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. Author manuscript; available in PMC: 2010 May 15.
Published in final edited form as: J Infect Dis. 2009 May 15;199(10):1536–1545. doi: 10.1086/598222

Rosiglitazone modulates innate immune responses to Plasmodium falciparum and improves outcome in experimental cerebral malaria

Lena Serghides 1,2, Samir N Patel 2,3, Kodjo Ayi 2,3, Ziyue Lu 2, D Channe Gowda 4, W Conrad Liles 1,2,3, Kevin C Kain 1,2,3
PMCID: PMC2854576  NIHMSID: NIHMS191159  PMID: 19392627

Abstract

For severe malarial syndromes such as cerebral malaria, adverse clinical outcomes are often immune-mediated rather than caused by the parasite directly; however, few therapeutics have been developed to modulate immunopathological host responses to infection. Here we report that PPARγ agonists (e.g. rosiglitazone), FDA-approved for unrelated conditions, modulate host response to malaria by enhancing phagocytic clearance of malaria-parasitized erythrocytes and by decreasing inflammatory responses to infection via inhibition of Plasmodium falciparum glycosylphosphatidylinositol-induced MAPK and NF-κB activation. We show that rosiglitazone modifies inflammatory response to experimental malaria infection in vivo and improves survival in experimental cerebral malaria in the Plasmodium berghei ANKA model even when initiated as late as day 5 post-infection. Furthermore, rosiglitazone reduces parasite burden in a CD36-dependent manner in the Plasmodium chaubaudi hyperparasitemia model. These data suggest that PPARγ agonists represent a novel class of host immunomodulatory drugs that may have application in the management of severe malaria syndromes.

Keywords: cerebral malaria, immunomodulation, innate immunity, murine malaria, inflammation, phagocytosis, adjunctive therapy, CD36, PPARγ, thiazolidinedione

Introduction

Plasmodium falciparum malaria is a major determinant of childhood mortality responsible for an estimated 1–3 million deaths annually [1–2]. Cerebral malaria (CM) is amongthe deadliest complications of falciparum malaria and affects an estimated 785,000 African children each year [1–2]. There is no specific therapy for CM and despite the use of rapidly active antimalarial therapy such as parenteral quinine or artesunate, mortality rates remain high [3–5]. This may be attributable to the observation that interventions for malaria are anti-parasitic, even though poor outcomes associated with CM appear to be mediated more by immunopathological host responses to infection than by the parasite per se [3–6].

Key events in the pathogenesis of severe and CM include: the sequestration of parasitised erythrocytes (PEs) within the cerebral microvasculature; dysregulated inflammatory responses to infection, contributing to immune-mediated tissue injury, endothelial activation and upregulation of sequestration receptors; and high parasite burdens that further enhance sequestration and immunopathology [7]. Defining the mechanisms underlying host response to malaria may identify novel targets for immunomodulation and interventions to improve the outcome of severe malaria syndromes.

Malaria-associated fatalities occur predominately in non-immune individuals. Survival in both human infections and murine models of malaria appears to be critically linked to the host’s ability to contain blood-stage parasite replication during the acute phase of infection [6]. Because malaria-specific immune responses are largely absent during acute infection, innate mechanisms appear to be essential in controlling parasite replication and decreasing the risk of progression to severe and fatal disease. Innate immunity therefore represents an attractive target for intervention [6].

Mononuclear phagocytes represent an essential first line of innate defence against malaria [6, 8–12]. Macrophage pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and scavenger receptors such as CD36, are important components regulating immune responses [13–14]. PRRs sense a wide range of microbial molecules and activate pro-inflammatory responses to infection. Parasites and parasite products, such as P. falciparum glycosylphosphatidylinositol (pfGPI) and hemozoin together with parasite DNA, induce the release of pro-inflammatory cytokines via interaction with PRRs, including TLR2, TLR9, and CD36 [11, 15–22]. Macrophages in general [6, 9–10] and macrophage CD36 in particular, have been shown to mediate clearance of PEs and contribute to control of acute blood-stage parasite replication and survival in experimental models of malaria [6, 11, 19–21].

