TLR9 and MyD88 are crucial for the development of protective immunity to malaria.
ABSTRACT Effective resolution of malaria infection by avoiding pathogenesis requires regulated pro- to anti-inflammatory responses and the development of protective immunity. TLRs are known to be critical for initiating innate immune responses, but their roles in the regulation of immune responses and development of protective immunity to malaria remain poorly understood. In this study, using wild-type, TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) mice infected with Plasmodium yoelii, we show that TLR9 and MyD88 regulate pro/anti-inflammatory cytokines, Th1/Th2 development, and cellular and humoral responses. Dendritic cells from TLR9(-/-) and MyD88(-/-) mice produced significantly lower levels of proinflammatory cytokines and higher levels of anti-inflammatory cytokines than dendritic cells from wild-type mice. NK and CD8(+) T cells from TLR9(-/-) and MyD88(-/-) mice showed markedly impaired cytotoxic activity. Furthermore, mice deficient in TLR9 and MyD88 showed higher Th2-type and lower Th1-type IgGs. Consequently, TLR9(-/-) and MyD88(-/-) mice exhibited compromised ability to control parasitemia and were susceptible to death. Our data also show that TLR9 and MyD88 distinctively regulate immune responses to malaria infection. TLR9(-/-) but not MyD88(-/-) mice produced significant levels of both pro- and anti-inflammatory cytokines, including IL-1β and IL-18, by other TLRs/inflammasome- and/or IL-1R/IL-18R-mediated signaling. Thus, whereas MyD88(-/-) mice completely lacked cell-mediated immunity, TLR9(-/-) mice showed low levels of cell-mediated immunity and were slightly more resistant to malaria infection than MyD88(-/-) mice. Overall, our findings demonstrate that TLR9 and MyD88 play central roles in the immune regulation and development of protective immunity to malaria, and have implications in understanding immune responses to other pathogens.
- SourceAvailable from: iai.asm.org[Show abstract] [Hide abstract]
ABSTRACT: Innate immune recognition of malaria parasites is the critical first step in the development of the host response. At present, Toll-like receptor 9 (TLR9) is thought to play a central role in sensing malaria infection. However, we and others have observed that Tlr9(-/-) mice, in contrast to mice deficient in the downstream adaptor, Myeloid differentiation primary response gene 88 (MYD88), exhibit few deficiencies in immune function during early infection with the malaria parasite Plasmodium chabaudi, implying that another MYD88-dependent receptor also contributes to the anti-malarial response. Here we use candidate-based screening to identify TLR7 as a key sensor of early P. chabaudi infection. We show that TLR7 mediates a rapid systemic response to infection through induction of cytokines such as type I interferons (T1IFN), interleukin 12, and interferon gamma. TLR7 is also required for induction of T1IFN by other species and strains of Plasmodium, including an etiological agent of human disease, P. falciparum, suggesting that malaria parasites harbor a common pathogen-associated molecular pattern (PAMP) recognized by TLR7. In contrast to the non-redundant requirement for TLR7 in early immune activation, sensing through both TLR7 and TLR9 were required for pro-inflammatory cytokine production and immune cell activation during the peak of parasitemia. Our findings indicate that TLR7 plays a central role in early immune activation during malaria infection, whereas TLR7 and TLR9 contribute combinatorially to immune responses as infection progresses.Infection and immunity 09/2013; · 4.21 Impact Factor
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ABSTRACT: Background Malaria is an extremely devastating disease that continues to affect millions of people each year. A distinctive attribute of malaria infected red blood cells is the presence of malarial pigment or so-called hemozoin. Hemozoin is a biocrystal synthesized by Plasmodium and other blood-feeding parasites to avoid the toxicity of free heme derived from the digestion of hemoglobin during invasion of the erythrocytes. Scope of Review Hemozoin is involved in several aspects of the pathology of the disease as well as in important processes such as the immunogenicity elicited. It is known that the once best antimalarial drug, chloroquine, exerted its effect through interference with the process of hemozoin formation. In the present review we explore what is known about hemozoin, from hemoglobin digestion, to its final structural analysis, to its physicochemical properties, its role in the disease and notions of the possible mechanisms that could kill the parasite by disrupting the synthesis or integrity of this remarkable crystal. Major Conclusions The importance and peculiarities of this biocrystal have given researchers cause to consider it as a target for new antimalarials and to use it through unconventional approaches for diagnostics and therapeutics against the disease. General Significance Hemozoin plays an essential role in the biology of malarial disease. Innovative ideas could use all the existing data on the unique chemical and biophysical properties of this macromolecule to come up with new ways of combating malaria.Biochimica et Biophysica Acta (BBA) - General Subjects 01/2014; · 3.85 Impact Factor
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ABSTRACT: The host response to infection requires an immune response to be strong enough to control the pathogen but restrained, to minimise immune-mediated pathology. The conflicting pressures of immune activation and immune suppression are particularly apparent in parasite infections, where co-evolution of host and pathogen has selected many different compromises between protection and pathology. Cytokine signals are critical determinants of both protective immunity and immunopathology and, in this review, we outline the key cytokines that are responsible for the protective and pathological immune responses elicited by parasite infections. We focus on the regulatory cytokine IL-10 and its role in protozoan and helminth infections. We discuss the sources and targets of IL-10 during parasite infection, the signals that initiate and reinforce its action, and its impact on the invading parasite, on the host tissue, and on coincident immune responses. This article is protected by copyright. All rights reserved.Parasite Immunology 03/2014; · 2.21 Impact Factor
The Journal of Immunology
TLR9 and MyD88 Are Crucial for the Development of
Protective Immunity to Malaria
Nagaraj M. Gowda,1Xianzhu Wu,1and D. Channe Gowda
Effective resolution of malaria infection by avoiding pathogenesis requires regulated pro- to anti-inflammatory responses and the
development of protective immunity. TLRs are known to be critical for initiating innate immune responses, but their roles in the
regulation of immune responses and development of protective immunity to malaria remain poorly understood. In this study, using
wild-type, TLR22/2, TLR42/2, TLR92/2, and MyD882/2mice infected with Plasmodium yoelii, we show that TLR9 and MyD88
regulate pro/anti-inflammatory cytokines, Th1/Th2 development, and cellular and humoral responses. Dendritic cells from
TLR92/2and MyD882/2mice produced significantly lower levels of proinflammatory cytokines and higher levels of anti-
inflammatory cytokines than dendritic cells from wild-type mice. NK and CD8+T cells from TLR92/2and MyD882/2mice
showed markedly impaired cytotoxic activity. Furthermore, mice deficient in TLR9 and MyD88 showed higher Th2-type and
lower Th1-type IgGs. Consequently, TLR92/2and MyD882/2mice exhibited compromised ability to control parasitemia and
were susceptible to death. Our data also show that TLR9 and MyD88 distinctively regulate immune responses to malaria
infection. TLR92/2but not MyD882/2mice produced significant levels of both pro- and anti-inflammatory cytokines, including
IL-1b and IL-18, by other TLRs/inflammasome- and/or IL-1R/IL-18R–mediated signaling. Thus, whereas MyD882/2mice
completely lacked cell-mediated immunity, TLR92/2mice showed low levels of cell-mediated immunity and were slightly more
resistant to malaria infection than MyD882/2mice. Overall, our findings demonstrate that TLR9 and MyD88 play central roles in
the immune regulation and development of protective immunity to malaria, and have implications in understanding immune
responses to other pathogens.The Journal of Immunology, 2012, 188: 5073–5085.
Malaria infection is characterized by dominant proinflammatory
responses with Th1 cell development during early stages of in-
fection that decrease as infection progresses with parallel increase
in production of anti-inflammatory responses (2–4). A robust
production of proinflammatory cytokine responses at initial stages
of infection is necessary for the efficient development of protec-
tive cell-mediated and humoral immune responses (2–4). In con-
trast, excessive and/or prolonged proinflammatory responses lead
to the development of severe malaria clinical conditions and fatal
outcomes (2, 3, 5). To overcome the detrimental effects of in-
flammation, as infection progresses, the proinflammatory respon-
ses are downregulated (2, 3, 5).
TLRs, a family of pathogen recognition molecules that sense
certain conserved structures of pathogens, play important roles in
alaria caused by infection with Plasmodium species
of protozoan parasites contributes substantially to the
health crisis and death tolls around the world (1).
initiating innate inflammatory responses to various pathogenic
infections, including malaria (6–8). In humans and mice together,
13 TLRs (TLR1 to TLR13) have been identified, and their ligand
recognition specificities have been studied extensively (8, 9). The
signal initiated upon TLRs sensing microbial components is
transmitted through their highly conserved cytoplasmic Toll/IL-1R
domains, which in most TLRs recruit a common adaptor protein
called MyD88 (7–9). Activation of MyD88 results in the recruit-
ment of several other proteins forming signaling complexes,
which activate MAPK and NF-kB pathways leading to the down-
stream cytokine production.
