Mild Plasmodium falciparum Malaria following an Episode of Severe Malaria Is Associated with Induction of the Interferon Pathway in Malawian Children

DISCUSSION

Understanding the factors leading to the acquisition of protection against severe clinical presentations of malaria in areas of endemicity may illuminate new approaches to both prevention and treatment. This is the first report that compares the transcriptomes of peripheral blood cells from children with severe malaria with the transcriptomes of cells from the same children during a subsequent mild malaria infection. These data identify an association of mild malaria with a type I IFN response.

In areas of high transmission, despite episodes of reinfection older children and adults generally suffer only mild or asymptomatic malaria infections and rarely develop severe disease (4, 9). To investigate the mechanism of these field observations and the immune responses associated with variation in disease severity, we carried out a subanalysis on patients enrolled in the Blantyre Malaria Research Project. This study protocol provided an opportunity to identify and study the same children during an episode of severe malaria and, 1 month later, with mild malaria.

During the initial severe disease presentation, the five patients who were part of this study displayed a broad range of abnormal laboratory findings and manifested clinical features characteristic of severe malaria (13, 20). During the second observed infection, patients were dramatically less ill, having no clinical evidence of severe disease. The illness in some was limited to a history of recent fever and vomiting, and they presented with an otherwise normal exam. No complete blood count was done during the outpatient visit, but the semiquantitative estimates of parasitemia revealed high-density parasitemias in several of the children. This may suggest the ability of these patients to mount a more effective host response despite high antigen loads during the mild malaria presentation.

Many of the genes upregulated during the subsequent mild malaria disease were related to the type I IFN pathway. The transcripts OAS1 and OAS3 that were upregulated in mild disease are part of the oligoadenylate synthase family of IFN-induced RNA polymerases (18). Other genes associated with mild disease included IFIT1, IFIT3, and MX1, which are also in the IFN-α/β signaling pathway (Ingenuity Pathway Analysis, version 9.0). The type I interferons IFN-α and IFN-β were originally described as central to effective antiviral immune responses (11). More recently, it has been shown that type I IFNs are also produced in response to Trypanosoma cruzi infection (34, 40). Additionally, a dominant type I IFN signature was identified from whole-blood transcriptional profiling in patients with active Mycobacterium tuberculosis (5).

Genetic studies of malaria-infected patients have found an association between mutations within IFN-α receptor (IFN-αR) and severe disease susceptibility, suggesting that the type I IFN pathway may play a role in severe disease (3, 17). IFN-α has been identified in a subset of malaria-infected patient sera (25, 29). A recent report finds that the mechanism of type I IFN immune induction is through host cell recognition of AT-rich parasite DNA motifs (30). However, the role of type I IFN in protection from severe malaria disease in human studies is unknown. Studies in the rodent model of experimental CM provide conflicting results regarding the role of type I IFN in disease outcomes. Plasmodium berghei ANKA-infected mice treated with 800,000 U of IFN-β demonstrated a 60% improvement in survival compared to untreated mice, without any differences in parasitemia (22). This IFN-β treatment led to reduction in CXCL9 and intracellular adhesion molecule-1 (ICAM-1) transcript levels in the brain, reduction of T-cell CXCR3 expression, and downregulation of serum tumor necrosis factor alpha (TNF-α), suggesting that the anti-inflammatory properties potentially accounted for improved survival. IFN-α has also been demonstrated to have a protective role in the rodent model of severe malaria. Administration of 25 days of IFN-α reduced the CM death rate in C57BL/6 P. berghei ANKA-infected mice from 87% to 6% (41). This protection was associated with reduction in peripheral parasitemia and also reduced upregulation of ICAM-1 expression in brain endothelial cells. In contrast, mice lacking the type I IFN receptor (IFN-αR1^−/−) were protected against CM in the P. berghei ANKA model in two studies (15, 30). These conflicting data may be due to the approaches taken; however, they suggest that this pathway is involved in mediating disease outcomes. Our data suggest that the type I IFN pathway is associated with a reduction in disease severity in the P. falciparum-infected human host, but more studies are needed. Identification of plasma α/β cytokines accompanying the type I IFN transcriptional signature during mild malaria would provide further evidence that this pathway is central to the host response during mild malaria infection compared to a severe disease presentation.

