Anti-erythrocyte antibodies may contribute to anaemia in Plasmodium vivax malaria by decreasing red blood cell deformability and increasing erythrophagocytosis

Abstract

BackgroundPlasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated.MethodsConcentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group.ResultsAnaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance.ConclusionsThese findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.

Effects of IgG and IgM autoantibodies on non-infected erythrocytes is related to ABO blood group in Plasmodium vivax malaria and is associated with anemia

“…Our defocusing microscopy and erythrophagocytosis results indicate that IgG antibodies purified from anemic patients with vivax malaria can decrease nRBCs membrane dynamics fluctuations and increase their in vitro phagocytic uptake, mainly of those from O phenotype. This clearance of non-infected erythrocytes is an important component of malaria-associated anemia as previously demonstrated [5, 17] and suggests that O individuals may be more prone to develop anemia during P. vivax infections. These data add new insights about the possible association between ABO blood groups and P. vivax -associated anemia.…”

“…Defocusing Microscopy (DM) is a non-invasive and powerful optical microscopy technique that allows the evaluation of morphological (volume, surface area, sphericity index, total 3-D shape), chemical (hemoglobin content and concentration) and mechanical properties (dynamic membrane fluctuation) of single cells using simple methods and easy setup [11]. DM was used to investigate the effects of different antibodies in the biomechanical properties of erythrocytes through measurement of membrane fluctuations amplitude of individual nRBCs from blood group A and O. DM experiments were performed in a Nikon Eclipse TI inverted microscope as previously described [5, 11]. To determine whether there is a difference between membrane rigidity of O and A erythrocytes after the addition of antibodies purified from different groups of study, we obtained the relative membrane fluctuation, which is calculated as the ratio between membrane fluctuation before and 30 min after antibody addition.…”

“…Blood samples were collected by venous puncture in heparin tubes from three healthy volunteers from each blood group (A or O) on the day of the erythrophagocytosis assay, which was performed as previously described [5]. Phagocytosed RBCs were counted on 400 macrophages.…”

“…One of the major complications of P. vivax infection is anemia [2, 3], which is thought to arise from the destruction of both infected and non-infected RBCs [4]. Recently we have demonstrated that autoantibodies from P. vivax -infected patients are able to opsonize nRBCs, decreasing their deformability and increasing their in vitro phagocytic uptake, which may contribute to vivax malaria-associated anemia [5]. However, the relationship between this clearance and ABO blood groups has yet to be investigated.…”

“…On the other hand, an association between ABO system and P. vivax infection has not been clearly defined and a few studies conducted in this field reveal that hemoglobin levels are lower in blood type O individuals, who are more likely to develop anemia [9, 10]. Taking into account the above-mentioned and our ability to study in vitro deformability and clearance of nRBCs [5, 11], we investigated here whether there is an association between ABO blood groups and IgG and IgM autoantibody-driven destruction of nRBCs in P. vivax infection.…”