic options

Proteomic Landscape of Neutrophils in Sickle Cell Anemia: An Unexpected Autoimmune Profile

Meeting: 60th Annual Meeting of the American-Society-of-Hematology (ASH)Location: San Diego, CADate: DEC 01-04, 2018Sponsor: Amer Soc HematolInternational audienceAbstract 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 <0.7). Interestingly, HC restored a normal expression pattern for most of the interferon signaling proteins. 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<0.001) and it is noteworthy that while 50% of SS patients have similar level of INFα compare to AA individuals the other 50% exhibit 10 to 1,000-fold increased levels (Fig. 2). 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