Genes and gene sets involved in a T cell response, such as T cell activation (GO 0042110), are also found to be associated with mild malaria. Cytotoxic T lymphocyte (CTL) granules contain perforin and granzyme A (27). These are key effector molecules for CTL-mediated cytolysis, and their transcripts perforin 1 (PRF1) and granzyme A (GZMA) are upregulated in mild malaria. Also upregulated in mild malaria is cathepsin W, a cysteine protease that is expressed in natural killer and CD8⁺ T cells and is released in target cell killing (33). Studies in the rodent model have identified T cell responses to be important in the liver stage of infection and in the control of the asexual stages (31, 39). Less is known about the role of T cells in human malaria infections. Recently, age-related qualitative differences in the T cell response to merozoite surface protein 1 were shown, which may contribute to age-related malarial immunity (10). Further investigation in the role of T cells in protection from severe disease in humans is needed.

There have been a number of studies that have identified biomarkers associated with severe malaria, including TNF-α, endothelial microparticles, and intracellular adhesion molecule-1, among many others (1). We identified six transcripts to be associated with severe disease, and some of these have been previously been reported in severe malaria. TK1 transcript was associated with severe malaria and was also found to be upregulated in peripheral whole-blood transcriptome in two CM-susceptible strains of mice (C57BL/6 and CBA/CaJ) compared to levels in CM-resistant BALB/c mice (23). Lipocalin 2, also known as human neutrophil lipocalin (HNL), is a component of the innate immune system. It is upregulated and secreted by immune cells after Toll-like receptor recognition of bacteria, with a bacteriostatic effect (12). The association of this transcript with severe malaria is consistent with a study in the Sudan that found plasma concentrations of HNL to be significantly elevated in patients with severe malaria compared to levels in patients with mild malaria (21). These results may lead to the identification of peripheral blood-based biomarkers of severe disease. A diagnostic biomarker would be clinically useful in view of the observation that the clinical diagnosis of CM can be incorrect up to 25% of the time (37).

CA1 was significantly upregulated in children during severe disease. CA1 is primarily expressed in human red blood cells, and one of its major functions is to maintain acid-base homeostasis (19). Acid-base status is often deranged in severe malaria, and four of five children had abnormally high levels of lactate during their severe disease admission (36). The upregulation of CA1 during severe malaria may reflect a host compensatory response to mediate the hyperlactemia. GPR89B was also associated with severe malaria. There are nearly 2,000 G protein-coupled receptor genes which are classified into over 100 subfamilies based on sequence, ligand structure, and function (16). The identification of peripheral blood cells expressing this protein, confirmation of protein expression, and characterization are under way to understand the role of this receptor in severe malaria.

We note that our study has potential limitations. A larger sample size may have facilitated the identification of additional genes or gene sets that are associated with severe malaria infection. The duration of infection at the time of sampling the peripheral blood transcriptomes could have differed between the severe and mild presentations. Children presenting with mild disease may have been studied earlier in the course of infection as they presented for a routine clinic visit which fortuitously detected the second infection, and the host transcriptional profile thus is related to timing of sampling rather than clinical phenotype. Studies of host response at multiple points over time postinfection could differentiate this possibility. Differences in transcriptional signatures can reflect differences in cellular composition or differences in transcript abundance. Peripheral blood contains a heterogeneous mixture of cells, and the two blood samples might differ in their cellular compositions. In a study of blood profiling of Kenyan children with mild malaria, there was a correlation of gene transcription and absolute neutrophil count (14). Thus, cell-specific transcriptomes would further define the host response on a cell phenotype level and could help determine the role of different white cell subsets in pathogenesis. Finally, through a comparison to an uninfected blood transcriptome as a baseline control, we can confirm that gene sets associated with mild malaria represent an upregulation during mild infection rather than a downregulation during severe disease.

Despite these limitations, we observed significant differences between our paired severe/mild transcriptomes. Type I IFN-related pathways and T cell biology were associated with mild malaria infection. The type I interferon data support some of the previously reported studies from animal models, and transcripts associated with severe disease replicate prior severe disease associations. To our knowledge, this is the first study comparing the transcriptional profiles of host peripheral blood cells during severe and subsequent mild disease and provides new paradigms of host response that can be explored to impact severe disease outcomes in malaria.