Am. J. Trop. Med. Hyg., 82(4), 2010, pp. 548–555
Copyright © 2010 by The American Society of Tropical Medicine and Hygiene
Cerebral malaria (CM) affects over half a million African
children per year and has a case fatality rate of 15% to
40%. 1 , 2 Cerebral malaria is defined as coma in the presence
of Plasmodium falciparum parasitemia, with no other cause of
coma identified. 3 For children who survive, 10–17% may suf-
fer from neurological sequelae such as epilepsy, cerebral palsy,
cortical blindness, and deafness, 2 and up to 21% may suffer
cognitive impairment. 4
Excessive serum levels of pro-inflammatory cytokines, par-
ticularly interferon-γ (IFN-γ) and tumor necrosis factor-α
(TNF-α), have been implicated in the pathogenesis of CM in
animal and human studies. 5 – 12 Human studies have shown an
association between CM and elevated IFN-γ 13 or TNF-α 6 , 8 , 14 , 15
levels, and our previous studies documented an association
between elevated IFN-γ levels and mortality in CM. 13 In addi-
tion, some human studies 16 and several animal studies 5 , 12 , 17
have provided pathological evidence that one or both cytok-
ines are involved in CM pathogenesis.
Toll-like receptors (TLRs) are mammalian pattern recog-
nition receptors that are found primarily in monocytes, mac-
rophages, and dendritic cells. These antigen-presenting cells
recognize and signal in response to microbial ligands that are
bound by the TLRs. As a result, the innate immune system
is triggered to produce specific cytokines to contain or elimi-
nate the microbial infection. In vitro evidence suggests that
TLR2, TLR4, and TLR9 recognize and signal in response to
P. falciparum ligands. Murine studies suggest that TLR2, and
to a lesser extent, TLR4 recognition of P. falciparum glyco-
phosphatidylinositol, are associated with increased production
of TNF-α, 18 and that TLR9 signals in response to hemozoin,
a by-product of host hemoglobin digestion by malaria para-
sites, and upregulates TNF-α, IL-12p40, monocyte chemoat-
tractant protein (MCP)-1, and IL-6 production by dendritic
cells. 19 Hemozoin acts as a carrier to facilitate entry of malar-
ial DNA into the host cell, where the latter can bind to, and
stimulate TLR9. 20 Human studies have shown that ligand in
P. falciparum schizont extract stimulates human plasmacytoid
dendritic cells to upregulate populations of γδ T cells, which
increase IFN-γ production by a TLR9-dependent pathway. 21
Toll-like receptor single nucleotide polymorphisms (SNPs)
or corresponding amino acid substitutions have been asso-
ciated with malaria manifestations and parasitemia in sev-
eral recent studies. 22 – 25 We focused on two studies, because
their results suggested an increase in clinical malaria sever-
ity and malaria-related complications in P. falciparum -infected
patients in Africa, where we have two study sites. In the first
study, TLR4 Asp299 Gly was associated with an increased risk
of maternal anemia, and TLR4 Asp299 Gly and the C allele
at TLR9 −1486 were associated with subsequent infant low-
birth weight in pregnant Ghanaian women with malaria. 22 In
the second, the risk of severe malaria in Ghanaian children
was increased 1.5-fold and 2.6-fold with TLR4 Asp299 Gly and
TLR4 Thr399 Ile , respectively. 23 Another study by Campino and
others, 24 based in The Gambia and Malawi, studied the effect
of TLR9 genetic variation on severe malaria. The TLR9 SNPs
included all 3 SNPs investigated in this study, plus the G2848A
SNP on exon 2 of the TLR9 gene. Although there was “no con-
vincing association” between TLR9 SNPs and malaria severity
in that study, the authors concluded that “ TLR9 expression is
potentially modulated through cis -regulatory variants, which
may lead to differential inflammatory responses to infection
These studies provided intriguing new information about
TLR SNPs and malaria disease severity, but did not further
investigate the potential mechanisms by which TLR SNPs
may mediate pathogenesis.
In this study, we hypothesized that TLR SNPs affect malaria
severity through inappropriate TLR signaling, causing down-
stream elevations in pro-inflammatory cytokine levels that in
turn result in more severe manifestations of malaria. To test the
relationship between TLR SNPs, cytokine production, and dis-
ease severity, TLR2, 4, and 9 SNP frequencies and serum levels
of IFN-γ and TNF-α, were compared in Ugandan children 3–12
years of age with CM and uncomplicated malaria (UM).
