Innate Sensing of Malaria Parasites PDF

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Innate sensing of malaria parasites

Ricardo T.Gazzinelli13, Parisa Kalantari1, Katherine A.Fitzgerald1 and
Douglas T.Golenbock1,2
Abstract | Innate immune receptors have a key role in immune surveillance by sensing
microorganisms and initiating protective immune responses. However, the innate immune
system is a classic double-edged sword that can overreact to pathogens, which can have
deleterious effects and lead to clinical manifestations. Recent studies have unveiled the
complexity of innate immune receptors that function as sensors of Plasmodium spp. in the
vertebrate host. This Review highlights the cellular and molecular mechanisms by which
Plasmodium infection is sensed by different families of innate immune receptors. We also discuss
how these events mediate both host resistance to infection and the pathogenesis of malaria.
Apicomplexa
A large group of protozoa that
includes the Plasmodium
genus. They possess an apical
complex structure that is
involved in penetrating host
cells.
Division of Infectious
Diseases and Immunology,
University of Massachusetts
Medical School,
0160502324 Worcester,
Massachusetts, USA.
2
Laboratrio de
Imunopatologia, Centro de
Pesquisa Ren Rachou,
Fundao Oswaldo Cruz,
30190002 Belo Horizonte,
Minas Gerais, Brazil.
3
Departamento de
Bioqumica e Imunologia,
Instituto de Cincias
Biolgicas, Universidade
Federal de Minas Gerais,
31270901 Belo Horizonte,
Minas Gerais, Brazil.
Correspondence to R.T.G.
e-mail: ritoga@cpqrr.fiocruz.br
doi:10.1038/nri3742
Published online
17 October 2014
1
Malaria is a disease of poverty and has a major negative

impact on political, social and economic stability, and
thereby hampers the development of less prosperous human
societies. In 2010, ~210 million people were infected with
Plasmodium and 1.2 million people, primarily children,
died of this devastating disease1. Although the exact numbers are uncertain, Plasmodiumfalciparum is responsible
for about two-thirds of all malaria infections and the vast
majority (9095%) of the infections that cause mortality in
sub-Saharan Africa. The remaining malaria infections are
due to Plasmodiumvivax infection in individuals living in
deprived areas of Latin America and Asia2 where the disease
has an enormous economic cost, although mortality is not a
substantial problem.
The Plasmodium genus belongs to the Apicomplexa
phylum, and the parasite has a complex life cycle3 (FIG.1).
A female Anopheles mosquito injects Plasmodium into the
skin, after which sporozoites enter the bloodstream, travel
to the liver and undergo asexual replication. The merozoites
derived from hepatocytes rapidly invade red blood cells
(RBCs), and repeated cycles of invasion, replication and
release from RBCs result in exponential growth of the
parasite population. More than a century ago, Camillo
Golgi proposed the malaria toxin hypothesis on the
basis that Plasmodium growth is synchronized and peaks
of fever typically coincide with the rupture of infected
RBCs during schizogeny, which indicates that a parasite
toxin is responsible for many of the symptoms and signs
of disease4. It is now clear that the mechanism underlying the malaria toxin hypothesis involves a deleterious
activation of innate immune cells by Plasmodium-derived
components (known as pathogen-associated molecular
patterns (PAMPs)) and host-derived components (known
as damage-associated molecular patterns (DAMPs)).
The discovery of various families of innate immune

receptors has enriched our understanding of how the
host senses microbial infections and initiates immune
responses58. Considerable progress has been made in
identifying the malaria parasite molecules that elicit
inflammation and the cognate host receptors that
respond to these signals9. These Plasmodium components
might be the long sought-after malaria toxin that causes
end-organ damage and potentially death. In this Review
we briefly summarize malaria-associated syndromes, and
then focus on recent studies that uncover the molecular
and cellular mechanisms by which Plasmodium infection
activates different innate immune receptors and related
signalling pathways. In the second part of this Review,
we discuss the biological relevance of these innate
immune receptors for host resistance to infection and
for the pathogenesis of malaria. Finally, we elaborate on
how this knowledge could be exploited to treat systemic
inflammation, avoid organ damage and prevent the most
devastating forms of this disease.
Pathogenesis and malaria-associated syndromes

Malaria is a multifactorial disease and its clinical outcome depends on many aspects, such as parasite and host
genetics, previous exposure to infection, age, nutritional
status, and geographic and socio-economic factors3,10,11. In
non-immune individuals, the first symptoms of malaria
which include fever, headache, muscle pain, chills, vomiting and lethargy followed by malarial paroxysms appear
between 715days post infection and are associated
with high levels of circulating cytokines. The activation of innate immune cells and consequent systemic
inflammation lead to the initial signs and symptoms
of malaria, and can also influence the development of
744 | NOVEMBER 2014 | VOLUME 14
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the more severe forms of the disease. If left untreated,
the uncomplicated symptomatic malaria caused by
P.falciparum can rapidly evolve into severe illness and
lethality. Children with severe malaria may develop anaemia, jaundice, respiratory distress in relation to metabolic
acidosis, and/or cerebral disease. In adults, multi-organ
involvement is also frequent, and impairment of kidney
a Plasmodium life cycle
b Pathogenesis of malaria
Asexual stage (bloodstream)

Parasite cycle in RBCs
Trophozoite
Ring
stage
Gametocytes
Schizont
Merozoite
Parasite sequestration
(brain and lungs)
Coagulation and
disruption of vascular
endothelial cells
Activation of vascular
endothelial cells
Vascular leakage and

perfusion abnormalities
Placental malaria
Sepsis (spleen)
Uptake of infected or
altered RBCs resulting
in macrophage activation
and cytokine production
Acute respiratory
distress
Destruction
of RBCs
Pro-inammatory
cytokines
Sporozoite infects
hepatocyte
Sexual stage (mosquito)

Parasite development in the mosquito
Salivary
gland
Sporozoites
Male
gamete
Leukocyte
inltration into the
tissue parenchyma
Cerebral malaria
Pro-inammatory
cytokines
P. vivax
Zygote
Tissue inammation
Fever
Merozoite
Ookinete
Endothelial dysfunction
Augmented expression of
adhesion molecules and
adherence of infected RBCs
Asexual stage (liver)

Infection and parasite replication
in hepatocytes
Hypnozoite
function may contribute to the development of metabolic

acidosis at later stages of infection. Importantly, individuals who are infected with malaria multiple times develop
natural acquired immunity; malaria parasitism is low in
hyper-immune individuals and consequently, there is
no deleterious activation of innate immune cells and the
infection is asymptomatic.
Impaired
erythropoiesis
(bone marrow)
Anaemia (bloodstream)
Adhesion and rupture of
infected and altered RBCs
Low levels
of RBCs
Renal impairment and

metabolic acidosis
Increased glycolysis
and lactic acid
accumulation
Hypoxia
Hyperventilation
Oocyst
Midget
lumen
Female
gamete
Figure 1 | Plasmodium life cycle and the pathogenesis of malaria.

a | After a mosquito bite, sporozoites travel to the liver to infect
hepatocytes and develop into approximately 30,000 merozoites that are
released in the bloodstream. Repeated cycles of red blood cell (RBC)
invasion, replication and merozoite release will result in the exponential
growth of the parasite population and lead to disease. Plasmodium vivax
sporozoites also differentiate into dormant hypnozoites that may cause
disease relapses162. Infected RBCs will circulate containing ring-stage
parasites, and a small proportion of merozoites will develop into male and
female gametocytes that infect mosquitoes, completing the parasite life
cycle. b|The removal of infected RBCs by splenic macrophages or the
uptake of free haemozoin results in the activation of innate immune
receptors and cytokine storm. The circulating cytokines will cause
paroxysms and induce the expression of adhesion molecules by