Several TLRs have been reported to recognize different com-
ponents of malaria parasites. TLR2 and TLR4 mediate the acti-
vation of macrophages by Plasmodium falciparum GPIs (10),
TLR4 recognizes heme and microparticles released from parasite-
infected erythrocytes (11, 12), and TLR9 is a receptor for the
activation of dendritic cells (DCs) by parasite DNA (13–15).
Additionally, in human and mouse malaria parasites, profilin has
been reported to activate DCs through TLR11 (16, 17). In mouse
malaria parasites, Plasmodium berghei, Plasmodium chabaudi
chabaudi AS, and Plasmodium yoelii, TLR2 and TLR9 have been
reported to be involved in the activation of innate immune system
(18–20). However, not much is known about the role of TLRs in
the regulation of innate and adaptive cellular and humoral im-
munity to malaria infection.
In addition to TLRs, a family of intracellular receptors called
nucleotide-binding oligomerization domain-like receptors senses
microbial components or the endogenous danger signals and
forms multiprotein complexes known as inflammasome (21). The
inflammasome is involved in the proteolytic cleavage of cell-
associated pro–IL-1b and pro–IL-18 into secreted IL-1b and IL-
18. The production of active IL-1b and IL-18 requires two signal
components, as follows: 1) TLR-dependent activation of cells that
leads to gene transcription and synthesis of pro–IL-1b and pro–IL-
Department of Biochemistry and Molecular Biology, Pennsylvania State University
College of Medicine, Hershey, PA 17033
1N.M.G. and X.W. contributed equally to this study.
Received for publication July 25, 2011. Accepted for publication March 18, 2012.
This work was supported by National Institute of Allergy and Infectious Diseases
Grant AI 41139, a National Institutes of Health grant, and a Pennsylvania Department
of Health grant.
Address correspondence and reprint requests to Prof. D. Channe Gowda, Department
of Biochemistry and Molecular Biology, Pennsylvania State University College of
Medicine, Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA
17033. E-mail address: firstname.lastname@example.org
The online version of this article contains supplemental material.
Abbreviations used in this article: DC, dendritic cell; FL-DC, FLT3 ligand-
differentiated DC; FLT3, Fms-like tyrosine kinase 3; GM-DC, GM-CSF–differenti-
ated DC; IRBC, infected RBC; WT, wild-type.
18, and 2) activation of caspase-1 of the inflammasome complex,
most likely by a second microbial stimuli, resulting in the cleav-
age of pro–IL-1b and pro–IL-18 into active cytokines. In malaria,
NALP3-mediated inflammasome has been reported to be involved
in hemozoin- and uric acid-induced maturation of pro–IL-1b and
pro–IL-18 (22, 23).
Among cells of the innate immune system, DCs play crucial
roles in TLR- and other pathogen-specific receptor-mediated
recognition, and initiation of innate immune responses and devel-
opment of adaptive immunity (24–26). At early stages of malaria
infection, DCs efficiently produce proinflammatory cytokines,
and, as the infection progresses, their ability to produce proin-
flammatory responses becomes low, but acquires increased ca-
pacity to produce anti-inflammatory responses (27). Additionally,
DCs activate NK cells, instruct T cells to induce programmed
Th1/Th2 responses, and initiate the development of cell-mediated
and humoral adaptive immunity (24, 25, 28, 29). Thus, DCs
provide a critical link between the innate and adaptive immune
responses and help shape the pathogen-specific adaptive immune
responses. TLRs and MyD88 are also expressed by T and B cells
and play important roles in the function of these cells (30–32).
For example, MyD88-mediated signaling is essential for T cell-
mediated resistance to Toxoplasma gondii (33), and TLR2-MyD88–
mediated signaling is necessary for CD8+T cell clonal expansion
and memory cell formation (34, 35). MyD88 has also been shown
to regulate virus-specific CD4+T cell responses (36), virus-in-
duced B cell activation, and Ab production and Ig class switching
to IgG2c (37, 38).
Of different immunostimulatory components of malaria para-
sites that activate TLRs (10–17), parasite protein-DNA complex/
nucleosome is the major factor that activates DCs through TLR9/
MyD88-mediated signaling and induces the production of proin-
flammatory responses (15, 39). Given that the cytokine milieu of
the initial immune responses determines the effectiveness of
adaptive immune responses, we hypothesized that TLR9 and
MyD88 play crucial roles in the regulation of Th1/Th2 develop-
ment and cellular and humoral adaptive immunity to malaria.
In this study, we tested this hypothesis by studying innate and
adaptive immune responses to the blood stage P. yoelii mouse
malaria infection. The results show that TLR9 and MyD88 are
critical for the robust proinflammatory responses, Th1 develop-
ment, and efficient cell-mediated and humoral immunity to
malaria infection. Hence, the deficiency in TLR9 and MyD88
resulted in the decreased capacity of DCs to produce proin-
flammatory cytokines with the increased ability to elicit anti-
inflammatory cytokine responses, impaired NK and CD8+T cell
cytotoxic activity, and increased Th2-type Ab responses. Conse-
quently, TLR92/2and MyD882/2mice harbored significantly
higher parasitemia and exhibit lower survival rates than wild-type
Materials and Methods
DMEM and RPMI 1640 medium were purchased from Mediatech (Man-
CA). FBS was purchased from Atlanta Biologicals (Lawrenceville, GA).
Collagenase D was from Roche Applied Science (Mannheim, Germany).
Nonessential amino acids and 2-ME for cell culturing, LPS from Salmo-
nella minnesota Re595 strain, and Percoll were from Sigma-Aldrich (St.
Louis, MO). CpG oligodeoxynucleotide1826 (simply referred to as CpG
in the figures) was from Coley Pharmaceuticals (Kanata, ON, Canada).
Pam3CSK4 was from Microcollections (Tu ¨bingen, Germany). Poly-
inosinic:polycytidylic acid, a synthetic analog of dsRNA (TLR3 li-
gand), was from InvivoGen (San Diego, CA). Chicken OVA peptide323–339
(OVA323–339) was from Peptides International (Louisville, KY). Isolymph
was from CTL Scientific Supply (Deer Park, NY). CytoTox 96 nonra-
dioactive cytotoxicity assay kit was from Promega (Madison, WI). Mouse
Ig isotyping ELISA kit and Cy5-conjugated annexin V were from BD
Biosciences (San Jose, CA). Target cells, YAC-1 (a murine lymphoma cell
line), and EL-4 (a mouse thymocyte cell line), for the measurement of NK
cell and CD8+T cell cytotoxic activity, were provided, respectively, by
N. Keasey and T. Schell (Pennsylvania State University College of Med-
Anti-mouse CD11c Ab (clone N418)-conjugated MicroBeads (catalog
130-052-001) for the isolation of DCs, anti-mouse CD90.2 Ab-conjugated
MicroBeads (catalog 130-049-101) for total T cell isolation, mouse CD8a
T cell isolation kit (catalog 130-090-859), and mouse NK cell isolation kit
(catalog 130-090-864) were purchased from Miltenyi Biotec (Auburn, CA).
Duoset ELISA kits for measuring mouse TNF-a, IL-12p40, IL-1b, IL-4,
IL-10, and IFN-g were from R&D Systems (Minneapolis, MN). Coating
and biotin-labeled detecting Abs against mouse IL-18 (clones 74 and 93-
10C) and mouse IL-18 standard for the measurement of IL-18 by ELISA
were purchased from MBL International (Woburn, MA).
The Abs used in this study were as follows: purified anti-mouse CD3ε Ab
(clone 17A2); anti-mouse CD16/32 mAb (clone 93); FITC-conjugated
mAbs against mouse CD3ε (clone 145-2C11), CD11c (N418), CD19
(eBio1D3), pan NK cell (DX5), and TNF-a (MP6-XT22); PE-conjugated
anti-mouse NK1.1 Ab (PK136); PE-Cy5–conjugated Abs against mouse
CD80 (16-10A1) and hamster IgG (eBio299Arm) isotype control, CD86
(GL1), and rat IgG2ak isotype control; PerCP-Cy5.5–conjugated anti-
CD8a mAb (53-6.7); allophycocyanin-conjugated Abs against mouse
CD3ε (clone 145-2C11), mouse CD40 (1C10), and mouse IFN-g (XMG1.2);
and rat IgG1k isotype control were from eBioscience (San Diego, CA).
PerCP-Cy5.5–conjugated anti-mouse IL-4 (11B11) and PE-conjugated
anti-mouse IL-12 Abs (clone 15.6) were from BD Biosciences (San
University College of Medicine, Hershey, has reviewed and approved the
protocols for use of animals in this study. The animal care was according to
the institutional guidelines of the Pennsylvania State University College of
WT, and TLR2, TLR4, TLR9, and MyD88 knockout mice, and OT-II
transgenic mice expressing TCR for OVA323–339peptide on CD4 T cells
were housed in a pathogen-free environment. All mice used in this study
were in C57BL/6J background.
