Macrophages are key in orchestrating immune responses to micro-environmental stimuli, sensed by a complex set of surface receptors. The human cell line THP-1 has a monocytic phenotype, including the ability to differentiate into macrophages, providing a tractable, standardised surrogate for human monocyte-derived macrophages. Here we assessed the expression of 49 surface markers including Fc, complement, C-type lectin and scavenger receptors; TIMs; Siglecs; and co-stimulatory molecules by flow cytometry on both THP-1 monocytes and macrophages and following macrophage activation with seven standard conditioning/polarizing stimuli. Of the 34 surface markers detected on macrophages, 18 altered expression levels on activation. From these, expression of 9 surface markers were consistently altered by all conditioning regimens, while 9 were specific to individual polarizing stimuli. This study provides a resource for the study of macrophages and highlights that macrophage polarization states share much in common and the differences do not easily fit a simple classification system.

In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.

Heterozygosity for Hemoglobin (Hb) S, sickle cell trait (SCT), affects over 40 million people and confers resistance to severe infection by Plasmodium falciparum. Homozygosity for HbS, or compound heterozygosity with certain other alleles of Hb, affects over 4 million individuals and causes sickle cell disease (SCD). Hemolytic anaemia is a prominent feature of SCD and is mainly extravascular, mediated by hepatic and splenic macrophages. No ligands for this process have been identified. As many macrophage phagocytic receptors recognise carbohydrates, we surveyed surface glycan expression by sickle cells using a panel of 8 lectins and flow cytometry. Most glycans were similar to those of healthy red blood cells (RBC), except much higher expression of terminal mannose. We investigated the structural basis for these residues using glycomic mass spectroscopy, which showed them to be N-linked high (Man5-9GlcNAc2) mannoses, a surprising conclusion as these are usually intermediates in the formation of complex glycans and not displayed on cell surfaces. High resolution microscopy revealed the mannose residues to be carried in discrete microdomains on the surfaces of sickle cells. These structures were absent on the surfaces of healthy RBC, instead being present in the membrane skeleton under the cell surface. Lectin blots and immunoprecipitation showed the mannoses to co-migrate predominantly with spectrin. We showed these mannose-bearing structures were able to stimulate phagocytosis of RBC by using a peripheral blood derived macrophage uptake assay. Sickle RBC were taken up at high rates compared to healthy RBC and this could be inhibited by congeners of mannose. We identified the importance of a cognate ligand (CD206: the mannose receptor) using blocking antibodies and knockdown of CD206 expression using siRNA. The in vivo and pathogenic relevance of mannose exposure was investigated by taking advantage of the heterogeneity of hemolysis in SCD. RBC with SCD (n=94), SCT (n=57) and healthy individuals (n=54) were assayed for mannose exposure by flow cytometry. SCT and healthy RBC showed no mannose exposure but high levels were found on HbSS RBC (p<0.0001). Co-incident inheritance of HbSS with higher HbF values and alleles encoding alpha-thalassaemia resulted in lower surface mannose values. Overall, markers of hemolysis (RBC count, haemoglobin, reticulocyte count) correlated well with mannose exposure (Spearman correlation coefficients -0.68, -0.40, 0.37; p=0.0001, 0.0032, 0.0063 respectively). Plasma LDH is a marker of intravascular hemolysis and correlated with overall hemolysis within SCD (r=-0.25, p=0.016), but not mannose exposure (r=0.14, p=0.19). Thus mannose exposure correlated only with extravascular hemolysis. Identification of a ligand pair mediating rapid clearance of sickle cells raised the possibility that they also mediate enhanced clearance of SCT RBC infected by malarial parasites. Indeed, P. falciparum cultures induced mannose expression at the pigmented trophozoite and schizont stages in infected HbAA RBCs, at levels corresponding to mild hemolysis in SCD. Mannose expression in infected HbAS RBCs was even higher, with levels corresponding to severe hemolysis in SCD. Infection with P. falciparum and selection for HbS arose only recently in human evolution, raising the question of what the physiological triggers for this mechanism are. Infection with malarial parasites causes oxidative stress. We therefore subjected healthy RBC to copper sulphate, which resulted in surface mannose exposure as well as uptake by macrophages. Oxidized SCT RBC displayed more mannose than oxidized healthy RBC. Thus, we have identified a new cell surface 'eat me' signalling mechanism that allows inspecting macrophages to engage with the rigid membrane skeleton and phagocytose the mannose displaying cell. The mechanism is stimulated by HbS: when present in high concentrations, the mechanism is activated constitutively, resulting in sickle cell anaemia. Heterozygosity for HbS is insufficient by itself to trigger mannose exposure. However, the mechanism is primed so that oxidative stress associated with infection by P. falciparum causes greater mannose display, increased parasitized cell clearance and protection against severe malaria. These findings should allow the design of inhibitors of sickle cell haemolysis and inducers of protection against malaria. Disclosures Cao: University of Aberdeen: Patents & Royalties. Barker:University of Aberdeen: Patents & Royalties. Vickers:University of Aberdeen: Patents & Royalties; GSK: Equity Ownership.

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.

In both sickle cell disease (SCD) and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man5-9GlcNAc2), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. Extravascular haemolysis in SCD correlates with high mannose glycan levels on RBCs. Infection of RBCs with Plasmodium falciparum expose surface mannose N-glycans on healthy RBCs, which occurred at significantly higher levels on RBCs from subjects with sickle cell trait compared to those lacking haemoglobin S. The glycans were associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans, a novel response to cellular stress, is the first molecular mechanism common to both the pathogenesis of SCD and resistance to severe malaria in sickle cell trait.