Single nucleotide polymorphisms of Toll-like receptors and susceptibility to infectious diseases

TLR-1

TLR-1 heterodimerizes with TLR-6 to mediate host responses to lipopeptides from different classes of pathogens. TLR-1 and TLR-6 SNPs, as well as their association with infectious diseases, is given in Table 1. The SNP TLR-1−7202A>G (rs5743551, occurring in the 5′-near gene of TLR-1), was associated strongly with a higher risk for death in sepsis and organ dysfunction in a large prospective cohort of patients with sepsis and septic shock 33 and patients with septic shock, bearing that homology in TLR-1 −7202G had a markedly higher prevalence of Gram-positive infection. TLR-1 1805G>T(Ser602Ile, rs5743618) is a non-synonymous polymorphism, affecting the transmembrane domain of TLR (Fig. 2) that presents a high level of linkage disequilibrium (LD) with TLR-1 −7202A>G. Carriers of the isoleucine allele demonstrated an increased cell surface expression of TLR-1 on peripheral monocytes 34, while carriers of the serine allele have decreased signalling ability and produce decreased IL-6 levels after lipopeptide stimulation 35. The SNP 1805G>T (Ser602Ile TLR-1) was shown to protect against leprosy among leprosy patients and asymptomatic control subjects recruited from areas where this disease was endemic. It was found that the 602Ser allele was significantly under-represented in the studied leprosy patient population. Interestingly, genotyping revealed that 602Ser was the most prevalent allele found in white individuals (75% frequency), with a decreased allele frequency observed in individuals of Turkish (43%) and African descent (26%) 34. These data indicate that more than half of white individuals are homozygous for the 602Ser allele. All 21 donors of East Asian ancestry genotyped in the same study were homozygous for the 602Ile allele; the absence of the 602Ser allele in this group suggests that any strong selection for the appearing 602 allele was linked to either genetic/environmental factors, geographically confined specific pathogens or past epidemics 34. Given the diverse range of pathogens recognized by TLR-1/TLR-2 heterodimers, the potential clinical impact of this SNP is high 36.

The TLR-1 Ser602Ile polymorphism has also been linked to urinary tract infections, with 1805TT (602Ile homozygous) showing protection from pyelonephritis when compared to the 1805GT (Ser602Ile heterozygous) or GG genotypes (Ser602 homozygous) 37, and protection from Chlamydia trachomatis infection in African American women with pelvic inflammatory disease 38.

Two TLR-1 polymorphisms, rs4833095 (Asn248Ser) and rs5743618 (Ser602Ile), were assessed among 302 primiparous Ghanaian women for their association with Plasmodium falciparum infection and manifestation; TLR-1 Asn248Ser variant was identified as being involved in the recognition of P. falciparum and indicated its role in susceptibility to and manifestation of malaria in pregnancy 39. Indeed, recent data demonstrate that the glycosylphosphatidylinositols (GPIs) of P. falciparum, which activate macrophages and produce inflammatory responses, are engaged preferentially by TLR-2–TLR-1 dimers 40,41.

Alleles of these SNPs (248Ser and 602Ile) were also associated with tuberculosis disease in African American patients 42. Asn248, which is common in European Americans, is a conserved residue in the extracellular domain of TLR-1 and TLR-6 (Fig. 2) and a putative glycosylation site; its replacement by Ser might result in altered glycosylation, potentially changing TLR-1 folding or function, e.g. in PAMP recognition or signal transduction 42.

Carriage of one of the key species in bacterial vaginosis, Atopobium vaginae, in the first half of pregnancy was associated significantly with the presence of TLR-1 743A>G variation (rs4833095) and associated marginally with the TLR-1 promoter −7202A>G variation 43. However, even if these associations constitute a genuine biological phenomenon, overall, the attributable risk of these polymorphisms appears to be limited 43.