MATERIALS AND METHODS
Study population and recruitment. The study was conducted
at our study site at the Mulago Hospital, in Kampala, Uganda,
TLR9 Polymorphisms Are Associated with Altered IFN-γ Levels
in Children with Cerebral Malaria
Nadia A. Sam-Agudu ,† Jennifer A. Greene ,† Robert O. Opoka , James W. Kazura , Michael J. Boivin , Peter A. Zimmerman ,
Melissa A. Riedesel , Tracy L. Bergemann , Lisa A. Schimmenti , and Chandy C. John *
University of Minnesota, Minneapolis, Minnesota; Case Western Reserve University, Cleveland, Ohio; Makerere University, Kampala, Uganda;
Michigan State University, East Lansing, Michigan
Abstract. Toll-like receptor (TLR) polymorphisms have been associated with disease severity in malaria infection,
but mechanisms for this association have not been characterized. The TLR2, 4, and 9 single nucleotide polymorphism
(SNP) frequencies and serum interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) levels were assessed in Ugandan
children with cerebral malaria (CM, N = 65) and uncomplicated malaria (UM, N = 52). The TLR9 C allele at −1237 and
G allele at 1174 were strongly linked, and among children with CM, those with the C allele at −1237 or the G allele at
1174 had higher levels of IFN-γ than those without these alleles ( P = 0.03 and 0.008, respectively). The TLR9 SNPs were
not associated with altered IFN-γ levels in children with UM or altered TNF-α levels in either group. We present the first
human data that TLR SNPs are associated with altered cytokine production in parasitic infection.
* Address correspondence to Chandy C. John, Center for Global
Pediatrics, 717 Delaware Street SE, Mail Code 1932, Minneapolis, MN
55414. E-mail: firstname.lastname@example.org
† Nadia Sam-Agudu and Jennifer Greene contributed equally to this
TLR POLYMORPHISMS AND IFN-g LEVELS IN MALARIA
from November 2003 to July 2004. This time period spanned one
major rainy season (March through May) and one minor rainy
season (November to December.) Children 3–12 years of age
were recruited as part of two studies assessing the complications
of CM. Eighty-six children with CM and 76 children with
UM were enrolled. Children with CM were enrolled if they
were admitted to Mulago Hospital and met the World Health
Organization (WHO) criteria for CM: coma (Blantyre coma
scale £ 2 or Glasgow coma scale £ 8), P. falciparum on blood
smear, and no other cause for coma. Lumbar punctures were
performed to rule out meningitis and encephalitis. Children
with UM were enrolled from the hospital’s acute care clinic
or another outpatient malaria clinic at the hospital. Children
were considered to have UM if they had signs and symptoms of
malaria (fever, chills, vomiting, headache), P. falciparum infection
on blood smear, and no evidence of malaria complications
(e.g., seizures, respiratory distress, severe anemia, or coma) or
other acute illness. Genetic testing was requested from study
participants on study follow-up testing 2 years after enrollment.
If consent was obtained from the study participant’s parent or
guardian, testing for TLR SNPs was performed on the filter
paper samples collected at initial enrollment. Sixty-five children
with CM and 52 children with UM consented to genetic studies.
Blood samples of 5 mL were obtained on enrollment.
Serum was frozen at −70°C until testing was performed.
Blood was collected on Whatman FTA filter paper (Whatman
Corporation, Florham Park, NJ) for future DNA extraction
and testing. Presence of P. falciparum was determined by light
microscopy of thick and thin blood smears, with two indepen-
dent readings, and a third independent reading if necessary to
resolve any discrepancies between the two initial readings.
Written informed consent was obtained from the parents or
guardians of study participants. Ethical approval for the study
was granted by the Institutional Review Boards for Human
Studies at Makerere University Faculty of Medicine, Case
Western Reserve University, Indiana Wesleyan University,
and the University of Minnesota.