Parasite infection, parasitemia, and survival rate
WT C57BL/6 mice were infected with cryopreserved nonlethal P. yoelii
(Py17XNL strain) parasites, and blood from these mice was used for
infecting experimental mice. Experimental mice were infected by i.p. in-
jection of 1 3 106infected erythrocytes from infected donor mice in 100
ml saline. Parasitemia was monitored on alternative days postinfection by
examining Giemsa-stained thin smears of tail blood on glass slides, and
results were expressed as percentage of parasite-infected erythrocytes.
Mice were monitored for survival twice per day. Blood was collected, and
sera were prepared and stored at 280˚C until used for cytokine and Ab
analyses by ELISA (15). Mice were also infected as above with P. berghei
NK65 strain. Infected RBCs (IRBCs) from P. yoelii- and P. berghei NK65-
infected mice were isolated and used for testing the Ag specificity of cy-
totoxic CD8+T cells.
Preparation of Fms-like tyrosine kinase 3 ligand- and
prepared by culturing mouse bone marrow cells in complete DMEM
supplemented with 15% of FLT3 ligand-containing conditioned medium,
which is obtained by culturing B16 cells expressing retrovirus-coded FLT3
ligand (15, 40).
GM-CSF–differentiated DCs (GM-DCs) were obtained by culturing
bone marrow cells from WT mice for 7 or 8 d in complete DMEM con-
taining 10% conditioned DMEM from the cultured GM-CSF–producing
5074TLR9 AND MyD88 REGULATE MALARIA IMMUNITY
Isolation of spleen and liver cells
Single-cell suspensions of mouse spleens were prepared, as described
previously (15), and used for the isolation of total T cells and CD8+T cells
by magnetic column separation (MACS). Total T cells from mouse spleens
were isolated using anti-mouse CD90.2 Ab-conjugated magnetic beads;
the purity of cells as assessed by flow cytometry after staining with anti-
CD3ε Ab was ∼90%. CD8+T cells were isolated using CD8a T cell
isolation kit; the purity of cells as analyzed by flow cytometry by stain-
ing with anti-CD3ε and anti-CD8a Abs was ∼92%.
DCs were isolated from the single-cell suspensions of mouse spleens,
prepared by digesting with 1 mg/ml collagenase D (15), by MACS after
staining with anti-mouse CD11c Ab-conjugated microbeads. The purity of
cells as assessed by flow cytometry after staining with anti-CD11c Ab was
For NK cell isolation, livers were flushed with 10 ml PBS (pH 7.2),
crushed, and filtered through a 70-mm strainer to obtain single-cell sus-
pensions. The cell suspensions were centrifuged on Isolymph cushions at
1200 3 g at 4˚C for 15 min; buffy coat on the top of Isolymph was col-
lected and washed; and NK cells were isolated by MACS using the mouse
NK cell isolation kit. The purity of cells by flow cytometry analysis after
staining with anti-mouse NK1.1 Ab was ∼60%.
Isolation of P. yoelii- and P. berghei-infected RBCs and
preparation of cell lysates
Erythrocyte pellets from blood samples of parasite-infected mice were
suspended in 2 vol of PBS (pH 7.2) and centrifuged on Isolymph cushions
at 1200 3 g at 4˚C for 15 min, and the buffy coat was removed. The
erythrocyte pellets were resuspended in 2 vol of PBS and centrifuged on
75% Percoll cushions at 1200 3 g at 4˚C for 15 min. The IRBCs on the top
of Percoll were collected and washed.
Lysates of P. yoelii IRBCs were prepared by alternative freezing and
thawing of IRBC suspensions three times, followed by sonication in water-
bath sonicator for 5 min. After centrifugation at 13,000 rpm in micro-
centrifuge for 10 min, the protein contents in the clear supernatants were
measured using Pierce micro bicinchoninic acid protein estimation kit
(Thermo Scientific, Rockford, IL), and used for Ab analysis in mice sera.
Stimulation of DCs and cocultures of DCs plus OT-II T cells
Mouse spleen DCs (1 3 105/well) from infected mice were seeded into 96-
well plates. Cells in 200 ml complete DMEM were stimulated with stan-
dard TLR ligands, as follows: Pam3CSK4(TLR2 ligand, 10 ng/ml), poly-
inosinic:polycytidylic acid (TLR3 ligand, 2 mg/ml), LPS (TLR4 ligand,
100 ng/ml), or CpG oligodeoxynucleotide (TLR9 ligand, 2 mg/ml). After
24 h, the culture supernatants were analyzed for cytokines by ELISA (15).
In coculturing experiments, spleen DCs (1 3 105/well) from infected mice
were either untreated or treated with 2 mg/ml OVA323–339peptide and
cocultured with spleen T cells (0.5 3 105/well) from the naive OT-II
transgenic mice that express OVA-specific TCR in 96-well U-bottom
plates containing 200 ml complete medium. OT-II T cells alone were
similarly stimulated with OVA323–339peptide as controls. After 72 h, the
culture supernatants were collected, and cytokines were analyzed by
Flow cytometry analysis of cytokine expression and
Todeterminethe celltypesthatproduce cytokinesincocultureexperiments,
cells were stimulated and treated with GolgiPlug (BD Biosciences) for 6 h.
Cells were harvested, treated with Fc block (anti-mouse CD16/32 Ab), and
stained for surface markers followed by intracellular staining with anti-
cytokine Abs (15). The survival of DCs was assessed by flow cytometry
using FACSCalibur (BD Biosciences) after staining with annexin V.
For the analysis of costimulatory molecules, total spleen cells from WT,
TLR92/2, and MyD882/2mice at 3 d postinfection were stained with dye-
conjugated Abs against costimulatory molecules. After washing, cells were
analyzed by flow cytometry, and the results were analyzed using CellQuest
software (BD Biosciences).
Restimulation of T cells from P. yoelii-infected mice
rest overnight in complete DMEM and plated (1 3 105cells/well) into 96-
well U-bottom plates. To each well was added FL-DCs (5 3 104/well) and
P. yoelii IRBCs (1.5 3 105/well) in 200 ml complete DMEM. After 72 h,
the culture supernatants were collected and cytokines were measured by
ELISA. Supernatants of T cells cocultured with WT FL-DCs in the ab-
sence of IRBCs or T cells alone cultured in the presence of IRBCs were
used as controls. Cytokine production by DCs and T cells was also ana-
lyzed by intracellular staining, as described above.
Cell cytotoxicity assay
The cytotoxic activity of spleen CD8+T cells was determined by measuring
the lysis of P. yoelii IRBC-pulsed GM-DCs and the anti-mouse CD3ε Ab-
redirected lysis of EL-4 cells (H-2bbackground) (42). The cytotoxic ac-
tivity of NK cells was measured by assaying the lysis of YAC-1 target cells
(43). Briefly, to different doses of spleen CD8+T cells in 96-well U-bottom
plates were added GM-DCs (1 3 104/well) that were previously pulsed
with P. yoelii or P. berghei IRBC lysates at DC to IRBC equivalent of 1:5
for 4 h. In separate sets of experiments, EL-4 cells were treated with 10
mg/ml anti-CD3ε Ab and washed with incomplete RPMI 1640 medium. To
different doses of spleen CD8+T cells plated in 96-well U-bottom plates
were added Ab-treated EL-4 target cells (1 3 104/well) in 100 ml RPMI
1640 medium. Similarly, to liver NK cells plated in 96-well U-bottom
plates were added YAC-1 cells (13 104/well) in 100 ml RPMI 1640 me-
dium. In each case, plates were centrifuged for 3 min at 300 3 g to fa-
cilitate cell-cell contact and incubated at 37˚C for 4 h. Target cell alone to
which control lysis solution was added served as positive controls. Culture
supernatants (50 ml each) were transferred into 96-well opaque plates, and
the extent of target cell lysis was measured using CytoTox 96 nonradio-
active cytotoxicity assay reagents, according to the manufacturer’s instruc-
EL-4 and YAC-1 cells used in the above assays were cultured in RPMI
1640 medium containing 10% FBS, 1% penicillin-streptomycin, 1%
nonessential amino acids, 1 mM sodium pyruvate, and 50 mM 2-ME, and
harvested at exponential growth phase.
Analysis of Ab responses in infected mice
The levels of total IgG in the sera of P. yoelii-infected mice were analyzed
by ELISA. Ninety-six–well microtiter plates were coated with IRBC
lysates (20 mg protein per ml) in 50 mM sodium bicarbonate buffer (pH
9.6) overnight at 4˚C, and blocked with 1% BSA in PBS. The plates were
incubated with serially diluted mouse sera at room temperature for 1 h.