TLR-2

TLR-2, as a heterodimer with TLR-1 or TLR-6, recognizes a large number of common bacterial motifs, including lipopeptides, peptidoglycan, glycosylphosphatidylinositol (GPI)-linked proteins and zymosan. Table 2 summarizes the TLR-2 SNPs that are described below. Two non-synonymous SNPs in TLR-2 have been linked to human diseases 44: first, a C>T transition in nucleotide 2029 (rs121917864), which replaces Arg677 with Trp (Arg677Trp), is common in African and Asian populations, but appears to be absent among white populations. In vitro, this SNP has been shown to inhibit both Mycobacterium leprae- and M. tuberculosis-mediated NF-κB activation and production 45. In a Korean and a Tunisian population, this SNP was associated, respectively, with leprosy 46 and susceptibility to tuberculosis 47; this agrees with data that patients carrying this allele show reduced basal and Mycobacterium-stimulated serum IL-12 levels, which is required for activation of the IFN-γ pathway and the induction of the T helper type 1 (Th1) response against intramacrophagic pathogens.

Another functional TLR-2 variant (rs5743708) consists of a G>A substitution at nucleotide 2251, which replaces Arg753 by Gln (Arg753Gln). This SNP maps in a region of highly conserved amino acids in the C-terminal end of TLR-2 (Fig. 2), is present in 3% of healthy white blood donor control subjects, is identified in patients with staphylococcal septic shock 48, is associated with increased risk of developing tuberculosis in a Turkish population 49 and has been shown to be associated with a significantly increased risk for development of infective endocarditis 50. Conversely, Arg753Gln was shown to occur at a significantly lower frequency in Lyme disease (LD) patients (especially late-stage disease) compared with matched controls 51, due possibly to a reduced signalling via TLR-2/TLR-1 51.

An immune response is elicited through TLR-2 by products of the symbiotic bacterium Wolbachia 52, which is present in filarial parasites Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus (the agents for lymphatic filariasis). A −196 to −173 deletion (del) polymorphism in the 5′ untranslated region of TLR-2 and two synonymous SNPs, 597C>T (rs3804099, Asn199Asn) and 1350C>T (rs3804100, Ser450Ser) in exon 3, are associated with asymptomatic bancroftian filariasis in Thailand by W. bancrofti 53, with the molecular mechanisms possibly involving changes in protein levels, mRNA structure, stability, kinetics of translation and alternative splicing 54.

Alternatively, synonymous SNPs might be proxies for non-examined polymorphisms. The 597C>T has been reported previously in connection with increased susceptibility to tuberculous meningitis 55.

TLR-3

TLR-3 recognizes dsRNA from viruses and also synthetic oligonoucleotides such as a synthetic dsRNA analogue, poly(I : C) (polyinosine : polycytidyllic acid). Analysis of a number of SNPs occurring on TLR-3 on 57 Japanese patients with Stevens–Johnson syndrome or toxic epidermal necrolysis with ocular surface complications revealed a strong association between two SNPs: 299698T>G (rs3775296, 5′UTR) and 293248A/G (rs3775290, exon 4, silent SNP Phe459Phe) of TLR-3 56. Two genetic variations in TLR-3 were shown to affect host susceptibility to enteroviral cardiomyopathies; first, the rare non-synonymous substitution TLR-3 Pro554Ser (rs121434431, 1660C>T), in patients with Coxsackievirus B3 myocarditis, and secondly, the common single nucleotide polymorphism, TLR-3 Leu412Phe, (rs3775291, 1235C>T), which was detected more frequently as homozygous for phenylalanine in the patient population by comparison with controls 57. The 554Ser variant was also related to HSV-1 encephalitis (HSV-1), which blunted TLR-3 signalling in response to infection with the virus and acted in a dominant-negative manner, suggesting that its presence could contribute to the host susceptibility to infection and possibly determine clinical outcome 58. The 412Phe mutation may confer resistance to HIV-1 infection 59. Leucine 412 is next to an asparagine, whose glycan moiety contacts the dsRNA 60 (Fig. 2). As the Asp413 mutation to alanine significantly reduces TLR-3 signalling 61, it is possible that Leu412Phe could either affect the glycosylation of asparagine 413 or hinder its glycan moiety interaction with dsRNA, thus explaining the reduced signalling activity of Phe-412 TLR-3 57. The crystal structure of TLR-3 identifies histidine 539 and asparagine 541 as critical for ds RNA binding 11 and this may justify the importance of Proline 554, which is very close to the RNA binding site; the change into a serine could either prohibit ligand-induced dimerization or prevent conformational changes necessary for downstream signalling.