DNA extraction. Genomic DNA was extracted from filter
paper samples according to instructions from the QIAamp
DNA mini kit (QIAGEN, Valencia, CA) We assessed for
common TLR2, TLR4, and TLR9 SNPs ( Table 1 ). These SNPs
were selected because they have been associated with malaria
severity in African populations 22 , 23 and with other infectious
and autoimmune diseases in different populations. 26 – 32
Polymerase chain reaction. Polymerase chain reaction
(PCR) was performed using a master mix consisting of 1×
PCR buffer, 125 μM dNTPs, 2.5 mM MgCl 2 , 125 nM primers,
and 0.8 units of Taq polymerase in a reaction volume of 25 μL.
The PCR primers and amplification conditions are listed in
Supplemental Table 1 (available online at www.ajtmh.org ).
The PCR products for SNP detection were analyzed on a 2%
agarose gel before the ligase detection reaction-fluorescent
microsphere assay (LDR-FMA ).
Cloning and sequencing. The PCR amplification products were
purified using the QIAquick PCR purification kit (QIAGEN,
Valencia, CA). Purified PCR products were sent to MWG Biotech
(High Point, NC) for sequencing. Sequences were analyzed using
the Sequencher software (Gene Codes Corporation, Ann Arbor,
MI). Sequenced DNA templates were subjected to PCR and
used in the LDR-FMA as positive controls.
SNP genotyping. Polymerase chain reaction products were
analyzed in an LDR-FMA divided into three steps: 1) ligation
of oligonucleotides to the SNP, 2) FlexMAP (Luminex Corp.,
Austin, TX ) microsphere hybridization, and 3) detection using the
Bioplex suspension array system, which includes a fluorescence
reader and the Bio-Plex Manager analytical software (Bio-
Rad Laboratories, Hercules, CA). This procedure is described
in detail elsewhere. 33 Common/conserved and allele-specific
probe sequences used in this assay are listed in Supplemental
Supplemental Table 2 (available online at www.ajtmh.org ).
A multiplex assay was used for simultaneous detection of TLR2
and TLR9 SNPs. The TLR4 SNPs were evaluated in a separate
multiplexed LDR. Equal volumes of each PCR product were
mixed, and 1 μL was added to the LDR. Conditions for the LDR
step of this assay are described elsewhere. 33 Mean fluorescence
intensity (MFI) values were used to calculate the allelic ratio for
each SNP by dividing the allele-specific MFI value by the sum
of the MFI values for that SNP (allele A/A + B = A n and allele
B/A + B = B n ), where A and B are the 2 alleles of a SNP. To
be homozygous for a particular allele, the allelic ratio must be
> 0.75. To be heterozygous, the ratio of the two alleles must be
between 0.25 and 0.75. Consequently, an allele included in a ratio
of < 0.25 is considered not present. Normalized values for A n and
B n were divided, and the quotient was log-transformed.
Cytokine testing. We compared serum IFN-γ and TNF-α
levels in children with TLR SNP variants because these
cytokines have been implicated in the development of CM
in previous studies (see Introduction). Serum samples for
cytokine measurement were obtained at the time of admission
(CM), outpatient treatment (UM), or enrollment (CC). Levels
of IFN-γ and TNF-α were also determined by microbead
suspension array technology using the Bioplex-Luminex
system (Austin, TX). 13 Results were interpolated from
5-parameter-fit standard curves generated with the relevant
recombinant human proteins (R&D Systems, Minneapolis,
MN). Samples were tested at a 1:8 dilution. Cytokine assay
results in the full cohort have previously been published. 13
Statistical analysis. Statistical analysis was performed with
Stata 10 software (StataCorp, College Station, TX) and R
version 2.8 ( http://www.r-project.org ). Genotype frequencies
in children with CM and UM were compared by the χ 2 test
or Fisher’s exact test as appropriate. Cytokine levels across
groups (common allele homozygotes, heterozygotes, rare allele
homozygotes) were compared using a log-additive model (in
R, linear model [lm] function). Linkage disequilibrium was
assessed by Lewontin’s D’ and the correlation coefficient r
statistics, calculated from estimates provided in Devlin and
Risch 34 requiring estimation of the two loci haplotype. The
χ 2 test was calculated from the statistic provided in Weir. 35
TLR2, TLR4, and TLR9 single nucleotide polymorphism (SNPs)
SNP LocationNucleotide change GenBank accession no.