Based on these observations, we hypothesized that pharmacological modulation of innate immunity, through CD36 and related PRR pathways, might increase parasite clearance, modify deleterious host inflammatory responses to infection and improve outcome. CD36 transcription is regulated by the nuclear receptor heterodimer peroxisome proliferator-activated receptor γ-retinoic X receptor (PPARγ-RXR) [23]. PPARγ-RXR is activated when either partner is ligand bound and regulates transcription of a variety of genes including PRRs [23–24]. PPARγ agonists have also been shown to modulate inflammatory responses, including decreasing the secretion of pro-inflammatory cytokines via inhibition of the activity of transcription factors such as AP-1 and NF-kB [25–28]. We postulated that FDA-approved PPARγ agonists could serve as immunomodulatory agents for the treatment of malaria via their potential to improve parasite clearance and regulate inflammatory responses to infection. Here we demonstrate in vitro and in two complementary models in vivo, that rosiglitazone, a PPARγ agonist [29], enhanced phagocytic clearance of parasites, regulated inflammatory responses to infection, and improved survival in a fatal model of experimental CM.

Methods

Parasites

P. falciparum cultures of the laboratory clone ItG were maintained and synchronized as described [19–20], were treated with mycoplasma removal agent (ICN, Costa Mesa, CA) and tested negative for mycoplasma by PCR analysis prior to use. Culture supernatants were collected, aliquoted, and frozen for subsequent use.

PbA (Malaria Resource Centre, Bethesda MD) and PccAS (provided by Dr. M. Stevenson, McGill University, Canada) were maintained by regular passage in naïve mice.

Phagocytosis assays

Phagocytosis assays were performed as previously described [19–21] (see supplemental methods for more detail). 1×106 PBMC or 2×105 murine macrophages were plated and treated with rosiglitazone or DMSO as a control for 48 h. Fc fragments (20μg/ml) were used to block Fc receptors. Various mAbs were used at 5μg/ml where appropriate. Synchronized parasites were layered on top for a final PE:monocyte ratio of 20:1. Hypotonic lysis was used to remove non-phagocytosed PEs. Phagocytosis was quantified microscopically counting the total number of PEs within 500 monocytes/macrophages.

Detection of CD36 surface expression by flow cytometry

Macrophages were treated with either rosiglitazone or DMSO as a control for 48 h, and surface CD36 was detected by staining with anti-CD36 mAbs. Isotypes controls were also performed. Cells were fixed in 1% paraformaldehyde/PBS and analyzed using the EPICS ELITE flow cytometer (Beckman-Coulter, France). Data were analysed using FlowJo software (TreeStar, Ashland, OR).

Isolation and purification of GPIs from P. falciparum

Protein-free pfGPIs were isolated and purified by HPLC as described [16–17] (see supplemental methods).

TNF assays

Human PBMC (5×105/well), or murine thioglycollate-elicited peritoneal macrophages (2×105/well) were treated with rosiglitazone or DMSO as a control, followed by the addition of HPLC-purified pfGPIs (200nM/ml), or no additions. Following 24 h incubation at 37°C, the supernatants were collected and assayed for TNF using ELISA.

Signal transduction assays

Thioglycollate-elicited murine peritoneal macrophages were pre-treated with rosiglitazone for 24 h and then stimulated with pfGPI for various periods of time. Cell lysates were collected, separated on 12% SDS-PAGE and transferred to PVDF membranes. Blots were probed with antibodies recognising total and phospho-specific ERK1/2, JNK, p38, and IκBα, followed by incubation with the appropriate HRP-conjugated secondary antibody, and developed using an ECL-based system [17].

Murine survival and serum cytokine studies

All experiments involving animals were reviewed and approved by the University of Toronto Animal Use Committee and were performed according to University’s animal ethics guidelines. Male 8–12 week old C57BL/6 mice (Charles River Laboratories) were used in most experiments. Cd36−/− C57BL/6 mice were bred in the animal facility at the University of Toronto. Mice were kept under pathogen-free conditions with a 12 h light cycle. One week prior to infection, or 1, 3, or 5 days post infection, mice were either fed a regular powder diet, or powder chow containing 50mg/kg of rosiglitazone in powder format. Infection was initiated by intraperitoneal injection of freshly isolated 1×106 P. berghei ANKA (PbA) or P. chabaudi chabaudi AS (PccAS) PEs per mouse. Mice on the rosiglitazone arm continued to receive the drug for the remainder of the experiment. The course of infection was monitored daily for 18 or 21 days by determining parasitemia on Giemsa stained thin blood smears.

