Apoptosis of non-parasitized red blood cells in malaria: a putative mechanism involved in the pathogenesis of anaemia

Abstract

Background: Severe anaemia is a common complication of Plasmodium falciparum malaria in hyperendemic regions. Premature elimination of non-parasitized red blood cells (nRBC) has been considered as one mechanism involved in the genesis of severe malaria anaemia. It has been reported that apoptosis can occur in RBC and, consequently, this cell death process could contribute to anaemia. This study was performed to evaluate the susceptibility of nRBC to apoptosis in a malaria anaemia murine model.

Methods: Balb/c mice were intraperitonially inoculated with 1 × 106 P. yoelii 17XL parasitized RBC (pRBC) and, then, parasitaemia and anaemia were monitored. Apoptosis in both pRBC and nRBC was assessed during early and late phases of infection by flow cytometry using Syto 16 and annexin V-PE double staining and forward scatter measurement.

Results: As expected, experimental infection of Balb/c mice with Plasmodium yoelii 17XL parasites was characterized by progressive increase of parasitaemia and acute anaemia, leading to death. Flow cytometry analysis showed that a number of pRBC was in the apoptotic process. It was noteworthy that the increase of nRBC apoptosis levels occurred in the late phase of infection, when anaemia degree was notably accentuated, while no significant alteration was observed in the early phase.

Conclusion: The increased levels of nRBC apoptosis herein firstly reported, in malaria infection could represent a putative mechanism worsening the severity of malarial anaemia.

Figure 1

Course of parasitaemia and progression of anaemia in P. yoelii 17XL infected Balb/c mice. Mice (n = 7) were inoculated with 10⁶ P. yoelii 17XL-pRBC and parasitaemia and anaemia degree were measured. In parallel, non-infected, age-matched mice were used as control (n = 5). (A) Individual parasitaemia in infected mice on days 3 and 5-8 post-infection. (B) Means ± SD of the number of RBC/μL of blood in infected (open circles) and non-infected (closed squares) mice on days 3, 5 and 7 post-infection. Data are representative of two separate experiments.

Figure 2

Phosphatidylserine (PS) exposure in pRBC and nRBC during P. yoelii 17XL infection. Balb/c mice (n = 5) were infected with 10⁶ P. yoelii 17XL-pRBC and, than, PS exposure was evaluated in the late stage of infection by flow cytometry using Syto 16 (FL-1) and annexin V-PE (FL-2) dual staining. In parallel, non-infected, age-matched mice were used as control (n = 4). (A) Representative flow cytometry analysis of pRBC exposing PS in infected mice. (B) Representative flow cytometry analysis of nRBC exposing PS in infected and non-infected mice. (C) Means ± SEM of levels of nRBC exposing PS in infected and non-infected mice detected ex-vivo and after 24 h culture. Data are representative of three separate experiments.

Figure 3

Cell shrinkage in nRBC during P. yoelii 17XL infection. Balb/c mice (n = 5) were infected with 10⁶ P. yoelii 17XL-pRBC and, than, RBC forward scatter was determined in the late stage of infection by flow cytometry. (A) Representative flow cytometry analysis of the nRBC forward scatter in infected and non-infected mice. (B) Means ± SEM of the geometric mean of the nRBC forward scatter in infected and non-infected mice detected ex-vivo and after 24 h culture. Data are representative of tree separate experiments.

Figure 4

Phosphatidylserine (PS) exposure and cell shrinkage in nRBC during early stage of P. yoelii 17XL infection. Balb/c mice (n = 5-7) were infected with 10⁶ P. yoelii 17XL-pRBC and, than, PS exposure and forward scatter was evaluated in the early stages of infection by flow cytometry. Data show means ± SEM of ex-vivo levels of nRBC exposing PS (A) and of the nRBC forward scatter (B) in infected and non-infected mice. Data are representative of two separate experiments.