Toll-like receptor polymorphisms have been associated with an altered risk of disease or disease severity in numerous infections,26,28–30,32,38
However, potential causal mechanisms for the associations between TLR SNPs and disease severity in malaria have not been described. In this study, we documented that TLR9 SNP genotypes are associated 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 studies 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 here13
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 pathogenesis 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 associated with increased IFN-γ levels in severe P. falciparum
infection. The lack of association between these TLR9 SNPs and altered IFN-γ levels in children with UM suggests that an additional factor may be required to alter IFN-γ levels in infected children.
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 others20
suggested that hemozoin is not a primary ligand for TLR9 but functions as a carrier for P. falciparum
DNA. Greater levels of hemozoin may for this reason 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 hypothesis. 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
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 quantitative 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 haplotype analysis suggested that both genotypes contributed to the differences in IFN-γ levels. TLR9 may also affect signaling 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 studies 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-specific 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 quinine to those of healthy community children (median level 0 pg/mL).13
This finding showed that serum IFN-γ levels in children 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 levels. Second, IFN-γ levels in children with CM were compared with those in children with UM and age-matched healthy community controls; the levels were higher in children with CM 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 undetectable levels after anti-malarial treatment of CM. Because the stimulation of TLR signaling may rely on multiple factors, 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 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 sample 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 confirmed 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 antagonists). More broadly, these findings support further study of the role played by TLR SNPs in human immune responses to other infections.