C ® A
G ® A
A ® G
C ® T
T ® C
T ® C
G ® A
SAM-AGUDU, GREENE AND OTHERS
The 2-locus haplotype for each person in the dataset and the
posterior probability of that haplotype were calculated using
the EM algorithm, 36 and associations between haplotype
and cytokine levels assessed by the methods of Schaid and
others. 37 To control the experiment-wise error rate, P values
were adjusted by the Bonferroni correction for multiple
comparisons. The primary analysis, comparison of IFN-γ
levels according to the 3 TLR9 SNPs, is a single experiment.
With this correction, at a false positive rate of 5%, P values of
< 0.017 were considered statistically significant.
Study participant characteristics. Sixty-five children with
CM and 52 with UM consented to genetic testing and had
samples from enrollment available for SNP genotyping. Clinical
and demographic characteristics of the study participants
are outlined in Table 2 . As compared with children with UM,
children with CM were younger and more often male. Fifty-
nine of 65 children with CM and 47 of 52 children with UM
had adequate serum for cytokine testing.
TLR2, 4, and 9 SNP frequencies. The TLR2 SNPs showed no
rare alleles (i.e., all children were CC for Pro 631His and GG
for Arg 753Gln). Rare alleles of the TLR4 SNPs (Asp299 Gly
and Thr399 Ile) occurred at a genotype frequency of < 13% in
children with CM or UM ( Table 3 ). In contrast, for the three
TLR9 SNPs assessed, rare allele genotype frequencies (C at
−1486, C at −1237, A at 1174) exceeded > 40% in children
with CM or UM ( Table 4 ). Overall, frequency of the C
allele at −1237 did not differ between children with CM and
children with UM, but children with CM were more frequently
homozygotes for the C allele (CC) than children with UM
(18.5% versus 5.8%, P = 0.05). In contrast, for the intronic
A allele at 1174 and the C allele at −1486, neither allele nor
homozygote frequency differed significantly between children
with CM and those with UM ( Table 4 ).
IFN-g and TNF-a levels in children with CM according to
TLR4 and TLR9 SNPs. The TLR4 Arg299Trp, which had a
genotype frequency of 12.3% in children with CM, was the
only TLR4 SNP variant with > 2% frequency in children with
CM. There were no homozygotes for this variant, and there
was no difference in IFN-γ or TNF-α levels in individuals
with or without the variant (data not shown). The TLR9 SNP
variants or rare alleles occurred in frequencies high enough
to allow comparison of IFN-γ and TNF-α levels according to
SNP variant. Children with CM and the rare C allele at −1237
had higher levels of IFN-γ than those without this allele, and
children with CM and the common G allele at 1174 had higher
levels of IFN-γ than those without this allele ( Figure 1 ). For
both the C allele at −1237 and the G allele at 1174, there was
a “dose-response” relationship, with increasing IFN-γ levels
in homozygotes as compared with heterozygotes ( Figure 1 ).
Fitting a log-additive model to test the association between
each of these polymorphisms with the response yields
significance levels of 0.03 for the C allele at −1237 and 0.008
for the G allele at 1174.
The C allele at −1237 was in linkage disequilibrium with the
G allele at 1174 in children with CM (Lewontin’s D' 0.885,
correlation coefficient 0.60, χ 2 = 23.35 P < 0.0001) or UM
(Lewontin’s D' 0.907, correlation coefficient 0.55, χ 2 = 16.08
P < 0.0001). Genotype frequencies and IFN-γ levels for the
various −1237 and 1174 genotype combinations in children
who had IFN-γ measured are shown in Table 5 . To assess if
the −1237 and 1174 SNPs were independently associated with
IFN-γ levels, the 2-locus haplotype was first estimated for each
child. In children with CM, −1237/1174 haplotype frequencies
were 0.019 (C/A), 0.381 (C/G), 0.389 (T/A), and 0.211 (T/G).
Patient demographic and clinical characteristics*
CM ( N = 65) UM ( N = 52) P
Age, years: mean (SD)
Sex: male (%)
Weight, kg: mean (SD)
Pre-presentation illness/fever duration (days): mean (SD)
Children with any antimalarial pretreatment (%)
No antimalarials/not sure
Parasite density/μL, median (interquartile range)
* CM = cerebral malaria; UM = uncomplicated malaria.
† Wilcoxon ranksum test.
‡ χ 2 test.
