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Hemoglobinopathies: Slicing the Gordian Knot of Plasmodium falciparum Malaria Pathogenesis

  • Steve M. Taylor ,

    steve.taylor@duke.edu

    Affiliations: Division of Infectious Diseases and International Health, Duke University Medical Center, Durham, North Carolina, United States of America, Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina, United States of America

  • Carla Cerami,

    Affiliation: Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, North Carolina, United States of America

  • Rick M. Fairhurst

    Affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America

Hemoglobinopathies: Slicing the Gordian Knot of Plasmodium falciparum Malaria Pathogenesis

  • Steve M. Taylor, 
  • Carla Cerami, 
  • Rick M. Fairhurst
PLOS
x

Abstract

Plasmodium falciparum malaria kills over 500,000 children every year and has been a scourge of humans for millennia. Owing to the co-evolution of humans and P. falciparum parasites, the human genome is imprinted with polymorphisms that not only confer innate resistance to falciparum malaria, but also cause hemoglobinopathies. These genetic traits—including hemoglobin S (HbS), hemoglobin C (HbC), and α-thalassemia—are the most common monogenic human disorders and can confer remarkable degrees of protection from severe, life-threatening falciparum malaria in African children: the risk is reduced 70% by homozygous HbC and 90% by heterozygous HbS (sickle-cell trait). Importantly, this protection is principally present for severe disease and largely absent for P. falciparum infection, suggesting that these hemoglobinopathies specifically neutralize the parasite's in vivo mechanisms of pathogenesis. These hemoglobin variants thus represent a “natural experiment” to identify the cellular and molecular mechanisms by which P. falciparum produces clinical morbidity, which remain partially obscured due to the complexity of interactions between this parasite and its human host. Multiple lines of evidence support a restriction of parasite growth by various hemoglobinopathies, and recent data suggest this phenomenon may result from host microRNA interference with parasite metabolism. Multiple hemoglobinopathies mitigate the pathogenic potential of parasites by interfering with the export of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of the host red blood cell. Few studies have investigated their effects upon the activation of the innate and adaptive immune systems, although recent murine studies suggest a role for heme oxygenase-1 in protection. Ultimately, the identification of mechanisms of protection and pathogenesis can inform future therapeutics and preventive measures. Hemoglobinopathies slice the “Gordian knot” of host and parasite interactions to confer malaria protection, and offer a translational model to identify the most critical mechanisms of P. falciparum pathogenesis.

Introduction

In the 4th century BC, Alexander the Great conquered the known Western world [1]. Prior to his conquests in Asia, he encountered the Gordian knot, a complex knot of bark affixing a mythic ox-cart to a post in the town of Gordium. Alexander—a pupil of Aristotle—set his mind to untangling the knot, but, like others before him, could not find the ends (and thus the means) to do so. Faced with this intractable problem, Alexander sliced through the Gordian knot with a stroke of his sword and freed the cart. As one of history's greatest military commanders, Alexander subsequently assembled and ruled an empire stretching from the Eastern Mediterranean to the Himalayas while remaining undefeated in battle. These military conquests were presaged by his “Alexandrian solution” to the Gordian knot, demonstrating decisiveness and imagination in the face of a complex and seemingly unsolvable problem.

Malaria is an ancient disease that has persisted to our modern age, intractably killing over 500,000 children in sub-Saharan Africa each year [2]. While current interventions are succeeding in reducing its morbidity in some contexts [3][5], further improvements in our fundamental understanding of the pathogenesis of Plasmodium falciparum malaria are clearly needed to identify the molecular and cellular targets of next-generation therapeutics and preventive measures. The mechanisms of falciparum malaria pathogenesis remain obscure owing to the complex tangle of parasite virulence factors, host susceptibility traits, and innate and adaptive immune responses that modulate the development of distinct malaria syndromes [6], [7].

We propose that hemoglobinopathies slice the Gordian knot of falciparum malaria pathogenesis to protect children from the severe, life-threatening manifestations of the disease. Most strikingly, heterozygous hemoglobin S (HbAS, or sickle-cell trait) and homozygous hemoglobin C (HbCC, or hemoglobin C disease) reduce the risk of severe falciparum malaria in sub-Saharan African children by 90% and 70%, respectively [8]. These structural hemoglobin variants do not protect from P. falciparum infection [8], suggesting they interfere with the specific molecular mechanisms responsible for the morbidity of falciparum malaria. By isolating these pathogenic processes and solving the Gordian knot of malaria pathogenesis, hemoglobinopathies offer an attractive “natural experiment” to identify the molecular correlates of clinical morbidity. These correlates may be amenable to exploitation by future parasiticidal, adjunctive, or preventive therapies, thereby yielding targets for a new “Alexandrian solution” to the world's falciparum malaria problem.

Here we review the proposed mechanisms by which hemoglobinopathies (and fetal hemoglobin) protect against falciparum malaria.

The Red Blood Cell and Plasmodium falciparum Parasites

The red blood cell (RBC) is critical for the propagation of malaria parasites (Figure 1A). After inoculation into a human by a mosquito and a brief, clinically silent incubation in the liver, P. falciparum parasites enter the erythrocytic stage of their life-cycle. It is during this time that parasites sequentially invade and egress from their host RBCs and cause the signs and symptoms of malaria. While developing within the RBC, the parasite traffics proteins to the RBC surface that mediate binding to extracellular host receptors and enable the parasite to sequester in the placenta, brain, and virtually every other organ. The attenuation of malaria by repeated, sub-lethal P. falciparum infections suggests a significant role for adaptive immunity, but the targets of this attenuating immune response remain largely obscure. Though this adaptive immunity can be protective, the development of maladaptive and dysregulated immune responses can also contribute to the pathogenesis of malaria.

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Figure 1. General mechanisms by which hemoglobinopathies may attenuate the pathogenesis of falciparum malaria.

(A) Restriction of red blood cell (RBC) invasion or intraerythrocytic growth, thereby suppressing parasite densities in vivo; (B) interference with parasite-derived mediators of pathogenesis, including those involved in the binding of parasite-infected RBCs (iRBCs) to extracellular host receptors; (C) modulation of innate host defenses to favor protective, anti-inflammatory responses over those that drive pathogenic, pro-inflammatory responses; (D) enhancement of adaptive cell-mediated and humoral immune responses that clear iRBCs from the blood.

http://dx.doi.org/10.1371/journal.ppat.1003327.g001

Variant RBCs are produced from some of the most common human genetic polymorphisms, and for over 60 years their widespread prevalence has been hypothesized to result from their evolutionary selection by severe, life-threatening falciparum malaria [9]. This natural selection is supported convincingly by clinical data for several common hemoglobin disorders (reviewed in [8]). Hemoglobin is the oxygen-carrying component and major protein of the RBC, and is normally formed as a tetramer of two α-globins and two β-globins which constitute adult hemoglobin A (HbA). The major hemoglobinopathies result from molecular lesions that either decrease the production of α- or β-globins (in α- and β-thalassemia, respectively) or encode single amino acid substitutions in β-globin (in HbS, HbC, and hemoglobin E [HbE]) (Table 1) [10]. The most severe hemoglobinopathies—HbSS homozygosity (sickle-cell disease) and the thalassemias major—are typically incompatible with life beyond early childhood without sophisticated medical care. Other hemoglobin traits such as HbAS, HbAC, HbCC, HbAE, HbEE, and the thalassemias minor are associated with essentially normal life-spans and far less directly attributable morbidity. Remarkably, these simple polymorphisms confer dramatic levels of protection from a complex disease: for HbAS, the substitution of glycine with valine at amino acid position 6 in only one of two β-globin chains reduces a child's risk of severe falciparum malaria by about 90% [8].

