Yves Colin

Recent genome-wide association and murine studies identified the human neutrophil antigen -3a/b polymorphism (HNA-3a/b) in SLC44A2 (rs2288904-G/A) as a risk factor in venous thromboembolism (VTE). The choline transporter-like protein CTL2 encoded by the SLC44A2 gene plays an important role in platelet aggregation and neutrophil interaction with the von Willebrand factor. By investigating alloantibodies to a high-prevalence antigen of unknown specificity, found in patients with a rare blood type, we showed that CTL2 is also expressed in red blood cells and carries a new blood group system. Furthermore, we identified three siblings of European ancestry who are homozygous for a large deletion in SLC44A2 , resulting in complete CTL2 deficiency. Interestingly, the first-ever reported CTL2-deficient individuals suffer from progressive hearing impairment, recurrent arterial aneurysms and epilepsy. In contrast to Slc44a2 ^-/- mice, CTL2 _null individuals showed normal platelet aggregation and do not suffer from any apparent hematological disorders. In addition, CD34 ⁺ cells from CTL2 _null patients undergo normal ex vivo erythropoiesis, indicating that CTL2 is not essential for erythroid proliferation and differentiation. Overall, our findings confirm the function of CTL2 in hearing preservation and provide new insights into the possible role of this protein in maintaining cerebrovascular homeostasis.

The tight regulation of intracellular nucleotides is critical for the self-renewal and lineage specification of hematopoietic stem cells (HSCs). Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside transporters in the differentiation of HSCs to the erythroid lineage and in red cell biology remains to be fully defined. Here, we show that the absence of the equilibrative nucleoside transporter (ENT1) in human red blood cells with a rare Augustine-null blood type is associated with macrocytosis, anisopoikilocytosis, an abnormal nucleotide metabolome, and deregulated protein phosphorylation. A specific role for ENT1 in human erythropoiesis was demonstrated by a defective erythropoiesis of human CD34+ progenitors following short hairpin RNA-mediated knockdown of ENT1. Furthermore, genetic deletion of ENT1 in mice was associated with reduced erythroid progenitors in the bone marrow, anemia, and macrocytosis. Mechanistically, we found that ENT1-mediated adenosine transport is critical for cyclic adenosine monophosphate homeostasis and the regulation of erythroid transcription factors. Notably, genetic investigation of 2 ENT1null individuals demonstrated a compensation by a loss-of-function variant in the ABCC4 cyclic nucleotide exporter. Indeed, pharmacological inhibition of ABCC4 in Ent1−/− mice rescued erythropoiesis. Overall, our results highlight the importance of ENT1-mediated nucleotide metabolism in erythropoiesis.

Background Gene expression profiling of renal allograft biopsies revealed the Duffy antigen receptor for chemokines (DARC) as being strikingly upregulated in antibody‐mediated rejection (ABMR). DARC has previously been shown to be associated with endothelial injury. This study aimed at assessing the value of DARC immunohistochemistry as diagnostic marker in ABMR. Methods The study was performed on 82 prospectively collected biopsies of a clinically well‐defined population (BORTEJECT trial, NCT01873157) of DSA positive patients with gene expression data available for all biopsies. Diagnostic histological assessment of biopsies was performed according to the Banff diagnostic scheme. Results DARC expression was focally accentuated, on peritubular capillaries (PTC) mostly in areas of interstitial fibrosis and/or inflammation. DARC positivity was associated with diagnosis of ABMR and correlated with DARC gene‐expression levels detected by microarray analysis. Still, as previously described, a substantial number of biopsies without signs of rejection showed DARC‐positive PTC. However, DARC positivity in patients with ABMR was associated with significantly reduced graft survival, as compared to patients with DARC‐negative ABMR or DARC‐positivity without ABMR. Conclusions In summary, the upregulation of DARC, detected by immunohistochemistry, is associated with but not specific for ABMR. We did not observe reduced graft survival in DARC positive patients.

