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Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Parroche et al. PNAS 2007. Azeen Hadadi & Emi Leonard. Host-derived or pathogen-derived?.

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Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9

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Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9

Parroche et al. PNAS 2007.

AzeenHadadi & Emi Leonard

  • Host-derived or pathogen-derived?

What component of the malaria-infected RBC is the “malaria toxin”?

  • 3.3 billion people live in areas in risk of malaria transmission

  • Affects 300-500 million people a year

  • In 2008, over 1 million people were killed

  • Young children & pregnant women are most affected, because of decreased immunity

Malaria Statistics

Falciparum malaria affects a greater proportion of RBCs than other types of malaria

It can be fatal within a few hours of the 1st symptoms

Responsible for over 90% of the deaths

Malaria Cycle

Malaria Symptoms

  • Exact cause of innate immune response to malaria is unknown

  • Innate immune system

    • Cells and mechanisms that non-specifically defend host against infection

    • Provides immediate defense but does not confer long-lasting or protective immunity (as opposed to adaptive system)

Innate Immune Response

Immune Response

  • Toll-Like Receptors (TLRs)

    • Receptor proteins that recognize microbes

    • After detection, TLRs induce MyD88 proteins which ultimately cause the release cytokines

  • Cytokines

    • Intercellular signaling molecules secreted by immune cells

    •  Recruit other immune cells to site of infection

  • Chloroquine– anti-malarial medication

  • Falciparum malaria, however, is resistant to this form of treatment

  • Artemisinins- combination therapy

  • No effective preventative vaccines are available

  • Chemotherapeutic treatment of large populations impossible due to socioeconomic conditions


  • 1978 Clark- endotoxin (LPS) may cause malaria and parasite death

  • 2004 Caulfield et al- undernutrition as underlying cause of malaria in children less than five years old

  • 2005 Coban et al- malarial hemozoin activates innate immune response

Past Research

  • The parasite digests hemoglobin in the food vacuole, which produces toxic hemes

  • As a form of protection, the parasite converts these hemes into insoluble, weakly magnetic crystals called hemozoins

Hemozoin (Hz)

Electron micrograph of crystals of hemozoin isolated from the malaria parasite Plasmodium falciparum

  • The rupture of parasitized erythrocytes is accompanied by the onset of symptoms like fever and rigors

    • These symptoms are caused by the systemic release of proinflammatory cytokines

  • Both natural and synthetic hemozoin (a.k.a. β-hematin) have been reported to induce these inflammatory responses both in vivo and in vitro


  • During infection, hemozoin concentration after erythrocyte rupture may be as high as 100 μg/mL

    • Liver and spleen quickly clear it from blood circulation because of its particulate form

  • Hemozoin has been suggested to be the cause of the inflammatory immune responses during the malaria infection


Toll-like Receptors

  • TLRs have been involved in the recognition of constituents of parasites

    • GPI (glycosylphophatidylinositol) anchors are the best studied parasitic molecules that engage TLRs

  • GPI from P. falciparum have been shown to interact with immune cells through the activation of TLR2 and TLR4

  • Therefore, it is likely that malaria pathogenesis involves the engagement of TLRs.


  • Parroche et al. hypothesized that TLRs recognize both synthetic and natural hemozoin, and thus cause the activation of cells in the innate immune system


  • Enzyme-linked ImmunosorbentAssay (ELISA) utilized to detect cytokine concentrations

  • Anti-cytokine antibodies coated in wells capture cytokines

  • Captured cytokines detected by biotin-conjugated antibodies and enzyme-labeled streptavidin

  • Colored enzyme substrate allows for quantification

Highly Pure β-Hematin Has No Intrinsic Stimulatory Activity

  • Crude bovine hemin- 65% pure

  • HPLC-purified β-hematin > 98% pure

  • Fms-like tyrosine kinase-3 ligand-derived dendritic mouse cells (FL-DCs)

  • Crude hemin stimulated FL-DCs to produce cytokines

  • Highly pure β-hematin did not

  • Therefore, "immune activity" of β-hematin caused by contaminants from crude bovine hemin

Natural HZ Activates Cells to Produce Cytokines Through TLR9 and MyD88 

  • Various concentrations of natural HZ was used to stimulate bone marrow-derived FL-DCs from WT & knockout mice

    • Strong stimulation of cytokines IL-12p40 & RANTES*

  • TLR2-null cells responded comparably to WT cells

  • Dendritic cells from TLR9 and MyD88 knockout mice failed to respond to HZ

  • Results confirm that natural HZ engages the TLR9/MyD88 pathway

    *Data not shown

Natural HZ engages the TLR9 Pathway

  • TLR9 binds unmethylated CpG DNA

  • The DNA ligands have been categorized into 3 classes:

