Background
Malaria is estimated to cause approximately 850,000 deaths per year with most mortality occurring before the age of 5 years [
1]. Since death occurs prior to the age of reproduction, the malaria parasite is thought to exert strong selective pressure on the human genome. The clearest example is the sickle cell trait where the heterozygote state provides protection against malaria whereas homozygosity for sickle cell trait (sickle cell disease) is uniformly fatal. The high prevalence of several independently arising alleles causing sickle cell trait in different populations implies a strong selective pressure on the human genome [
2]. While the immunologic host determinants of susceptibility to malaria remain incompletely understood, family-based longitudinal studies have demonstrated that susceptibility to clinical malaria is partially heritable [
3].
The role of innate immunity and, specifically, innate immune responses in host defence against malaria has been reviewed previously [
4]. Common genetic variations in TLR genes have been associated with disease susceptibility in humans to a wide variety of pathogens [
5]. Polymorphisms in innate sensing pathways have also been implicated in host susceptibility to malaria [
6]. There are several components of the malaria parasite that have been implicated as pathogen-associated molecular patterns (PAMPs) recognizable by the innate immune system, including glycosylphosphatidylinositol (GPI) anchors on the surface membranes of infected red blood cells (RBCs) and DNA-haemozoin complexes within the cytoplasm [
7,
8]. GPI initiated signals are thought occur via TLR1/2 or TLR2/TLR-6 heterodimers and DNA-hemozoin complexes via TLR9 [
8‐
10]. TLR1 is part of a complex of TLR receptors that includes TLR1, TLR6, and TLR10, and is thought to have arisen via a gene duplication event [
11].
Since
Plasmodium is a well-described cause of the sepsis syndrome, the possible contribution of
TLR polymorphisms to clinical outcomes in malaria also has potential explanatory power [
12,
13]. In a cohort of patients with sepsis and septic shock from an area with no ongoing malaria transmission, it has been shown that common variation in the gene for TLR1, the G (minor) allele of
TLR1
7202A/G (rs5743551) is associated with a large increase in the severity of organ dysfunction and an increased risk of death [
14]. Others have observed similar biologic effects of this hyperfunctional variant and have shown this allele to be associated with increased risk for clinical tuberculosis, leprosy and frequency of leprosy reversal reactions [
15,
16]. The minor allele of
TLR1
7202A/G has been also been associated with increased Interleukin-6 (IL-6) production and decreased regulatory T cell (CD4+, CD25+) mediated suppression of proliferation utilizing in vitro culture systems [
17]. The
TLR1
7202A/G (minor allele) is a non-coding SNP that sits within the 5′ upstream region of the TLR1 gene and, thus, cannot have a direct effect on TLR1 protein function. It has been previously demonstrated, however, that the rs5743551 SNP is associated with increased secretion of soluble mediators of inflammation in response to stimulation with Pam3CysSerLys4 (Pam3CSK4), a specific ligand for TLR1/TLR2 heterodimers [
14]. One potential explanation for the observed hyper responsiveness is that
TLR1 rs5743551 is in LD with several coding polymorphisms that strongly increase cell surface expression of TLR1.
These data suggest that polymorphisms in TLR1 have functional consequences in immunologic signalling and influence the host response to a broad range of microbial pathogens. Given the potential role of TLR1 in inflammatory responses to malaria, it was hypothesized that the presence of the minor TLR1
A7202G
allele would be associated with malaria severity and the magnitude of parasitaemia.
Discussion
Early host innate immune inflammatory responses are likely to play an important role in determining the clinical course after infection with
P. falciparum. It was determined that
TLR1 polymorphisms previously demonstrated to be associated with functional changes in immunologic signaling were associated with different levels of host control of
P. falciparum in patients with clinical malaria. In the studied patient population, the GG (minor) allele of rs5743551 was associated with a parasite density roughly half of that of patients with the TT (major) allele. The G (minor) allele has been associated with increased inflammatory cytokine production in response to bacterial lipopeptides that are recognized by TLR1/2 heterodimers [
14]. The direction of this association with the G (minor) allele in the studied population of patients with clinical malaria, however, is opposite the direction in sepsis patients where the presence of the T (major) allele was associated with improved clinical outcomes. Furthermore, the density of the pathogen is different in patients with the G (minor) allele with respect to malaria and sepsis, as carriers of the minor G allele were more likely to have blood cultures with bacterial growth (a proxy for degree of bacterial burden) compared to carriers of the major T allele whereas the minor G allele was associated with fewer parasites per millilitre of blood. This implies that the influence of innate signaling molecules on the ability of the host to control an infectious process is more than simply the degree of inflammatory mediators produced. The G (minor) allele may have persisted in the population because early inflammation may be important to control parasite replication during acute malaria infection, whereas hyperresponsiveness to other PAMPS is detrimental in other disease states (e.g. bacterial sepsis).