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Immunology
endothelial cells, which mediate parasite sequestration.
The| sequestration
of infected RBCs disrupts blood flow, promotes blood clots, injures
endothelial cells and ruptures vascular walls, leading to the extravasation
of vascular content and local tissue inflammation. These mechanisms
contribute to acute respiratory distress, cerebral malaria or placental
malaria. The sequestration of infected reticulocytes is less intense163,
and these syndromes are not common in P.vivax malaria65. Haemolysis
of infected and bystander (uninfected) RBCs, uptake of altered RBCs by
splenic macrophages and cytokine-induced impairment of erythropoiesis
cause anaemia. Free haemoglobin catalyses oxidative damage, hypoxia
and lactic acidosis, promoting metabolic acidosis, which is aggravated by
the altered renal function that is observed in patients with malaria.
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the more severe forms of the disease. If left untreated,
the uncomplicated symptomatic malaria caused by
P.falciparum can rapidly evolve into severe illness and
lethality. Children with severe malaria may develop anaemia, jaundice, respiratory distress in relation to metabolic
acidosis, and/or cerebral disease. In adults, multi-organ
involvement is also frequent, and impairment of kidney
a Plasmodium life cycle
b Pathogenesis of malaria
Asexual stage (bloodstream)

Parasite cycle in RBCs
Trophozoite
Ring
stage
Gametocytes
Schizont
Merozoite
Parasite sequestration
(brain and lungs)
Coagulation and
disruption of vascular
endothelial cells
Activation of vascular
endothelial cells
Vascular leakage and

perfusion abnormalities
Placental malaria
Sepsis (spleen)
Uptake of infected or
altered RBCs resulting
in macrophage activation
and cytokine production
Acute respiratory
distress
Destruction
of RBCs
Pro-inammatory
cytokines
Sporozoite infects
hepatocyte
Sexual stage (mosquito)

Parasite development in the mosquito
Salivary
gland
Sporozoites
Male
gamete
Leukocyte
inltration into the
tissue parenchyma
Cerebral malaria
Pro-inammatory
cytokines
P. vivax
Zygote
Tissue inammation
Fever
Merozoite
Ookinete
Endothelial dysfunction
Augmented expression of
adhesion molecules and
adherence of infected RBCs
Asexual stage (liver)

Infection and parasite replication
in hepatocytes
Hypnozoite
function may contribute to the development of metabolic

acidosis at later stages of infection. Importantly, individuals who are infected with malaria multiple times develop
natural acquired immunity; malaria parasitism is low in
hyper-immune individuals and consequently, there is
no deleterious activation of innate immune cells and the
infection is asymptomatic.
Impaired
erythropoiesis
(bone marrow)
Anaemia (bloodstream)
Adhesion and rupture of
infected and altered RBCs
Low levels
of RBCs
Renal impairment and

metabolic acidosis
Increased glycolysis
and lactic acid
accumulation
Hypoxia
Hyperventilation
Oocyst
Midget
lumen
Female
gamete
Figure 1 | Plasmodium life cycle and the pathogenesis of malaria.

a | After a mosquito bite, sporozoites travel to the liver to infect
hepatocytes and develop into approximately 30,000 merozoites that are
released in the bloodstream. Repeated cycles of red blood cell (RBC)
invasion, replication and merozoite release will result in the exponential
growth of the parasite population and lead to disease. Plasmodium vivax
sporozoites also differentiate into dormant hypnozoites that may cause
disease relapses162. Infected RBCs will circulate containing ring-stage
parasites, and a small proportion of merozoites will develop into male and
female gametocytes that infect mosquitoes, completing the parasite life
cycle. b|The removal of infected RBCs by splenic macrophages or the
uptake of free haemozoin results in the activation of innate immune
receptors and cytokine storm. The circulating cytokines will cause
paroxysms and induce the expression of adhesion molecules by

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Immunology
endothelial cells, which mediate parasite sequestration.
The| sequestration
of infected RBCs disrupts blood flow, promotes blood clots, injures
endothelial cells and ruptures vascular walls, leading to the extravasation
of vascular content and local tissue inflammation. These mechanisms
contribute to acute respiratory distress, cerebral malaria or placental
malaria. The sequestration of infected reticulocytes is less intense163,
and these syndromes are not common in P.vivax malaria65. Haemolysis
of infected and bystander (uninfected) RBCs, uptake of altered RBCs by
splenic macrophages and cytokine-induced impairment of erythropoiesis
cause anaemia. Free haemoglobin catalyses oxidative damage, hypoxia
and lactic acidosis, promoting metabolic acidosis, which is aggravated by
the altered renal function that is observed in patients with malaria.

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Sporozoites
The Plasmodium stage that
develops in the salivary glands
of the mosquito and infects
humans during the blood meal.
When they are in the
bloodstream, the sporozoites
are transported to the liver
where they invade hepatocytes
for the first round of replication
in the vertebrate host.
Merozoites
The pathogenic Plasmodium
stage that infects red blood
cells (RBCs) and rapidly
reproduces asexually, forming
schizonts that contain multiple
parasites. RBCs are destroyed
by the end of this process,
which releases many
merozoites that will infect
other host cells.
The three main pathophysiological events that

occur during malaria infection are: the release of proinflammatory cytokines; adhesion of Plasmodiuminfected RBCs to capillaries and venules; and the rupture and removal of parasitized and altered RBCs by
splenic macrophages (FIG.1). These three events are
connected and are responsible for the main syndromes
that are associated with malaria, which include systemic
inflammation, anaemia, metabolic acidosis, as well as
cerebral and placental malaria3,10,11 (TABLE1).
Splenic macrophages and monocytes are main contributors to the cytokine storm that is observed during
acute malaria episodes. As the microcirculatory beds
filter out altered RBCs, innate immune cells in the
spleen clear Plasmodium-infected RBCs12,13. Splenic
macrophages have a central role in sensing and phagocytosing altered RBCs, and they are exposed to a high
number of parasites. As a consequence, at least in mice,
these cells respond by producing large amounts of proinflammatory mediators1416. However, the rupture and
removal of infected RBCs by splenic macrophages is
not sufficient to explain the pronounced decrease in
the number of RBCs3,12,13,17. Instead, the suppressive
effect of pro-inflammatory cytokines on erythropoiesis
is thought to contribute to the severe anaemia that is
observed in the advanced stages of malaria18,19.
As parasite growth may be synchronized, the release

of pyrogenic cytokines such as interleukin1
(IL1) and tumour necrosis factor (TNF) is
cyclic 20,21, and paroxysms occur every 48hours in
P.vivax and P.falciparum infections. Fever can aid
host defence by delaying the growth of pathogens that
have strict temperature preferences. Indeed, deliberate infection with P.vivax was used to induce high
fever to eliminate Treponema pallidum infection in
patients with neurosyphilis22. However, fever is also
associated with signs of malaria illness, such as chills,
rigours, low blood pressure, headache, excessive
perspiration and hyperpyrexia11,17,23,24.
Another key event in malaria pathophysiology is the
sequestration of parasitized RBCs into different organs
including the lungs, brain, liver, kidneys and placenta25.
Exposure to circulating cytokines in particular TNF
and interferon (IFN) or components released
from Plasmodium-infected RBCs leads to enhanced
expression of adhesion molecules by endothelial
cells3,10,17. Also, members of the diverse family of molecules related to P.falciparum erythrocyte membrane
protein1 (PfEMP1)26 are expressed on the surface of
infected RBCs and bind to adhesion molecules that are
distributed among different host organs and tissues, such
as CD36, intercellular adhesion molecule 1 (ICAM1),
Table 1 | Malaria-associated syndromes

Syndrome
Clinical features
Mechanism
Refs
Anaemia
Low haemoglobin levels and RBC

counts
Pallor
Lethargy often with jaundice
Rupture of infected RBCs

Removal of infected and altered RBCs by splenic macrophages
Inhibition of erythropoiesis by cytokines
Systemic
inflammation
Alterations in body temperature