    A-class oligonucleotides

    -strong inducers of Type I Interferons

    B-class oligonucleotides

    -virtually no IFN-inducing activity

    C-class oligonucleotides

    -features of both A- & B- classes

HZ Failed to Induce Production of Cytokine IFNα

  • WT FL-DCs were also stimulated for 24 hours with medium alone or medium plus the indicated ligand

  • Interferon α (IFNα) released into the medium was measured (ELISA)

  • Natural HZ failed to induce production of IFNα

    • suggesting that the TLR9 ligand is B-class CpG DNA or is not CpG DNA

Natural HZ Stimulates FL-DCs in a DNase-Sensitive Fashion

  • Activity of HZ was abolished by nuclease digestion

    • HZ crystals remained intact, though

  • Inhibition of S7 nuclease (with EDTA) inactivation of HZ as a TLR ligand

  • RNase = no effect on HZ activity

  • When HZ mixed with glycerol & sonicated, associated genomic DNA could be observed on an ethidium-stained gel*

  • TLR9-inducing activity of HZ is due to contaminating DNA

    *Data not shown

The Ectodomain of TLR9 Binds Directly to the Surface DNA on HZ

  • TLR9-Fc and TLR2-Fc (Fc portion of mouse IgG2a fused to protein) used to assess ligand binding

  • HZ or S-7 nuclease treated-HZ coated 96-well fluorimeter dishes

  • Anti-mouse IgG Alexa Fluor 488 pAb used to detect binding

  • Demonstrated binding of TLR9,  not TLR2 to untreated HZ

  • S-7 nuclease treated HZ did not bind TLR9

    • TLR9 binds surface DNA not HZ

  • Positive controls confirmed results

The DNA on the Surface of HZ is Malarial in Origin

  • PCR

    • HZ is template; human, mouse and Plasmodiumprimers

  • HZ DNA template only significantly replicated by Plasmodiumprimers

  • positive controls confirmed

  • DNA on HZ is malarial in origin

Does Malarial DNA Activate TLR9?

  • Malaria genome highly AT-rich

  • Tested malarial DNA as stimulant of FL-DCs

    • failed to activate cells even at 50 μg/mL*

  • TLR9 localized in endocytic compartment when DNA is internalized by dendritic cells (Latz et al)

  • genomic DNA not efficiently internalized into cells

  • Is malarial DNA not being properly internalized?

    *Data not shown

Malarial DNA Binds TLR9 and HZ Traffics DNA into a TLR9-Positive Compartment

  • DOTAP- reagent that targets nucleotides into endosomal compartment

  • malaria DNA + DOTAP strongly activated cells to secrete cytokines

  • HZ/DOTAP didn't activate FL-DCs in TLR9-/- mouse cells*

  • malaria DNA + HZ is as potent as malaria DNA + DOTAP

  • HZ as effective as DOTAP at targeting malarial DNA to endosomal compartment

    *Data not shown


  • Fever in malaria associated with the rupture of infected RBCs and the release of merozoites.

  • Because the malaria parasite is coated with GPI anchors and GPI anchors are an established TLR2 ligand, many have thought this group of molecules represents the malaria toxin.

  • Parrocheet al. found the role of TLR2 in mice malaria to be minor.


  • Initially thought synthetic HZ was the cytokine inducer, because it seemed less likely to be contaminated with endotoxin & other biologically active molecules than the natural molecule.

    • 2 sources of synthetic HZ (hemin chloride source & other cleaned natural source) have no immunomodulatory activity.

  • Pure HZ crystal by itself cannot activate a TLR and ultimately trigger a cytokine immune response


  • HZ functions to internalize malaria DNA into an intracellular compartment where it may be sensed by TLR9

  • HZ has carrier properties, and transforms malaria DNA from an otherwise harmless molecule to one with the ability to potently generate cytokines.

  • Despite the AT-rich nature of the malaria genome, several "classic" CpG motifs were found in the malaria genome.

    • 269 sequences resembling the CpG B-class motif

  • Oligonucleotides from malaria CpG rich motifs are highly immunostimulatory

Possible Further Research

  • Is the hemozoin-DNA complex the "malaria toxin" in humans?

  • TLR9 inhibitors as possible method to determine significance of malarial DNA and TLR9 in humans

  • Investigate new drugs for the prevention of hemozoin formation

  • Vaccine


  • Data not shown

  • Natural hemozoin prepared were not pure crystals (contaminated with proteins)

  • Reference to previously unpublished work

  • Failure to fully explain Figure 1C

  • Order of information presented

Recent Research

  • Sacarlal et al (2009) developed malaria vaccine RTS,S/AS02A and demonstrated its effectiveness and safety

  • Dondorp et al (2009)- P. falciparum is developing resistance to artesunate

  • Marshall et al (2009)- Control malaria with transgenic mosquitoes

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