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
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 Cycle 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
Innate Immune Response • Exact cause of innate immune response to malaria is unknown • Innate immune system o Cells and mechanisms that non-specifically defend host against infection o Provides immediate defense but does not confer long-lasting or protective immunity (as opposed to adaptive system)
Immune Response • Toll-Like Receptors (TLRs) Receptor proteins that recognize microbes o After detection, TLRs induce MyD88 proteins which ultimately cause the release cytokines o
• Cytokines o o
Intercellular signaling molecules secreted by immune cells Recruit other immune cells to site of infection
Treatments • 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
Past Research • 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
Hemozoin (Hz) • 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
Electron micrograph of crystals of hemozoin isolated from the malaria parasite Plasmodium falciparum http://en.wikipedia.org/wiki/File:Hemozoin_crystals.jpg
Introduction • The rupture of parasitized erythrocytes is accompanied by the onset of symptoms like fever and rigors o
These symptoms are caused by the systemic release of proinflammatory cytokines
• Both natural and synthetic hemozoin (a.k.a. βhematin) have been reported to induce these inflammatory responses both in vivo and in vitro
Introduction • During infection, hemozoin concentration after erythrocyte rupture may be as high as 100 μg/mL o 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 o
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.
Hypothesis • Parroche et al. hypothesized that TLRs recognize both synthetic and natural hemozoin, and thus cause the activation of cells in the innate immune system
ELISA • Enzyme-linked Immunosorbent Assay (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
http://www.microscopesblog.com/2009/08/elisa.htm
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 ligandderived dendritic mouse cells (FLDCs) • Crude hemin stimulated FL-DCs to produce cytokines • Highly pure β-hematin did not • Therefore, "immune activity" of βhematin 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 o 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 o 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 o 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 o
TLR9 binds surface DNA not HZ
• Positive controls confirmed results
The DNA on the Surface of HZ is Malarial in Origin • PCR o HZ is template; human, mouse and Plasmodium primers • HZ DNA template only significantly replicated by Plasmodium primers • 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 o 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
Discussion • 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.
• Parroche et al. found the role of TLR2 in mice malaria to be minor.
Discussion • 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. o
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
Summary • 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. o 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
Critiques • 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