TLR4 SNP genotype frequencies in children with CM vs. UM*
CM ( N = 65)
UM ( N = 52)
No. (%) P †
Asp299Gly (A ® G)AA
Thr399Ile (C ® T)
* SNP = single nucleotide polymorphism; CM = cerebral malaria; UM = uncomplicated
† Fisher’s exact test, comparing proportions in the three groups.
TLR9 SNP genotype frequencies in children with CM vs. UM*
TLR9 SNPCM ( N = 65) No. (%) UM ( N = 52) No. (%) P †
* SNP = single nucleotide polymorphism; CM = cerebral malaria; UM = uncomplicated
† Fisher’s exact test, comparing proportions in the three groups.
TLR POLYMORPHISMS AND IFN-g LEVELS IN MALARIA
Using the method of Schaid and others, 37 significant differ-
ences were seen in IFN-γ levels between the four haplotypes
(χ 2 = 8.34, P = 0.039). Haplotype scores for specific −1237/1174
haplotypes were 2.56 for C/G ( P = 0.01), −2.33 for T/A ( P =
0.02), and 0.05 for T/G ( P = 0.96). A C/A haplotype-specific
score was not calculated because this haplotype was very infre-
quent. The IFN-γ levels did not differ according to the pres-
ence of the C allele at −1486 in children with CM ( Figure 1 ).
The TNF-α levels did not differ according to any TLR9 SNP
genotype in children with CM ( Figure 2 ).
IFN-g and TNF-a levels in children with UM according
to TLR4 and TLR9 SNP genotypes. The IFN-γ and TNF-α
levels did not differ according to any TLR9 SNP genotype in
children with UM ( Figures 3 and 4 ). Less than 10% of children
with UM had the TLR4 Asp299 Gly variant, and there was no
difference in IFN-γ ( P = 0.57) or TNF-α ( P = 0.84) levels in
children with as compared with those without this variant.
Toll-like receptor polymorphisms have been associated
with an altered risk of disease or disease severity in numerous
infections, 26 , 28– 30 , 32 , 38 including malaria. 22 , 23 However, potential
causal mechanisms for the associations between TLR SNPs
Figure 1. Interferon-γ (IFN-γ) levels by TLR9 single nucleotide
polymorphism (SNP) in children with cerebral malaria (CM).
Figure 2. Tumor necrosis factor-α (TNF-α) levels by TLR9 single
nucleotide polymorphism (SNP) in children with cerebral malaria (CM).
SAM-AGUDU, GREENE AND OTHERS
and disease severity in malaria have not been described. In
this study, we documented that TLR9 SNP genotypes are asso-
ciated with altered serum IFN-γ levels in children with CM.
This study is the first to report TLR SNP-related alterations in
cytokine levels in humans in response to a parasitic infection,
and suggests a mechanism by which TLR SNPs may relate
to disease severity in P. falciparum infection. Previous stud-
ies have documented elevated IFN-γ levels in children with
severe as compared with uncomplicated malaria, 13 , 39 and an
earlier report of the cohort of children described here 13 showed
a further increase in IFN-γ levels in children with CM who die
as compared with survivors. In a human study, it is difficult
to assess definitively whether IFN-γ levels are part of patho-
genesis of disease or an epiphenomenon, but several studies
have implicated a causal role for IFN-γ in a mouse model of
CM pathogenesis. 7 , 10 , 17 , 40 , 41 If elevated IFN-γ levels contribute
to the pathogenesis of human CM, then children with the C
allele at TLR9 −1237 or the G allele at TLR9 1174 may be
more likely to develop CM because these alleles are associ-
ated with increased IFN-γ levels in severe P. falciparum infec-
tion. The lack of association between these TLR9 SNPs and
altered IFN-γ levels in children with UM suggests that an addi-
tional factor may be required to alter IFN-γ levels in infected
Hemozoin is a potential co-factor that could functionally
affect TLR9 response to P. falciparum infection. The TLR9 is
located in the endosomal/lysosomal compartment of dendritic
and other cell types, and typically recognizes nucleic acids
common in microbes, particularly CpG DNA. 42 , 43 Plasmodium
falciparum , although possessing an AT-rich genome, 44 has
potentially stimulatory CpG motifs and may require a carrier
to transport its DNA to the endosomal compartment. A recent
study by Parroche and others 20 suggested that hemozoin is not
a primary ligand for TLR9 but functions as a carrier for P. fal-
ciparum DNA. Greater levels of hemozoin may for this rea-
son lead to increased TLR9 activation. Several studies have
documented that hemozoin load is higher in children with
severe malaria as compared with uncomplicated malaria. 45 – 47
A difference in hemozoin level may have allowed greater
endosomal transport of parasite DNA and TLR9 activation
in children with CM relative to those with UM. Unfortunately,
microscope slides were not placed in long-term storage so we
were not able to assess hemozoin load and test this hypoth-
esis. Future studies will assess the interactions between TLR9
SNPs, hemozoin load, and IFN-γ levels.
