br Material and Methods br Results br

Material and Methods
Results
Discussion Translational research is limited in SCA very young infants either because diagnosis is delayed or because, in case of neonatal diagnosis, parental approval for clinical trials is extremely difficult to obtain at a very young age in the setting of such a severe disease. Here, we confirm in a larger cohort of SCA infants aged less than 6months, the very early onset of haemolytic anaemia contrasting with the slow decline of HbF. These results, consistent with previous reports (Maier-Redelsperger et al., 1994; Steinberg et al., 2014), illustrate the pitfall of considering elevated HbF level as protective. Measuring global HbF level by HPLC or enumerating F cells overlooks the content of HbF at a cellular level because the number of F-cells with polymer-inhibiting concentrations of HbF is a more important determinant than the concentration of HbF in the hemolysate or the total number of F-cells. However, quantitative methods for measuring the amount of HbF in each F-cell (HbF/F-cell) and plotting the distribution of HbF among F-cells are not available (Steinberg et al., 2014). Measuring HbF does not address therefore the heterogeneous distribution of HbF within RBCs and hence subpopulations of RBCs prone to polymerisation, sickling and haemolysis because of their very low content in HbF. These numerically small subpopulations may in fact play an important pathophysiological role. Cytometric analysis of SCA RBCs indeed evidenced reticulocytes overexpressing the erythroid adhesion molecules: Lu/BCAM, ICAM-4 and LFA-3, whilst no difference was evidenced on mature red cells. Overexpression of Lu/BCAM and ICAM-4 has been demonstrated in SCA (El Nemer et al., 1998; Parsons et al., 1999) and, importantly, functional activation by phosphorylation (Gauthier et al., 2005) results in increased RBC adhesion to vascular endothelium and resistance to high shear-stress forces, thereby contributing to VOC genesis (Kaul et al., 2006). Lu/BCAM is the carrier of the Lutheran and the BCAM d-glucose and the unique erythroid receptor for laminin α5 chain, a major component of the sub endothelial extracellular matrix. It is expressed both on erythroid and endothelial cells and is a receptor for α4β1 integrin expressed on sickle reticulocytes and leukocytes (El Nemer et al., 2007; Bartolucci et al., 2010). ICAM-4 carries the antigens of the Landsteiner–Wiener (LW) blood group system and is a ligand for a large repertoire of integrins including αVβ3 present on endothelial cells. ICAM-4 undergoes phosphorylation on serine along the PKA-dependent pathway. In addition to its adhesive interaction with endothelial cells, ICAM-4 was shown to play a critical role in vaso-occlusion in murine models through its interaction with αVβ3 (Delahunty et al., 2006). Conversely, no data is available on the role of LFA-3 in erythroid cells, but growing evidence points to LFA-3 as a novel functional marker involved in regulating the self-renewal of tumour-initiating cells in colorectal cancer (Xu et al., 2014). Interestingly, LFA-3 was found on the surface of exosomes released by rat reticulocytes pointing out a mechanism through which it is down regulated during terminal erythroid maturation (Vidal, 2010). Known adhesion markers previously identified on SCA reticulocytes i.e., CD36 and CD49d (α4β1/Very Late Antigen-4) (Swerlick et al., 1993; Joneckis et al., 1993) were not significantly increased in our SCA infant cohort (Fig. 1). This discrepancy may be explained by the overall relatively low percentage of reticulocytes, as opposed to adults, and is consistent with the findings of Odièvre et al. in SCA children (Odievre et al., 2008). Recent publications have pointed to the significance of an elevated reticulocyte count in association with disease severity (Meier et al., 2013, 2014). In this study we confirm the very early onset of reticulocytosis in SCA infants and demonstrate the presence of circulating stress reticulocytes with a specific adhesion molecule profile, overexpressing the adhesion molecules Lu/BCAM, ICAM-4 and LFA-3. This numerically small subpopulation of reticulocytes expressing Lu/BCAM, ICAM-4 and LFA-3 could play a role in early infancy notably in splenic injury as these cells would be prone to splenic trapping in the open circulation of the filtering beds, through adhesion to laminin and/or the spleen-specific endothelial cells of the venous sinus (Brousse et al., 2014). Ongoing longitudinal analysis will confirm if this idiosyncratic erythroid adhesion molecule profile in infants indeed correlates with increased haemolysis and adhesion, functional activation and disease severity.