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
http://setyoufreenews.blogspot.com/2010_11_19_archive.htm
What component of the malariainfected RBC is the “malaria toxin”?
• Host-derived or pathogen-derived?
http://www.doh.state.fl.us/environment/
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
http://www.cdc.gov/malaria/malaria_worldwide/impact.html
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
http://www.ibtimes.com/articles/76908/20101028/scientists-stop-malaria-by-immunizing-mosquitoes.htm
Malaria Symptoms
http://royaldutchshellplc.com/2006/11/05/houston-chronicle-oil-companies-tackle-malaria-in-africa
/
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
http://www.nature.com/nrmicro/journal/v6/n1/fig_tab/nrmicro1815_F2.html
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
http://www.jhsph.edu/publichealthnews/articles/2007/jacobs_lorena_mosquito.htm