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Table 1. The major hemoglobinopathies: epidemiology, molecular pathology, and clinical phenotype.

http://dx.doi.org/10.1371/journal.ppat.1003327.t001

The current understanding of falciparum malaria pathogenesis suggests four general hypotheses for investigating the nature of malaria protection by hemoglobinopathies (Figure 1): 1) restriction of RBC invasion or intraerythrocytic parasite growth, 2) interference with parasite-derived mediators of pathogenesis, 3) modulation of innate host responses, and 4) enhancement of the host's adaptive immune clearance of parasite-infected RBCs (iRBCs). While these mechanisms may be occurring simultaneously in vivo, we review the evidence for each of them separately.

Do Hemoglobinopathies Restrict P. falciparum Invasion of or Growth in RBCs?

Numerous investigations of the invasion and growth of P. falciparum in RBCs containing variant hemoglobins rapidly followed the development of in-vitro cultivation systems by Trager and Jensen, and Haynes et al. in 1976 (Table 2) [11], [12]. Reductions in RBC invasion have been reported for a variety of hemoglobinopathies including α-thalassemia trait [13], HbH disease [14], [15], HbEE [13], [15], HbAE [15], and the compound heterozygous β-thalassemia/HbE disorder [13], [15], [16]; reductions in the intraerythrocytic growth or maturation of parasites have been reported for HbH disease [14], [16], β-thalassemia minor [16], HbSS [17], [18], HbAS [17], HbCC [19][21], HbEE [22], HbAE [16], and HbF [23][26]. In addition to these positive findings, conflicting data have been reported from many of these investigations (see Table 2).

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Table 2. Studies of P. falciparum invasion of and development in RBCs containing hemoglobin variants.

http://dx.doi.org/10.1371/journal.ppat.1003327.t002

For HbS-containing RBCs specifically, several reports have implicated enhanced sickling of iRBCs as a mechanism of malaria protection. Luzzatto et al. [27] and Roth et al. [28] separately reported increased sickling of HbAS iRBCs at low oxygen tension compared to HbAA iRBCs. Similarly, Friedman [29] described comparable parasite growth rates in HbAA, HbAS, and HbSS RBCs at high oxygen tension (18%), but sickling and destruction of parasites in HbAS and HbSS RBCs at lower oxygen tensions (1%–5%) that more closely mimic the micro-aerophilic environment of post-capillary venules in vivo. Conversely, exposure of iRBCs with either α- or β-thalassemia traits to high oxygen tensions restricted parasite growth, suggesting a reduced ability to tolerate oxidative stress [24].

A recent study proposes a novel mechanism of P. falciparum growth inhibition in HbS-containing RBCs. Both HbAS and HbSS RBCs manifest host microRNA (miRNA) profiles that are distinct from those of HbAA RBCs [30], [31]. Employing multiple independent approaches, LaMonte et al. [32] describe the translocation of several host RBC miRNAs into P. falciparum parasites, as well as the fusion of these human miRNAs with extant parasite mRNA transcripts to subsequently inhibit the translation of enzymes that are critical for parasite development. Specifically, the host miRNAs miR-451 and let-7i were significantly more abundant in HbAS and HbSS RBCs, and were associated with attenuated parasite growth in these cells. In addition, the inhibition of these two miRNAs by experimental transfection of RBCs with antisense oligonucleotides partially restored parasite growth. Downstream, miR-451 appears to fuse with transcripts of the regulatory subunit of the parasite's cAMP-dependent protein kinase (PKA-R) to reduce its translation, thereby upregulating activity of its substrate PKA and ultimately disrupting multiple parasite developmental pathways. Thus, the aberrant miRNA profile of HbS-containing RBCs may modulate the intraerythrocytic maturation of P. falciparum in a way that restricts parasite growth.

Do Hemoglobinopathies Interfere with Intrinsic Pathogenic Mechanisms of P. falciparum Malaria?

Two major pathogenic phenotypes of iRBCs have been described: those that mediate binding of iRBCs to endothelial receptors (“cytoadherence”) [33] and those that mediate binding of iRBCs to uninfected RBCs (“rosetting”) [34], [35]. Both adherence phenotypes are conferred by the expression of P. falciparum erythrocyte membrane protein 1 (PfEMP1) [36][38], a family of highly variant proteins that are concentrated in protuberant structures called “knobs” on the iRBC surface. Different PfEMP1 variants mediate the binding of iRBCs to microvascular endothelial cells (via CD36, ICAM-1, etc.) [39], placental syncytiotrophoblasts (via chondroitin sulfate A) [40], [41], and uninfected RBCs (via complement receptor 1, A and B blood group antigens, and heparin sulfate-like antigens) [42][44]. Other pathogenic mechanisms that may be associated with disease include the production of cytokines in response to P. falciparum glycosylphosphatidylinositol (PfGPI) [45] and parasite-derived uric acid [46], direct hemolysis due to parasite egress from RBCs, and PfEMP1-mediated suppression of inflammatory cytokines (discussed below) [47].

A series of investigations suggests that the weakening of cytoadherence interactions partially governs malaria protection by the hemoglobinopathies. Early studies by Udomsangpetch et al. [48] described impaired rosetting and binding to human umbilical-vein endothelial cells by α- and β-thalassemic iRBCs, although many of these RBCs contained additional hemoglobin mutations that may have influenced this phenotype. Additionally, impaired rosetting and cytoadherence were not clearly associated with reductions in the levels of surface antigens implicated in binding interactions. Similarly, Fairhurst et al. [21] found that the density of PfEMP1-laden knobs was markedly lower on the surface of HbAC and HbCC iRBCs (compared to HbAA iRBCs) despite comparable total iRBC levels of knob-associated histidine-rich protein (KAHRP), a major parasite-produced component of knobs. Further investigation of this phenomenon found that HbAC and HbCC markedly impaired the binding of iRBCs to human microvascular endothelial cells (HMVECs) under both static and physiologic flow conditions [49]. Subsequent investigations also found significant reductions in the binding of HbAS iRBCs [50], HbF-containing iRBCs [51], and α-thalassemic iRBCs [52] to HMVECs. Taken together, these reports support a common pathway for reducing the pathogenicity of parasites infecting hemoglobinopathic RBCs, whereby aberrant surface expression of PfEMP1 attenuates the binding of iRBCs to host cells within microvessels [53].

