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

1. 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 http://setyoufreenews.blogspot.com/2010_11_19_archive.html

2. Host-derived or pathogen-derived? What component of the malaria-infected RBC is the “malaria toxin”? http://www.doh.state.fl.us/environment/medicine/arboviral/Malaria.html

3. 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 http://www.cdc.gov/malaria/malaria_worldwide/impact.html

4. 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 http://www.ibtimes.com/articles/76908/20101028/scientists-stop-malaria-by-immunizing-mosquitoes.htm

5. Malaria Symptoms http://royaldutchshellplc.com/2006/11/05/houston-chronicle-oil-companies-tackle-malaria-in-africa/

6. 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

7. 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 http://www.nature.com/nrmicro/journal/v6/n1/fig_tab/nrmicro1815_F2.html

8. 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 Treatments

9. 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

10. 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 http://en.wikipedia.org/wiki/File:Hemozoin_crystals.jpg

11. 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 Introduction

12. 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 Introduction

13. 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.

14. 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

15. ELISA • 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 http://www.microscopesblog.com/2009/08/elisa.html

16. 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

17. 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

19. 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

20. 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

21. 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

23. 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

25. 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

26. 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

27. 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

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

29. 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. • 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

30. 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. • 269 sequences resembling the CpG B-class motif • Oligonucleotides from malaria CpG rich motifs are highly immunostimulatory

31. 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

32. 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

33. 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 http://www.jhsph.edu/publichealthnews/articles/2007/jacobs_lorena_mosquito.html