Evidence from transfected animal and human cells shows
that TLR SNPs can affect signaling and ultimately, cytokine
production in response to infection. 26 , 38 , 48 , 49 These exaggerated
responses may involve several mechanisms, including changes
in ligand binding sites on the receptor, thereby affecting ligand
affinity and strength of stimulation, changes in transcription
factor binding sites on the TLR promoter, 50 or qualitative/
quantitative changes in the TLR protein. In regard to TLR9
SNP genotypes, the C allele at −1237, in the promoter region,
has been shown to affect promoter activity, 51 most likely by
modifying a potential binding site for the transcription factor
NF-κB. 50 NF-κB is a complex of proteins that remains in cells in
an inactive state and is rapidly activated by a series of cascade
events after ligands are bound to TLRs. Qualitative or quanti-
tative changes in NF-κB activation may in turn lead to altered
transcription regulation of inflammatory cytokine genes, which
could lead to alterations in the production of cytokines such
as IFN-γ. The 1174 SNP is located in an intron in the TLR9
gene. Though located in a non-coding region, variants in such
an SNP could affect signaling by creating alternative splicing
sites and consequently, affecting the protein product. In this
study, the rare C allele at −1237 and the common G allele at
1174 were associated with increasing IFN-γ levels, and hap-
lotype analysis suggested that both genotypes contributed to
the differences in IFN-γ levels. TLR9 may also affect signal-
ing to dendritic cells and thus affect activation of T regulatory
cells (Tregs), as has been recently demonstrated in a murine
model. 52 A recent study showed that individuals infected with
malaria have upregulation of TLR-9 and increased IFN-γ, and
that TLR-9 knockout mice have significantly reduced levels
of IFN-γ in response to Plasmodium chabaudi infection as
compared with wild-type mice, 53 supporting the importance of
TLR-9 in IFN-γ production in malaria infection. Further stud-
ies are required to determine the specific effects of these SNP
genotypes on TLR9 signaling.
This study measured serum levels of IFN-γ rather than
IFN-γ produced by parasite-stimulated or antigen-stimulated
mononuclear cells. Serum levels may be considered non-spe-
cific because it cannot be determined if documented IFN-γ
levels are seen solely in response to P. falciparum . The design
of this study supports the contention that the levels of IFN-γ
are largely P. falciparum specific. First, IFN-γ levels in children
with CM were measured on admission and 72 hours after anti-
malarial therapy was initiated. As previously reported, IFN-γ
levels in children with CM decreased after treatment with qui-
nine to those of healthy community children (median level 0
pg/mL). 13 This finding showed that serum IFN-γ levels in chil-
dren with CM did not appear to be elevated for reasons other
than P. falciparum infection: once P. falciparum infection was
treated, serum IFN-γ returned to very low or undetectable lev-
els. Second, IFN-γ levels in children with CM were compared
with those in children with UM and age-matched healthy com-
munity controls; the levels were higher in children with CM
Multivariate analysis: cross-tabulation of TLR9 −1,237 and 1,174 genotypes and corresponding interferon-γ (IFN-γ) levels in children with cerebral
* IFN-γ levels shown on the right half of the table are median (range) values for individuals with that genotype (e.g., first row, second column, individuals with a 1174 GA and −1237 TT genotype
have a median IFN-γ level of 52.5 pg/mL, with a range from 0 to 500.7 pg/mL). For genotypes where there are only two individuals (e.g., 1174 GG/−1237 TT), the median value given the value half-
way between the two values for that genotype.
−1237/1174 genotypes, N
Median IFN-γ levels (range) (pg/mL), for all possible −1237/1174 genotypes
TLR POLYMORPHISMS AND IFN-g LEVELS IN MALARIA
than both of the latter groups, consistent with the notion that
IFN-γ levels were specifically elevated in the context of CM.