A recent study supports this candidate mechanism of malaria protection. Cyrklaff et al. [54] investigated the protein-trafficking network of the iRBC and demonstrated that the parasite remodels the RBC's actin cytoskeleton to enable the export of parasite-derived proteins to knobs on the iRBC surface. In HbSC and HbCC iRBCs, this actin cytoskeleton is disrupted and the export of parasite proteins to surface knobs is relatively inhibited, possibly due to the inhibition of actin polymerization by hemichromes. These forms of oxidized, denatured hemoglobin are known to accumulate in HbS- and HbC-containing RBCs, thus providing a potential link between hemoglobin instability and abnormal PfEMP1/knob display. Further studies are needed to explore the impact of this phenomenon upon both in-vitro measures of parasite virulence—including PfEMP1 expression and iRBC binding to host cells—and in-vivo measures of malaria severity.

Do Hemoglobinopathies Impact the Innate Host Defense Responses to P. falciparum?

There is an emerging recognition of the impact of aberrant host responses in the pathogenesis of malaria, particularly severe falciparum malaria (reviewed in [55][57]). Studies of adjunctive interventions to modulate this response in humans have not yielded sustained successes [58], but experiments in murine models continue to demonstrate benefit [59], and new modalities remain under active investigation [58], [60], [61].

The innate host defense response encompasses myriad stereotypical pathways that are activated by microorganisms and orchestrated to mitigate insults while minimizing collateral toxicity (reviewed in [62]). Typically initiated by the recognition of pathogen-associated molecular patterns (PAMPs) by Toll-like receptors (TLRs) on leukocytes, these responses subsequently progress through: 1) a pro-inflammatory phase marked by release of cytokines, activation of endothelial cells, and recruitment of circulating and locally resident immune effector cells; 2) a counter-regulatory phase in which tissue-protective molecules such as erythropoietin [63], [64], heme oxygenase-1 (HO-1) [65], [66], and angiopoietin-1 [67] are deployed to limit inflammatory damage; and 3) a repair phase mediated by vascular- and tissue-specific stem cells [68], [69]. These phases result from host and pathogen factors that collectively balance these pro-inflammatory and counter-regulatory responses.

In falciparum malaria, these innate immune responses are potently initiated through the activation of TLRs on leukocytes by both PfGPI [70] and hemozoin (the product of heme polymerization) [71], [72], as well as by microvascular inflammation caused by PfEMP1-mediated binding of iRBCs to endothelium (see above) [73]. In murine models, the outcome of malaria is sensitive to experimental manipulation of multiple host innate response molecules, such as tumor necrosis factor (TNF), interferon-γ [74], and erythropoietin [75], suggesting their role in mediating differential infection outcome. In human studies, severe malaria has been associated with increased angiopoietin-2, decreased angiopoietin-1, and decreased endothelial nitric oxide levels [76][78], and the upregulation of counter-regulatory molecules including HO-1 and erythropoietin [78][80].

Few studies have investigated the impact of hemoglobinopathies on these responses, though recent murine and human studies have shed light on a possible role for HO-1. Normally, HO-1 catabolizes and thus mitigates the cytotoxicity of free heme, which is released by the degradation of the RBC's hemoglobin. Recent studies in murine models identified HO-1 as a protective counter-regulatory molecule in sepsis [81] and malaria [82], [83]. In addition, a recent study by Cunnington et al. has demonstrated that when HO-1 is upregulated in response to hemolysis during murine Plasmodium infection, resistance to non-typhoidal Salmonella disease is abrogated [84]. In combining mouse models of human cerebral malaria and of human sickle-cell trait, Ferreira et al. [85] demonstrated that mice carrying RBCs with human HbS were protected from cerebral malaria. Furthermore, they provided evidence that this protection is associated with chronically elevated production of HO-1 and with reduced production of inflammatory cerebral chemokines during infection. However, the interpretation that HO-1 activity may mediate HbS-associated protection from malaria in mice is complicated by elegant recent work on severe malaria in humans [86]. In Gambian children, the association of HO-1 levels with severe malaria was variable, and HO-1 promoter polymorphisms that confer higher constitutive levels of HO-1 were associated with increased risk of severe malaria. These findings, gathered mostly from patients with HbAA, suggest that HO-1 may be either protective or deleterious across a wide spectrum of levels in vivo.

Despite the paucity of investigations of hemoglobinopathies and innate host defenses, a separate line of investigations of nitric oxide (NO) and severe malaria highlights the importance of interactions between iRBC and endothelium in the pathogenesis of severe malaria. As noted above, severe falciparum malaria is associated with low NO bioavailability [87], [88], and a polymorphism in the nitric oxide synthase 2 (NOS2) promoter—which increases NO production and is prevalent in Kenyan and Tanzanian children—was associated with substantial protection from severe malaria [89]. Data support diverse roles for NO in mediating parasite death [90] and in acquiring immune memory [91]. NO also manifests anti-inflammatory activity by reducing the expression of host receptors used by iRBCs to bind microvascular endothelial cells [92]. Indeed, the addition of NO to an in-vitro model of endothelial binding downregulated the endothelial expression of ICAM-1 and VCAM-1, and attenuated the cytoadherence of iRBCs under flow conditions [93]. Thus, both hemoglobinopathies and increased NO production are associated with protection from severe malaria in vivo, and both are also associated with reduced binding of iRBCs to endothelium in vitro. These findings suggest that similar molecular phenomena—the disruption of iRBC–endothelium interactions—may also occur in vivo.

Do Hemoglobinopathies Enhance the Adaptive Immune Response to P. falciparum?

Evidence from field studies supports an association between several hemoglobinopathies, adaptive immunity, and protection from malaria [94], [95], though investigations of these relationships are complicated by the absence of reliable correlates of immune protection.

A central role for antibodies in malaria immunity is suggested by the ability of polyclonal IgG from malaria-immune adults to clear parasitemias in children with malaria [96]. Several field studies have investigated differences in antigen seroreactivity in children with hemoglobinopathies. Cross-sectional studies of children with hemoglobinopathies in Nigeria [97], the Gambia [98], Cameroon [99], Gabon [100], [101], and Burkina Faso [102] have yielded inconsistent results, with some demonstrating higher seroreactivity to specific or variant surface P. falciparum antigens of heterologous parasites in HbAS children, and others reporting no differences. These findings were explored by measuring seroreactivity to a panel of 491 P. falciparum proteins in Malian children before and after a well-defined malaria transmission season [103]; though IgG responses to antigens were enhanced after the transmission season, there were no qualitative or quantitative differences in antigen recognition between HbAA, HbAS, and HbAC children. Given the methodological strengths of this study and the broad spectrum of antigens investigated, it seems clear that these hemoglobinopathies do not generally enhance the acquisition of antibodies to P. falciparum antigens. It remains to be fully investigated whether they enhance IgG recognition of specific variants of PfEMP1 or other surface antigens that are known to play a role in malaria immunity.