After washing, the plates were incubated with 1:3000 diluted HRP-
conjugated goat anti-mouse IgG for 1 h, followed by SureBlue tetrame-
thylbenzidine peroxidase substrate solution (Kirkegaard & Perry Labora-
tories, Gaithersburg, MD). The reaction was stopped by adding 2 N
H2SO4, and absorbance at 450 nm was measured.
The Ab isotypes in the sera from P. yoelii-infected mice were analyzed
using the mouse Ig isotyping ELISA kit, according to the manufacturer’s
The data were plotted as mean values 6 SD or SEM. Statistical analysis of
data was performed by one-way ANOVA, followed by the Newman–Keuls
test. GraphPad Prism software version 3.0 was used for the analysis. A p
value ,0.05 was considered statistically significant.
TLR9 and MyD88 regulate malaria-induced DC functions
Malaria infection is characterized by a robust proinflammatory
cytokine production during the early stages of infection that
declines as infection progresses with parallel gradual increase in
the production of anti-inflammatory cytokines (2–4). The initial
cytokine responses play important roles in the regulation and
development of immunity to malaria (2–5). Although several
TLRs, including TLR9, TLR2, and TLR4, have been shown to
recognize various components of malaria parasites and induce
proinflammatory cytokines (10–17), which of these TLRs play key
roles in the generation of protective immunity to malaria remains
unknown. Because DCs are central to the development of innate
and adaptive immunity to malaria (2–4), we first studied the roles
of TLRs and their key shared adaptor protein MyD88 in the
regulation of pro- and anti-inflammatory cytokine responses by
DCs isolated from the spleen of WT, TLR22/2, TLR42/2,
TLR92/2, or MyD882/2mice infected with P. yoelii. At the early
stages of infection (5 d postinfection), when the parasite is
establishing a stable infection (see later), DCs obtained from WT,
TLR22/2, and TLR42/2mice, but not those from TLR92/2and
The Journal of Immunology5075
MyD882/2mice, efficiently produced TNF-a and IL-12 (Fig. 1).
At the later stages (10 and 17 d) of infection, that is, when par-
asites grew exponentially, the levels of these cytokines produced
by DCs from the WT, TLR22/2, and TLR42/2mice were
markedly declined. In parallel, however, DCs from these mice at
10 and 17 d postinfection produced increased levels of IL-4 as
compared with DCs from the infected mice at 5 d postinfection.
The expression of IL-10 was also modestly increased in DCs from
mice at 10 d postinfection compared with cells from mice at
5 d postinfection, but decreased considerably in DCs from mice at
17 d postinfection when parasitemia was high (Fig. 1). Further-
more, DCs produced high levels of cytokines in response to
standard immunostimulatory ligands in TLR-specific manner
(Supplemental Fig. 1). Together these results indicated that at the
early stages of infection, DCs produce mainly proinflammatory
responses, which are known to instruct the immune system to
develop effective Th1 adaptive immunity for parasite clearance,
whereas at later stages of infection these cells produce primarily
anti-inflammatory cytokines, presumably leading to Th2 respon-
In contrast to infected WT, TLR22/2, and TLR42/2mice, DCs
from infected TLR92/2and MyD882/2mice produced low levels
of TNF-a and IL-12, and higher levels of IL-10 and IL-4, at all
stages of infection (Fig. 1). Notably, at early stages of infection,
TNF-a and IL-12 levels produced by DCs from infected TLR92/2
or MyD882/2mice were comparable to those produced by DCs
from the infected WT, TLR22/2, and TLR42/2mice at later
stages (10 and 17 d) of infection. Collectively, these results
revealed that TLR9- and MyD88-mediated signaling dominantly
drives proinflammatory cytokine responses to malaria parasites
during the early stages of infection, and thus, the deficiency in
TLR9 or MyD88 leads to type 2 cytokine/anti-inflammatory
responses by DCs. The results are consistent with our recent re-
port that TLR9 is the major sensor of P. falciparum that mediates
proinflammatory responses (15).
Given that cytokine responses by DCs from infected TLR92/2
and MyD882/2mice were markedly different from those by DCs
from infected WT mice, it was of interest to determine whether
DCs from these mice differ in their activation status. Therefore,
we evaluated maturation of DCs in the infected animals by ana-
lyzing surface expression of costimulatory molecules. Spleen DCs
from WT, TLR92/2, and MyD882/2mice at 3 d postinfection
exhibited significantly increased levels of CD40, CD80, and
CD86 than DCs from naive WT mice (Supplemental Fig. 2).
These results demonstrate that, regardless of the critical require-
ment of TLR9 and MyD88 for the production of proinflammatory
cytokines by DCs, malaria parasites can activate and induce
maturation of DCs in TLR9- and MyD88-independent manner.
These results agree with the ability of DCs from infected TLR92/2
and MyD882/2mice to efficiently produce anti-inflammatory
responses (see Fig. 1).
To further understand the role of TLR9 and MyD88 in malaria
parasite-inducedfunctionofDCs, weassessedthe abilityofexvivo
DCs to induce cytokine responses in T cells. DCs from the spleens
of mice at 5, 10, and 17 d postinfection were cocultured with OT-II
T cells and treated with OVA peptide, and the levels of TNF-a, IL-
12, IFN-g, IL-4, and IL-10 in the culture supernatants were ana-
lyzed by ELISA. The cytokine levels produced by the cocultures
were significantly higher than those produced by the control DCs
(p , 0.05 to , 0.001) (Fig. 2A–E). Intracellular staining with
anti-cytokine Abs and flow cytometry analysis showed that DCs
from mice at both 5 and 10 d postinfection can efficiently induce
the production of TNF-a and IFN-g in OT-II T cells (Fig. 2F);
cells were gated as indicated in Supplemental Fig. 3A. DCs from
mice at 5 d postinfection induced substantially higher levels of
TNF-a and IFN-g production by OT-II T cells than DCs from
mice at 10 d postinfection. These results are consistent with
a previous report, based on ELISA analysis of cytokines produced
by cocultures, that the ability of DCs from malaria-infected mice
to induce T cells to produce proinflammatory responses decreases
as the infection progresses (27). However, in contrast (27), DCs
from mice at both 5 and 10 d postinfection were unable to induce
detectable levels of IL-4 and IL-10 by OT-II T cells (Supplemental
Fig. 3B and data not shown), even though the cocultures produced
substantially higher levels of these cytokines than DCs from
control mice. Therefore, it appears that, in vivo, besides DCs,
cytokine milieu and other APCs such as macrophages and mast
cells influence T cells to produce IL-4 and IL-10. Macrophages
uptake IRBCs efficiently and produce IL-10 and IL-4 (44) (X. Wu,
N.M. Gowda, and D.C. Gowda, unpublished results). Consistent
with the fact that T cells produce little or no IL-12 (45), the ex-
pression of this cytokine by OT-II T cells was not evident.
However, interestingly, the cocultures produced significantly
higher levels of IL-12 than control DCs (Fig. 2C). Therefore, the
observed increased production of IL-4, IL-10, and IL-12 by
cocultures, even though OT-II T cells were unable to produce
anti-inflammatory cytokine production by DCs in
malaria parasite-infected mice. Spleen DCs, isolated
from P. yoelii-infected WT, TLR22/2, TLR42/2,
TLR92/2, and MyD882/2mice at 5, 10, and 17 d
postinfection were cultured in 96-well plates. After
24 h, TNF-a (A), IL-12 (B), IL-10 (C), and IL-4 (D)
released into the culture media were analyzed by
ELISA. Spleen DCs from naive WT mice were ana-
lyzed as controls. Experiments were performed three
times, and, each time, ELISA was performed in
duplicates. The results of a representative experiment
are shown. Error bars represent mean values 6 SD.
The letters a, b, and c denote the statistical significance
between the levels of cytokines produced by DCs from
the indicated gene knockout mice and those produced
by DCs from the corresponding infected WT mice.
ap , 0.001,bp , 0.01,cp , 0.05.
TLR9 and MyD88 regulate pro- and
5076 TLR9 AND MyD88 REGULATE MALARIA IMMUNITY
these cytokines, suggested that OT-II T cells in turn influenced
DCs to induce increased expression of cytokines and/or enabled
DCs to survive longer, leading to increased production of cyto-
kines. The influence of T cells in inducing DCs to produce higher
levels of cytokines is also evident from enhanced production of
TNF-a and IL-4 by DCs cocultured with OT-II T cells as com-
pared with DCs cultured alone (Supplemental Fig. 3C). To de-
termine the possibility that ligation of T cells leads to increased
life span of DCs in coculture, we assessed the duration of survival
of cocultured DCs by measuring the levels of cell death at dif-
ferent time points by flow cytometry after staining with annexin V.