Tachycardia
Rapid breathing, often with
impaired gas exchange
Arterial hypotension
High levels of lactate in the blood
Altered mental status
Parasite activation of splenic macrophages leads to a systemic inflammatory

response (cytokine storm) that is associated with dysfunction of cardiovascular,
respiratory, renal, neurological, hepatic, and/or coagulation systems
Sepsis-like syndrome leads to acute respiratory distress syndrome
Parasitized RBC sequestration causes a disruption of the alveolarcapillary
interface, resulting in leukocyte infiltration, and oedema in the interstitium
and alveoli, culminating in severe hypoxaemia
3,10,11,13,
1719,23,
24,31
Metabolic
acidosis
Hyperventilation
Hypoxia
Headache
Altered mental status
Nausea
Vomiting
Abdominal pain
Muscle weakness
Metabolic acidosis is due to increased glycolysis and the accumulation of

lactic acid, in particular during hypoxia caused by anaemia and/or blood
vessel obstruction by sequestering parasites
Renal dysfunction and insufficient clearance of lactic acid. The lower blood pH
stimulates the brainstem to increase the respiratory rate to expel more carbon
dioxide, resulting in hypocapnia, which causes vasoconstriction and cerebral
hypoxia, thus exacerbating cerebral disease
3,10,11,17,
23, 24,31
Cerebral
malaria
Lethargy
Unbalanced movements
Seizures
Impaired consciousness
Coma
Neurological sequelae
Activation of endothelial cells by circulating pro-inflammatory mediators or by

parasite components
Enhanced expression of adhesion molecules in endothelial cells, parasite
sequestration, obstruction of blood flow, intravascular coagulation, disruption
of the endothelial barrier integrity, and local tissue inflammation
3,10,11,17,
25,28,29,31
Placental
malaria
Placental insufficiency
Fetal growth retardation
Low birth weight
Premature delivery
Stillbirth
Miscarriage
Neonatal and maternal morbidity
and mortality
Parasite sequestration and deposition of haemozoin in the intervillous space

of the placenta results in the activation of placental macrophages, production
of chemokines, recruitment of monocytes, intravascular macrophage
differentiation, and production of pro-inflammatory cytokines that are
deleterious to the fetus
10,25,30,31
1013,18,19
RBC, red blood cell.
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Schizogeny
The replicative process of
malaria parasites, either in
hepatocytes or red blood cells,
that involves nuclear division
without cytoplasmic
segmentation, followed by a
cell budding to form the
progeny called merozoites.
Pathogen-associated
molecular patterns
(PAMPs). Molecular structures
that are normally abundant in
certain pathogens and are
detected by innate immune
receptors known as pattern
recognition receptors. PAMPs
are often used as vaccine
adjuvants.
Malaria-associated
syndromes
A collection of signs and
symptoms that are
characteristic of a specific
manifestation of malaria,
such as anaemia, respiratory
distress or cerebral malaria.
Paroxysms
In malaria, paroxysms refer to
sudden attacks of disease that
are characterized by high fever,
chills and various other
symptoms. This sudden
worsening of malaria
symptoms is cyclic and
coincides with the synchronous
rupture of Plasmodiuminfected red blood cells.
Glycosylphosphatidylinositol
anchors
(GPI anchors). Glycolipid
structures that link surface
proteins to the surface
membrane of eukaryotic cells.
In protozoa, most surface
proteins are linked via GPIs,
and free GPIs are also
expressed at the surface.
Haemozoin
The crystalline product
resulting from the digestion of
haemoglobin during different
intraerythrocytic Plasmodium
stages.
Toll-like receptors
(TLRs). A family of pattern
recognition receptors that
sense pathogen-associated
molecular patterns and initiate
pro-inflammatory responses
during microbial infection.
platelet endothelial cell adhesion molecule 1 (PECAM1),

complement receptor 1, heparan sulphate and chondroitin sulphate A. Hence, the expression of specific
PfEMPs and adhesion molecules by host cells is the
main determinant of parasite tissue tropism and
pathogenicity 27. For instance, CD36 and ICAM1 are
important for parasite sequestration in the brain25,28,29,
and heparan sulphate and chondroitin sulphate A are
the main ligands for infected RBCs in the placenta30.
Non-adherent parasitized RBCs are rapidly cleared in
the spleen, whereas parasitized RBCs that are adhered
to endothelial cells are protected12,13. However, adherence of infected RBCs triggers coagulation by activating
thrombin which catalyses fibrin deposition in blood
vessels and amplifies inflammation and thereby disrupts endothelial barrier integrity and favours local
tissue inflammation 31,32. The obstruction of capillaries and venules, and local inflammation contribute to the development of acute respiratory distress
syndrome and metabolic acidosis, as well as cerebral
and placental malaria. Of note, high levels of nitric
oxide protect against vascular dysfunction and severe
forms of malaria are associated with low availability of
nitricoxide33,34.
In conclusion, systemic inflammation is a central
event in the malaria-associated syndromes. Hence,
defining the mechanism by which parasite components
activate innate immune cells is crucial to understanding
the pathophysiology of Plasmodium infection.
Sensors of Plasmodium PAMPs

PAMPs are microbial structures that are detected
by pattern recognition receptors (PRRs) 8,35. Three
P. falciparum PAMPs have been studied in detail
namely, glycosylphosphatidylinositol anchors (GPI
anchors), haemozoin and immunostimulatory nucleic
acid motifs (TABLE2). The Toll-like receptors (TLRs)5 (FIG.2)
detect PAMPs at the membrane of endosomes or at the
cell surface, whereas RIGI-like receptors (RLRs)6 and
NOD-like receptors (NLRs)7 are cytosolic. Activated
PRRs trigger distinct transcriptional programmes and
induce multiple downstream pathways that are involved
in pathogen clearance. However, excessive activation is
deleterious as it can cause systemic inflammation and
disease. The molecular structures of both PRRs and
PAMPs are highly conserved among vertebrate hosts
and certain categories of pathogens, respectively. In
this section, we discuss experiments carried out in
mouse models or invitro systems using human cells
that were instrumental in identifying the host PRRs that
are activated by PlasmodiumPAMPs.
GPI anchors. GPI anchors link most surface proteins
to the protozoan plasma membrane and are therefore
essential for parasite viability 36. Importantly, protozoan
GPI anchors are potent stimulators of cytokine synthesis by macrophages9. This activity is determined by
their fine structure, which includes the number and
variation of carbohydrate units; the lipid inositol portion (glycerol versus ceramide); and the number, length
and degree of saturation of the hydrocarbon chains36.
GPI anchors from P.falciparum merozoites have either

two or three fatty acyl chains and trigger the phosphory
lation of mitogen-activated protein kinases and inhibitor
of nuclear factorB (NF-B) family members through
the activation of TLR2TLR6 or TLR1TLR2 hetero
dimers and, to a lesser extent, TLR4 homodimers37,38
(FIG.2; TABLE2). In mouse macrophages, the activation of
TLRs induces the production of nitric oxide and the synthesis of pro-inflammatory cytokines, such as TNF and
IL139,40. In human umbilical vascular endothelial cells,
TLR activation by GPI anchors induces the expression
of ICAM1, vascular cell adhesion molecule 1 (VCAM1)
and Eselectin, as well as the production of nitric oxide41.
Haemozoin. During the intraerythrocytic stage, parasites
digest haemoglobin as a source of amino acids, which in
turn leads to the generation of potentially toxic proto
porphyrin metabolites42,43. To survive, the parasite uses
a detoxification enzyme that polymerizes haem and
results in the formation of haemozoin crystals in the
food vacuole of the parasite42. The parasitized RBCs are
phagocytosed by macrophages, neutrophils and dendritic cells (DCs) in various organs such as the spleen,
liver and brain. The quantity of haemozoin that is deposited within phagocytes reflects the parasite burden, and
coincides with periodic fevers and high circulating levels
of IL1 and TNF; therefore, haemozoin may be used as
a biomarker of malaria severity 43. Importantly, invitro
and invivo studies have demonstrated that haemozoin
activates both mouse and human monocytes and macro
phages to produce pro-inflammatory cytokines such as
TNF and IL1, and certain chemokines4449. By contrast,
mouse but not human macrophages produce nitric oxide
when stimulated with IFN and haemozoin50.
Although some studies indicate that haematin (synthetic haemozoin) crystals are pro-inflammatory, this
matter becomes more complex when the strong adsorptive nature of haemozoin is taken into consideration.
Natural haemozoin produced in P.falciparum cultures is
normally bound to proteins, lipids and nucleic acids that
may be of parasite or host origin45,51,52. The DNA binding
seems to require certain proteins45,53. The identity of the
crucial components of native haemozoin aggregates that
trigger inflammation is still a matter of intense debate.
It has been shown that synthetic haemozoin binds to,
induces conformational changes in and activates TLR9 on
mouse DCs and macrophages, and also on human B lymphocytes48,54,55. In addition, purified synthetic haemozoin
can induce NOD-, LRR- and pyrin domain-containing3
(NLRP3)dependent secretion of IL1 by lipopoly
saccharide (LPS)-primed mouse macrophages and the
human THP1 monocytic cell line46,47,56. In our view,
however, haemozoin bound to P.falciparum nucleic acids
traffics into phagolysosomes and the cytosol of host cells,
where parasite DNA activates endosomal TLR9, inflamma
somes and other cytosolic sensors (FIG.2). The activation of
inflammasomes requires two signals: first, a priming signal which can result from TLR9 activation leads to
the transcription of the gene encoding proIL1, as well
as those encoding inflammasome components; second,
a signal which can be provided by, for example, the