Antibody-mediated phagocytosis of iRBCs is believed to be an important effector mechanism in protection from malaria. Investigations with normal RBCs have demonstrated that monocytes preferentially phagocytose iRBCs compared to uninfected RBCs, and that this preference is potentiated by the binding of IgG to iRBCs [104]. Additionally, polyclonal IgG from hyper-immune sera binds more avidly to both α-thalassemic iRBCs [105], [106] and β-thalassemic iRBCs [105] compared to non-thalassemic iRBCs, suggesting that this mechanism may preferentially clear iRBCs harboring hemoglobin variants. Indeed, Ayi et al. [107] demonstrated that ring-parasitized HbAS, β-thalassemic, and HbH RBCs had higher levels of membrane-bound hemichromes, C3c fragments, and aggregated band 3 proteins, and were phagocytosed more readily than ring-parasitized HbAA RBCs. While these data suggest that hemoglobinopathies functionally enhance the clearance of iRBCs, the precise mechanism of this enhancement remains obscure, evidence for this role in parasite clearance by other hemoglobinopathies is lacking, and the correlation between this mechanism and protection from clinical disease has not been specifically investigated.

Multiple lines of evidence support the hypothesis that P. falciparum parasites interfere with the acquisition of immunologic memory responses that contribute to subsequent control of parasitemia (reviewed in [108]). Several mechanisms are supported by murine and human studies, including the depletion by circulating parasites of dendritic cells [109], parasite-specific CD4+ T cells [110], [111], and memory B cells [112] by either soluble factors [111] or interactions between iRBCs and antigen-presenting cells [113]. It is unknown whether hemoglobinopathies impact the efficiency or magnitude of the cellular and molecular mechanisms that suppress immune memory directed at P. falciparum parasites.

An Integrated Hypothesis

In this review, we have artificially partitioned the evidence for diverse mechanisms of protection, but pathogenic pathways overlap substantially, and it is similarly likely that protective mechanisms in vivo also involve multiple pathways. As noted above, field evidence indicates that hemoglobinopathies do not impair parasite infection but instead attenuate malaria; this pattern suggests that protection from malaria syndromes is not mediated against the pre-erythrocytic stages of the P. falciparum life-cycle, and that hemoglobinopathies may influence the transition from parasite infection to disease.

Embroidering the positive and negative evidence summarized above suggests a model of malaria protection in which hemoglobinopathies impair the parasite's trafficking of PfEMP1 and other knob proteins to the iRBC surface [21], possibly due to the association of hemoglobinopathies with elevated hemichrome levels [49], [54]. Abnormal PfEMP1/knob display weakens the molecular interactions involved in the cytoadherence [49] and rosetting [48], [49] of iRBCs in microvessels. The attenuation of these host–parasite interactions not only mitigates microvascular obstruction and ischemia, but also impairs the activation of endothelial cells and limits the elaboration of inflammatory mediators including TNF [114]. Additionally, given the role of PfEMP1 in downregulating the release of pro-inflammatory cytokines like IL-12 [113] and IFN-γ [47] from PBMCs, abnormal PfEMP1/knob display may inhibit the parasite's ability to blunt both innate and adaptive immune responses. Finally, this attenuation of host-cell injury, coupled with mechanisms of parasite growth restriction in hemoglobinopathic iRBCs, may be involved in prolonging the asymptomatic phase of parasitemia. This delay in developing symptoms (and thus delay in seeking antimalarial treatment) may offer a greater amount of time for erythrocytic-stage antigens and markers of RBC senescence to be exposed to the immune system, thereby enhancing both the acquisition and maintenance of the adaptive and memory immune responses that ultimately protect individuals from developing the deadliest complications of P. falciparum infection. This model is based upon currently available data largely obtained from in-vitro experimental and in-vivo animal model studies, and most commonly for HbAS and α-thalassemia. Clearly, many opportunities exist to interrogate these phenomena in translational studies involving human populations that carry diverse hemoglobinopathies (Box 1).

Box 1. Questions for future translational investigations

  1. Do microRNAs impact the maturation of parasites in HbC, HbE, α-thalassemic, or HbF RBCs? Is their profile or impact in HbAS RBCs modified by the presence of α-globin deletions?
  2. Does microRNA manipulation of HbS-containing iRBCs impact their ability to cytoadhere to human microvascular endothelial cells or rosette with uninfected RBCs?
  3. How does the presence of α-thalassemia modify the effects of HbS, HbC, or HbE on parasite growth, maturation, microvascular adhesion, or endothelial cell activation?
  4. Do hemoglobinopathies quantitatively or qualitatively restrict the expression of specific variants of PfEMP1?
  5. Does reduced binding to human endothelial cells in vitro correlate with reductions in markers of endothelial activation in vivo?
  6. How do other malaria-protective polymorphisms, such as type O blood group antigen and glucose-6-phosphate dehydrogenase (G6PD) deficiency, interact with co-inherited hemoglobinopathies in mitigating pathogenesis?
  7. How do hemoglobinopathies impact the resting endogenous levels of angiopoietin-1, angiopoietin-2, and heme oxygenase-1?
  8. Do hemoglobinopathies augment cell-mediated immunity to P. falciparum antigens?
  9. Do hemoglobinopathies quantitatively or qualitatively enhance the acquisition of antibodies to specific variants or domains of PfEMP1?
  10. Do hemoglobinopathies impair the mechanisms by which the iRBC and the parasite interfere with the acquisition of immune memory?

Conclusions

In the spirit of Alexander, we propose that hemoglobinopathies may be nature's “Alexandrian solution” to the problem of understanding fundamental aspects of falciparum malaria. This bold slice through the Gordian knot of malaria pathogenesis represents a unique opportunity to isolate and identify the molecular correlates of falciparum malaria pathogenesis in humans in vivo, and to translate these findings into future interventions to prevent, treat, and eliminate this ancient and intractable scourge.