The aforementioned SNPs in relation to respective infectious diseases are summarized in Table 3.

TLR-4

The fact that TLR-4 is the receptor recognizing LPS has stimulated studies on its role in human disease. Two polymorphisms in TLR-4 exist with population frequencies >5% and are reported to be up to 18% 42 (Table 4). These are a 1063A>G transition, resulting in an aspartic acid substitution by glycine at amino acid location 299, Asp299Gly (rs4986790), and a 1363C>T transition conferring a threonine substitution by isoleucine at amino acid location 399, Thr399Ile (rs4986791). Individuals with Asp299Gly and/or Thr399Ile polymorphisms had a blunted response to inhaled LPS in humans 62, and this early finding triggered a number of studies in search of associations between these two polymorphisms and infectious diseases. Today, there are contradictory conclusions on the role of Asp299Gly on susceptibility to Gram-negative bacterial infections 63, due possibly to the fact that many studies looked only at the effect of either the Asp299Gly or Thr399Ile polymorphisms separately, neglecting the fact that these SNPs exist in a co-segregated (299Gly/399Ile) manner, which implies that a total of four haplotypes (wild-type/wild-type, 299Gly/wild-type, 399Ile/wild-type and 299Gly/399Ile) are represented in the population 64. Additionally, the discrepancies may be attributed to the use of small sample sizes, different stimulatory methods or the use of in-vitro systems which may not mimic primary cells conditions 36. Different studies have investigated the link between Asp299Gly SNP and sepsis. Two studies have demonstrated a link between this SNP and increased risk of septic shock due to infection by Gram-negatives 65,66; the haplotype Asp299Gly/Thr399Ile had a higher prevalence of Gram-negative infections only in the latter study, but had little, if any, effect on susceptibility to septic shock 66. The single TLR-4 Asp299Gly haplotype has been shown to alter the cytokine response to LPS, thus possibly affecting susceptibility to Gram-negative infection 67, and has also been associated with an increased incidence of systemic inflammatory response syndrome 68. Taken together, these results suggest that the Asp299Gly haplotype may predispose to septic shock, but this impact may be restricted to Gram-negative infections, because it has been shown not to impact polymicrobial sepsis 69. The effect of the rare Thr399Ile haplotype on function and susceptibility remains unclear due to its scarcity in the population 64.

Both the TLR-4-Asp299Gly and the TLR-4-Thr399Ile variants confer increased risk of severe malaria, respectively, in Ghanaian children, linking these SNPs to disease manifestation 70, while TLR-4 299Gly granted a protective effect against mortality due to P. falciparum cerebral malaria in Africa 42, where the TLR-4 299Gly allele is highly prevalent: in some populations, up to 15%. Despite this protective effect, its fixation (an increase in allele frequency of 100%) in Africa has been hindered by the deleterious effect of its protein product on the severity of Gram-negative infection, which may also be the cause of the near-complete elimination of 299Gly polymorphism from Europe and Asia 42.

The strongest association between TLR-4 polymorphisms and disease susceptibility has been reported in respiratory syncytial virus (RSV) infection, where infants heterozygous for Asp299Gly and Thr399Ile showed increased susceptibility to infection 71.

The presence of TLR-4-Asp299Ile and TLR-4-Thr399Ile in unrelated donors has been associated with an increased risk of invasive aspergillosis (IA) among recipients of haematopoietic cell transplants 72, and Asp299Gly has been associated significantly with chronic cavitary pulmonary aspergillosis 73.

Read et al. supported that there was no association between the Asp299Gly polymorphism and the susceptibility or severity of meningococcal infection, as the allele frequency of the Asp299Gly polymorphism was 5·9, 6·5 and 4·1% among blood donors, patients with microbiologically proven meningococcal disease and patients who died of meningococcal disease 74. More recently, the results of another study suggested that the heterozygous TLR- Asp299Gly genotype is linked to an increased mortality in children with invasive meningococcal disease 75.

