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

What component of the malaria- infected RBC is the “malaria toxin”? Host-derived or pathogen-derived? medicine/arboviral/Malaria.html

Malaria Statistics 3.3 billion people live in areas in risk of malaria transmission Affects 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 1 st symptoms Responsible for over 90% of the deaths

Malaria Symptoms /

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) o Receptor proteins that recognize microbes o After detection, TLRs induce MyD88 proteins which ultimately cause the release cytokines Cytokines o Intercellular signaling molecules secreted by immune cells o 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

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. β- hematin) 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

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 Highly Pure β-Hematin Has No Intrinsic Stimulatory Activity

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