References

  1. 1. Yenne B (2010) Alexander the Great: lessons from history's undefeated general. New York: Palgrave Macmillan.
  2. 2. World Health Organization (2012) World malaria report: 2012. Available: http://www.who.int/malaria/publications/world_malaria_report_2012/en/index.html. Accessed 19 March 2013.
  3. 3. Bejon P, Lusingu J, Olotu A, Leach A, Lievens M, et al. (2008) Efficacy of RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N Engl J Med 359: 2521–2532. doi: 10.1056/nejmoa0807381
  4. 4. The RTS,S Clinical Trials Partnership (2011) First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children. N Engl J Med 365: 1863–1875. doi: 10.1056/nejmoa1102287
  5. 5. O'Meara WP, Bejon P, Mwangi TW, Okiro EA, Peshu N, et al. (2008) Effect of a fall in malaria transmission on morbidity and mortality in Kilifi, Kenya. Lancet 372: 1555–1562. doi: 10.1016/s0140-6736(08)61655-4
  6. 6. Miller LH, Baruch DI, Marsh K, Doumbo OK (2002) The pathogenic basis of malaria. Nature 415: 673–679. doi: 10.1038/415673a
  7. 7. Amaratunga C, Lopera-Mesa TM, Tse JG, Mita-Mendoza NK, Fairhurst RM (2010) In:Kaufmann S, Rouse B, Sacks D, editor. Immunology of infectious diseases. Washington, DC: American Society of Microbiology Press.
  8. 8. Taylor SM, Parobek CM, Fairhurst RM (2012) Haemoglobinopathies and the clinical epidemiology of malaria: a systematic review and meta-analysis. Lancet Infect Dis 12: 457–468. doi: 10.1016/s1473-3099(12)70055-5
  9. 9. Haldane JB (1949) The rate of mutation of human genes. Hereditas 35: 267–273. doi: 10.1111/j.1601-5223.1949.tb03339.x
  10. 10. Weatherall DJ, Provan AB (2000) Red cells I: inherited anaemias. Lancet 355: 1169–1175. doi: 10.1016/s0140-6736(00)02073-0
  11. 11. Trager W, Jensen JB (1976) Human malaria parasites in continuous culture. Science 193: 673–675. doi: 10.1126/science.781840
  12. 12. Haynes JD, Diggs CL, Hines FA, Desjardins RE (1976) Culture of human malaria parasites Plasmodium falciparum. Nature 263: 767–769. doi: 10.1038/263767a0
  13. 13. Bunyaratvej A, Butthep P, Sae-Ung N, Fucharoen S, Yuthavong Y (1992) Reduced deformability of thalassemic erythrocytes and erythrocytes with abnormal hemoglobins and relation with susceptibility to Plasmodium falciparum invasion. Blood 79: 2460–2463.
  14. 14. Ifediba TC, Stern A, Ibrahim A, Rieder RF (1985) Plasmodium falciparum in vitro: diminished growth in hemoglobin H disease erythrocytes. Blood 65: 452–455.
  15. 15. Chotivanich K, Udomsangpetch R, Pattanapanyasat K, Chierakul W, Simpson J, et al. (2002) Hemoglobin E: a balanced polymorphism protective against high parasitemias and thus severe P falciparum malaria. Blood 100: 1172–1176.
  16. 16. Brockelman CR, Wongsattayanont B, Tan-ariya P, Fucharoen S (1987) Thalassemic erythrocytes inhibit in vitro growth of Plasmodium falciparum. J Clin Microbiol 25: 56–60.
  17. 17. Pasvol G, Weatherall DJ, Wilson RJ (1978) Cellular mechanism for the protective effect of haemoglobin S against P. falciparum malaria. Nature 274: 701–703. doi: 10.1038/274701a0
  18. 18. Pasvol G (1980) The interaction between sickle haemoglobin and the malarial parasite Plasmodium falciparum. Trans R Soc Trop Med Hyg 74: 701–705. doi: 10.1016/0035-9203(80)90182-0
  19. 19. Friedman MJ, Roth EF, Nagel RL, Trager W (1979) The role of hemoglobins C, S, and Nbalt in the inhibition of malaria parasite development in vitro. Am J Trop Med Hyg 28: 777–780.
  20. 20. Olson JA, Nagel RL (1986) Synchronized cultures of P falciparum in abnormal red cells: the mechanism of the inhibition of growth in HbCC cells. Blood 67: 997–1001.
  21. 21. Fairhurst RM, Fujioka H, Hayton K, Collins KF, Wellems TE (2003) Aberrant development of Plasmodium falciparum in hemoglobin CC red cells: implications for the malaria protective effect of the homozygous state. Blood 101: 3309–3315. doi: 10.1182/blood-2002-10-3105
  22. 22. Nagel RL, Raventos-Suarez C, Fabry ME, Tanowitz H, Sicard D, et al. (1981) Impairment of the growth of Plasmodium falciparum in HbEE erythrocytes. J Clin Invest 68: 303–305. doi: 10.1172/jci110248
  23. 23. Pasvol G, Weatherall DJ, Wilson RJ, Smith DH, Gilles HM (1976) Fetal haemoglobin and malaria. Lancet 1: 1269–1272. doi: 10.1016/s0140-6736(76)91738-4
  24. 24. Friedman MJ (1979) Oxidant damage mediates variant red cell resistance to malaria. Nature 280: 245–247. doi: 10.1038/280245a0
  25. 25. Pasvol G, Weatherall DJ, Wilson RJ (1977) Effects of foetal haemoglobin on susceptibility of red cells to Plasmodium falciparum. Nature 270: 171–173. doi: 10.1038/270171a0
  26. 26. Wilson RJ, Pasvol G, Weatherall DJ (1977) Invasion and growth of Plasmodium falciparum in different types of human erythrocyte. Bull World Health Organ 55: 179–186.
  27. 27. Luzzatto L, Nwachuku-Jarrett ES, Reddy S (1970) Increased sickling of parasitised erythrocytes as mechanism of resistance against malaria in the sickle-cell trait. Lancet 1: 319–321. doi: 10.1016/s0140-6736(70)90700-2
  28. 28. Roth EF Jr, Friedman M, Ueda Y, Tellez I, Trager W, et al. (1978) Sickling rates of human AS red cells infected in vitro with Plasmodium falciparum malaria. Science 202: 650–652. doi: 10.1126/science.360396
  29. 29. Friedman MJ (1978) Erythrocytic mechanism of sickle cell resistance to malaria. Proc Natl Acad Sci U S A 75: 1994–1997. doi: 10.1073/pnas.75.4.1994
  30. 30. Chen SY, Wang Y, Telen MJ, Chi JT (2008) The genomic analysis of erythrocyte microRNA expression in sickle cell diseases. PLoS One 3: e2360 doi:10.1371/journal.pone.0002360.
  31. 31. Sangokoya C, Telen MJ, Chi JT (2010) microRNA miR-144 modulates oxidative stress tolerance and associates with anemia severity in sickle cell disease. Blood 116: 4338–4348. doi: 10.1182/blood-2009-04-214817
  32. 32. Lamonte G, Philip N, Reardon J, Lacsina JR, Majoros W, et al. (2012) Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance. Cell Host Microbe 12: 187–199. doi: 10.1016/j.chom.2012.06.007
  33. 33. Baruch DI, Gormely JA, Ma C, Howard RJ, Pasloske BL (1996) Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1. Proc Natl Acad Sci U S A 93: 3497–3502. doi: 10.1073/pnas.93.8.3497