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Table 2 | Malaria PAMPs and their role in pathogenesis
Parasite
component
Properties
Receptor activation
Pathways
Role in pathogenesis
Refs
GPI anchors and

GIPLs
GPIs serve as a bridge

between surface proteins
and the protozoan plasma
membrane
The free forms of GPI
anchors (GIPLs) are present
in the protozoan surface
membranes
TLR1TLR2 heterodimer
(activated by GPI anchors
containing three fatty
acid chains)
TLR4 homodimer
TLR2TLR6 heterodimer
(activated by GPI anchors
containing two fatty acid
chains)
MYD88NFB
Induces macrophage release

of pro-inflammatory mediators
(for example, TNF and nitric
oxide)
Induces the expression
of adhesion molecules on
endothelial cells
3741
Haemozoin
A detoxification crystal
formed by haem released
from haemoglobin, which is a
main nutrient for the parasite
during the erythrocytic stage
TLR9 (induced by
haemozoin bound to
parasite DNA)
NLRP3, AIM2 and other
cytosolic sensors (owing
to haemozoin-induced
liberation of phagosome
contents including DNA)
Mediates induction of
MYD88NFB
pro-inflammatory cytokine
Assembly of
production by DCs and
NLRP3 and AIM2
macrophages
inflammasomes
Promotes caspase 1 activation
STINGIRF3
and cleavage of proIL1
Mediates induction of typeI
IFN production
4455
TLR9
MYD88NFB
Induces the release of

pro-inflammatory cytokines by
DCs and macrophages
DNA
Immunostimulatory The P. falciparum genome
CpG motifs
contains ~300CpG motifs
The P.vivax genome
contains ~2,500CpG motifs
45,62,63
ATrich motif
P.falciparum DNA contains

~ 6,000 ATrich motifs
P.vivax DNA contains
~5,000 ATrich motifs
Undefined
STINGIRF3
Induces typeI IFN production
64
Unknown motif
DNA from different

Plasmodium species
AIM2
Assembly of AIM2
inflammasomes
Promotes caspase 1 activation

and cleavage of proIL1
53
Unknown motif
Single-stranded RNA from

different Plasmodium species
TLR7
MYD88NFB
Undefined
Unknown motif
RNA from different

Plasmodium species
MDA5MAVS
TypeI IFN
Early activation of innate

immune responses
Induces typeI IFN production
in hepatocytes
RNA
66,68
66,67,69
AIM2, absent in melanoma 2; CpG, a cytosine and a guanine separated by one phosphate; DC, dendritic cell; GIPL, glycoinositol phospholipids; GPI, glycosylphosphatidylinositol; IFN, interferon; IL, interleukin; IRF3, IFN-regulatory factor 3; MAVS, mitochondrial antiviral signalling protein; MDA5, melanoma differentiationassociated protein 5; MYD88, myeloid differentiation primary response protein 88; NFB, nuclear factorB; NLRP3, NOD-, LRR- and pyrin domain-containing 3;
P.falciparum, Plasmodium falciparum; P. vivax, Plasmodium vivax; STING, stimulator of IFN genes protein; TLR, Toll-like receptor; TNF, tumour necrosis factor.
Cytosolic sensors
crystalline structure of haemozoin that destabilizes the

lysosomal membranes and causes the release of their contents into the cytosol46,47,53,5658. Shio etal.46 also found that
haemozoin-induced, NLRP3dependent IL1 production
by mouse macrophages and the THP1 cell line is mediated
by the tyrosine kinases SYK and LYN, as well as cathepsin
B. Furthermore, this production is blocked by inhibiting
phagocytosis, K+ efflux or the generation of reactive oxygen species46,47. Importantly, haemozoin that is carrying
DNA can activate the DNA sensor absent in melanoma2
(AIM2) to form inflammasome specks, and specks of
NLRP3, AIM2 and NLRP12 inflammasomes are also found
in circulating monocytes during malaria in humans16,53.
Thus, haemozoin has emerged as a key component
of innate immune activation by malaria parasites.
Different families of pattern

recognition receptors located
in the cytoplasm that are often
activated by DNA and RNA,
and that induce the production
of typeI interferons.
Plasmodial DNA. As is the case for other protozoa,

DNA is an important activator of innate immune
responses by Plasmodium parasites in both mice and
humans45,5964. Of particular interest, the P.falciparum
Inflammasomes
Molecular platforms formed
by members of the NOD-like
receptors family, which activate
caspase 1 to cleave proIL1
and proIL18 and release the
active form of these cytokines.
genome contains the highest AT content (82%)

described in nature64. In silico analyses indicate that the
P.falciparum genome contains approximately 300CpG
(cytosine and guanine separated by one phosphate)45
and 6,000 ATrich64 immunostimulatory motifs. Despite
the lower AT content (56%), the P.vivax genome has a
similar number of ATrich stimulatory motifs (~5,500)
and has approximately 2,000CpG motifs45,64. The higher
CpG content may explain the lower pyrogenic threshold
of P.vivax infection65.
When they are in the phagolysosomes, the immuno
stimulatory CpG motifs of Plasmodium DNA activate
TLR9 (FIG.2; TABLE2). Phagocytosis of intact infected
RBCs results in TLR9 engagement, presumably as a result
of parasite degradation and the presence of parasite DNA
in the phagolysosome. In addition, parasite DNA can be
carried to the inner cellular compartments by haemozoin or protein aggregates45,53,63. If released in the host cell
cytosol, in addition to activating AIM2 inflammasomes,
DNA from schizonts also induces typeI IFN release in
REVIEWS
Urate
crystals
Sporozoite
Microvesicles
Infected RBC
Haemozoin
dsDNA
Hepatocyte
Haem
GPI
TLR1
Macrophage
or DC
TLR9
Merozoite
MYD88
IRAK4
RNA
MYD88
TLR4
TLR2
MAL
MAL
TRAM
MYD88
MYD88
TRIF
IRAK1
IRAK1
TBK1
TRAF6
TRAF6
IKK
TRAF6
TLR7
?
IB
RNA
p65 p50
Phagolysosome
dsDNA
MDA5
NF-B
AT-rich DNA
TRAF3
IKK IRAK1
GTP cGAS
ATP
MAVS
NLRP3
Mitochondrion
NLRP12
AIM2
ASC
23 cGAMP
Pro-caspase 1
Pro-caspase 1
TBK1
IRF7 IRF7
STING
ER
Active caspase 1
IRF3 IRF7
Type I
IFNs
Cytosol
Nucleus
IL-1
IL-18
Pro IL-1
Pro IL-18
Type I
IFNs
TBK1
Pro-inammatory
cytokines
IRF3 IRF3
Type I
IFNs
IRF3 IRF3
Nucleus
Figure 2 | Innate sensors of Plasmodium PAMPs and malaria DAMPs.