It is known that annexin V binds phosphatidylserine expressed on
the surface of apoptotic cells and the annexin V-binding assay is
used for measuring the extent of cell death (46, 47). The results
showed that the levels of annexin V-positive DCs were substan-
tially higher in control DC culture than that in coculture (Fig. 2G).
Together the above data clearly demonstrated that ligation of OT-
II T cells leads to increased life span of DCs, and hence, increased
production of cytokines by DCs. These results are consistent with
the previous report that ligation of CD40 and CD40L leads to
increased production of IL-12 by DCs (48, 49).
Furthermore, in three independent experiments, we consistently
found that OT-II T cells cocultured with spleen DCs from infected
TLR92/2and MyD882/2mice produced noticeably lower levels
of IFN-g than OT-II cells cocultured with DCs from infected WT
mice, although there was no difference in the level of TNF-a
production (Fig. 2F). However, in all three groups, OT-II T cells
cocultured with DCs from mice at 5 d postinfection produced
markedly higher levels of TNF-a and IFN-g than OT-II T cells
cocultured with DCs from mice at 10 d postinfection. Thus, these
infected mice induced cytokine ex-
pression by OT-II T cells. Spleen
T cells from naive OT-II transgenic
mice were cocultured with spleen
DCs from P. yoelii-infected WT,
TLR22/2, TLR42/2, TLR92/2, and
MyD882/2mice at the indicated
days postinfection in the presence of
OVA323–339peptide. After 72 h, the
culture supernatants were collected
and assayed for TNF-a (A), IFN-g
(B), IL-12 (C), IL-10 (D), and IL-4
(E) by ELISA. Cocultures not trea-
ted with OVA peptide were used as
controls. Data are a representative of
three independent experiments, each
performed in duplicates. Note: 5-
d DC plus T, 10-d DC plus T, and
17-d DC plus T refer to cocultures of
spleen DCs from mice at 5, 10, and
17 d postinfection and OT-II T cells
from naive mice. The letters, a and
b, indicate the statistical significance
between the levels of cytokines
produced by OT-II T cells activated
with DCs from the indicated gene
knockout mice and those produced
by OT-II T cells activated with DCs
from the corresponding infected WT
mice.ap , 0.001,bp , 0.01. (F) OT-
II T cells and DCs from mice at 5
and 10 d postinfection were cocul-
tured for 6–12 h in the presence of
OVA peptide and then added Gol-
giPlug. Production of TNF-a and
IFN-g by T cells was analyzed by
flow cytometry after intracellular
staining with anti-cytokine Abs.
Histograms show the percentage of
positive OT-II T cells for each cy-
tokine. (G) Spleen DCs from mice at
5 d postinfection were cultured ei-
ther alone or with OT-II T cells in
presence of OVA peptide. After 24
or 48 h, DCs were surface stained
with annexin Vand analyzed by flow
cytometry. Histograms indicate the
percentage of annexin V-positive cells
in gated DCs in coculture (thin lines)
versus control DCs alone (thick lines).
DCs from malaria-
The Journal of Immunology 5077
results demonstrated that DCs are programmed to induce a strong
Th1 development at early stages of infection in a TLR9- and
MyD88-dependent manner, and that the ability of DCs to induce
Th1 polarization decreases as the infection progresses.
TLR9 and MyD88 differentially regulate cytokine production in
response to malaria infection
In addition toDCs, various other cell types,includingTand B cells,
produce cytokines, contributing to serum cytokine profiles of
malaria-infected mice. This is especially the case at later stages of
infection. To determine the role of TLR9 and MyD88 on cytokine
production in vivo in response to malaria infection, we analyzed
cytokine responses in P. yoelii-infected WT, TLR22/2, TLR42/2,
TLR92/2, and MyD882/2mice. The serum TNF-a, IL-12, IL-10,
and IL-4 profile in WT, TLR22/2, TLR42/2, and TLR92/2mice
at the early stages of infection was similar to those produced by
DC ex vivo (Fig. 3), that is, higher levels of proinflammatory cy-
tokines, including IFN-g, and lower Th2-type/anti-inflammatory
cytokines by WT, TLR22/2, and TLR42/2mice, and vice versa
by TLR92/2mice. However, at the later stages of infection,
whereas the profiles of TNF-a and IL-10 in the sera of infected
WT, TLR22/2, and TLR42/2mice were comparable to those
produced by ex vivo DCs, the serum levels of IL-12 and IL-4 were
substantially increased in all these mice.
Notably, in contrast to cytokine profiles observed by DC ex vivo
(see Fig. 1), the infected TLR92/2mice presented a distinct cy-
tokine profile at the later stages of infection, that is, significantly
higher levels of IL-12 and IL-4 compared with WT, TLR22/2, and
TLR42/2mice (Fig. 3). Furthermore, interestingly, in the case of
infected MyD882/2mice, the levels of both pro- and anti-
inflammatory cytokines were markedly low at all stages of in-
fection, despite the fact that ex vivo DCs produced significantly
higher levels of IL-10 and IL-4 than DCs from the infected WT,
TLR22/2, and TLR42/2mice (compare Figs. 1 and 3). Because
cytokine profiles of DCs from WT, TLR22/2, and TLR42/2mice
were more or less comparable to those observed in vivo in these
mice at early stages of infection (compare Figs. 1 and 3), it
appears that, during early stages of infection, DCs are the major
contributors to the cytokine profiles observed in infected mice.
However, at later stages of infection, it appears that other cell
types, such as T and B cells, contribute to the production of
cytokines in a TLR9-independent and MyD88-dependent manner.
Thus, the results demonstrate that MyD882/2mice, but not
TLR92/2mice, have intrinsic defect in producing pro- and anti-
inflammatory cytokines in response to malaria infection (Fig. 3),
although DCs deficient in TLR9 and MyD88 produce similar
cytokine profiles (see Fig. 1).
TLR9 and MyD88 contribute to cytokine production by T and
B cells in response to malaria infection
To test the prediction that, at late infection stages, T and B cells
significantly contribute to the observed serum cytokine profiles
(see above), we first analyzed cytokines produced by T cells. In-
tracellular staining showed that T cells from WT, TLR92/2, and
MyD882/2mice at 10 and 17 d postinfection produced substantial
levels of IFN-g and IL-4 (Fig. 4A, 4B). The proinflammatory
cytokine expression by T cells from the infected TLR92/2and
MyD882/2mice was appreciably lower than that by T cells from
WT mice. We next analyzed cytokine responses by T cells isolated
from mice at 10 d postinfection after restimulation with P. yoelii
IRBC Ags presented by in vitro generated DCs. Previously, we
showed that IRBCs can induce efficient production of cytokines
by FL-DCs and spleen DCs, and that the cytokine profiles pro-
duced by these cells are similar (15). In this study, we used FL-
DCs from WT mice as APCs for the restimulation of T cells from
infected mice. Cytokine profiles produced by T cells restimulated
with IRBC-treated FL-DCs were similar to those produced by
unstimulated ex vivo T cells (Supplemental Fig. 4A, and compare
Supplemental Fig. 4A with Fig. 4B). Together, the above results
indicate that cytokine production by T cells in response to malaria
mice differentially produce cytokines. WT, TLR22/2,
TLR42/2, TLR92/2, and MyD882/2mice (n = 5 in
each group) were infectedwithP. yoelii. Bloodfrom the
infected mice at 5, 10, and 17 d postinfection was col-
lected, and sera prepared. TNF-a (A), IFN-g (B), IL-12
(C), IL-10 (D), and IL-4 (E) present in the pooled sera
were measured by ELISA. Experiments were per-
formed twice, and, each time, ELISAwas performed in
duplicates.Thelettersa,b,andc representthe statistical
significance between the levels of cytokines produced
by the indicated gene knockout mice and those pro-
duced by the corresponding infected WT mice.ap ,
0.001,bp , 0.01,cp , 0.05.
5078TLR9 AND MyD88 REGULATE MALARIA IMMUNITY
infection is to a certain extent TLR9 and MyD88 dependent, and
that the cytokines produced by T cells contribute significantly to
the serum cytokine profiles of parasite-infected WT, TLR92/2,
We next measured cytokine levels in the cocultures of spleen
T cells from WT, TLR92/2, and MyD882/2mice at 10 and
17 d postinfection and FL-DCs from WT mice, in which T cells
were restimulated with P. yoelii IRBCs presented by FL-DCs. The
profiles of TNF-a, IFN-g, IL-12, IL-4, and IL-10 produced were
nearly comparable to those of serum cytokines in the respective
infected mice (Fig. 4C, 4E, see Fig. 3). The levels of cytokines
produced by the cocultures were significantly higher than those
secreted by the IRBC-stimulated WT DCs alone (Fig. 4C, 4E).
