Severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes

Abstract

Severe malarial anemia (SMA) is the most frequent life-threatening complication of malaria and may contribute to the majority of malarial deaths worldwide. To explore the mechanisms of pathogenesis, we developed a novel murine model of SMA in which parasitemias peaked around 1.0% of circulating red blood cells (RBCs) and yet hemoglobin levels fell to 47% to 56% of baseline. The severity of anemia was independent of the level of peak or cumulative parasitemia, but was linked kinetically to the duration of patent infection. In vivo biotinylation analysis of the circulating blood compartment revealed that anemia arose from accelerated RBC turnover. Labeled RBCs were reduced to 1% of circulating cells by 8 days after labeling, indicating that the entire blood compartment had been turned over in approximately one week. The survival rate of freshly transfused RBCs was also markedly reduced in SMA animals, but was not altered when RBCs from SMA donors were transferred into naive recipients, suggesting few functional modifications to target RBCs. Anemia was significantly alleviated by depletion of either phagocytic cells or CD4+ T lymphocytes. This study demonstrates that immunologic mechanisms may contribute to SMA by promoting the accelerated turnover of uninfected RBCs.

Figure 1.

Severe malarial anemia at low parasite burden in rodent malaria infections. (A) Representative data from 3 infected semi-immune BALB/c mice showing parasitemia (•) and Hb levels (▴) following infection with 10⁴ P berghei. (B) Parasitemia (•) and Hb levels (▴) of naive inoculum control BALB/c mice. (C) Lack of correlation between peak parasitemia and minimum Hb levels in infected semi-immune mice. (D) Parasitemia (•), reticulocyte levels (○), and Hb (▴) of semi-immune mice normalized to the day of patency. (E) Mean parasitemias (•), reticulocyte levels (○), and Hb (▴) in naive adult rats infected with P berghei. n = 14 ± SEM for immune mice; n = 6 ± SEM for naive mice and naive rats.

Figure 2.

Erythropoietic response to severe malarial anemia in semi-immune mice. (A) Correlation between peak reticulocyte levels and minimum Hb levels during SMA in semi-immune mice (n = 14). (B) Hb (▴ and ▵) and reticulocyte (• and ○) levels ± SEM in mice with SMA (▴ and •, n = 4) or nonmalarial PHZ-induced anemia (▵ and ○, n = 6). Mice were selected for equivalent levels of anemia and data are normalized to the day of peak anemia (day = 0). ^**P < .01; ^***P < .001.

Figure 3.

Accelerated RBC turnover during severe malarial anemia. (A) Survival of the circulating RBC population biotinylated on the day of patency in semi-immune mice infected with P berghei (▪) and Hb levels (▵). Basal turnover of biotinylated RBCs in noninfected mice is shown in inset (□). Cell survival was monitored by flow cytometry and is expressed as a percentage change in total number of initial biotinylated RBCs. n = 4 ± SEM. (B) Representative flow cytometry profile of biotinylated RBCs in (i) an infected semi-immune mouse and (ii) naive resting control mouse on days 0 (day of labeling) and 8 after patency. (C) Survival of circulating RBC populations in naive rats, biotinylated on day 6 (•), day 10 (□), and day 13 (▴) after patency. Basal turnover of biotinylated RBCs in uninfected rats is shown in inset (○). Cell survival was monitored by flow cytometry and is expressed as a percentage change in total number of initial biotinylated RBCs. n = 3 ± SEM per group.

Figure 4.

RBC fate is not determined by changes to target cells. (A) Naive RBCs were labeled with CSFE (•) or biotin (○) and adoptively transferred into infected semi-immune mice on day 2 or day 4 after patency, respectively. Survival was monitored by flow cytometry and is expressed as a percentage change in total number of transferred RBCs; n = 6 ± SEM. The inset shows the percentage change in Hb (▴) in semi-immune mice over the same period of days after patency. (B) Comparison of rate of the survival of naive CSFE-labeled (•), naive biotin-labeled (○), and resident host biotin-labeled (▪) cells in semi-immune mice. Values are expressed as percentage change in the total number of cells present from day 4 after patency (100%). n = 6 ± SEM. (C) RBCs from (i) noninfected naive mice, (ii) nonanemic semi-immune mice on the day of patency, and (iii) anemic semi-immune mice on day 4 after patency were biotinylated in vivo and adoptively transferred into naive recipients. Survival was monitored by flow cytometry. Plots show circulating RBC populations in the recipients on the day of transfer (day 0) and day 6 after transfer. Survival rate values show the mean percentage survival of cells from each donor group on day 6 after transfer. n = 5 ± SEM.

Figure 5.

Depletion of immune effector cells alleviates SMA in semi-immune mice. (A) Parasitemia and (B) Hb levels, in phagocyte-depleted semi-immune mice (open symbols) and control semi-immune mice (closed symbols), normalized to the day of patency. n = 9 ± SEM. (C) Parasitemia and (D) Hb levels, in CD4⁺ cell–depleted (open symbols) and control (closed symbols) mice, normalized to day of patency. Mean individual minimal Hb levels for CD4⁺ cell–depleted (open bars) and controls (closed bars) are shown in the inset. n = 8 ± SEM for the CD4⁺ T-cell–depleted and n = 7 ± SEM for control groups. ^*P < .05; ^**P < .01; ^***P < .001.