RNA derived from merozoites translocates from the parasitophorous
vacuole into the cytoplasm of hepatocytes (left panel) and activates
melanoma differentiation-associated protein 5 (MDA5), which signals
through mitochondrial antiviral signalling protein (MAVS) to induce the
production of typeI interferons (IFNs). Haemozoin traffics Plasmodium
nucleic acids into the phagolysosomes (right panel). Toll-like receptor 7
(TLR7) and TLR9 are activated by parasite RNA and dsDNA, respectively,
whereas parasite glycosylphosphatidylinositol (GPI) anchors activate
TLR1TLR2 heterodimers. Haem and host cell-derived microvesicles
activate TLR4. TLRs signal through myeloid differentiation primary
response protein 88 (MYD88), which eventually leads to the activation
nuclear factorB (NFB) and the induction of pro-inflammatory cytokines.
Haemozoin crystals destabilize phagolysosomes, which leads to the
release of their contents into the host cell cytoplasm and the activation of
NOD-, LRR- and pyrin domain-containing 3 (NLRP3) and NLRP12 that
assemble into inflammasomes. The IFN-inducible protein absent in
melanoma 2 (AIM2), which is activated by parasite

forms
Nature dsDNA,
Reviewsalso
| Immunology
inflammasomes. Inflammasomes recruit pro-caspase 1 either directly
(NLRP12) or indirectly (NLRP3 and AIM2) via the adaptor molecule ASC.
This leads to the activation of caspase 1, and the consequent cleavage
and secretion of interleukin1 (IL1). Cyclic GMPAMP synthase (cGAS)
generates the second messenger cyclic GMPAMP (cGAMP), which binds
and activates stimulator of IFN genes protein (STING) that in turn
activates serine/threonine-protein kinase TBK1. TBK1mediated
phosphorylation of IFN-regulatory factor 3 (IRF3) leads to the expression
of IFN. In addition, Plasmodium ATrich motifs activate an unknown
receptor that also converges on the STINGTBK1IRF3 pathway.
Question marks indicate that some components of these pathways
remain to be characterized. DC, dendritic cell; ER, endoplasmic
reticulum; IB, inhibitor of NFB; IKK, IB kinase; IRAK, IL1 receptorassociated kinase; MAL, MYD88 adaptor-like protein; TRAF, TNF
receptor-associated factor; TRAM, TRIF-related adaptor molecule;
TRIF, TIR domain-containing adaptor protein inducing IFN.

REVIEWS
a TLR-independent manner in both human and mouse
phagocytes64,66 (FIG.2; TABLE2). A potentially novel cytosolic DNA sensor seems to detect Plasmodium DNA
as TLR9, DNA-dependent activator of IFN-regulatory
factors (DAI; also known as ZBP1), RNA polymeraseIII, IFNinducible protein 16 (IFI16), retinoic acidinducible gene I (RIGI) and mitochondrial antiviral
signalling protein (MAVS) could not recognize the
P.falciparum ATrich hairpin motifs64. Nevertheless, it
was shown that the unidentified receptor for plasmodial
ATrich DNA signals through stimulator of IFN genes
protein (STING; also known as TMEM173), serine/
threonine-protein kinase TBK1 and IFN-regulatory
factor 3 (IRF3), which leads to typeI IFN production.
The role of cyclic GMPAMP synthase (cGAS) in this
pathway remains to be determined.
Plasmodial RNA. Recent studies indicate that
RNA derived from either mouse parasites (such as
Plasmodiumberghei and Plasmodiumyoelii) or human
parasites (such as P.falciparum) activate PRRs6668 (FIG.2;
TABLE2). A pronounced signature of IFN-inducible genes
is observed in hepatocytes soon after mice are infected
with sporozoites67,69. The invasion of hepatocytes by
sporozoites activates a typeI IFN response via melanoma differentiation-associated protein 5 (MDA5; also
known as IFIH1) through MAVS and the transcription
factors IRF3 and IRF7 (REF.67). In addition, the erythro
cytic stage of P.yoelii induces the production of typeI
IFN invivo, which is dependent on RNA polymerase III,
MDA5 and MAVS, but not RIGI66. In addition, the RNA
sensor TLR7 has a role in the initial activation of innate
immunity in a mouse model of malaria68. In all of these
studies, the cytosolic sensors and the typeI IFN response
had a modest but significant role in controlling parasite
growth and parasitaemia66,67,69.
Malaria DAMPs
DAMPs are endogenous components released from
stressed, damaged or dying cells that activate PRRs
and inflammasomes, regardless of whether the cells are
infected70. Uric acid, microvesicles and haem have been recognized as important malaria DAMPs7173 (FIG.2; TABLE3).
In addition to the association with severe disease, malaria
DAMPs possess intrinsic pro-inflammatory activity,
which is in part mediated by the activation of PRRs7173.
Microvesicles
Vesicles derived from platelets,
red blood cells, endothelial
cells and leukocytes that serve
as a communication system
between host cells, and display
important pro-inflammatory
activity.
Nucleic acids and urate crystals. Circulating human

DNA is found in physiological conditions and is
thought to be derived from both active release from
living cells and from the breakdown of dying cells74.
However, DNA is found at elevated levels in both infectious and sterile inflammatory diseases74. In autoimmune diseases, such as systemic lupus erythematosus
and psoriasis, high levels of circulating RNA and DNA
activate nucleic acid sensors and induce inflammation6,75. Increased levels of circulating human DNA are
also associated with symptomatic P.vivax infection in
humans76. However, the relevance of this finding to
the inflammatory response observed during malaria
remains to be determined.
Nucleic acids are also pro-inflammatory owing to

their degradation products. Uric acid a byproduct
of purine metabolism is released in large quantities
from dying cells and forms monosodium urate crystals
when present at saturating concentrations in body fluids. Importantly, high levels of uric acid and the purine
derivative hypoxanthine accumulate in infected RBCs
during severe forms of human and mouse malaria, and
they are released into the circulation when infected RBCs
are ruptured72,77,78. Hypoxanthine is then converted into
uric acid and urate crystals, and is internalized by phagocytes, thereby contributing to NLRP3 activation and the
inflammatory response during malaria58 (FIG.2; TABLE3).
Microvesicles. Host cell-derived microvesicles function
as a communication system between host cells and they
have immune-modulatory effects71. Pathogen-derived
microvesicles contribute to inflammation by delivering
components, such as antigens and TLR agonists, to antigenpresenting cells. Indeed, microvesicles derived from
Plasmodium-infected human or mouse RBCs are internalized by macrophages and trigger a cytokine response via
TLR4 activation79,164. However, the immunostimulatory
components of these microvesicles have not been identified71,79. Importantly, microvesicles derived from platelets,
endothelial cells and leukocytes are also present at elevated
levels during P.vivax and P.falciparum malaria, and
their frequency correlates with the severity of disease80,81.
Whether the circulating microvesicles are a cause or a
consequence of systemic inflammation in human disease
remains to be determined. Nevertheless, studies carried
out both in cell lines and in mice suggest that these hostderived microvesicles contribute to the overall inflammatory
response and the pathogenesis of malaria79,82.
Haem. Haemolysis and the release of haemoglobin is an
important aspect of malaria pathophysiology. The oxidation of free haemoglobin leads to the release of the haem
moiety that catalyses the formation of reactive oxygen
species, which results in oxidative stress and, consequently, microvascular damage83. Haem also promotes
inflammation by activating endothelial cells, which leads
to the expression of adhesion molecules, increased vascular permeability and finally, tissue infiltration of leuko
cytes83. Although haem activates TLR4 in mice73, it is
probably the excess of free haem that leads to inflammation, primarily by inducing oxidative damage, cell death
and tissue injury 83. This toxic effect is prevented by haem
oxygenase1, which degrades haem and thereby attenuates
the signs of severe malaria in mice84,85. However, a recent
study suggests that carboxyhaemoglobin a haem oxygenase 1 byproduct may exacerbate organ dysfunction
in severe human disease86. Nevertheless, in both mice and
humans, increased circulating levels of ferritin which
stores iron seem to protect the host from malaria87.
Immunity to Plasmodium infection