  34. 34. Carlson J, Helmby H, Hill AV, Brewster D, Greenwood BM, et al. (1990) Human cerebral malaria: association with erythrocyte rosetting and lack of anti-rosetting antibodies. Lancet 336: 1457–1460. doi: 10.1016/0140-6736(90)93174-n
  35. 35. Kaul DK, Roth EF Jr, Nagel RL, Howard RJ, Handunnetti SM (1991) Rosetting of Plasmodium falciparum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions. Blood 78: 812–819.
  36. 36. Su XZ, Heatwole VM, Wertheimer SP, Guinet F, Herrfeldt JA, et al. (1995) The large diverse gene family var encodes proteins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell 82: 89–100. doi: 10.1016/0092-8674(95)90055-1
  37. 37. Smith JD, Chitnis CE, Craig AG, Roberts DJ, Hudson-Taylor DE, et al. (1995) Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell 82: 101–110. doi: 10.1016/0092-8674(95)90056-x
  38. 38. Baruch DI, Pasloske BL, Singh HB, Bi X, Ma XC, et al. (1995) Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82: 77–87. doi: 10.1016/0092-8674(95)90054-3
  39. 39. Raventos-Suarez C, Kaul DK, Macaluso F, Nagel RL (1985) Membrane knobs are required for the microcirculatory obstruction induced by Plasmodium falciparum-infected erythrocytes. Proc Natl Acad Sci U S A 82: 3829–3833. doi: 10.1073/pnas.82.11.3829
  40. 40. Fried M, Duffy PE (1996) Adherence of Plasmodium falciparum to chondroitin sulfate A in the human placenta. Science 272: 1502–1504. doi: 10.1126/science.272.5267.1502
  41. 41. Salanti A, Staalsoe T, Lavstsen T, Jensen AT, Sowa MP, et al. (2003) Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria. Mol Microbiol 49: 179–191. doi: 10.1046/j.1365-2958.2003.03570.x
  42. 42. Rowe JA, Moulds JM, Newbold CI, Miller LH (1997) P. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 388: 292–295. doi: 10.1016/s0887-7963(98)80055-2
  43. 43. Carlson J, Wahlgren M (1992) Plasmodium falciparum erythrocyte rosetting is mediated by promiscuous lectin-like interactions. J Exp Med 176: 1311–1317. doi: 10.1084/jem.176.5.1311
  44. 44. Chen Q, Barragan A, Fernandez V, Sundstrom A, Schlichtherle M, et al. (1998) Identification of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as the rosetting ligand of the malaria parasite P. falciparum. J Exp Med 187: 15–23. doi: 10.1084/jem.187.1.15
  45. 45. Schofield L, Hackett F (1993) Signal transduction in host cells by a glycosylphosphatidylinositol toxin of malaria parasites. J Exp Med 177: 145–153. doi: 10.1084/jem.177.1.145
  46. 46. Lopera-Mesa TM, Mita-Mendoza NK, van de Hoef DL, Doumbia S, Konate D, et al. (2012) Plasma Uric Acid Levels Correlate with Inflammation and Disease Severity in Malian Children with Plasmodium falciparum Malaria. PLoS One 7: e46424 doi:10.1371/journal.pone.0046424.
  47. 47. D'Ombrain MC, Voss TS, Maier AG, Pearce JA, Hansen DS, et al. (2007) Plasmodium falciparum erythrocyte membrane protein-1 specifically suppresses early production of host interferon-gamma. Cell Host Microbe 2: 130–138. doi: 10.1016/j.chom.2007.06.012
  48. 48. Udomsangpetch R, Sueblinvong T, Pattanapanyasat K, Dharmkrong-at A, Kittikalayawong A, et al. (1993) Alteration in cytoadherence and rosetting of Plasmodium falciparum-infected thalassemic red blood cells. Blood 82: 3752–3759.
  49. 49. Fairhurst RM, Baruch DI, Brittain NJ, Ostera GR, Wallach JS, et al. (2005) Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria. Nature 435: 1117–1121. doi: 10.1038/nature03631
  50. 50. Cholera R, Brittain NJ, Gillrie MR, Lopera-Mesa TM, Diakite SA, et al. (2008) Impaired cytoadherence of Plasmodium falciparum-infected erythrocytes containing sickle hemoglobin. Proc Natl Acad Sci U S A 105: 991–996. doi: 10.1073/pnas.0711401105
  51. 51. Amaratunga C, Lopera-Mesa TM, Brittain NJ, Cholera R, Arie T, et al. (2011) A role for fetal hemoglobin and maternal immune IgG in infant resistance to Plasmodium falciparum malaria. PLoS One 6: e14798 doi:10.1371/journal.pone.0014798.
  52. 52. Krause MA, Diakite SA, Lopera-Mesa TM, Amaratunga C, Arie T, et al. (2012) alpha-Thalassemia impairs the cytoadherence of Plasmodium falciparum-infected erythrocytes. PLoS One 7: e37214 doi:10.1371/journal.pone.0037214.
  53. 53. Fairhurst RM, Bess CD, Krause MA (2012) Abnormal PfEMP1/knob display on Plasmodium falciparum-infected erythrocytes containing hemoglobin variants: fresh insights into malaria pathogenesis and protection. Microbes Infect 14: 851–862. doi: 10.1016/j.micinf.2012.05.006
  54. 54. Cyrklaff M, Sanchez CP, Kilian N, Bisseye C, Simpore J, et al. (2011) Hemoglobins S and C interfere with actin remodeling in Plasmodium falciparum-infected erythrocytes. Science 334: 1283–1286. doi: 10.1126/science.1213775
  55. 55. Schofield L, Grau GE (2005) Immunological processes in malaria pathogenesis. Nat Rev Immunol 5: 722–735. doi: 10.1038/nri1686
  56. 56. Clark IA, Alleva LM, Mills AC, Cowden WB (2004) Pathogenesis of malaria and clinically similar conditions. Clin Microbiol Rev 17: 509–539 table of contents. doi: 10.1128/cmr.17.3.509-539.2004
  57. 57. Hunt NH, Grau GE (2003) Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol 24: 491–499. doi: 10.1016/s1471-4906(03)00229-1
  58. 58. John CC, Kutamba E, Mugarura K, Opoka RO (2010) Adjunctive therapy for cerebral malaria and other severe forms of Plasmodium falciparum malaria. Expert Rev Anti Infect Ther 8: 997–1008. doi: 10.1586/eri.10.90
  59. 59. Achtman AH, Pilat S, Law CW, Lynn DJ, Janot L, et al. (2012) Effective adjunctive therapy by an innate defense regulatory peptide in a preclinical model of severe malaria. Sci Transl Med 4: 135ra164. doi: 10.1126/scitranslmed.3003515
  60. 60. Casals-Pascual C, Idro R, Picot S, Roberts DJ, Newton CR (2009) Can erythropoietin be used to prevent brain damage in cerebral malaria? Trends Parasitol 25: 30–36. doi: 10.1016/j.pt.2008.10.002
  61. 61. Weinberg JB, Lopansri BK, Mwaikambo E, Granger DL (2008) Arginine, nitric oxide, carbon monoxide, and endothelial function in severe malaria. Curr Opin Infect Dis 21: 468–475. doi: 10.1097/qco.0b013e32830ef5cf
