Plasmodium falciparum, but not P. vivax, can induce erythrocytic apoptosis

Abstract

Background: Apoptosis can occur in red blood cells (RBC) and seems to be involved in hematologic disorders related to many diseases. In malaria it is known that parasitized RBC (pRBC) is involved in the development of anemia and thrombosis; however, non-parasitized RBC (nRBC) apoptosis could amplify these malaria-associated hematologic events. In fact, in experimental malaria, increased levels of apoptosis were observed in nRBC during lethal Plasmodium yoelii 17XL infection, but in human malaria erythrocytic apoptosis has never been studied. The present study was performed to investigate if nRBC apoptosis also occurs in P. vivax and P. falciparum infections.

Findings: Apoptosis of nRBC was evaluated in blood samples of P. vivax malaria patients and clinically healthly individuals living in Manaus, Brazil, both ex vivo and after incubation of RBC for 24 h. Additionally, the capacity of plasma from P. vivax or P. falciparum patients was tested for induction of in vitro apoptosis of normal RBC from a clinically healthy individual living in a non-endemic malaria region. Apoptosis was detected by flow cytometry using annexin V staining. In contrast to experimental malaria that significantly increased the levels of apoptotic nRBC both ex-vivo and after 24 h of incubation, no significant alteration on apoptotic nRBC rates was detected in P. vivax infected patients when compared with non-infected control individuals. Similar results were observed when plasma of these P. vivax patients was incubated with normal RBC. Conversely, plasma from P. falciparum-infected subjects induced significant apoptosis of these cells.

Conclusions: Apoptosis of normal RBC can be induced by plasma from individuals with P. falciparum (but not with P. vivax) malaria. This finding could reflect the existence of erythrocytic apoptosis during infection that could contribute to the pathogenesis of hematological and vascular complications associated with falciparum malaria.

Figure 1

Representative flow cytometry analysis of erythrocytic apoptosis induced by plasma. Red blood cells (RBC) from a healthy individual were incubated for 48 h in plasma from control mice (A), P. yoelii-infected mice (B, early and; C, late stage of infection), healthy control individuals (D) or malaria patients infected with P. vivax (E) or P. falciparum (F). Apoptosis of RBC was detected using annexin V staining.

Figure 2

Induction of apoptosis in normal red blood cells (RBC) by plasma from P. falciparum patients. RBC from a healthy individual were incubated for 24h and 48h in the presence of plasma samples from non-infected control healthy individuals or from malaria patients infected with P. vivax (Pv) or P. falciparum (Pf). Apoptosis was detected by flow cytometry using annexin V staining. Statistical difference was tested by non-parametric Mann Whitney test.

Figure 3

Cell membrane integrity and cell volume in erythrocytic apoptosis induced by plasma. RBC from a healthy individual were incubated for 48 h in the presence of plasma from non-infected control healthy individuals or malaria patients. (A-C) Representative flow cytometry analysis of cell membrane integrity accessed by calcein-AM and annexin V-APC double staining in RBC incubated with plasma from non-infected individuals (A) or from P. vivax (B) or P. falciparum (C) patients. (D) Forward scatter of RBC exposing (AnV+) or not (AnV-) phosphatidylserine after incubation with plasma from control non-infected individuals or patients infected by P. vivax (Pv) or P. falciparum (Pf). Statistical difference in (D) was tested by non-parametric Mann Whitney test.