After multiple malaria infections, most individuals living in hyperendemic areas develop natural acquired
immunity and the main surrogate marker of protection is the high level of circulating merozoite-specific
REVIEWS
Table 3 | Malaria DAMPs
Host
component
Properties
Receptors
Pathways
Role in pathogenesis
Refs
Uric acid
Uric acid is released from

dying cells and at high
concentrations, forms
monosodium urate crystals
that incite inflammation
NLRP3
Assembly
of NLRP3
inflammasomes
Caspase 1
activation and
cleavage of
pro-IL1
Release of active IL1
72,77,78
Microvesicles
Microvesicles derived from

platelets, endothelial cells,
leukocytes and infected
RBCs are pro-inflammatory
TLR4
MYD88NFB
Induces the release of inflammatory

mediators by macrophages
Induces the expression of adhesion
molecules on endothelial cells
71,7982
Haem
The oxidation of free

haemoglobin released from
infected RBCs produces
haem
TLR4
MYD88NFB
Induces the release of inflammatory

mediators by macrophages
TLR4-independent
induction of
oxidative stress
Catalyses the
formation of
reactive oxygen
species
Directly activates endothelial cells

promoting the expression of adhesion
molecules, increased vascular
permeability and consequent tissue
infiltration of leukocytes
Causes membrane damage, cell death,
tissue necrosis and inflammation
8387
TLR4-independent
induction of haem
oxygenase 1
Degrades toxic
haem
Haem oxygenase 1 exerts

anti-inflammatory effects and promotes
tissue repair
8387
73
DAMP, damage-associated molecular pattern; IL, interleukin; MYD88, myeloid differentiation primary response protein 88; NF-B, nuclear factorB;
NLRP3, NOD-, LRR- and pyrin domain-containing 3; RBC, red blood cell; TLR4, Toll-like receptor 4.
antibodies3,10,88. When infected, hyper-immune individuals will develop low parasitaemia and asymptomatic
infection. The merozoite-specific antibodies are thought
to mediate host resistance to infection by blocking parasite invasion of RBCs, or by opsonizing infected RBCs
and promoting phagocytosis and parasite elimination
by macrophages88,89. In addition, studies carried out in
mice and humans indicate that both sporozoite-specific
neutralizing antibodies and cytotoxic CD8+ Tcells are
important components of immune-mediated resistance
to the hepatocytic stage of the Plasmodium life cycle88,89.
However, it is not clear whether sporozoite-specific
immune responses contribute to natural acquired immunity or whether they are only relevant to vaccine-induced
protection88. Different studies have disclosed mechanisms
by which innate immunity influences the development of
acquired immunity, but the role of innate immune receptors in this process is still elusive89. The studies discussed
above have unveiled the complexity of innate immune
receptors that function as sensors of malaria parasites and
we are beginning to understand how they mediate host
resistance to Plasmodium infection. In this section, we
discuss the known mechanisms of host defence that are
triggered by the activation ofPRRs.
Innate immunity. Sporozoites stimulate the expression of typeI IFN-inducible genes in hepatocytes during Plasmodium replication inside a parasitophorous
vacuole67,69 (FIG.3). TypeI IFN-induced proteins mediate the recruitment of natural killer (NK) cells and
NKTcells, which are important sources of IFN, and
trigger the expression proteins that mediate control of
parasite replication, such as those that are involved in the
production of nitric oxide and other toxic components

that interfere with parasite schizogeny in the liver 67,69.
However, a single sporozoite will generate thousands of
merozoites and typeI IFN-mediated parasite control is
inefficient67,69. The parasites that survive immunological
control in hepatocytes are released into the bloodstream
where they initiate the erythrocytic cycle and cause
full-blown malaria in non-immune individuals.
TLRs, NFB-inducible pro-inflammatory cytokines
and IFN-inducible genes are upregulated during the erythrocytic cycle in both human and mouse
malaria15,64,90,91. IFN also has an important role in priming innate immune cells and promoting pro-inflammatory
responses, and in activating the effector functions of
macrophages that mediate resistance to infection9295,165.
Of note, treatment with IL12 during either the hepatocytic or erythrocytic stages of malaria protects mice in a
manner dependent on IFN, TNF and nitric oxide96,97.
However, both antibody-mediated and T cell-mediated
immunity are ultimately required for effective control of
parasite burden and malaria89,98.
Bridging innate and acquired immunity. DCs have a central role as a bridge between innate and acquired immunity 99. As mentioned above, parasite DNA activates DCs
via TLR9 and cytosolic sensors to produce cytokines that
mediate host resistance to infection14,62,66,100104. An important mechanism of resistance to Plasmodium infection
in mice is the myeloid differentiation primary response
protein 88 (MYD88)dependent production of IL12 by
DCs, and the subsequent release of IFN by NKcells100104.
This process results in the polarization of CD4+ T helper 1
(TH1) lymphocytes that produce IFN89,97, which activates

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Immunologically silent (asymptomatic)
Leukocyte adhesion
and inltration
MDA5 Chemokines
Adhesion
MAVS
molecules
iNOS
Merozoite
Infected
RBC
Type I
IFNs
DNA
Dysfunction of
endothelial wall
IFN
TH1 cell
Haemozoin
Type I IFNs
STING
DNA and
RNA sensors
Phagolysosome
Chemokines and
adhesion molecules
B cell
IL-1
TNF
Bacteria
Antibodies
CD8+
CD4
T cell
IL-12
TLR1
TLR4
MAL TRAM
MAL
MYD88 MYD88 TRIF
Phagolysosome
MYD88
TLR2
MHC
class II
TLR9
TLR7
TNF
receptor
MHC
class I
Fever
Blood vessel
NK cell
NKT cell
T cell
RNA
c Disease
MYD88
IB
p65 p50
TLR9
Pro-caspase 1
88
TLR7
IB
p65 p50
NF-B
Inammasome
assembly
YD
88
RNA
b Acquired immunity
Hepatocyte
YD
a Innate immunity
Cytokine storm and local tissue inammation (severe malaria)
Pro-IL-1
Active
caspase 1
Macrophage or DC
Innate sensing
IFN priming
TLR hyperresponsiveness
Nature
Reviews
| Immunology
Figure 3 | Pattern recognition receptors and the pathophysiology of malaria. a | Plasmodium
RNA
translocates
from
the parasitophorous vacuole to the hepatocyte cytosol and activates melanoma differentiation-associated protein 5 (MDA5),
which induces the production of typeI interferons (IFNs). Macrophages and dendritic cells (DCs) phagocytose either free
haemozoin or infected red blood cells (RBCs). In the phagolysosomes, nucleic acids are released from destroyed parasites
orhaemozoin and activate Toll-like receptors (TLRs). The release of the nuclear factorB (NFB) subunits p50 and p65
induces the expression of interleukin12 (IL12) that promotes production of IFN by CD4+ Tcells, CD8+ Tcells and natural
killer (NK) cells. Haemozoin also destabilizes the membrane of phagolysosomes, which leads to the release of crystals and
parasite DNA into host cell cytoplasm, and the activation of inflammasomes and DNA sensors. b|TypeI IFN-inducible
proteins promote the recruitment of leukocytes (such as NKTcells) and control parasite replication in the liver. TypeI IFNs
also interfere with DC function. IFN has multiple effects in phagocytic cells, including the induction of IL12 production
that favours the differentiation of T helper 1 (TH1) lymphocytes. The IFN-inducible IgG isotypes opsonize infected RBCs
that are internalized and destroyed by activated macrophages. c | IFN priming makes macrophages prone to produce
extreme levels of cytokines upon a secondary microbial challenge. Circulating cytokines lead to fever and promote parasite
sequestration by inducing the expression of adhesion molecules on endothelial cells. The sequestration of infected RBCs
results in an alteration in blood flow, rupture of the vascular wall, extravasation of vascular content and leukocyte migration
into the parenchyma, leading to organ-specific syndromes such as cerebral malaria, respiratory distress and placental
malaria. IB, inhibitor of NFB; iNOS, inducible nitric oxide synthase; MAVS, mitochondrial antiviral signalling protein;
MYD88, myeloid differentiation primary response protein 88; STING, stimulator of IFN genes protein; TRAM, TRIF-related
adaptor molecule; TRIF, TIR domain-containing adaptor protein inducing IFN; TNF, tumour necrosis factor.
effector functions, maintains the pool of effector memory