  62. 62. Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140: 805–820. doi: 10.1016/j.cell.2010.01.022
  63. 63. Villa P, Bigini P, Mennini T, Agnello D, Laragione T, et al. (2003) Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis. J Exp Med 198: 971–975. doi: 10.1084/jem.20021067
  64. 64. Siren AL, Fratelli M, Brines M, Goemans C, Casagrande S, et al. (2001) Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress. Proc Natl Acad Sci U S A 98: 4044–4049. doi: 10.1073/pnas.051606598
  65. 65. Wagener FA, Eggert A, Boerman OC, Oyen WJ, Verhofstad A, et al. (2001) Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase. Blood 98: 1802–1811. doi: 10.1182/blood.v98.6.1802
  66. 66. Kapturczak MH, Wasserfall C, Brusko T, Campbell-Thompson M, Ellis TM, et al. (2004) Heme oxygenase-1 modulates early inflammatory responses: evidence from the heme oxygenase-1-deficient mouse. Am J Pathol 165: 1045–1053. doi: 10.1016/s0002-9440(10)63365-2
  67. 67. Kim H, Higgins S, Liles WC, Kain KC (2011) Endothelial activation and dysregulation in malaria: a potential target for novel therapeutics. Curr Opin Hematol 18: 177–185. doi: 10.1097/moh.0b013e328345a4cf
  68. 68. Koh SH, Noh MY, Cho GW, Kim KS, Kim SH (2009) Erythropoietin increases the motility of human bone marrow-multipotent stromal cells (hBM-MSCs) and enhances the production of neurotrophic factors from hBM-MSCs. Stem Cells Dev 18: 411–421. doi: 10.1089/scd.2008.0040
  69. 69. Erbayraktar Z, Erbayraktar S, Yilmaz O, Cerami A, Coleman T, et al. (2009) Nonerythropoietic tissue protective compounds are highly effective facilitators of wound healing. Mol Med 15: 235–241.
  70. 70. Krishnegowda G, Hajjar AM, Zhu J, Douglass EJ, Uematsu S, et al. (2005) Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 280: 8606–8616. doi: 10.1074/jbc.m413541200
  71. 71. Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, et al. (2007) Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc Natl Acad Sci U S A 104: 1919–1924. doi: 10.1073/pnas.0608745104
  72. 72. Jaramillo M, Plante I, Ouellet N, Vandal K, Tessier PA, et al. (2004) Hemozoin-inducible proinflammatory events in vivo: potential role in malaria infection. J Immunol 172: 3101–3110. doi: 10.4049/jimmunol.172.5.3101
  73. 73. Tripathi AK, Sha W, Shulaev V, Stins MF, Sullivan DJ Jr (2009) Plasmodium falciparum-infected erythrocytes induce NF-kappaB regulated inflammatory pathways in human cerebral endothelium. Blood 114: 4243–4252. doi: 10.1182/blood-2009-06-226415
  74. 74. Clark IA, Hunt NH, Butcher GA, Cowden WB (1987) Inhibition of murine malaria (Plasmodium chabaudi) in vivo by recombinant interferon-gamma or tumor necrosis factor, and its enhancement by butylated hydroxyanisole. J Immunol 139: 3493–3496.
  75. 75. Kaiser K, Texier A, Ferrandiz J, Buguet A, Meiller A, et al. (2006) Recombinant human erythropoietin prevents the death of mice during cerebral malaria. J Infect Dis 193: 987–995. doi: 10.1086/500844
  76. 76. Yeo TW, Lampah DA, Gitawati R, Tjitra E, Kenangalem E, et al. (2008) Angiopoietin-2 is associated with decreased endothelial nitric oxide and poor clinical outcome in severe falciparum malaria. Proc Natl Acad Sci U S A 105: 17097–17102. doi: 10.1073/pnas.0805782105
  77. 77. Conroy AL, Phiri H, Hawkes M, Glover S, Mallewa M, et al. (2010) Endothelium-based biomarkers are associated with cerebral malaria in Malawian children: a retrospective case-control study. PLoS One 5: e15291 doi:10.1371/journal.pone.0015291.
  78. 78. Lovegrove FE, Tangpukdee N, Opoka RO, Lafferty EI, Rajwans N, et al. (2009) Serum angiopoietin-1 and -2 levels discriminate cerebral malaria from uncomplicated malaria and predict clinical outcome in African children. PLoS One 4: e4912 doi:10.1371/journal.pone.0004912.
  79. 79. Clark IA, Awburn MM, Harper CG, Liomba NG, Molyneux ME (2003) Induction of HO-1 in tissue macrophages and monocytes in fatal falciparum malaria and sepsis. Malar J 2: 41. doi: 10.1186/1475-2875-2-41
  80. 80. Casals-Pascual C, Idro R, Gicheru N, Gwer S, Kitsao B, et al. (2008) High levels of erythropoietin are associated with protection against neurological sequelae in African children with cerebral malaria. Proc Natl Acad Sci U S A 105: 2634–2639. doi: 10.1073/pnas.0709715105
  81. 81. Larsen R, Gozzelino R, Jeney V, Tokaji L, Bozza FA, et al. (2010) A central role for free heme in the pathogenesis of severe sepsis. Sci Transl Med 2: 51ra71. doi: 10.1126/scitranslmed.3001118
  82. 82. Pamplona A, Ferreira A, Balla J, Jeney V, Balla G, et al. (2007) Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria. Nat Med 13: 703–710. doi: 10.1038/nm1586
  83. 83. Seixas E, Gozzelino R, Chora A, Ferreira A, Silva G, et al. (2009) Heme oxygenase-1 affords protection against noncerebral forms of severe malaria. Proc Natl Acad Sci U S A 106: 15837–15842. doi: 10.1073/pnas.0903419106
  84. 84. Cunnington AJ, de Souza JB, Walther M, Riley EM (2012) Malaria impairs resistance to Salmonella through heme- and heme oxygenase-dependent dysfunctional granulocyte mobilization. Nat Med 18: 120–127. doi: 10.1038/nm.2601
  85. 85. Ferreira A, Marguti I, Bechmann I, Jeney V, Chora A, et al. (2011) Sickle hemoglobin confers tolerance to Plasmodium infection. Cell 145: 398–409. doi: 10.1016/j.cell.2011.03.049
  86. 86. Walther M, De Caul A, Aka P, Njie M, Amambua-Ngwa A, et al. (2012) HMOX1 gene promoter alleles and high HO-1 levels are associated with severe malaria in Gambian children. PLoS Pathog 8: e1002579 doi:10.1371/journal.ppat.1002579.
  87. 87. Lopansri BK, Anstey NM, Weinberg JB, Stoddard GJ, Hobbs MR, et al. (2003) Low plasma arginine concentrations in children with cerebral malaria and decreased nitric oxide production. Lancet 361: 676–678. doi: 10.1016/s0140-6736(03)12564-0
  88. 88. Yeo TW, Lampah DA, Gitawati R, Tjitra E, Kenangalem E, et al. (2007) Impaired nitric oxide bioavailability and L-arginine reversible endothelial dysfunction in adults with falciparum malaria. J Exp Med 204: 2693–2704. doi: 10.1084/jem.20070819
