In this study, we identified over 300 potential biomarkers of ECM detectable in the circulation by comparing the whole-blood transcriptional profiles of resistant BALB/c mice to those of two susceptible strains of mice (C57BL/6 and CBA/CaJ) during the symptomatic phase of disease. While the major objective of this study was to identify biomarkers that may be of prognostic/diagnostic value, we note that the transcriptional profile of the peripheral blood captures the molecular and immunological events occurring in the brain and spleen during ECM. To our knowledge, this is the first study that measured the transcription profile of whole blood during the ECM phase in mice; previous studies were performed with brain and spleen samples.
Several important clinical and biological observations were made in this study. We noted that the clinical features of ECM are slightly different between C57BL/6 and CBA/CaJ mice. While C57BL/6 mice slowly progress into a state of coma, CBA/CaJ mice experience a more sudden onset of disease with the obvious signs of seizure prior to coma. Based on the subtle genetic differences, it is reasonable to argue that the distinct clinical features induced by P. berghei ANKA infection in these two inbred mouse strains are regulated by their host genetic factors. It is important to note that the spectrum of clinical features seen in young African children experiencing CM is also known to vary greatly, which is generally attributed to host genetics. Our study has allowed us to discern some of the host factors that may be responsible for these various clinical features observed during ECM. We also determined if anemia correlated with the pathogenesis of ECM by comparing the hematocrits of susceptible mice (C57BL/6 and CBA/CaJ) exhibiting symptoms of ECM to that of resistant BALB/c mice. We found that on day 6 postinfection, all three strains of mice had a similar hematocrit, suggesting that ECM occurs independently of anemia.
Although there was no significant difference in the hematocrits in moribund C57BL/6 and CBA/CaJ versus resistant BALB/c mice, our microarray data suggest that erythropoiesis is comparatively dysfunctional during ECM, with at least 23 genes related to erythropoiesis downregulated by greater than 4-fold in moribund C57BL/6 and CBA/CaJ mice. Erythropoiesis, the differentiation of hematopoietic stem cells into mature red blood cells, is characterized by the extrusion of nuclei from orthochromatic erythroblasts to form enucleated reticulocytes and subsequent mature red blood cells (17
). Previously, we reported simultaneous downregulation of transcription but upregulation of translation of the hemoglobin-α gene in brain tissue of mice with ECM (32
). Based on these results, we hypothesized that low levels of hemoglobin-α RNA were a result of a paucity of (nucleated) immature erythrocytes within the brain due to decreased production of erythrocytes, while elevations in hemoglobin-α protein were caused by an abundance of mature erythrocytes due to excess hemorrhaging within the brain. The consistent transcriptional repression of genes associated with erythroid differentiation observed in the present study may reflect a lack of young nucleated cells of the erythroid lineage in the peripheral blood and suggests the importance of suppressed erythropoiesis during ECM. However, we want to emphasize that since this study included only one ECM-resistant strain of mice, it is possible that robust infection-induced erythropoiesis may be a feature of the BALB/c strain of mice rather than a phenotype unique to ECM-resistant mice. Future studies on additional strains of ECM-resistant mice will be needed to fully address this question.
In a previous study, transcriptional analysis of uninfected versus P. berghei
ANKA-infected C57BL/6 mice prior to the onset of symptoms demonstrated suppressed erythropoiesis in the bone marrow and spleen of mice on days 1, 3, and 5 of infection that translated into reduced levels of peripheral reticulocytes (36
). These results indicate that dysfunctional erythropoiesis may occur early in the pathogenesis of ECM. An earlier study that examined bone marrow aspirates of nine patients with cerebral malaria in Thailand suggested that dyserythropoiesis may also be a feature of human CM (44
). In this study, microscopic analysis of the bone marrow aspirates presented evidence of morphological abnormalities of erythroblasts that the authors speculated could be caused by local release of malaria toxin, hypoxia, or disturbed macrophage function induced by concomitant cytoadherence of parasitized erythrocytes to endothelial cells in the bone marrow sinusoids. Indeed, recent in vitro
studies indicate that hemozoin can directly inhibit erythropoiesis by induction of apoptosis of erythroid precursors, and macrophages actually reduce this inhibitory effect of hemozoin on erythroid development by preventing contact between hemozoin and erythroid cells (23
Interestingly, murine studies have shown that treatment of mice with erythropoietin reduces mortality from ECM (20
). Although the primary function of erythropoietin is regulation of erythrocyte production, these studies have focused on its neuroprotective capacity to prevent hypoxia and apoptosis within the brain. However, a strong association between high levels of plasma but not CSF erythropoietin and a reduced risk of neurological sequelae and death was recently reported in African children with CM (6
). Our microarray data suggest that suppressed erythropoiesis does not alter the hematocrit during ECM. These results may be explained by an earlier observation showing that reticulocytes are only a small fraction of total erythrocytes during a P. berghei
ANKA infection (36
). However, it is plausible that the absence of young erythrocytes may contribute to decreased brain oxygenation, which may augment symptoms and potentially result in neurological sequelae. Future studies will be needed to determine whether dysfunctional erythropoiesis during CM has a causative role in the pathogenesis of disease.
