Malaria - Emi Leonard

July 23, 2018 | Author: Anonymous | Category: N/A
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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

What component of the malariainfected 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 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

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) 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

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

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

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