Investigations into the role of TLR signalling in malaria infection using murine models have revealed conflicting results. Both TLR1 and TLR6 signal via forming heterodimers with TLR2 [
9,
10] All TLRs except for TLR3 signal via the adaptor protein MyD88 and disruption in MyD88 and TLR2 disrupts signaling cascades generated by TLR1 and TLR6 [
27]. During infection with
Plasmodium berghei ANKA strain (a model for cerebral malaria),
Tlr2−/− and
Myd88−/− mice have been shown by some groups to be protected from development of cerebral malaria, although the effects of TLR2 have not been consistently observed and may depend on the genetic background of the mice [
28‐
31]. Infection with
Plasmodium yoelii, a model of acute uncomplicated malaria, had either a strong phenotype with impaired survival in
Tlr2−/−, and
Myd88−/− mice or no observable phenotypic difference [
32,
33]. It is likely that there are multiple innate mechanisms that contribute to the host response against malaria infection, and single knockout inbred mice may not recapitulate the complexity of human disease.
In an effort to clarify the role of TLRs in human malaria, several studies have used genetic approaches attempting to detect associations between common genetic variants in the genes for TLRs and clinical outcomes in patients with malaria. In a group of malaria cases from northern Thailand with confirmed
P. falciparum infection, none of the genetic variants tested were significantly associated with development of complicated malaria (Table
3), whereas increasing copy numbers of the major T allele of
TLR1 rs5743551 demonstrated a trend towards higher odds of complicated disease (p = 0.13). Notably, while this polymorphism is in high linkage disequilibrium (LD) with
TLR1 rs5743551 in Caucasian subjects, it was determined that in the Southeast Asian subjects from Northern Thailand that
TLR1 rs5743618 is in very weak LD with
TLR1 rs5743551 and is found at very low frequency [
14].
The finding that
TLR1 rs5743618 is associated with higher parasitaemia on day zero in an Asian population is similar to an investigation by a Brazilian group that demonstrated an association between the G allele of
TLR1 rs5743618 and increased risk of developing symptomatic malaria after infection with
Plasmodium [
33]. The Brazilian group did not genotype
TLR1 rs5743551 in their study, so direct comparisons between their study and the studied population cannot be made. However, taken together, these studies suggest that genetic variation in
TLR1 affects host susceptibility to malaria disease severity, while variation in
TLR6 is less likely to play a significant role in these processes. This association is further supported by a study in a population in Cameroon that reported a different SNP in
TLR1 (rs4833095) was also associated with increased parasite burden, whereas a SNP in
TLR6 was not associated with any clinical phenotype [
34].
Stronger evidence that
TLR1 rs5743551 may influence host responses to malaria is provided by the finding of a significant association between the copy number of the T allele of this polymorphism and increased parasitaemia measured on day 0 of enrollment. Subjects carrying the TT genotype had a nearly two-fold higher parasitaemia than subjects carrying the CC genotype (Table
3). Segregated analysis by ethnicity showed that the strongest effect of the T (minor) allele was present in the subjects of Mon ethnicity. This finding differs from those of the Brazilian group who found no association between
TLR1 variation and parasitaemia. In contrast, the population in Cameroon demonstrated a similar association with
TLR1 polymorphism associated with differences in parasite load. Explanations for the differing findings may be due to the different
TLR1 variants tested but could also include marked differences in environment (Brazil versus Thailand), and underlying population genomic differences. Furthermore, in the Brazilian study a substantial minority of patients were infected with
P. vivax (39 %), whereas the population in Cameroon was similar to the studied population in that it was a population uniformly infected with
P. falciparum. Several groups have demonstrated different host response to the various
Plasmodium species. For example, higher levels of IL-10 have been documented in humans subjects infected with
Plasmodium vivax when compared to individuals infected with
P. falciparum [
31‐
33].
Conclusions
The particular strengths of the present study include detailed clinical information on a population uniformly infected with PCR confirmed P. falciparum. The current study is the first to document an association of TLR1 rs5743551 with a clinical outcome in an Asian population with malaria. TLR6 has never been strongly implicated in the host response to malaria and no association with TLR6 SNPs and clinical outcome in malaria were found.
These data demonstrate that the G allele of rs5743551 in
TLR1 is associated with the host control of parasitaemia in Asian patients with acute febrile malaria. This assertion is consistent with the demonstrated ability of malarial GPI to activate cells through TLR2/TLR1 heterodimers [
4]. From an evolutionary standpoint, this suggests that G allele of
TLR1 rs5743551 may persist in higher frequencies in non-Caucasian populations because of a beneficial effect on host fitness in environments with high prevalence of malaria. This is the first plausible explanation for the relatively high frequency of an allele that is associated with worse outcomes in tuberculosis, sepsis, and leprosy [
14‐
16]. While parasitaemia is generally correlated with complicated disease, high parasitaemia alone is neither necessary nor sufficient to account for poor patient outcomes and other host factors likely influence clinical outcomes [
24]. These findings stress the complex interplay between the human genome and endemic pathogens.
Authors’ contributions
WOH, LKE, KCK, MWW, and WCL planned and designed the study. WOH, MWW, and WCL created the first draft of the manuscript. MWW and SHB performed the SNP genotyping. MWW performed the initial statistical analysis. SK helped to enroll the cohort included in the study. All authors read and approved the final manuscript.