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, 9 (1), 18965

A Primate Model of Severe Malarial Anaemia: A Comparative Pathogenesis Study

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A Primate Model of Severe Malarial Anaemia: A Comparative Pathogenesis Study

Amber I Raja et al. Sci Rep.

Abstract

Severe malarial anaemia (SMA) is the most common life-threatening complication of Plasmodium falciparum infection in African children. SMA is characterised by haemolysis and inadequate erythropoiesis, and is associated with dysregulated inflammatory responses and reduced complement regulatory protein levels (including CD35). However, a deeper mechanistic understanding of the pathogenesis requires improved animal models. In this comparative study of two closely related macaque species, we interrogated potential causal factors for their differential and temporal relationships to onset of SMA. We found that rhesus macaques inoculated with blood-stage Plasmodium coatneyi developed SMA within 2 weeks, with no other severe outcomes, whereas infected cynomolgus macaques experienced only mild/ moderate anaemia. The abrupt drop in haematocrit in rhesus was accompanied by consumption of haptoglobin (haemolysis) and poor reticulocyte production. Rhesus developed a greater inflammatory response than cynomolgus macaques, and had lower baseline levels of CD35 on red blood cells (RBCs) leading to a significant reduction in the proportion of CD35+ RBCs during infection. Overall, severe anaemia in rhesus macaques infected with P. coatneyi has similar features to SMA in children. Our comparisons are consistent with an association of low baseline CD35 levels on RBCs and of early inflammatory responses with the pathogenesis of SMA.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Manifestation of severe malaria during blood-stage P. coatneyi infection in rhesus and cynomolgus macaques. (A) The study schedule indicates sample collection timepoints and the number of animals per species during the course the infection. (B) Clinical indicators of severe malaria and (CH) laboratory indicators of severe malaria in rhesus (n = 4) and cynomolgus (n = 4) macaques infected with blood-stage P. coatneyi. For the graphs in the left panel (CE) values above the dashed line represent severe malaria and for the graphs in the right panel values below the dashed line represent severe malaria. Error bars show the SEM for each group. Using linear mixed-effects models the (C) parasitaemias of infected rhesus and cynomolgus macaques were compared between days 4 and 11, when all animals remained alive (p = 0.56), and days 12 and 15, when the number of animals per group was decreasing (**p < 0.001).
Figure 2
Figure 2
Deep tissue parasite accumulation and haemozoin deposition patterns of P. coatneyi in infected rhesus and cynomolgus macaques. (A) Mature parasite accumulation and (B) deposition of parasite pigment was assessed using H&E staining of organs from infected rhesus and cynomolgus macaques. H&E stained spleens from one representative (C) rhesus and (D) cynomolgus macaque. IBA1 stained spleens from one representative (E) rhesus and (F) cynomolgus macaque to assess whether parasite pigment was within the cytoplasm of macrophages. Myeloperoxidase stained spleens from one representative (G) rhesus and (H) cynomolgus macaque to assess association of parasite pigment and neutrophils. Arrowheads indicate areas of haemozoin pigment. Images (C) and (D) were taken at 4X magnification (scale bars represent 200 µm), (E) and (G) were taken at 40X magnification (scale bars represent 20 µm), and (F) and (H) were taken at 20X magnification (scale bars represent 50 µm).
Figure 3
Figure 3
Haematological indicators during early blood-stage P. coatneyi infection. (A) Haptoglobin, (B) reticulocyte production index and (C) erythropoietin levels in rhesus (n = 4) and cynomolgus (n = 4) macaques infected with blood-stage P. coatneyi parasites. Error bars show SEM for each group. Haptoglobin data were analysed using an unpaired, two-tailed t-test, with *indicating p values < 0.05. Reticulocyte production index and log10 erythropoietin were compared between species on days 4 to 11 using linear mixed-effects models that adjusted for linear time trends and day 0 levels, and allowed for individual-specific random effects.
Figure 4
Figure 4
Complement regulatory protein (CD35) on RBCs during the early course of P. coatneyi blood-stage infection. Blood samples were collected and assessed during blood-stage P. coatneyi infection in rhesus (n = 4) and cynomolgus (n = 4) macaques for (A) the median fluorescence intensity (MFI) of RBCs, (B) the MFI normalised to the baseline of each animal, (C) the percentage of CD35+ RBCs normalised to the baseline of each animal and (D) the percentage of CD35+ reticulocytes (CD71+). Error bars show SEM for each group. Data in graph (C) were analysed using an unpaired, two-tailed t-test, with *indicating p values < 0.05.
Figure 5
Figure 5
Cytokine and chemokine response during early blood-stage P. coatneyi infection in rhesus and cynomolgus macaques. Plasma samples were collected from rhesus and cynomolgus macaques on day 0, 4, 7, 9 and 11 post blood-stage P. coatneyi infection and were assessed for (A) MIP-1α, (B) IP-10, (C) GM-CSF, (D) IL-23, (E) TNFα and (F) TNFα: IL-10 ratio levels. Error bars show SEM for each group. Log10 biomarker levels were compared between species on days 4 to 11 using linear mixed-effects models that adjusted for linear time trends and day 0 levels, and allowed for individual-specific random effects.
Figure 6
Figure 6
Immune activation of CD4 and CD8 T cells during early blood-stage P. coatneyi infection in rhesus and cynomolgus macaques. Blood samples were collected from rhesus and cynomolgus macaques on day 0, 4, 7, 9 and 11 post blood-stage P. coatneyi infection. The percentage of (A) CD4 and (B) CD8 T cells expressing CD69. Error bars show SEM for each group.

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References

    1. World Health Organization. Malaria: Fact sheet. (World Health Organization 2017).
    1. World Health Organization Severe malaria. Tropical Medicine & International Health. 2014;19:7–131. doi: 10.1111/tmi.12313_2. - DOI - PubMed
    1. Slutsker LE, Taylor TJ, Wirima J, Steketee RW. In-hospital morbidity and mortality due to malaria-associated severe anaemia in two areas of Malawi with different patterns of malaria infection. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1994;88:548–551. doi: 10.1016/0035-9203(94)90157-0. - DOI - PubMed
    1. Abdalla S, Weatherall DJ, Wickramasinghe SN, Hughes M. The anaemia of P. falciparum malaria. British journal of haematology. 1980;46:171–183. doi: 10.1111/j.1365-2141.1980.tb05956.x. - DOI - PubMed
    1. Wickramasinghe SN, Abdalla S, Weatherall DJ. Cell cycle distribution of erythroblasts in P. falciparum malaria. Scandinavian journal of haematology. 1982;29:83–88. doi: 10.1111/j.1600-0609.1982.tb00567.x. - DOI - PubMed
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