Furthermore, in the case of T cells from WT mice at
10 d postinfection, the cocultures produced significantly higher
levels of TNF-a, IFN-g, and IL-12 than the case of TLR92/2
duction of cytokines by Tand B cells. Total spleen cells
from WT, TLR92/2, and MyD882/2mice at 10 and
17 d postinfection were cultured for 4 h in the presence
of GolgiPlug, surface stained with anti-mouse CD3ε
Ab followed by intracellular staining using anti-cyto-
kine Abs, and analyzed by flow cytometry. (A) Cells
were selected by side scattering (SSC) and forward
scattering (FSC) and were gated for total T cells
(CD3ε+; B) and B cells (CD19+; G). (B) Percentage of
positive cells for each cytokine in gated T cells is
shown in the histograms. Cells from naive WT mice
were analyzed as controls. Isolated spleen T cells from
WT, TLR92/2, and MyD882/2mice at 10 d (C, D) and
17 d (E, F) postinfection were cocultured with WT
FL-DCs in the presence (C, E) or absence (D, F) of
P. yoelii IRBCs [(D) and (F) are controls for (C) and (E),
respectively]. In parallel, WT FL-DCs alone were also
stimulated with IRBCs as controls for cocultures. After
72 h, cytokines secreted into the culture medium were
analyzed by ELISA. The letters a, b, and c represent
statistical significance between the levels of cytokines
in indicated groups of infected mice.ap , 0.001,bp ,
0.01,cp , 0.05. (G) The cultured total spleen cells were
surface stained for B cells using anti-mouse CD19 Ab,
followed by intracellular staining using Abs against
mouse IL-12 and IL-4. Histograms show the percentage
of B cells positive for each cytokine. B cells from naive
WT mice were similarly stained and analyzed as con-
TLR9 and MyD88 are critical for pro-
The Journal of Immunology 5079
T cells. However, in the case of restimulated TLR92/2T cells, the
cocultures produced significantly higher levels of IL-4 and IL-10
(Fig. 4C, 4D). In the case of T cells from TLR92/2mice at
17 d postinfection, the cocultures produced significantly higher
levels of TNF-a, IL-12, and IL-4 than the cocultures of T cells
from WT mice and FL-DCs (Fig. 4E, 4F). Compared with these
results, in the case of T cells from MyD882/2mice at 10 and
17 d postinfection, the cocultures produced substantially lower
levels of both pro- and anti-inflammatory cytokines. The cytokine
responses in the cocultures represents those produced by both T
cells and FL-DCs (Supplemental Fig. 4). Note that IL-12 is pro-
duced predominantly, if not exclusively, by DCs in the cocultures.
Together, the above results confirm that DC and T cell interactions
lead to cytokine production by both cell types in response to
malaria parasites in a TLR9- and MyD88-dependent manner.
We also assessed cytokine responses by B cells at 10 and
17 d postinfection by intracellular staining and flow cytometry.
Although B cells are known to produce a wide range of cytokines,
including TNF-a, IFN-g, IL-12, IL-10, and IL-4, we analyzed IL-
12 and IL-4 production as representatives of proinflammatory and
type 2 cytokine responses, respectively. B cells from infected WT,
TLR92/2, and MyD882/2mice produced appreciable levels of
both IL-12 and IL-4. However, the levels of cytokines produced by
B cells from MyD882/2mice were lower than those produced by
WT and TLR92/2mice (Fig. 4G). Although detailed analysis of
cytokine responses by B cells from WT and TLR knockout mice
has not been done in the current study, the above results suggest
that B cells produce substantial levels of cytokines in response to
malaria infection, contributing to the serum cytokine profiles of
Overall, the results of the above analyses demonstrate that the
cytokines produced by Tand B cells from infected WT, TLR92/2,
and MyD882/2mice contribute substantially to the serum cyto-
kine profiles of the respective mice. The results further demon-
strate that TLR9 and MyD88 distinctively regulate cytokine
responses by T and B cells in response to malaria infection.
TLR9-independent/MyD88-dependent and/or IL-1R/IL-18R–
mediated signaling also contribute to cytokine responses to
Whereas the malaria parasite-induced function of DCs was de-
pendent on both TLR9 and MyD88 (see Fig. 1, and also see Ref.
15), the serum cytokine responses to malaria infection were sig-
nificantly independent of TLR9, but largely dependent on MyD88
(see Fig. 3). To gain insight into how TLR9 and MyD88 differ-
entially regulate immune responses to malaria, we performed the
following studies. The malaria parasite hemozoin has been re-
ported to activate inflammasome-mediated signaling to induce the
production of IL-1b (22, 23). To determine the contribution of
inflammasome-mediated signaling, we measured the levels of IL-
1b and IL-18 in the sera of P. yoelii-infected mice. Both IL-1b and
IL-18 were produced throughout the course of infection (Fig. 5).
The production of IL-1b decreased as the infection progressed,
whereas that of IL-18 increased with increasing parasitemia.
Furthermore, although the production of both cytokines was de-
pendent, to some extent, on TLR9 at early stages of infection, it
was mostly independent of TLR9 at later stages of infection (Fig.
5). These results suggested that inflammasome also plays an im-
portant role in immune responses to malaria infection. Further-
more, because infected MyD882/2, but not TLR92/2mice
showed markedly low levels of IL-1b and IL-18, collectively, the
above results suggested that TLR9-independent/MyD88-depen-
dent and/or IL-1R/IL-18R–mediated signaling is also involved in
the inflammatory cytokine responses to malaria parasites. Because
several cell types, including monocytes and NK, T, and B cells,
have IL-1R/IL-18R (50), which trigger signaling through MyD88
(51), it is likely that high levels of IL-18 produced during later
stages of infection induce IL-12 and IL-4 production by these
cells. This explains the increased production of IL-12 and IL-4 by
infected TLR92/2mice. Furthermore, because production of IL-
1b and IL-18 by inflammasome-mediated signaling requires the
pathogen recognition receptor-mediated expression of pro–IL-1b
and pro–IL-18, it appears that TLRs other than TLR9 also con-
tribute to certain extent in these cytokine responses. Because se-
rum cytokine profiles of infected TLR22/2and TLR42/2mice
were similar to those of the infected WT mice (see Fig. 3), despite
parasites having TLR2 and TLR4 as well as TLR11 ligands (10–
17), it appears that collective signaling strength of TLR2, TLR4,
and TLR11 is responsible for the MyD88-dependent cytokine-
inducing activity seen in TLR9-deficient mice.
TLR9 and MyD88 are essential for the development of
cell-mediated immunity to malaria
As shown in Fig. 1, at the early stages of infections, proin-
flammatory cytokine responses to malaria parasites by DCs are
essentially dependent on TLR9 and MyD88, whereas, as the in-
fection progresses, TLR9 and MyD88 differentially regulate these
cytokine responses in vivo. To determine whether TLR9 and
MyD88 similarly or differentially regulate cell-mediated immu-
nity to malaria parasites, we measured cytotoxic activity of NK
and CD8+T cells from infected WT, TLR92/2, and MyD882/2
mice at different stages of infection. It has been shown that GM-
DCs efficiently internalize IRBCs by the scavenger receptor
(CD36)-mediated and nonspecific phagocytosis and present Ags
to T cells (52–54). Therefore, we used IRBC-pulsed GM-DCs as
target cells for measuring T cell cytotoxic activity, and the cyto-
toxic activity of NK cells from infected mice was measured using
YAC-1 cells as a target. Both NK and CD8+T cells from infected
WT mice exhibited noticeable levels of cytotoxic activity to target
cells even at 5 d postinfection, and the activity was substantially
increased at later stages of infection (Fig. 6). In contrast, NK and
TLR9 independent but MyD88 dependent. Sera from P. yoelii-infected
mice at 5, 10, and 17 d postinfection were prepared as outlined in legends
to Fig. 3. The levels of IL-1b (A) and IL-18 (B) in pooled sera were
measured by ELISA. The letters, b and c, denote the statistical significance
between the levels of IL-1b and IL-18 produced by the indicated mice and
those produced by the corresponding infected WT mice.bp , 0.01,cp ,
Production of IL-1b and IL-18 during malaria infection is
5080TLR9 AND MyD88 REGULATE MALARIA IMMUNITY
CD8+T cells from infected MyD882/2mice exhibited little or no
cytotoxic activity at all stages of infection, and cells from infected
TLR92/2mice exhibited low levels of cytotoxic activity. To fur-
ther confirm the cytotoxic activity of CD8+T cells, we also
measured their ability to lyse anti-CD3ε–redirected EL-4 target
cells. As in the case of IRBC-internalized DCs, EL-4 cells were
efficiently lysed by WT CD8+T cells, but not by CD8+T cells
from MyD882/2mice; cells from TLR92/2mice showed low
levels of cytolytic activity (data not shown). These results dem-
onstrate that both TLR9 and MyD88 play important roles in NK
and T cells acquiring cytotoxic effector activity. These results,
together with the data that proinflammatory cytokine responses by
DCs at early stages of infection are critically dependent on both
TLR9 and MyD88 (see Fig. 1), demonstrate that DCs contribute
predominantly to the development of cytotoxic activity of NK and
CD8+T cells to malaria. Furthermore, the observed differences,
albeit low, in the cytotoxic activity of TLR9- and MyD88-deficient
cells suggest that TLR9-independent/MyD88-dependent and/or
IL-1R/IL-18R–mediated signaling also plays a role to a certain
extent in the development of cell-mediated immunity.
