Toll-like receptors (TLRs) recognise a diverse array of pathogens and initiate intracellular signalling via their Toll/Interleukin-1 receptor (TIR)-domains, leading to differential effects on gene expression and the generation of an inflammatory host response. TLR2 has been shown to recognise, among other agonists, lipoteichoic acid, lipoarabinomannan and mycobacterial lipopeptides1
, whilst TLR4 recognises lipopolysaccharide (LPS)7,8,9
. Both TLRs 2 and 4 sense P. falciparum
glycosylphosphatidylinositol (GPI) (Aucan et al.
. The adaptor protein TIRAP (TIR domain-containing adaptor protein), also known as MAL (MYD88 adapter-like), is essential for MYD88-dependent signalling downstream of TLR2 (with TLR1 and TLR6 as co-receptors) and TLR41,5
. Following stimulation of TLR2 or TLR4, TIRAP triggers a signalling cascade which culminates in the activation of the transcription factor NF-κB and the subsequent activation of pro-inflammatory genes. Based on the central position of TIRAP in the TLR2 and TLR4 pathways, and knowledge of the microbial components which associate with these TLRs, we hypothesised that genetic variation at TIRAP
might underlie susceptibility to common infectious diseases.
TIRAP spans 14500 base pairs on Chr11q24.2 and encodes a protein of 221 amino acids. Analyzing thirty-three SNPs in TIRAP
and surrounding region in multiple populations found TIRAP
S180L consistently associated with disease (Supplementary Note
, Fig. 1
, Table 1
S180L genotyping was performed in a total of 6106 individuals with four different diseases: two United Kingdom (UK) populations of European ancestry with invasive pneumococcal disease (IPD); a Kenyan population with bacteraemia; three populations with malaria, (from The Gambia, Kenya, and Vietnam); and two populations with tuberculosis (from Algeria as well as the West African populations of The Gambia, Guinea-Bissau and the Republic of Guinea).
In the UK population, heterozygosity at TIRAP S180L was associated with protection from invasive pneumococcal disease (3×2 χ2=8.72, P=0.013, ). An excess of mutant homozygotes amongst IPD cases () was also observed in this UK population. TIRAP S180L was then examined in a separate group of UK individuals with thoracic empyema and a second control group. Although no association was observed between genotype and susceptibility to thoracic empyema overall (n=584, 3×2 χ2=0.63, P=0.73), analysis of the small subgroup of individuals with pneumococcal empyema revealed a non-significant trend towards association (3×2 χ2=5.05, P=0.080; ). Interestingly, an excess of mutant homozygotes was again observed amongst this second group of IPD cases ().
TIRAP S180L genotype frequencies in individuals with infectious disease and controls
We then studied TIRAP S180L in a second population with invasive bacterial disease, comprising Kenyan children with well-defined bacteraemia. Although the mutant allele was found to be less common in the Kenyan population than in UK individuals, the same pattern of association was observed. The TIRAP S180L heterozygotes were significantly more common amongst community controls (5.9%), compared to individuals with bacteraemia (2.4%) (2×2 χ2=9.05, P=0.003; ). The heterozygote protective effect of the S180L locus was also significant within the subgroup of 164 Kenyan children with pneumococcal bacteraemia (Fexact=0.024, ), thus replicating the findings in the UK studies.
In the Gambian malaria case-control study, TIRAP S180L heterozygosity demonstrated a significant protective effect against both general malaria (Wald=8.35, P=0.004, ) and severe malaria (Wald=8.706, P=0.003, ). This result was replicated in a second malaria case-control study, this time in a Vietnamese population whose design included only cases of severe malaria: TIRAP S180L heterozygotes were again found to be more prevalent among the controls (4.7%) compared to the severe malaria cases (1.1%) (Fexact=0.046, ). An attempt to replicate this in a third malaria case-control study (from Kenya) demonstrated similar results (2×2 χ2 =3.28, P=0.035; ).
Finally, the possible effect of the TIRAP S180L polymorphism on susceptibility to tuberculosis was examined in 1280 individuals from The Gambia, Guinea-Bissau and the Republic of Guinea (collectively known as the EUTB West African study), again with ethnically matched controls. The TIRAP S180L heterozygous genotype was found to be protective against clinical tuberculosis (Wald=6.23, P=0.013). This finding was then replicated in two family-based studies on tuberculosis: from the EUTB West African study, as well as from Algeria. In both studies, the variant TIRAP S180L was found to be under-transmitted in subjects with clinical tuberculosis compared to subjects free from clinical tuberculosis (TRANSMIT P=0.075 and P=0.038 respectively; ).
