Team: Red cell tissue biology

Website: http://www.u1134.inserm.fr

Towards a better understanding of RBC filtration by the human spleen

The spleen contributes to the efficient circulation of RBC in small vessels, a process essential for life. Stiff or sticky RBC are indeed cleared by the spleen before they would clog small vessels in other organs. We have explored how the human spleen controls the quality of RBC with a focus on unique mechanical challenges exerted by splenic microcirculatory structures. We study RBC deformation & circulation with a panel of reference tools and with innovative methods (Buffet et al. 2006 ; Deplaine et al. 2011 ; Safeukui et al. 2012 ; Picot, Ndour et al. 2015).

Based on observations in human spleens perfused ex-vivo, we have defined the functional filtering unit of the spleen, the “splenon” (Buffet et al. 2011) and provided the first quantification of retention thresholds (Safeukui, Buffet et al. 2012; Safeukui, Buffet et al. 2013). In the slow circulation, in a sequential quality control, macrophages check for surface abnormalities first, and this process is followed by the sensing of RBC mechanical alterations by splenic slits in the wall of sinuses. We have developed a microsphere-based filtration method (« microsphiltration ») that mimics the mechanical sensing of RBC by the spleen (Deplaine & Safeukui et al. 2011), and have recently adapted this method to microplates for simultaneous analysis of multiple samples and for drug screening (Duez, Holleran et al. 2015 ; Duez, Carucci et al. Submitted). We collaborate closely with colleagues at MIT and Brown University who developed a relevant modeling of RBC crossing splenic slits based on our observations of human spleens (Pivkin et al. 2016). We also use microfluidic chips to analyse the ability of RBC from different subpopulations to cross splenic slits. In collaboration with the Team of Olivier Hermine at Imagine Institute these explorations can be extended to mice (Duez et al. 2015).

Clearance of parasitized RBC by the human spleen – Pathogenesis and treatment of malaria

Plasmodium falciparum develops in RBC in which it triggers molecular and mechanical alterations. We have contributed to show that the innate mechanical retention of RBC infected with P. falciparum plays an important role in the pathogenesis (reviewed in Ndour et al 2015) and transmission of malaria (Tiburcio et al. 2012). We have also studied the spleen-specific “pitting” process whereby undeformable bodies or dead parasites are expelled from the RBC without cell lysis (Jauréguiberry & Ndour et al. 2014; Ndour et al. 2015). We have shown that pitting of RBC containing dead parasites is a major parasite clearance mechanism involved in post-treatment anemia (Jauréguiberry & Ndour et al. 2014; Jaureguiberry et al. 2015, Fernandez-Arias et al. 2016).This approach has recently enabled the dipstick-based prediction of Post-Artesunate Delayed Hemolysis (PADH) – an adverse event that affects more than 15% of patients treated for severe malaria with artemisinins (Ndour et al. 2017). Recently, in collaboration with a team from the Institut pour la Recherche et le Développement (IRD), we have collected preliminary information on RBC deformability in subjects belonging to different tribes in the North of Benin (Henry et al. 2016). Interestingly Fulani, who display an innate protection against malaria but a higher frequency of anemia and splenomegaly, had more deformable RBC in circulation than subjects from other tribes. This suggests a more stringent filtration of RBC by the spleen in Fulani which would be consistent with their innate protection against malaria (more effective clearance of parasitized RBC from the circulation) and their propensity to be anemic with larger spleens (more RBC are removed from the circulation by the spleen and retained in the organ).

Protective and pathogenic roles of the spleen in RBC diseases and transfusion – Hyposplenism Transfusion

A powerful way to determine the function of an organ – here the quality control operated by the spleen on RBC – is to assess what happens when this organ is absent (or dysfunctional). Despite the severe consequences of splenectomy or asplenia in the long term (Sabatini 2011 Lancet, Jais Thorax 2005, Kristinsson Haematologica 2014), the spleen is one of the few organs that can be entirely removed (or that can become severely dysfunctional) without immediate major impact on survival. We have shown that the deformability of RBC is mildly but significantly altered in splenectomized patients, even in those without preexisting RBC disease (Prendki et al. 2012).
With core specialists of sickle cell disease (Brousse et al. 2014) we have proposed a novel hypothesis whereby the progressive loss of splenic filtration results in the presence in circulation of RBC subpopulations associated with complications of the disease. We have recently analysed splenic retention of RBC from sickle cell trait carriers infected or not with P. falciparum (Diakité et al. 2016). Taken together, these observations strongly suggest that the spleen removes stiff, potentially pathogenic, RBC from the circulation, a protective role that comes at the expense of anemia and sometimes splenomegaly.

In close collaboration with Pascal Amireault from Team 1 we have uncovered a subpopulation of small spherocytic RBC that appear in RBC concentrates stored for several weeks before transfusion (Roussel, Dussiot et al. 2017). These small cells are expected to be retained in the spleen, and – if left in circulation when the spleen is saturated or dysfunctional – to impair the microcirculation, potentially leading to severe complications. The double edge role of the spleen (i.e. protective and pathogenic) could therefore also impact the outcome of transfusion. The spleen would be protective by removing the population of abnormal small RBC that accumulate during storage but this would come at the expense of a reduced transfusion yield.

Optimizing the management of leishmaniasis – Clinical research

In his previous positions at Institut Pasteur and Pitié Salpêtrière Hospital, the team leader has contributed, as PI or collaborator of clinical trials, to the development and use of efficient local treatments of cutaneous leishmaniasis (Ben Salah et al. 2013 & 2015, Morizot et al. 2013, Blum et al. 2014). Because experts in neglected tropical diseases are rare, he will maintain a 10-20% activity in this field as consultant physician at Institut Pasteur, member of the Scientific Advisory Committee of Drugs for Neglected Disease Initiative (on personal time), and invited expert at WHO workshops (same). This experience in clinical research has been helpful to implement the medical component of the main project on red blood cells (RBC).