T cells and promotes long-term resistance to Plasmodium
infection105. The TH1lymphocytes also direct the IgG2c
isotype switch and the secretion of protective merozoitespecific antibodies98. However, the lack of a functional
gene encoding MYD88 or IL12 only partially affects the
development of TH1 cellmediated immune responses,
which indicates that other PRRs and cytokines (such as
IL18) are also involved in the induction of IFN production by NK cells and Tcells, and in promoting host
resistance to mouse malaria106.
DCs are also sources of typeI IFNs, but the role of

these cytokines in host resistance to Plasmodium infection is less well understood. On the one hand, IFN
mediates resistance to Plasmodiumchabaudi infection in mice by generating new DCs in a manner that
is dependent on uric acid and FMS-related tyrosine
kinase3 ligand (FLT3L)107. On the other hand, typeI
IFNs modulate DC function and impair the development of protective T lymphocytes during mouse
malaria108,109. These contrasting effects may be dependent on parasite strain. Importantly, in mice, virulent
REVIEWS
Plasmodium strains subvert the function of DCs, and
thereby impair the production of IL12 and the development of T cell-mediated immunity 104,110. Consistent
with this, the function of human DCs is impaired when
they are exposed to infected RBCs or haemozoin111,112.
Furthermore, patients with symptomatic P.falciparum
or P.vivax malaria have decreased levels of circulating
DCs113. Hence, modulation of DC function is an important mechanism of parasite escape from host immune
responses.
Adjuvant
An agent that is mixed with an
antigen to increase the immune
response to that antigen
following immunization.
Reticulocyte
Immature red blood cells
(RBCs) that correspond to
1% of total circulating RBCs
in humans.
Vaccine-induced immunity. After a mosquito bite, only

a few sporozoites invade hepatocytes, thus a vaccine that
blocks this obligatory step of the Plasmodium life cycle
may induce sterile immunity. Indeed, experimental
inoculation of irradiated sporozoites or intact sporozoites in healthy individuals receiving chloroquine is
very effective as a vaccine against Plasmodium infection in humans114,115. The potential of such a vaccine is
currently being investigated further in clinical trials.
Does the high level of protection that is observed in
response to the administration of attenuated sporozoites reflect efficient activation of the innate immune
system? The ability of sporozoite RNA to trigger a typeI
IFN gene signature in an MDA5-dependent and MAVSdependent manner may help to answer this question.
If this is important for the development of sporozoitespecific immunity, the use of poly I:C (a double-stranded
RNA and potent inducer of typeI IFN) may be a preferable adjuvant for a vaccine against the hepatocytic stage
of malaria. In addition, RTS,S the most advanced
vaccine against malaria uses the circumsporozoite
protein, which is the dominant protein found on the
sporozoite surface116. Although sterile immunity was
not achieved in a Phase III trial of this vaccine, immunized children experienced a ~50% reduction in the
incidence of clinical infections116. However, in another
study with similar first year efficacy (43.6%), the level
of protection dropped to 0.4% during the fourth year
(with 32.1% overall across the four-year study), which
indicates that the RTS,S vaccine needs considerable
improvement 117.
An alternative approach is an anti-malaria vaccine
that targets the erythrocytic stage of the Plasmodium
life cycle. For instance, vaccination with a synthetic
segment of Plasmodium GPI anchor, which is a potent
TLR2 activator, was shown to prevent inflammationinduced pathology and mortality in a mouse model of
malaria118. Although it does not block transmission,
natural acquired immunity controls the exponential
parasite growth in RBCs, and prevents the excessive
activation of innate immune cells, as well as clinical
signs of disease. Consistent with this, vaccination with
either merozoite surface protein 1 (MSP1) in combination with MSP2 or the apical membrane antigen 1
induces a small, but significant reduction of clinical cases caused by parasite strains carrying the vaccine alleles of the merozoite antigens119,120. The main
candidate for a P.vivax-specific vaccine is the Duffybinding protein, which is expressed by merozoites and is
necessary for reticulocyte invasion121.
However, antigen variation among different parasite

isolates and the induction of protective levels of parasitespecific antibodies are major obstacles to overcome88.
The latter issue reflects the growing interest in innate
immunity in the malaria field and the search of novel
vaccine adjuvants that work through PRRs. For instance,
the choice of adjuvant formulation substantially
affected the efficacy of the RTS,S vaccine in a PhaseII
clinical trial122. In addition, the identification of functionally important malaria PAMPs may lead to the development of new immunological adjuvants. For example,
Plasmodium-derived GPI anchors could be used as a
vaccine adjuvant. Furthermore, highly pure synthetic
haemozoin is an effective adjuvant in animal models54,123,
and is a potential vaccine vehicle owing to its ability to
bind proteins, DNA and lipids. Hence, haemozoin could
be used to deliver antigen and adjuvants to lysosomes
and the host cell cytoplasm to promote the activation
of intracellular PRRs and antigen presentation via the
exogenous and endogenous pathways53. Furthermore,
both CpG motifs and ATrich motifs that are found in
the Plasmodium genome may be developed as adjuvants
that induce DCs to express costimulatory molecules,
such as IL12 and typeI IFNs45,64, which would favour
the development of protective B cell-mediated and
Tcell-mediated immune responses.
Implications for malaria pathogenesis

A hallmark of the innate immune response during both
human and mouse malaria is pro-inflammatory priming15,16,124126; at very early stages of infection, Plasmodium
parasites stimulate the production of IFN by NK cells
and NKT cells15,69,127. IFN pre-activates the innate
immune cells to respond to minute amounts of microbial
products, and this enhanced ability to respond to microorganisms during immunosurveillance protects against
infectious insults. However, this pro-inflammatory priming means that the innate immune system can overreact
leading to a deleterious response (FIG.3). In this section,
we discuss the importance of different innate immune
receptors and the cytokine storm in the pathogenesis of
malaria. By reviewing immunogenetic and field studies
conducted in patients with malaria, we provide evidence
that these mechanisms are relevant to human disease.
MYD88 and TLRs. Bacterial superinfections in children with malaria have recently been recognized as
important cofactors for severe disease65,128130. The risk
of developing severe P.falciparum malaria is estimated
to increase 8.5fold in children with bacteraemia131,
and casecontrol and longitudinal studies indicate
that children undergoing malaria episodes have an
increased susceptibility to infection with non-typhoidal
Salmonella species and other bacteria132,133. Consistent
with this, Plasmodium-infected mice are highly susceptible to infection with non-typhoidal Salmonella species.
It has been reported that malaria-induced immunemodulatory haem oxygenase 1 and IL10 dampen the
effector functions of neutrophils and macrophages134,135.
Similarly, impaired neutrophil function has been
described both in P.falciparum and P.vivax malaria126,136.