Permanent availability of red blood cells (RBCs) for transfusion depends on refrigerated storage, during which morphologically altered RBCs accumulate. Among these, a subpopulation of small RBCs, comprising type III echinocytes, spheroechinocytes, and spherocytes and defined as storage-induced microerythrocytes (SMEs), could be rapidly cleared from circulation posttransfusion. We quantified the proportion of SMEs in RBC concentrates from healthy human volunteers and assessed correlation with transfusion recovery, investigated the fate of SMEs upon perfusion through human spleen ex vivo, and explored where and how SMEs are cleared in a mouse model of blood storage and transfusion. In healthy human volunteers, high proportion of SMEs in long-stored RBC concentrates correlated with poor transfusion recovery. When perfused through human spleen, 15% and 61% of long-stored RBCs and SMEs were cleared in 70 minutes, respectively. High initial proportion of SMEs also correlated with high retention of RBCs by perfused human spleen. In the mouse model, SMEs accumulated during storage. Transfusion of long-stored RBCs resulted in reduced posttransfusion recovery, mostly due to SME clearance. After transfusion in mice, long-stored RBCs accumulated predominantly in spleen and were ingested mainly by splenic and hepatic macrophages. In macrophage-depleted mice, splenic accumulation and SME clearance were delayed, and transfusion recovery was improved. In healthy hosts, SMEs were cleared predominantly by macrophages in spleen and liver. When this well-demarcated subpopulation of altered RBCs was abundant in RBC concentrates, transfusion recovery was diminished. SME quantification has the potential to improve blood product quality assessment. This trial was registered at www.clinicaltrials.gov as #NCT02889133.

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors more than 150 proteins to the cell surface. Pathogenic variants in several genes that participate in GPI biosynthesis cause inherited GPI deficiency (IGD) disorders. Here, we reported that homozygous null alleles of PIGG, a gene involved in GPI modification, are responsible for the rare Emm-negative blood phenotype. Using a panel of K562 cells defective in both the GPI-transamidase and GPI remodeling pathways, we demonstrate that the Emm antigen, whose molecular basis has remained unknown for decades, is carried only by free GPI and that its epitope is composed of the second and third ethanolamine of the GPI backbone. Importantly, we show that the decrease in Emm expression in several IGD patients is indicative of GPI defects. Overall, our findings establish Emm as a novel blood group system and have important implications for understanding the biological function of human free GPI.

Background: Red blood cells (RBC) change upon hypothermic conservation, and storage for 6 weeks is associated with the short‐term clearance of 15% to 20% of transfused RBCs. Metabolic rejuvenation applied to RBCs before transfusion replenishes energetic sources and reverses most storage‐related alterations, but how it impacts RBC circulatory functions has not been fully elucidated.Study Design and Methods: Six RBC units stored under blood bank conditions were analyzed weekly for 6 weeks and rejuvenated on Day 42 with an adenine‐inosine–rich solution. Impact of storage and rejuvenation on adenosine triphosphate (ATP) levels, morphology, accumulation of storage‐induced microerythrocytes (SMEs), elongation under an osmotic gradient (by LORRCA), hemolysis, and phosphatidylserine (PS) exposure was evaluated. The impact of rejuvenation on filterability and adhesive properties of stored RBCs was also assessed.Results: Rejuvenation of RBCs restored intracellular ATP to almost normal levels and decreased the PS exposure from 2.78% to 0.41%. Upon rejuvenation, the proportion of SME dropped from 28.2% to 9.5%, while the proportion of normal‐shaped RBCs (discocytes and echinocytes 1) increased from 47.7% to 67.1%. In LORCCA experiments, rejuvenation did not modify the capacity of RBCs to elongate and induced a reduction in cell volume. In functional tests, rejuvenation increased RBC filterability in a biomimetic splenic filter (+16%) and prevented their adhesion to endothelial cells (−87%).Conclusion: Rejuvenation reduces the proportion of morphologically altered and adhesive RBCs that accumulate during storage. Along with the improvement in their filterability, these data show that rejuvenation improves RBC properties related to their capacity to persist in circulation after transfusion.

Although most individuals with sickle cell disease (SCD) live in sub‐Saharan Africa, the natural history of the disease on this continent remains largely unknown. Intravascular haemolysis results in activation of circulating blood cells and release of microparticles (MPs) that exert pro‐inflammatory effects and contribute to vascular damage. We designed a case‐control study nested in the CADRE cohort (Coeur‐Artère‐DRÉpanocytose, clinical trials.gov identifier NCTO3114137) and based on extreme phenotypes, to analyse blood cell‐derived MPs in 232 adult SS patients at steady state in Bamako and Dakar. Thirty‐six healthy adult controls matched by age and sex were recruited in Bamako. The MPs concentrations were higher in SS patients compared to AA controls with a predominance of erythrocyte‐ and reticulocyte‐derived MPs. These erythroid‐derived MPs were significantly lower in patients with retinopathy (P = 0·022). Reticulocyte‐derived MPs were significantly negatively and positively associated with a history of priapism (P = 0·020) and leg ulcers (P = 0·041) respectively. We describe for the first time the comparative patterns of plasma MPs in healthy subjects and patients with SCD living in sub‐Saharan Africa and exhibiting various complications. Because our present results show no clear pattern of correlation between erythroid MPs and the classical hyper‐haemolytic complications, we hypothesise a weak relevance of the hyper‐haemolysis versus hyper‐viscous paradigm in Africa.