In some experiments mice were sacrificed on day 3, 5 and 7 and peripheral blood was collected by cardiac puncture. Serum was stored at −80°C and later analyzed for levels of TNF and TGF-β by ELISA (R&D Systems, Minneapolis, MN).

Statistical analysis

All in vitro experiments were performed in duplicate or triplicate and repeated at least three times. Data are mean ± SD, unless otherwise noted. Statistical significance was determined by ANOVA with a post-hoc Tukey-Kramer test, or Mann-Whitney Rank Sum test, depending on whether the data were normally distributed or not. Normality was assessed using the Kolmogorov-Smirnov test. Survival studies for PbA infections were done using 5 mice per group and repeated three times. Survival studies for PccAS were done using 6–10 mice per group and repeated once. Statistical significance for was determined by log-rank test. Parasitemia time courses were analysed using two-way ANOVA with Bonferroni post-test. Statistical analyses were performed using GraphPad Prism software.

Results

Rosiglitazone upregulates CD36, increases phagocytosis of P. falciparum parasitized erythrocytes and inhibits pfGPI-induced TNF secretion

Failure to control acute blood-stage parasite replication and dysregulated inflammatory responses to malaria infection is associated with a poor clinical outcome. We hypothesized that therapeutic interventions to modify these components of malaria pathogenesis would be of clinical benefit. Macrophage-mediated uptake of PEs, primarily via engagement of scavenger receptors such as CD36, has been shown to contribute to control of acute infection [6, 9–11, 19–20]. To determine whether rosiglitazone could upregulate CD36 surface expression and PE phagocytosis, human and thioglycollate-elicited murine peritoneal macrophages were treated with increasing concentrations of rosiglitazone. Rosiglitazone induced a dose-dependent increase in CD36 surface expression (figure 1A–B, D–E) associated with a dose-dependent increase in phagocytosis of non-opsonised PEs (figure 1C and F). Phagocytosis was significantly inhibited by monoclonal antibody blockade of CD36, suggesting CD36-dependence.

Figure 1. Rosiglitazone upregulates CD36 surface expression and CD36-mediated clearance of P. falciparum parasitized erythrocytes.

Figure 1

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Human (A–C) and murine (D–F) macrophages treated with rosiglitazone for 48 h were assessed for CD36 surface expression by flow cytometry, and for ability to phagocytose non-opsonized P. falciparum PEs. Histograms of CD36 fluorescence intensity are shown for human (A) and for mouse macrophages (D) respectively. Macrophages treated with 100 μM rosiglitazone are shown in black; vehicle control-treated macrophages are shown in gray. Isotype control-stained macrophages are shown in light grey. CD36 geometric mean fluorescence intensity (GMFI) is shown for human (B) and for murine macrophages (E), respectively. Phagocytosis of non-opsonized PEs by human (C) and murine (F) macrophages are shown. Hatched bars indicate uptake by macrophages pre-treated with 5μg/ml of monoclonal anti-CD36 antibody. Data are depicted as means +/− SD of a representative experiment performed in triplicate. All experiments were repeated at least 3 times. For (C) * p=0.02, p=0.0023, p=0.0005 for DMSO (vehicle) vs. 10 μM, 50 μM, and 100 μM respectively. For (F) * p=0.008, p=0.0034 for DMSO (vehicle) vs. 50 μM, and 100 μM respectively. All statistical comparisons were performed by ANOVA with post-hoc two-tailed Tukey-Kramer.

To determine whether rosiglitazone could modulate malaria-induced inflammatory responses, human PBMCs and murine thioglycollate-elicited peritoneal macrophages were incubated with increasing concentrations of rosiglitazone and HPLC-purified pfGPI [11, 16–17]. Rosiglitazone inhibited pfGPI-induced TNF secretion by both human (figure 2A) and murine (figure 2B) cells in a dose-dependent manner.