Third, serum IFN-γ levels, though related to severity of disease,
were not related to length of the primary disease symptoms of
CM (coma, fever) or UM (fever) (John CC, unpublished data).
Taken together, these findings document that in children in
this community, serum IFN-γ levels are very low in healthy
children, are elevated with increasing severity of malaria but
not with duration of symptoms, and decrease to low or unde-
tectable levels after anti-malarial treatment of CM. Because
the stimulation of TLR signaling may rely on multiple fac-
tors, including the type of cell stimulated, hemozoin level, and
parasite strain (if there is variance in CpG DNA abundance
between strains), a precise in vitro model that mimics in vivo
disease may be difficult to construct. This study may therefore
provide a more accurate reflection of the in vivo process than
an in vitro cell culture-based model.
There was no relationship between TLR9 SNPs and TNF-α
levels in children with CM or UM. Although TNF-α has been
associated with disease severity in other studies of children
with CM, 6 , 8 , 14 , 15 TNF-α levels in children with CM and UM were
Figure 4. Tumor necrosis factor-α (TNF-α) levels by TLR9 sin-
gle nucleotide polymorphism (SNP) in children with uncomplicated
malaria (UM). Cytokine levels in Figures 1 – 4 were compared across
the three groups using a log-additive model (see Methods). The lines
shown represent the median cytokine level for each group.
Figure 3. Interferon-γ (IFN-γ) levels by TLR9 single nucleotide
polymorphism (SNP) in children with uncomplicated malaria (UM).
SAM-AGUDU, GREENE AND OTHERS
similar in this study cohort. 13 The lack of association between
TNF-α level and disease severity in this cohort may explain the
lack of association of TNF-α levels with specific TLR9 SNPs.
Alternatively, TNF-α levels may be associated with SNPs not
yet described and not assessed in the current study. The sam-
ple size of this study could detect only large differences in
TLR9 SNP allele frequency between children with CM versus
UM, though the difference in homozygote frequency for the C
allele at −1237 approached statistical significance. In addition,
because the study assessed only CM and UM, we could not
assess whether these TLR9 SNPs were associated with altered
IFN-γ levels in other forms of severe malaria such as severe
malarial anemia. Future studies will evaluate the frequency of
TLR9 SNP alleles in a larger cohort, including individuals with
severe malarial anemia, and examine whether hemozoin levels
correlate with differences in IFN-γ production in children with
severe versus uncomplicated malaria.
In conclusion, we presented the first human data to
show that TLR SNPs are associated with altered cytokine
responses in parasitic infection, and specifically documented
a relationship between TLR9 SNPs and serum levels of the
pro-inflammatory cytokine IFN-γ in children with CM. If con-
firmed by other studies, these findings may partially explain
the increased risk of CM in some children and could lead to
assessment of specific adjunct therapies (e.g., TLR9 antago-
nists). More broadly, these findings support further study of
the role played by TLR SNPs in human immune responses to
Received August 11, 2009. Accepted for publication December 9, 2009.
Note: Supplemental tables can be found at www.ajtmh.org .
Acknowledgments: We thank our study team of medical officers,
nurses, data entry clerks, and office staff for their effort, and the
patients and families for their participation in the study. This study
was supported in part by grants from the National Institutes of Health
Fogarty International Center (TW006794) and National Institute of
Neurological Disorders and Strokes (5R01NS055349-02) to CCJ, a
Fulbright African Regional Research Award to MJB and National
Institutes of Health grants AI46919 and TW007872 to PAZ.
Authors’ addresses: Nadia A. Sam-Agudu, Melissa A. Riedesel, and
Chandy C. John, Center for Global Pediatrics, Minneapolis, MN.
Jennifer A. Greene, James W. Kazura, and Peter A. Zimmerman,
Center for Global Health and Diseases, Wolstein Research Building,
Cleveland, OH. Robert O. Opoka, Mulago Hospital, Department of
Paediatrics and Child Health, Kampala, Uganda. Tracy L. Bergemann,
Division of Biostatistics, School of Public Health, Minneapolis, MN.
Lisa A. Schimmenti, Pediatric Genetics, Minneapolis, MN.
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