The gene encoding the Duffy blood group protein (Fy, CD234; additional designations Duffy Antigen Receptor of Chemokines [DARC] and Atypical Chemokine Receptor 1 [ACKR1]) is characterized by a SNP in a GATA-1 transcription factor binding site associated with the erythrocyte silent (ES) phenotype. FY ^ES homozygous people are viewed to be highly resistant to blood stage infection with Plasmodium vivax . Increasingly, however, studies are reporting P. vivax infections in Fy-negative individuals across malarious African countries where FY ^ES approaches genetic fixation. This suggests that P. vivax has evolved a Fy-independent RBC invasion pathway, or that the GATA-1 SNP does not abolish Fy expression. Here, we tested the second hypothesis through binding studies to erythroid lineage cells using recombinant P. vivax Duffy binding protein, the parasite’s invasion ligand and Fy6-specific antibodies. We first observed variable Fy expression on circulating RBCs, irrespective of FY genotype; FY ^ES RBCs were periodically Fy-positive. Furthermore, during the in vitro erythroid differentiation of CD34+ cells and on ex vivo bone marrow samples, we observed Fy expression on erythroid precursor cells from FY ^ES people. Finally, the Fy6-specific nanobody, CA111 was used to capture Fy from the surface of FY ^ES RBCs. Our findings reveal that the GATA-1 SNP does not fully abolish Fy expression and provide insight on potential susceptibility of Fy-negative people to vivax malaria. Significance: Duffy blood group negativity results from a single nucleotide polymorphism (SNP) in the gene promoter, and reaches genetic fixation in many African ethnicities. Because the Duffy protein (Fy) is an important contact point during Plasmodium vivax human red blood cell invasion, Fy-negativity is considered to confer resistance to P. vivax malaria. With recent studies in African countries reporting P. vivax infection in Fy-negative people, we studied Fy expression across erythroid development. Here we report that the FY promoter SNP does not abolish Fy protein expression in erythroid progenitors developing in the bone marrow. These results further emphasizes the importance of reticulocytes as targets for P. vivax blood stage infection and propose a mechanism for P. vivax infections in Fy-negative people.

Red blood cells (RBC) ability to circulate is closely related to their surface area-to-volume ratio. A decrease in this ratio induces a decrease in RBC deformability that can lead to their retention and elimination in the spleen. We recently showed that a subpopulation of “small RBC” with reduced projected surface area accumulated upon storage in blood bank concentrates, but data on the volume of these altered RBC are lacking. So far, single cell measurement of RBC volume has remained a challenging task achieved by a few sophisticated methods some being subject to potential artifacts. We aimed to develop a reproducible and ergonomic method to assess simultaneously RBC volume and morphology at the single cell level. We adapted the fluorescence exclusion measurement of volume in nucleated cells to the measurement of RBC volume. This method requires no pre-treatment of the cell and can be performed in physiological or experimental buffer. In addition to RBC volume assessment, brightfield images enabling a precise definition of the morphology and the measurement of projected surface area can be generated simultaneously. We first verified that fluorescence exclusion is precise, reproducible and can quantify volume modifications following morphological changes induced by heating or incubation in non-physiological medium. We then used the method to characterize RBC stored for 42 days in SAG-M in blood bank conditions. Simultaneous determination of the volume, projected surface area and morphology allowed to evaluate the surface area-to-volume ratio of individual RBC upon storage. We observed a similar surface area-to-volume ratio in discocytes (D) and echinocytes I (EI), which decreased in EII (7%) and EIII (24%), sphero-echinocytes (SE; 41%) and spherocytes (S; 47%). If RBC dimensions determine indeed the ability of RBC to cross the spleen, these modifications are expected to induce the rapid splenic entrapment of the most morphologically altered RBC (EIII, SE, and S) and further support the hypothesis of a rapid clearance of the “small RBC” subpopulation by the spleen following transfusion.