Figure 2. Rosiglitazone inhibits pfGPI-induced signalling and TNF production.

Figure 2

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Human PBMC (A) and murine macrophages (B) were treated with rosiglitazone (10 to 100 μM) or DMSO (vehicle control) plus pfGPI (200 nM/ml). TNF levels were assessed by ELISA 24 h post-treatment. Data are displayed as means +/− SD of a representative experiment.

Experiments were repeated at least 3 times with similar results. In (A) * p=0.0003, and p=0.0002 for DMSO vs. 50 μM and 100 μM respectively (n=4). In (B) * p=0.0075, p=0.0002 and p=0.00018 for DMSO vs.10 μM, 50 μM and 100 μM respectively (n=6). All statistical comparisons were performed by ANOVA with a post-hoc two-tailed Tukey-Kramer.

Murine macrophages were pre-treated with rosiglitazone or DMSO (control) for 24 h, then incubated with pfGPI (200 nM/ml) for 30 minutes (C), or for the specified times (D). Cell lysates were analyzed for phospho-ERK1/2, phospho p38, phospho JNK, and IκBα degradation. Total p38 and total ERK1/2 were used to assess equal loading. Experiments were repeated 3 times.

pfGPI promotes cytokine induction via TLR2-dependent activation of MAP kinase signaling pathways and NF-κB [11, 16–17]. To investigate the mechanisms involved in rosiglitazone-mediated inhibition of the pfGPI-stimulated inflammatory response, we examined the effects of rosiglitazone on pfGPI-induced signaling pathways. pfGPI induced phosphorylation of JNK, ERK1/2, and p38, and promoted the degradation of IκBα, a precursor step to NF-κB activation (figure 2) [16–17]. Treatment with rosiglitazone resulted in a dose-dependent inhibition of the phosphorylation of JNK, ERK1/2 and p38 (figure 2C). In addition, rosiglitazone inhibited pfGPI-induced IκBα degradation (figure 2D). These observations indicate that rosiglitazone reduces pfGPI-induced signaling and pro-inflammatory cytokine production in vitro.

Rosiglitazone modulates innate immune responses to malaria and improves infection outcome in vivo

To extend our results to the in vivo setting, we examined the efficacy of rosiglitazone in modulating innate responses in murine models of malaria. Since no single experimental murine model of malaria exists that encompasses all clinical disease aspects of P. falciparum infection in humans, we investigated the efficacy of rosiglitazone in two complementary models. To examine the ability of rosiglitazone in modulating host immunopathology we utilized the P. berghei ANKA (PbA) model of experimental CM [8]. To examine the ability of rosiglitazone to enhance parasite clearance and reduce parasite burden, we utilized the P. chabaudi chabaudi AS (PccAS) model of hyperparasitemia [6, 8].

i. Rosiglitazone modifies inflammatory responses and improves survival in an experimental model of cerebral malaria

Similar to P. falciparum infection in humans, mice susceptible to PbA (e.g. C57BL/6) develop symptoms of CM including a cytokine-associated encephalopathy, neurological symptoms and acidosis culminating in a fatal outcome [8]. PbA-associated CM is characterized by an unbalanced cytokine response to infection with elevated levels of pro-inflammatory cytokines and inadequate induction of regulatory cytokines [6, 8]. We utilized this model to investigate whether rosiglitazone could favorably modulate host inflammatory responses to infection and confer protection to experimental CM.

Control and rosiglitazone-treated C57BL/6 mice were infected with 1×106 PbA PEs, and assessed daily for parasitemia, serum TNF and TGF-β levels, and twice daily for morbidity and survival (figure 3). All control mice developed neurological signs including limb paralysis, movement disorder, ataxia, convulsions and coma characteristic of CM and succumbed to infection between days 6–10. In contrast, rosiglitazone-treated mice had a significantly improved survival rate compared to controls (43% vs 0%, p=0.0073; figure 3A). The rosiglitazone-treated mice that did succumbed to their infection also displayed neurological signs. Parasitemia levels did not differ between control and rosiglitazone-treated mice (figure 3B) consistent with previous studies showing that dysregulated cytokine responses, rather than parasitemia per se, are responsible for mortality during the acute phase of PbA infection [8, 30].