Transmission Disequilibrium Test (TDT) results of TIRAP S180L in West Africa and Algeria
When all the study groups are combined and stratified for the different populations, there is a clear association between heterozygosity at the TIRAP S180L locus and protection against multiple infectious diseases (N=6106, P≤9.6×10-8). To our knowledge, there are no other samples where we (or others) have tried and failed to replicate this finding. Studies on the functional effect of the S180L variant were consequently undertaken.
We first assayed the effect of the S180L polymorphism on TLR2 signalling using transfected murine embryonic fibroblasts (MEFs) from Tirap knockout mice (). Treatment of wild type MEFs with the TLR2 ligand Malp2 induced I-κBα degradation at 30 and 50 minutes (first panel). This effect was absent in Tirap-deficient MEFs (second panel). However, transfection of Tirap knock-out cells with the wild-type 180-Ser fully reconstituted TLR2 signalling, with degradation of IκB-α occurring at 30 and 50 minutes post-treatment (third panel). Transfection with the mutant 180-Leu failed to result in IκB-α degradation following TLR2 stimulation (fourth panel). Both wild-type 180-Ser and variant 180-Leu were expressed at similar levels in the cells (not shown).
Figure 1 Macrophage-activating lipopeptide 2 (Malp2) induced, TLR2 signalling assay as measured by degradation of IκB-α at various time points. First panel: Malp2 (5nM) -stimulated wild-type murine embryonic fibroblast (MEF) cells. Second panel: (more ...)
A similar lack of reconstitution was observed when IL-6 was measured, as shown in . Both LPS and Malp2 induced IL-6 in wild-type MEFs causing a 4-fold increase over untreated cells (first histobar in each set). The effect of both stimuli was however abolished in Tirap-deficient cells (second histobar in each set). Transfection of the Tirap-deficient cells with a plasmid encoding wild-type Tirap 180-Ser reconstituted the effect of both LPS and Malp2, leading to a 3-fold stimulation over untreated cells (third histobar in each set). The variant Tirap 180-Leu was however unable to reconstitute the effect of either stimulus (fourth histobar in each set). Both wild-type 180-Ser and variant 180-Leu were expressed at similar levels in the cells (lower panel). We also tested the functional consequences of co-expression of wild-type 180-Ser and variant 180-Leu in Tirap-deficient cells. As shown in , expression of wild-type 180-Ser Tirap activated an NF-κB-linked reporter gene (second histobar). The variant 180-Leu form was however inactive in cells lacking endogenous Tirap (third histobar), attesting further to the functional deficiency of this form of Tirap. Importantly, the variant form inhibited the ability of the wild-type form of Tirap to activate NF-κB (fourth histobar). This suggests that TIRAP signalling will be directly impaired in heterozygotes, since the variant might interfere with signalling by the wild-type form.
Figure 2 Wild-type MEFs or Tirap knockout MEFs transfected with empty vector or plasmids encoding Tirap 180-Ser or Tirap 180-Leu as indicated, were treated with LPS (1μg/ml) or Malp2 (5nM). After 24h, supernatants were removed and assayed for IL-6 by ELISA. (more ...)
Figure 3 Tirap knockout MEFs were transfected with 100ng empty vector, or plasmids encoding Tirap 180-Ser or Tirap 180-Leu (50ng of each, supplemented to 100ng with 50ng of empty vector), or a combination of plasmids encoding Tirap 180-Ser and Tirap 180-Leu (50ng (more ...)
Wild-type TIRAP has been shown previously to interact with TLR211
. We generated models of the wild-type and mutant protein in order to assess the effect of the S180L mutation on TIRAP structure. According to our model (), serine 180 is located on the edge of a surface-exposed loop opposite the BB-loop, the region that contains a critical proline residue that has been shown for other TIR containing proteins to form a point of contact with downstream signalling molecules. The model predicts that the electrostatic surface potential does not differ significantly between the wild-type and mutant protein. However the leucine residue appears to be more exposed ( upper panel). Previous modelling and interaction studies have suggested that TIRAP may bind to TLR2 via a region termed the DD-loop11
. In our current model, Serine 180 is located in close proximity to this DD-loop ( lower panel). We therefore performed an in vitro
binding assay in order to assess the effect of the mutation on the TIRAP: TLR2 interaction. As shown in upper panel, a GST-tagged version of the TIRAP 180-Leu containing allotype, unlike wild-type TIRAP, failed to bind to TLR2 suggesting that the defect in reconstitution of signalling by TIRAP S180L occurs as a result of TLR2 not recruiting the variant. In addition to TLR2, TIRAP has also been shown to bind to both itself and MyD884
. We therefore tested the effect of the S180L mutation on these interactions. As shown in , middle and lower panel, respectively, the S180L mutation had no effect on the interaction of TIRAP with both itself and MyD88.