To determine whether the observed T cell cytotoxic activity was
Ag specific, we analyzed the ability of CD8+T cells from P. yoelii-
infected WT mice to lyse GM-DCs pulsed with P. berghei IRBCs;
GM-DCs pulsed with P. yoelii IRBCs were used as a control. The
cytotoxic activity against P. berghei Ags was ∼20% of that against
P. yoelii Ags. These results agree with the fact that many proteins
of P. yoelii and P. berghei are homologous (55), and strongly
suggest that the P. yoelii-induced cytotoxic activity of CD8+
T cells is largely, if not completely, Ag specific.
TLR9 and MyD88 modulate Ab responses to malaria infection
To determine whether TLR9 and MyD88 also contribute to the
humoral responses to malaria infection, we analyzed total Ab
responses and IgG subclasses in sera of parasite-infected mice. As
shown in Fig. 7, TLR92/2and MyD882/2mice produced similar
levels of Ab titer and similar Ab subclass profiles. Furthermore,
interestingly, WT mice showed significantly lower levels of total
IgG than TLR92/2and MyD882/2mice (Fig. 7A); this appears to
be in response to the significantly lower levels of parasitemia in
WT mice than in TLR92/2and MyD882/2mice. Ig subclass
analysis showed significantly higher levels of IgG1 in the sera of
parasite-infected TLR92/2and MyD882/2mice than in the sera
of WT mice (Fig. 7B). In contrast, levels of IgG2a and IgG2b
were significantly higher in infected WT mice than in TLR92/2
and MyD882/2mice. The observed high levels of Th1-type Abs
in WT mice and higher level of Th2-type Abs in TLR92/2and
MyD882/2mice are consistent with robust Th1 responses driven
by WT DCs and significantly higher type 2 cytokine responses by
TLR92/2and MyD882/2DCs. These results are consistent with
and T cell cytotoxic activity. Liver NK cells (A–C) and spleen CD8+
T cells (D–F) from P. yoelii-infected WT, TLR92/2, and MyD882/2mice
at 5, 10, and 17 d postinfection were cocultured, respectively, with YAC-1
and P. yoelii IRBC-pulsed GM-DCs as target cells, as described in
Materials and Methods. The percentages of target cell lysed were plotted.
Data shown are a representative of two independent experiments. (G) Plots
of percentage of cell lysis of GM-DCs pulsed with either P. yoelii (closed
circle) or P. berghei (open circle) IRBCs by spleen CD8+T cells from WT
mice at 10 d postinfection. The letters, b and c, represent the statistical
significance between the cytotoxic activity of NK and T cells from the
infected TLR92/2mice and that of NK and T cells from the infected WT
mice.bp , 0.01,cp , 0.05.
TLR9 and MyD88 are essential for the development of NK
parasites. Total IgG levels (A) and Ab isotypes (B) in the pooled sera of P.
yoelii-infected WT, TLR92/2, and MyD882/2mice at 40 d postinfection
were analyzed by ELISA. Sera were 1:3 serially diluted for total IgG
analysis, and Ab isotype analysis was performed using 1:8100 diluted sera.
Serum from naive WT mice was used as a control. The letters a, b, and c
represent statistical significance between the levels of total IgG or Ab
isotypes in sera of infected TLR92/2or MyD882/2mice and those in sera
of infected WT mice. Data shown are a representative of two independent
experiments.ap , 0.001,bp , 0.01,cp , 0.05.
TLR9 and MyD88 play roles in Ab responses to malaria
The Journal of Immunology5081
the previous report that, in the case of P. chabaudi chabaudi AS
infection, MyD882/2mice produced higher levels of IgG1 than
WT and TLR22/2mice (18). Overall, the above results demon-
strate that TLR9 and MyD88 significantly regulate humoral
responses to malaria parasites that are most likely driven by DCs
at early stages of infection.
TLR9 and MyD88 significantly contribute to
Finally, we assessed the contributions of TLRs and MyD88 to the
development of overall protective immunity to malaria by mea-
suring the progression of infection in WT, TLR22/2, TLR42/2,
TLR92/2, and MyD882/2mice and by assessing the survival of
infected animals. In all mouse types studied, parasitemia remained
low during the first week of infection and thereafter parasites grew
exponentially, reaching peak parasitemia between 18 and 20 d
postinfection (Fig. 8A, 8B). After 3 wk, parasitemia rapidly de-
creased in survived mice, and infection was completely cleared at
the end of the fourth week. During the exponential growth, par-
asitemia was 2-fold higher in MyD882/2than in WT, TLR22/2,
and TLR42/2mice. TLR92/2mice showed noticeably lower levels
of parasitemia than MyD882/2mice, but exhibited considerably
higher parasitemia than WT, TLR22/2, and TLR42/2mice. Com-
pared with WT mice, both MyD882/2and TLR92/2mice were
more susceptible to infection, and 80–90% of mice eventually
succumbed to death (Fig. 8C, 8D). These results are consistent
with the higher cytotoxic activity and Th1-type Ab responses by
WT mice than TLR92/2and MyD882/2mice. Thus, collectively,
our results demonstrate that TLR9 and MyD88 regulate innate
immune responses as well as cellular and humoral immunity to
malaria infection, contributing to the development of protective
The results presented in this work allow us to make two important
conclusions that have broader implications in understanding the
molecular mechanisms involved in the regulation of innate and
adaptive immune responses to malaria. For one, TLR9 and MyD88
play central roles in the regulation of pro- and anti-inflammatory
responses and Th1 and Th2 responses, and in the development of
cytotoxic effector function and Ab responses to malaria infection.
This conclusion is supported by the following findings. The de-
ficiency in either TLR9 or MyD88 but not that in TLR2 or TLR4
leads to the following: 1) markedly decreased production of
proinflammatory cytokines with concomitant increase in the pro-
ductionoftype 2/anti-inflammatorycytokinesbyDCs; 2)increased
commitment to Th2 development; 3) impaired NK and T cell
cytolytic activity; 4) significantly lower levels of Th1-type Ab
responses and increased Th2-type Abs; and 5) significantly higher
levels of parasitemia and increased susceptibility to malaria. Sec-
ondly, the inflammasome as well as TLR9-independent/MyD88-
dependent- and/or IL-1R/IL-18R–mediated signaling also con-
tribute substantially to the development of protective immunity to
malaria. This conclusion is evident from our findings that infected
TLR92/2mice produced higher levels of IL-1b and IL-18 than
infected MyD882/2mice. Furthermore, NK and CD8+T cells
from infected TLR92/2mice exhibit cytotoxic activity, albeit at
marginal levels, whereas the corresponding cells from the infected
MyD882/2mice showed little or no cytotoxic activity (see Fig. 6).
Thus, our study provides valuable insights into role of TLRs in the
regulation of innate and adaptive immune responses to malaria
Our data also clearly demonstrate that TLR9 and MyD88 play
critical roles in the development of protective immunity to malaria
infection. Studies have shown that protective immunity to malaria
involves strong early proinflammatory cytokine responses and Th1
polarization, leading to effective cell-mediated and humoral
responses (2–4). In contrast, higher anti-inflammatory and type 2
cytokine responses at the early stages of infection result in in-
ability to control infection, leading to pathogenesis (2–4). In
agreement with this notion, in the current study, parasite-infected
WT mice that were protected from infection, but not susceptible
TLR92/2and MyD882/2mice, produced high levels of proin-
flammatory cytokines and low levels of type 2/anti-inflammatory
cytokines, and were strongly Th1 polarized, elicited efficient NK
and T cell cytotoxic activity, and produced higher levels of pro-
tective Th1-type (56), opsonizing IgG2a and IgG2b, which are
likely to aid effective parasite clearance. In contrast, the deficiency
in TLR9 resulted in substantially lower levels of proinflammatory
cytokines and higher levels of type 2/anti-inflammatory cytokines
at early stages of infection, increased production of both pro- and
anti-inflammatory responses at later stages of infection, and sig-
deficient mice exhibit higher parasitemia and mortality
than infected WT, TLR22/2, and TLR42/2mice. WT,
TLR22/2, TLR42/2, TLR92/2, and MyD882/2mice
(n = 10 in each case) were infected with P. yoelii. (A
and B) Blood parasitemia was measured every 2
d postinfection, and mean parasitemia in survived ani-
mals is plotted. (C and D) The infected mice were
monitored twice daily, and their survival rates are
plotted. Experiments were performed twice. The letters
a, b, and c denote the statistical significance of para-
sitemia and survival rates between TLR92/2and
MyD882/2mice and infected WT mice.ap , 0.001,
bp , 0.01,cp , 0.05.