Figure 3. Rosiglitazone improves survival and modulates inflammation in a murine model of cerebral malaria.

Figure 3

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C57BL/6 mice receiving either rosiglitazone (50 mg/kg of chow) or no additions to their chow were infected with 1×106 P. berghei ANKA PEs via intra-peritoneal injection. (A) Survival was assessed twice daily. Significant differences between survival in the control mice vs. rosiglitazone-treated mice were assessed by Log rank test, p=0.0073 (n=15 per group). (B) Parasitemia was assessed daily and did not differ between groups. Serum TNF and TGF-β levels were assessed on days 3, 5, and 7 (C–D). Data represent means +/− SD (n=5 per group). * p=0.0022 assessed by a two-tailed Mann-Whitney Rank Sum test.

During acute infection, early pro-inflammatory responses, in particular IFN-γ and TNF, are required to facilitate parasite clearance, but must be balanced, in part by anti-inflammatory/immunoregulatory cytokines such as TGF-β and IL-10, in order to limit host-mediated immunopathology [31–35]. To assess the effect of rosiglitazone on PbA-induced inflammatory responses we examined serum levels of TNF and TGF-β on day 3, 5 and 7 of infection. Circulating levels of TNF were significantly lower in rosiglitazone-treated mice compared to control mice (figure 3C). Moreover, the TNF/TGF-β ratio was significantly decreased in rosiglitazone-treated mice on all days assessed (figure 3D). Taken together, these data demonstrate that rosiglitazone treatment alters host inflammatory responses to PbA infection and enhances survival to experimental cerebral malaria in vivo.

To determine at what point during the course of infection rosiglitazone might still confer enhanced survival, we repeated the PbA infection in C57BL/6 mice commencing treatment at either 1 week prior to infection, or at 1, 3, or 5 days post-infection (figure 4). As before, all control mice succumbed to their infection (before day 9) and mice receiving rosiglitazone prior to infection were significantly protected (60% vs. 0% survival, p=0.0017). Initiating rosiglitazone 1 or 3 days post-infection enhanced survival to similar levels as that of pre-treatment (40% survival for day 1, 50% survival for day 3 vs. 0% for control, p=0.0142 and p=0.0028 respectively; no significant difference for pre-treat vs. day 1 or day 3, p=0.3885 and p=0.7311 respectively). Mice starting rosiglitazone therapy as late as on day 5 post-infection were still significantly protected against CM (10% vs. 0% survival, p=0.016), but fared significantly worse than the pre-treated mice (60% vs. 10% survival, p=0.041). These data indicate that therapeutic administration of rosiglitazone as late as day 5 post-infection enhances survival to experimental CM.

Figure 4. Therapeutic administration of rosiglitazone as late as 5 days post-infection enhances survival.

Figure 4

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C57BL/6 mice received rosiglitazone (50mg/kg of chow) at either 1 week pre-infection (A), or at 1 day (B), 3 days (C), or 5 days (D) post-infection. Control mice received no additions to their chow. Mice were infected with 1×106 P. berghei ANKA PEs via intra-peritoneal injection. Survival was assessed daily until day 21 post-infection. Significant differences in survival between control and rosiglitazone treated mice were assessed by Log rank test, n=10 per group. In (A) p=0.0017; in (B) p=0.0142; in (C) p=0.0028; in (D) p=0.016. Control treated mice are shown in black and rosiglitazone treated mice in grey.

ii. Rosiglitazone improves parasite clearance in a CD36-depedent manner, in a hyperparasitemia model of malaria

The acute phase of PccAS infection is characterized by hyperparasitemia that results in death if parasite replication is not adequately contained [6, 8]. Macrophages have been shown to be key effector cells in the control of hyperparasitemia [9, 10]. Early control of parasite replication is dependent on innate mechanisms involving scavenger receptors including CD36, and is largely independent of opsonins including parasite-specific IgG and complement [9–11, 36–38].