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Plasmodium infection also lowers the threshold
for bacteria-induced septic-shock. Malaria loads the
gun and bacteria pull the trigger is an expression that
reflects the mechanism by which Plasmodium infection enhances sensitivity to microbial infection. By promoting TLR hyperresponsiveness and the assembly of
inflammasomes, IFN priming lowers the threshold for
septic shock in mice by more than 100fold15,16. Although
IFN-deficient mice infected with P.chabaudi are highly
resistant to LPS challenge, the role of TLR9 and IL12 in
pro-inflammatory priming is partial, which suggests that
other PRRs and IFN-inducing cytokines also contribute
to this response15,16.
In addition, MYD88deficient mice infected with
P.chabaudi are more resistant to systemic manifestations, such as cytokinaemia, changes in body weight,
temperature and liver damage14,137. Similarly, in the
P.berghei model of placental malaria, mice lacking functional MYD88 had reduced levels of IL6 and TNF, and
unaltered fetal body weight, contrasting with the lower
body weight of fetuses from infected wild-type mice138.
Furthermore, inflammatory infiltrates and cytokinaemia are reduced and pathology of experimental cerebral
malaria is attenuated in MYD88deficient mice103,139.
Although the role of TLR2 and TLR4 in Plasmodium
infection is controversial14,66,102,106,139,140, the data indicating a role for TLR9 in experimental cerebral malaria
are more consistent. Therapy with the TLR antagonist
E6446, which binds both DNA and RNA within lyso
somes, is highly effective at protecting mice against
experimental cerebral malaria 127. Since protection
against experimental cerebral malaria is only partial
in TLR9deficient mice, it is possible that the E6446 is
also blocking the activation of TLR7, and other cytosolic
DNA and RNA sensors53,64,68.
Hence, PRRs promote the development of cerebral
malaria and sepsis-like syndrome by stimulating the
production of extreme levels of the same cytokines that
are involved in host resistance to Plasmodium infection. For instance, the development of experimental
cerebral malaria is attenuated in mice deficient for
functional genes encoding IFN-inducible adhesion
molecules, such as CXC-chemokine ligand 9 (CXCL9)
and CXCL10, which are important for the recruitment
of CD8+ Tcells to the central nervous system141,142.
Furthermore, treatment with TNF-specific monoclonal
antibodies prevents the development of cerebral malaria
in mice143. However, in small clinical trials, treatment
with TNF-specific antibodies or the anti-inflammatory
drug pentoxifylline was not shown to be effective in
preventing human disease caused by P. falciparum144,145,
and the role of cytokines and inflammation in cerebral
malaria is still a matter of debate28,29.
Inflammasomes and IL1. Despite strong invitro
evidence that haemozoin triggers the formation of
NLRP3 inflammasomes, different studies show that
the lack of functional NLRP3, ASC, caspase 1, IL18
or IL1 receptor has only a minor impact (or no
impact) on the control of parasitaemia and the development of mouse cerebral malaria16,46,140. Thus, the
role of inflammasomes, IL1 and IL18 in host resistance to Plasmodium infection is questionable16,46,140.
Nevertheless, in both humans and mice, Plasmodium
infection results in expression of an inflammasome
gene signature, the formation of NLRP3, NLRP12
and AIM2 inflammasomes, and caspase 1 activation16.
In addition, NLRP3, ASC and caspase 1 were shown
to have an important role in mediating haemozoininduced local inflammation, and may be involved in
organ damage observed during malaria. During mouse
malaria, inflammasomes and IL1 are also key players in enhanced sensitivity to bacteria-induced septic
shock16. As a result, hypersensitivity to bacterial superinfection is prevented by treating mice with an IL1R
antagonist 16. Furthermore, as IL1 is highly pyrogenic,
inflammasomes are probably important mediators of
malaria paroxysms in humans10,11,65.
Cytosolic sensors and typeI IFNs. The importance of
nucleic acid sensors and typeI IFN release is now being
appreciated in the context of various infectious diseases,
beyond their traditional roles in antiviral immunity. In
a mouse model of cerebral malaria using the ANKA
strain of P.berghei mice deficient for the typeI IFN
receptor, IRF3 or IRF7 exhibited less severe symptoms
and delayed mortality compared with infected wild-type
mice64,66,108,146,147. TypeI IFNs dampen the development
of TH1 cell responses that control of parasitaemia at a
very early stage of infection and thereby cause a worsening the disease67,69,84,109. However, in another study, it
was shown that therapy with IFN protects mice from
cerebral malaria, suggesting that the involvement typeI
IFNs in this process is more complex 148.
Genetics and human disease. Various studies have
associated single nucleotide polymorphisms (SNPs) in
inflammatory genes with resistance or susceptibility
to P.vivax or P.falciparum malaria. Although most of
these studies are inconclusive because they used a small
cohort of patients, protection against severe disease has
been associated with SNPs in the genes encoding TNF149,
IFNAR1 (a subunit of the typeI IFN receptor)146,150, IL12
(REF.151), IFN152,153, IL1154, CD40 ligand (CD40L; also
known as TNFRSF5)155 and inducible nitric oxide synthase (also known as NOS2)156. Several SNPs identified
in the genes encoding TLRs, such as TLR4 (REF.157) and
TLR9 (REF.158), were associated with low birth weight
and maternal anaemia. SNPs in TLR9 are also associated
with increased levels of IFN in children with cerebral
malaria159 and increased parasitaemia in patients with
P.vivax malaria160. Furthermore, hemizygosity for a
SNP in the gene encoding the TLR2 and TLR4 MYD88
adaptor-like protein (MAL; also known as TIRAP) may
protect against death due to malaria161.
Concluding remarks
More than 150years after the discovery of Plasmodium
by Laveran in 1861, malaria remains a devastating disease that accounts for ~1 million deaths every
year. Although there are tools with which to eliminate the disease, the eradication of malaria will be
REVIEWS
extraordinarily expensive in the absence of an effective
vaccine. Years of research on malaria pathogenesis have
culminated in the consensus that the clinical manifestations are mostly owing to a deleterious activation of
innate immune cells. It is now clear that Plasmodium
infection activates innate immune cells through signalling pathways downstream of PRRs, but further investigation is required to develop a better understanding
of the biological role of specific receptors in malaria.
We highlight studies indicating the crucial role of
haemozoin in the activation of TLRs, inflammasomes
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Acknowledgements
The authors thank M. Trombly for critically reviewing this

manuscript. The authors are also grateful to all of the members and collaborators of the R.T.G., K.A.F. and D.T.G. laboratories for their invaluable contributions to the work that is
reviewed here. R.T.G. is a recipient of a Scholar Fellowship
from Coordenao de Aperfeioamento de Pessoal de Ensino
Superior (CAPES), Brazil, and the David Rockefeller Center
for Latin American Studies at Harvard School of Public
Health, USA. The R.T.G. laboratory in Brazil is funded by the
National Institute of Science and Technology for Vaccines,
Conselho Nacional de Desenvolvimento Cientfico e
Tecnolgico (CNPq) and Fundao de Amparo a Pesquisa de
Minas Gerai (Fapemig). D.T.G. is a recipient of a Visiting
Scientists Fellowship from CNPq. K.A.F., D.T.G. and R.T.G. are
funded by the US National Institutes of Health.
Competing interests statement
The authors declare no competing interests.


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REVIEWS

Innate sensing of malaria parasites

Malaria is a disease of poverty and has a major negative

The discovery of various families of innate immune

Pathogenesis and malaria-associated syndromes

744 | NOVEMBER 2014 | VOLUME 14

a Plasmodium life cycle

Asexual stage (bloodstream)

Vascular leakage and

Sexual stage (mosquito)

Asexual stage (liver)

function may contribute to the development of metabolic

Renal impairment and

Figure 1 | Plasmodium life cycle and the pathogenesis of malaria.

paroxysms and induce the expression of adhesion molecules by

NATURE REVIEWS | IMMUNOLOGY

VOLUME 14 | NOVEMBER 2014 | 745

a Plasmodium life cycle

Asexual stage (bloodstream)

Vascular leakage and

Sexual stage (mosquito)

Asexual stage (liver)

function may contribute to the development of metabolic

Renal impairment and

Figure 1 | Plasmodium life cycle and the pathogenesis of malaria.

paroxysms and induce the expression of adhesion molecules by

NATURE REVIEWS | IMMUNOLOGY

VOLUME 14 | NOVEMBER 2014 | 745

The three main pathophysiological events that

As parasite growth may be synchronized, the release

Table 1 | Malaria-associated syndromes

Low haemoglobin levels and RBC

Rupture of infected RBCs

Alterations in body temperature

Parasite activation of splenic macrophages leads to a systemic inflammatory

Metabolic acidosis is due to increased glycolysis and the accumulation of

Activation of endothelial cells by circulating pro-inflammatory mediators or by

Parasite sequestration and deposition of haemozoin in the intervillous space

RBC, red blood cell.

746 | NOVEMBER 2014 | VOLUME 14

platelet endothelial cell adhesion molecule 1 (PECAM1),

Sensors of Plasmodium PAMPs

GPI anchors from P.falciparum merozoites have either

NATURE REVIEWS | IMMUNOLOGY

VOLUME 14 | NOVEMBER 2014 | 747

GPI anchors and

GPIs serve as a bridge

Induces macrophage release

Induces the release of

P.falciparum DNA contains

Induces typeI IFN production

DNA from different

Promotes caspase 1 activation

Single-stranded RNA from

RNA from different

Early activation of innate

crystalline structure of haemozoin that destabilizes the

Different families of pattern

Plasmodial DNA. As is the case for other protozoa,

genome contains the highest AT content (82%)

748 | NOVEMBER 2014 | VOLUME 14

Figure 2 | Innate sensors of Plasmodium PAMPs and malaria DAMPs.