  89. 89. Hobbs MR, Udhayakumar V, Levesque MC, Booth J, Roberts JM, et al. (2002) A new NOS2 promoter polymorphism associated with increased nitric oxide production and protection from severe malaria in Tanzanian and Kenyan children. Lancet 360: 1468–1475. doi: 10.1016/s0140-6736(02)11474-7
  90. 90. Rockett KA, Awburn MM, Cowden WB, Clark IA (1991) Killing of Plasmodium falciparum in vitro by nitric oxide derivatives. Infect Immun 59: 3280–3283.
  91. 91. Rockett KA, Awburn MM, Rockett EJ, Cowden WB, Clark IA (1994) Possible role of nitric oxide in malarial immunosuppression. Parasite Immunol 16: 243–249. doi: 10.1111/j.1365-3024.1994.tb00346.x
  92. 92. De Caterina R, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, et al. (1995) Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest 96: 60–68. doi: 10.1172/jci118074
  93. 93. Serirom S, Raharjo WH, Chotivanich K, Loareesuwan S, Kubes P, et al. (2003) Anti-adhesive effect of nitric oxide on Plasmodium falciparum cytoadherence under flow. Am J Pathol 162: 1651–1660. doi: 10.1016/s0002-9440(10)64299-x
  94. 94. Williams TN, Mwangi TW, Roberts DJ, Alexander ND, Weatherall DJ, et al. (2005) An immune basis for malaria protection by the sickle cell trait. PLoS Med 2: e128 doi:10.1371/journal.pmed.0020128.
  95. 95. Gong L, Maiteki-Sebuguzi C, Rosenthal PJ, Hubbard AE, Drakeley CJ, et al. (2012) Evidence for both innate and acquired mechanisms of protection from Plasmodium falciparum in children with sickle cell trait. Blood 119: 3808–3814. doi: 10.1182/blood-2011-08-371062
  96. 96. Cohen S, Mc GI, Carrington S (1961) Gamma-globulin and acquired immunity to human malaria. Nature 192: 733–737. doi: 10.1038/192733a0
  97. 97. Cornille-Brogger R, Fleming AF, Kagan I, Matsushima T, Molineaux L (1979) Abnormal haemoglobins in the Sudan savanna of Nigeria. II. Immunological response to malaria in normals and subjects with sickle cell trait. Ann Trop Med Parasitol 73: 173–183.
  98. 98. Allen SJ, Bennett S, Riley EM, Rowe PA, Jakobsen PH, et al. (1992) Morbidity from malaria and immune responses to defined Plasmodium falciparum antigens in children with sickle cell trait in The Gambia. Trans R Soc Trop Med Hyg 86: 494–498. doi: 10.1016/0035-9203(92)90083-o
  99. 99. Le Hesran JY, Personne I, Personne P, Fievet N, Dubois B, et al. (1999) Longitudinal study of Plasmodium falciparum infection and immune responses in infants with or without the sickle cell trait. Int J Epidemiol 28: 793–798. doi: 10.1093/ije/28.4.793
  100. 100. Luty AJ, Ulbert S, Lell B, Lehman L, Schmidt-Ott R, et al. (2000) Antibody responses to Plasmodium falciparum: evolution according to the severity of a prior clinical episode and association with subsequent reinfection. Am J Trop Med Hyg 62: 566–572.
  101. 101. Cabrera G, Cot M, Migot-Nabias F, Kremsner PG, Deloron P, et al. (2005) The sickle cell trait is associated with enhanced immunoglobulin G antibody responses to Plasmodium falciparum variant surface antigens. J Infect Dis 191: 1631–1638. doi: 10.1086/429832
  102. 102. Verra F, Simpore J, Warimwe GM, Tetteh KK, Howard T, et al. (2007) Haemoglobin C and S role in acquired immunity against Plasmodium falciparum malaria. PLoS One 2: e978 doi: 10.1371/journal.pone.0000978.
  103. 103. Tan X, Traore B, Kayentao K, Ongoiba A, Doumbo S, et al. (2011) Hemoglobin S and C heterozygosity enhances neither the magnitude nor breadth of antibody responses to a diverse array of Plasmodium falciparum antigens. J Infect Dis 204: 1750–1761. doi: 10.1093/infdis/jir638
  104. 104. Celada A, Cruchaud A, Perrin LH (1982) Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes. Clin Exp Immunol 47: 635–644.
  105. 105. Luzzi GA, Merry AH, Newbold CI, Marsh K, Pasvol G, et al. (1991) Surface antigen expression on Plasmodium falciparum-infected erythrocytes is modified in alpha- and beta-thalassemia. J Exp Med 173: 785–791. doi: 10.1084/jem.173.4.785
  106. 106. Williams TN, Weatherall DJ, Newbold CI (2002) The membrane characteristics of Plasmodium falciparum-infected and -uninfected heterozygous alpha(0)thalassaemic erythrocytes. Br J Haematol 118: 663–670. doi: 10.1046/j.1365-2141.2002.03610.x
  107. 107. Ayi K, Turrini F, Piga A, Arese P (2004) Enhanced phagocytosis of ring-parasitized mutant erythrocytes: a common mechanism that may explain protection against falciparum malaria in sickle trait and beta-thalassemia trait. Blood 104: 3364–3371. doi: 10.1182/blood-2003-11-3820
  108. 108. Langhorne J, Ndungu F, Sponaas A-M, Marsh K (2008) Immunity to malaria: more questions than answers. Nature Immunol 9: 725–732. doi: 10.1038/ni.f.205
  109. 109. Urban BC, Mwangi T, Ross A, Kinyanjui S, Mosobo M, et al. (2001) Peripheral blood dendritic cells in children with acute Plasmodium falciparum malaria. Blood 98: 2859–2861. doi: 10.1182/blood.v98.9.2859
  110. 110. Xu H, Wipasa J, Yan H, Zeng M, Makobongo MO, et al. (2002) The mechanism and significance of deletion of parasite-specific CD4(+) T cells in malaria infection. J Exp Med 195: 881–892. doi: 10.1084/jem.20011174
  111. 111. Sun T, Holowka T, Song Y, Zierow S, Leng L, et al. (2012) A Plasmodium-encoded cytokine suppresses T-cell immunity during malaria. Proc Natl Acad Sci U S A 109: E2117–2126. doi: 10.1073/pnas.1206573109
  112. 112. Wykes MN, Zhou YH, Liu XQ, Good MF (2005) Plasmodium yoelii can ablate vaccine-induced long-term protection in mice. J Immunol 175: 2510–2516. doi: 10.4049/jimmunol.175.4.2510
  113. 113. Urban BC, Ferguson DJ, Pain A, Willcox N, Plebanski M, et al. (1999) Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400: 73–77.
  114. 114. Wu Y, Szestak T, Stins M, Craig AG (2011) Amplification of P. falciparum Cytoadherence through induction of a pro-adhesive state in host endothelium. PLoS One 6: e24784 doi:10.1371/journal.pone.0024784.
  115. 115. Santiyanont R, Wilairat P (1981) Red cells containing hemoglobin E do not inhibit malaria parasite development in vitro. Am J Trop Med Hyg 30: 541–543.