We used the PccAS model of malaria to examine the efficacy of rosiglitazone in facilitating clearance of PEs and reducing parasite burden in vivo. Control and rosiglitazone-treated C57BL/6 mice were infected with 1×106 PccAS PEs. Parasitemia, morbidity and mortality were assessed daily starting at day 5 post-infection. C57BL/6 mice are innately resistant to PccAS infection [6, 8] and accordingly, survival rates did not differ significantly between rosiglitazone and control-treated mice (90% vs. 80% respectively). However rosiglitazone had a significant effect on parasitemia (p<0.0001, by two-way ANOVA). Parasitemia was lower in rosiglitazone-treated mice at all times post day 8 and was significantly lower on days 11 and 12 post-infection (mean (SEM) day 11: control 41.9% (3.84%) vs. rosiglitazone 31.0% (4.61%), P=0.029; day 12: control 41.3% (2.48%) vs. rosiglitazone 26.5% (3.61%), P=0.0007, Student’s t).

To determine if the rosiglitazone-induced reduction in parasite burden observed in the PccAS model was dependent on CD36, we repeated the infection using control and rosiglitazone-treated Cd36−/− mice. Although these mice are on the resistant C57BL/6 genetic background, lack of CD36 has previously been shown to render these mice more susceptible to PccAS infection [11]. Both mortality rates (50% control and rosiglitazone) and parasitemia levels did not differ significantly between control and rosiglitazone-treated mice. In summary, rosiglitazone treatment in the absence of CD36 did not decrease parasite burden or improve survival in PccAS-infected mice, suggesting that CD36 was required for a beneficial effect.

Discussion

Host immunopathological responses are important contributors to severe and fatal outcomes in a number of life threatening infectious disease syndromes such as CM [3, 39]. Despite this, few therapeutics have been developed to modulate deleterious host immune response to infection. Here we show that rosiglitazone, an FDA-approved PPARγ agonist, enhanced macrophage phagocytosis of PEs in vitro and reduced parasitemia in vivo, modified innate inflammatory responses to malaria in vitro and in vivo, and conferred improved survival in an experimental CM model. In experiments mimicking clinical conditions, rosiglitazone enhanced survival even when initiated up to 5 days after infection, at a time when symptoms of CM are manifesting. This suggests that rosiglitazone may have utility in the treatment of established infections.

For both human infections and murine models of malaria, survival is linked to the ability of the host to generate a regulated inflammatory response and control parasite replication during the acute stage of infection [6]. Excessive or dysregulated inflammatory responses to infection have been consistently implicated in malaria-associated immunopathology [6, 31, 34–35]. In this study we demonstrate that rosiglitazone inhibited pfGPI-induced signaling and TNF secretion in vitro and modulated innate inflammatory responses, particularly the balance between pro- and immunoregulatory cytokines, in mice infected with PbA. Malaria GPI is thought to be a major pro-inflammatory mediator, and CD36 cooperates with TLR2 in recognizing and initiating responses to it. The CM syndrome observed in PbA infection occurs even in the absence of CD36 [40], and therefore the beneficial actions of rosiglitazone observed in this model may be independent of its effects on CD36 expression. Instead, rosiglitazone may exert anti-inflammatory properties via direct transcription regulation, by inhibition of the activity of transcription factors such as AP-1 and NF-kB [25–28].

Studies in humans and mice also support an important role for mononuclear phagocytes in the clearance of blood-stage parasites and early control of parasite burden during acute infection [6, 9–11, 19–21]. Here we demonstrate that rosiglitazone upregulated macrophage CD36 expression and CD36-mediated uptake of P. falciparum PEs in vitro. Using the in vivo PccAS model of hyperparasitemia, we demonstrate that rosiglitazone treatment resulted in a CD36-dependent decrease in parasite burden. In mice sufficient for CD36, rosiglitazone significantly reduced parasitemia. Notably, rosiglitazone failed to decrease parasitemia when administered to CD36-deficient mice, suggesting that the effects of rosiglitazone on parasitemia are CD36-dependent and may be attributable to an increase in CD36-mediated clearance. In the PbA model of experimental CM, rosiglitazone did not affect parasitemia. However, unlike P. falciparum and P. chaubaudi, it is unclear whether P. berghei PEs are cleared by CD36-mediated phagocytosis [11, 42].