P. yoelii-infected TLR9- and MyD88-
5082TLR9 AND MyD88 REGULATE MALARIA IMMUNITY
nificantly impaired NK and CD8+T cell cytotoxic activity. The
MyD882/2deficiency, in contrast, caused low levels of both
proinflammatory and anti-inflammatory cytokine responses at all
stages of infection and completely impaired NK and CD8+T cell
cytotoxic activity. Furthermore, TLR9 or MyD88 deficiency sig-
nificantly limited the production of Th1-type IgGs, which are
known to be protective (56), while increasing the levels of Th2-
type IgGs. The proinflammatory cytokines and strong Th1 polar-
ization are known to increase macrophage phagocytic activity and
proliferation of cytotoxic CD8+T cells, and promote the pro-
duction of opsonizing Abs (57, 58). All of these can contribute to
efficient parasite clearance, thereby providing protection against
infection in WT mice. Effective clearance of IRBC-internalized
macrophages, DCs, and other phagocytic cells is also likely to be
important for allowing the immune system to function efficiently
against malaria infection; this function is severely compromised in
TLR92/2and MyD882/2mice. Thus, our conclusion that TLR9
and MyD88 are crucial for the development of protective immu-
nity to malaria is supported by the observed markedly lower
ability of TLR92/2and MyD882/2mice to control parasitemia
and substantially higher susceptibility to death compared with WT
mice. Our conclusion also agrees with the results of recent studies
that TLR9 provides protection against cerebral malaria in P.
berghei ANKA-infected mice (59), MyD88 protects P. chabaudi
chabaudi-infected mice from malaria illness (18), and MyD88 is
also involved in the control of early parasitemia in nonlethal P.
yoelii infection (60).
The results of this study further demonstrate that TLR9-
independent/MyD88-dependent and/or IL-1R/IL-18R–mediated
signaling were also significantly involved in the regulation of
immune responses to malaria infection. This is evident from our
observation that P. yoelii-infected MyD882/2mice produced
markedly low levels of both pro- and anti-inflammatory cytokines,
whereas the infected TLR92/2mice produced substantial levels of
both types of cytokines (see Fig. 3). Furthermore, in a preliminary
study using FL-DCs, we noted that DCs deficient in TLR9 pro-
duced appreciable levels of TNF-a and IL-12 upon stimulation
with P. yoelii-infected erythrocytes, whereas DCs deficient in
MyD88 were unable to produce these cytokines (N.M. Gowda,
X. Wu, and D.C. Gowda, unpublished results). Malarial GPIs,
microparticles from IRBCs, and heme released from the parasite-
infected erythrocytes have been shown to activate macrophages
through TLR2 or TLR4 (10–12). However, the effects of TLR2 or
TLR4 were not evident either from the serum cytokine profiles of
the infected mice or from the cytokine pattern produced by DCs
ex vivo (see Figs. 1–3). Compared with other protozoan parasites
such as Trypanosoma and Leishmania, malaria parasites express
relatively low levels of GPIs (61, 62), and also it is possible that
levels of microparticles released by parasite-infected erythrocytes
are low. Furthermore, although malaria parasite’s profilin has been
reported to activate DCs via TLR11 to produce IL-12 (16, 17), its
activity has been reported to be low (63). Therefore, it appears that
individually these receptors are unable to activate cells to a sig-
nificant extent to induce strong immune responses, but collective
signaling by TLR2, TLR4, and TLR11 significantly activates the
innate immune system in TLR92/2mice in vivo to induce cyto-
kine responses by DCs and other cells. Moreover, the production
of IL-1b and IL-18 at high levels by P. yoelii-infected TLR92/2
mice, but not by MyD882/2mice (see Fig. 5), suggests that
MyD88-dependent, inflammasome- and IL-1R/IL-18R–mediated
signaling also contributes substantially to the activation of cells of
the immune system, especially during later stages of infection
when IL-18 is produced at higher levels. Therefore, cytokines
produced in response to MyD88-mediated signaling, involving
TLR2, TLR4, TLR11, and/or IL-1R/IL-18R, collectively account
for the observed high levels of serum cytokines in P. yoelii-
infected TLR92/2mice. This explains why parasite-infected
MyD882/2mice produce only marginal levels of cytokines.
The results presented in this work additionally demonstrate that
IL-18, which has been shown to play an important role in malaria
infection (60, 64–66), is produced in a TLR9-independent and
MyD88-dependent manner. IL-18 is known to activate NK and
T cells through its receptor-mediated signaling to produce IFN-g,
contributing to Th1 cell development (50, 67). Additionally, IL-18
enhances NK cell cytotoxicity, Fas ligand-mediated Th1 cell cy-
totoxicity, and proliferation of activated T cells (67, 68). In the
parasite-infected mice, parallel to IL-18 induction, IL-12 is pro-
duced at increased levels as infection progresses (see Figs. 3C,
5B). IL-12 and IL-18 have shared functions and, thus, they syn-
ergistically and independently activate NK, T, and B cells through
their receptors to produce cytokines (50, 69, 70). IL-18 is also
known to upregulate IL-12R, and IL-12 upregulates IL-18R (70).
Thus, during malaria infection, IL-18 appears to collaborate with
IL-12 in the development of Th1 cells and enhancement of NK
and T cell cytotoxic activity by promoting cell proliferation. This
is apparent from the observed high levels of proinflammatory
cytokines in mice at later stages of infection and increased cyto-
toxic activity of NK and T cells as infection progresses. Previous
studies have shown that IL-18 and IL-12 are protective to P. yoelii
and P. chabaudi malaria (71–73). IL-18 has also been shown to be
protective against severe malaria in human (64–66). Based on
these observations, it is expected that TLR92/2mice should be
protected from malaria infection as they produce high levels of IL-
18 and IL-12. However, IL-18 is also known to induce anti-
inflammatory cytokines and Th2 responses depending on the cy-
tokine milieu of the system (74). Therefore, unusually high levels
of IL-4 seen in the infected TLR92/2mice in parallel with in-
creased production of IL-18 are most likely due to the IL-18–
mediated Th2 responses. Because the deficiency in TLR9 caused
Th2-biased responses, IL-18 can efficiently augment Th2 re-
sponses in TLR92/2mice. Therefore, it appears that IL-18 by
efficiently driving both Th1 and Th2 responses neutralizes the
protective effect of Th1 responses by Th2 responses in TLR92/2
mice. Thus, this explains why TLR92/2mice, similar to MyD882/2
mice, have impaired cell-mediated and protective Ab responses,
and thus, are more susceptible to malaria infection than WT mice.
Overall, results of the current study demonstrate that initial
robust proinflammatory cytokines are critical for the development
of protective immunity to malaria. DCs are the major cells of the
innate immune system, which play a crucial role in the initiation of
innate immune responses and development of adaptive immunity
by connecting the innate immune arm to that of adaptive immune
system. This is evident from our observation that DCs from the
infected WT mice produced substantially high levels of proin-
flammatory cytokines at early stages of infection that correlated
with high levels of NK and CD8+T cell cytotoxicity and protec-
tive Th1 Ab production. In contrast, DCs from TLR92/2and
MyD882/2mice produced low levels of proinflammatory cyto-
kines that resulted in complete absence or marginal levels of cy-
totoxic activity and decreased Th1 Ab production.
Finally, our observations have broader implication in under-
standing the roles of TLRs in the regulation of innate and adaptive
immunity in other pathogenic infections as well. Although TLRs
have been recognized to be crucial for producing proinflammatory
responses during infections by diverse group of pathogens such as
bacteria, fungi, and parasites, the roles of these receptors in the
regulation of immune responses remain poorly understood. The
The Journal of Immunology 5083
in the regulation of innate and adaptive immunity to various
pathogenic microorganisms. Detailed understanding of the TLR-
dependent immune regulations is likely to provide strategies for
the development of therapeutics or vaccines against pathogenic
We thank Drs. Shizuo Akira and Tatoshi Uematsu (Research Institute for
Microbial Diseases, Osaka University, Osaka, Japan) for providing TLR
and MyD88 knockout mice; Dr. Glenn Dranoff (Dana-Farber Cancer Insti-
tute and Harvard University Medical School) for giving FLT3 ligand-
expressing B16 cells; Drs. Christopher Norbury and Todd Schell (Depart-
ment of Microbiology and Immunology, Hershey Medical Center, Hershey,
X63 melanoma cells and EL-4 cells; and Dr. Nikki Keasey (Department of
Hematology/Oncology, Hershey Medical Center, Hershey, PA) for provid-
ing YAC-1 cell line.
The authors have no financial conflicts of interest.
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