Figure 4 Molecular models of wild-type and mutant TIRAP. The computational utility Molecular Operating Environment (MOE2006.02) (www.chemcomp.com) was used to generate the models. Electrostatic surface potentials are highlighted in blue (positive) and red (negative) (more ...)
Figure 5 HEK-293 cells (1×106) were transfected with 3 μg of Flag-tagged TLR2, HA-Tirap or AU1-tagged MyD88. Lysates were incubated with purified glutathione-coupled GST, GST-Tirap and GST-Tirap S180L for 2 hrs. After washing, the complex was analysed (more ...)
Studies in Tirap-deficient mice have demonstrated impaired cytokine responses and NF-κB activation following stimulation with ligands at TLR4 and TLR2, as well as the TLR2 co-receptors TLR1 and TLR65,6
. Unlike bacterial components1,12,13
, the interaction of malaria parasites with TLRs is less well-described, although it has been demonstrated that P. falciparum
-derived GPI is a ligand for both TLR2 and TLR410
(Aucan et al.
unpublished data). The association between TIRAP
variants and malaria in Gambian and Vietnamese populations presented here provides further evidence for a role of the TLR pathway in malaria pathogenesis. In addition, a role for the TLR2 system in host defence against malaria is also suggested from mutations in CD36, which associate with susceptibility to severe malaria14
. Recently, CD36 has been shown to be a co-receptor for TLR215
. Taken with the TIRAP
variant presented here, the TLR2 system is clearly important for the host response in malaria.
On the basis of our co-transfection experiments (), the protective heterozygote state is likely to be associated with attenuated signaling and reduced NF-κB activation. This finding is consistent with increasing evidence that an excessive host inflammatory response may render individuals more vulnerable to developing severe forms of malaria and bacterial disease16,17,18
. On the other hand, too little signaling would lead to an inadequate anti-pathogen response. This provides the opportunity for intermediate genotypes regulating signaling to be of optimal protective value. The mutant homozygotes are too rare to assess in African and Vietnamese populations, although in each of the UK studies there is a significant association with increased susceptibility to IPD amongst mutant homozygotes. This may have occurred by chance through analysis of the relatively small number of mutant homozygote individuals, or alternatively it may reflect a true heterozygote protective effect. Heterozygote protection against infectious disease is well recognized with examples such as sickle cell trait and malaria, prion protein gene variation and spongiform encephalopathy, and HLA and HIV/AIDS disease progression19,20,21
. If the TIRAP
180-Leu homozygous state does indeed confer increased susceptibility to IPD, the mechanism by which this occurs is unclear, although we speculate that it may result in severely impaired or abolished signaling and increased susceptibility to severe infection, as has also been demonstrated in patients with rare functional mutations in IRAK4
in the same pathway22,23
. We suggest that heterozygosity at S180L may therefore confer a protective phenotype characterized by intermediate levels of pathway activation and an ‘optimal’, balanced inflammatory response. Additional host and microbial factors such as pathogen virulence and dose and prior immunity might be expected to interact with this model, as described in murine models of pneumococcal susceptibility24
, and the optimal level of inflammatory response to a given exposure will likely vary depending on such factors.
The rarity of the mutant allele in African populations is also noteworthy. The burden of infectious disease mortality is very high in African populations, and this may strongly select against mutant homozygote individuals and act to reduce the 180-Leu allele frequency. The observed allele frequency may reflect a balance between protection afforded to heterozygotes and increased susceptibility to infectious disease in mutant homozygotes. It is also possible that the Leu allele may be subject to ongoing selective pressure and has not yet achieved fixation in the African population. Furthermore, it is conceivable that TIRAP S180L may influence susceptibility to other common infectious and inflammatory causes of death and so be subject to multiple selective pressures which may differ between human populations and ecological settings. Investigation of the role of this functional variant of TIRAP in susceptibility to other diseases may shed light on this area. We report here the first single nucleotide change that appears to impact on numerous major infectious diseases, roughly halving the risk in heterozygous individuals. This should represent a major evolutionary pressure as these diseases together account for over five million deaths each year in the developing world.