The contribution of CD36 to pathogenesis or conversely protection during malaria infection has been controversial. CD36 was initially identified as a sequestration receptor for PEs, leading to the assumption that it contributes to the pathogenesis of CM; however several lines of evidence would appear to challenge this premise [11, 9–21, 40–43]. CD36 expression in the brain is low to absent, thus direct cytoadherence to endothelial CD36 is unlikely to account for cerebral sequestration, although it has been proposed that adhesion may be mediated via bridging through platelet CD36 [44–45]. CD36 is highly expressed in microvascularendothelium of skin and adipose tissue and may direct parasites to these non-vital sites andaway from cerebral microvasculature [20–21]. Thishypothesis is supported by studies demonstrating significantly higher binding to CD36occurs in cases of non-severe disease; by reports showing that protection against CM observed in individuals with Southeast Asian ovalocytosis is associated with increased adhesion of parasitized ovalocytic erythrocytes to CD36 [41–42]; and by populationdata linking CD36 deficiency with an increased susceptibilityto CM [43]. In contrast, recent reports examining other erythrocyte disorders associated with protection from severe disease including sickle cell and thalassemia, have reported decreased CD36 mediated adhesion due to altered PfEMP-1 expression on PEs [46]. However these were in vitro studies performed in the absence of serum and direct in vivo evidence from human infections is at present lacking. While the precise contribution of CD36 to protection or pathogenesis will require additional investigation, our in vitro and in vivo data support a beneficial role for PPARγ agonists in acute malaria infection that is at least partly mediated by the modulation of innate immune responses including those involving macrophage CD36.

Effects of rosiglitazone on modifying sequestration of PEs to endothelium are of potential concern. Although PPARγ agonists have been shown to upregulate both CD36 and ICAM-1 expression, these agonists had minimal effects on the expression of sequestration receptors on endothelial cells and did not upregulate endothelial cell adhesion of PEs [47].

There is an urgent need for adjunctive therapeutic interventions that improve the outcome of CM. However, given that the current costs of new drug discovery exceed $750 million per new chemical entity, development of new therapeutics for diseases with a primary burden in the developing world faces sizable economic barriers [48]. High-throughput screening of FDA-approved drugs for novel indications represents one potential approach to expedite drug discovery and overcome financial obstacles. Another strategy, as was exploited in this study, is to identify disease critical pathways and make use of available drugs predicted to act on these pathways through known interactions with transcriptional response elements or other cellular targets.

The data presented in this manuscript suggest that PPARγ agonists such as rosiglitazone may have clinical utility as an adjunct to standard therapy for human falciparum malaria. However recent systematic reviews have highlighted discordance between treatment outcomes in animal models and human clinical trials [49–50]. Therefore we have extended our animal studies and recently completed a randomized, double-blind, placebo-controlled treatment trial of P. falciparum malaria acquired on the Thai-Myanmar border to investigate the efficacy of rosiglitazone as adjunctive therapy in falciparum malaria (manuscript submitted). In this randomized clinical trial, rosiglitazone treatment significantly improved parasite clearance, lowered parasite burden, and decreased levels of inflammatory biomarkers associated with adverse clinical outcomes.

In summary, these data provide direct in vitro and in vivo evidence that rosiglitazone can modulate host response and improve outcome during experimental CM and suggest a potential role for this class of immunomodulators in the management of severe and CM.

Supplementary Material

Supplementary Data

Acknowledgments

Funding. This work was supported by a CIHR Team Grant in Malaria (KCK), operating grant MT-13721 (KCK), Genome Canada through the Ontario Genomics Institute (KCK), the McLaughlin-Rotman Centre and the McLaughlin Centre for Molecular Medicine (KCK, WCL), and a CIHR Canada Research Chair (KCK, WCL), and NIAID, NIH grant AI41139 (DCG).

Portions of this work have been presented as a late breaker presentation at the 55th meeting of the American Society of Tropical Medicine and Hygiene, on November 2006, in Atlanta, Georgia.

Footnotes

Potential conflicts of interest. The University Health Network holds Intellectual Property pertaining to the use of PPARγ-RXR agonists for the treatment of inflammatory states. The authors declare there are no other competing financial interests.

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