Exteriorisation of Mannoses on Human Erythrocyte Membrane Skeleton Provides 'Eat Me' Signals for Oxidatively Damaged Cells to be Cleared By Macrophages: A Pathway Mediating Hemolysis in Sickle Cell Disease
Authors: Vickers Mark A , Barker Robert N , Cao Huan , Wassall Heather J , Forrester Megan A , Hall Lindsay S , Moss Michael , Henderson Sadie , Konieczny Gabriela , Beverly Minter , Shepherd Jenna , Haslam Stuart M , Rees David C , Wilson Heather M , Tampakis Dimitrios , Williams Maria-Louise 
Published: January 2020

Abstract
The disposal of unwanted or dying cells is a key biological process driven by the display of 'eat me' signals that are recognised by phagocytes. Although these markers for uptake are known to include certain lipids and proteins, the role of glycans, which are abundant on cell surfaces, remains poorly characterized. Here, an unbiased glycomic survey by mass spectroscopic analyses of glycosidase (PNGase F) digested human red blood cell (RBC) membranes identified novel N-linked high mannose structures, which are sequestered inside healthy cells with spectrin, the major protein of the internal membrane skeleton, but exteriorised when cells are damaged as a dominant signal for uptake by macrophages. A panel of lectin probes was used to demonstrate that the mannose species were available as discrete patches on the surface of RBC that had been stressed by oxidation, but undetectable on unpermeabilized untreated cells. High mannoses co-localized with alpha-spectrin on lectin/Western blots and this signal was degraded by prior incubation with glycosidases. Super resolution microscopy showed co-localisation of mannose with spectrin in membrane protrusions of oxidized RBC. Co-localization of spectrin with N-linked mannose and exteriorisation on effete cells were also observed in nucleated cells. The mannose displayed on oxidized RBC represents a novel 'eat-me' signal for human cells, since congeners of mannose inhibited uptake by human monocyte-derived macrophages. The mannose receptor (CD206) was identified as an important receptor in this system by the use of blocking antibody and siRNA mediated knock-down.
The mechanisms underlying the hemolysis characteristic of sickle cell disease (SCD) remain to be fully defined. We therefore investigated the contribution of this new pathway. RBC from patients with SCD demonstrated remarkably high levels of surface mannose, and super resolution microscopy showed the expression again to be limited to discrete patches and co-localized with spectrin in a disrupted membrane skeletal structure. The importance of mannose exposure to uptake of sickle cells by macrophages was confirmed by inhibition by congeners of mannose and blocking antibody to CD206.
The prevalence of SCD is high because of selective pressure caused by the resistance of subjects with sickle cell trait to malarial parasites, which are mainly cleared by splenic macrophages. We therefore investigated whether RBC from subjects with sickle cell trait expressed surface mannose. We show that such RBC express modestly raised levels (p&lt;.01, n=10 (Wilcoxon rank sum)). This excess surface mannose should predispose to parasitized RBC being cleared more rapidly in infected individuals and may cause the increasingly recognised clinical manifestations of sickle cell trait.
We also investigated whether the degree of mannose exposure correlated with peripheral blood count parameters. The Pearson correlation coefficients of log transformed surface mannose expression with hemoglobin levels, red cell counts and reticulocyte counts were -0.83, -0.84 and 0.73 respectively (all p values &lt;0.0001)(n=70, 30 patients with homozygous SS, 10 with AS, 30 with AA); it was noted that cells from patients taking hydroxycarbamide, who had concomitant alpha-thalassaemia or SC disease exhibited intermediate levels.
In summary, our results reveal a previously undescribed cryptic mannose exteriorization pathway, which mediates disposal of oxidatively damaged cells. The pathway is constitutively activated in SCD, where it mediates haemolysis and the degree of activation correlates well with clinical phenotypes of SCD and sickle cell trait. It thus represents a new mechanism for possible future therapeutic intervention.

Disclosures
Vickers: University of Aberdeen: Patents & Royalties: About to apply for patent. Barker: University of Aberdeen: Employment, Patents & Royalties: About to apply for patent. Cao: University of Aberdeen: Patents & Royalties: About to apply for patent.