We also found that several genes related to platelet and clotting functions were significantly downregulated during ECM. Numerous studies have implicated a role for platelets in the pathogenesis of ECM (40
), and recent studies have examined the role of platelets in human CM. In a study of Malawian children, platelet accumulation in the microvessels of brain tissue was found to be significantly greater in patients with CM than those with severe malaria anemia or nonmalarial encephalopathy (12
). In addition, all Malawian pediatric patients with CM had low platelet counts with the degree of thrombocytopenia significantly correlating with parasitemia (42
). Indeed, previous clinical studies have indicated that retinal hemorrhaging (a possible indicator of thrombocytopenia) is significantly associated with the severity of CM in humans (25
), and malaria retinopathy is currently being explored as a promising tool for the differential diagnosis of CM (2
Interestingly, CD8β1 is significantly upregulated (3.9 ± 0.5; P
< 0.0005) in moribund C57BL/6 mice but markedly downregulated (−7.5 ± 1.2; P
< 0.0003) in moribund CBA/CaJ mice compared to resistant BALB/c mice. This suggests that the CD8+
T-cell population produced during a lethal P. berghei
ANKA infection that culminates in ECM may differ phenotypically in susceptible C57BL/6 and CBA/CaJ strains of mice. Alternatively, it is possible that absence of CD8β1 in the circulation may result from preferential sequestration of CD8β1+
T cells within the brain by CBA/CaJ mice. Previously, Randall et al. reported that CD8β1 is significantly upregulated in brain tissue of moribund C57BL/6 but not CBA P. berghei
ANKA-infected mice (34
). In murine studies conducted by Grau et al. in the early 1980s, depletion of CD8+
T cells with an antibody that targets CD8β1 did not prevent the onset of ECM in P. berghei
ANKA-infected CBA mice (13
). However, it was recently shown that anti-CD8α successfully inhibits the progression of ECM in CBA mice (34
). Together, these studies indicate that pathogenic CD8+
T cells may differ phenotypically among susceptible strains of mice.
The primary objective of this study was to identify biomarkers detectable in the circulation that could be used for the prognosis and differential diagnosis of CM. Efforts are under way to identify host biomarker-based correlates of CM. For example, previous studies have assessed the prognostic and diagnostic potential of angiopoietin 1, a marker of the resting endothelium, and angiopoietin 2, a marker of endothelial activation, for CM (26
). In a study of Ugandan children and Thai adults, Lovegrove et al. demonstrated that angiopoietin-1 levels are decreased and angiopoietin-2 levels are increased in CM versus uncomplicated malaria and healthy controls and the angiopoietin 2/angiopoietin 1 ratio was useful in predicting survival in African children with CM.
Among the greater than 300 circulatory biomarkers of ECM identified by microarray in our study, we selected a subset of biomarkers for further evaluation by quantitative real-time PCR (qRT-PCR). Differential expression of six molecules (c1qb, nccrp1, psca, DnaJC15, gsto1, and tk1) in susceptible (C57BL/6) versus resistant (BALB/c) mice was assessed by qRT-PCR. Of these molecules, expression of c1qb, DnaJC15, and tk1 was significantly higher (as defined by a P value of ≤0.05) in moribund C57BL/6 mice compared to resistant BALB/c mice, with fold increases of 17.3 ± 1.3, 3.7 ± 0.04, and 1.89 ± 0.005, respectively. Nccrp1, psca, and gsto1 had fold increases in expression of 7.1 ± 0.05, 5.7 ± 0.3, and 3.6 ± 0.5, respectively, which approached statistical significance (as defined by a P value of ≤0.1). These results demonstrate that circulatory host biomarkers identified by genome-wide gene expression profiling studies can be used as a diagnostic tool for ECM. Overall, our ability to detect the host biomarkers in blood that are closely associated with the clinical state of ECM offers the prospect of developing diagnostic tests for the prognosis and differential diagnosis of CM in hospital settings in countries where malaria is endemic. While some biologically relevant ECM biomarkers identified in our studies could be validated in clinical studies, extensive gene expression-profiling studies of blood samples from children during different clinical states of CM are needed to identify the biomarkers of human CM.