The TLR-4 polymorphisms Asp299Gly and Thr399Ile were shown to be associated with elevated viral load in HIV-infected individuals 76, possibly through a more central role of TLR-4 on the modification of the innate response at the colonized mucosal surfaces and the host's ability to fight invasive pathogens, and not due to an interaction of TLR-4 with the pathogen 77. The TLR-4 polymorphisms Asp299Gly and Thr399Ile have also been shown to be associated with resistance to infection by Legionella pneumophila, an intracellular Gram-negative bacterium with an unusual LPS structure that is recognized primarily by TLR-2 rather than TLR-4, suggesting that Legionella stimulates TLR-4 in an unusual fashion that differs from other Gram-negative bacteria and may be cell-specific 78.

The crystal structure of the TLR-4 Asp299Gly/Thr399Ile has been solved as a complex with MD-2 and LPS 79, and its comparison to the wild-type TLR-4/MD-2/LPS complex structure demonstrated that the overall arrangements of the two complexes were similar and that topical differences were present only around the Asp299Gly SNP site, which induces a structural change modulating the surface properties of TLR-4. This effect may be more apparent upon stimulation of TLR-4 by ligands with weak agonistic activity. The impact of the Thr399Ile change was minor, as almost no structural differences were observed 79.

TLR-5

A cytosine–thymidine transition polymorphism at base pair 1174 in TLR-5 (rs5744168) changes the arginine at amino acid 392 to a stop codon and results in a truncated TLR-5, which lacks the whole transmembrane domain as well as the 198 amino acid-long signalling cytoplasmic tail. This polymorphism acts in a dominant fashion with respect to the wild-type allele and is associated with susceptibility to infection with a flagellated organism, L. pneumophila, in humans 80 without, however, rendering human carriers universally susceptible to infection with flagellated bacteria. The frequency of this SNP was not significantly different in patients with typhoid and matched control subjects and did not have any measurable effect on clinical parameters associated with typhoid fever 81. Interestingly, the TLR-5₃₉₂STOP, a null variant, is present at frequencies ranging from 10% in Europeans to up to 23% in some populations, which suggests the TLR-5 function being partially compensated by other genes and to TLR-5 being functionally redundant 82. Alternatively, pathogen recognition may occur on many levels and this is a plausible, previously unappreciated feature of the innate immune system. The identification of a second flagellin receptor, Ipaf (presently called NLCR4) 83,84, is consistent with this idea, while the fact that these two genes follow different downstream paths and sense different types of bacteria may indicate co-operation between them in the recognition of flagellated bacteria, rather than a complete functional redundancy.

TLR-6

TLR-6 forms heterodimers with TLR-2 (like TLR-1) to mediate host responses against lipopeptides from different organisms (Table 1). There is little information on functional studies on TLR-6 polymorphisms and association studies with infections. In one study, Kesh et al. 85 examined the association between SNPs in TLR-1, TLR-4 and TLR-6 genes and the risk of developing IA in 127 allogeneic haematopoietic stem cell transplant recipients consisting of 22 patients with IA and 105 unaffected control subjects. Ser249Pro polymorphism was found to potentiate IFN-γ release following injection of the bacillus Calmette–Guérin (BCG) vaccine 86 TLR-6 SNPs have been also associated with non-infectious diseases, but these are outside the scope of this review.

TLR-7/TLR-8

TLR-7 and TLR-8 recognize viral single-stranded RNA. Multiple uridine-rich oligoribonucleotides derived from HIV-1 virus have been demonstrated to activate human TLR-7 and TLR-8, and the TLR-7/TLR-8 ligation to the viral RNA is suggested to affect susceptibility to or course of the infectious disease 63. Data on the impact of TLR-7/8 SNPs on HIV infection remain scarce. The presence of the most frequent TLR-7 polymorphism, TLR-7 Gln11Leu (rs179008), was associated with higher viral loads and accelerated progression to advanced immune suppression in HIV patients 87. Conversely, presence of the most frequent TLR-8 polymorphism, TLR-8 1A>G, Met1Val (rs3764880) was shown to confer a significantly protective effect regarding progression of the disease 88. These polymorphisms are also related to chronic hepatitis C virus (HCV) infection, with TLR-7 Gln11Leu SNP not affecting HCV viral load, but decreasing IL-29/IFN-λ expression and being associated with disease-induced portal lymphoid aggregates 89. Wang and co-workers demonstrated that the TLR-8 129G>C (rs3764879, 5′ near gene) and TLR-8 1G>A variants were in complete linkage disequilibrium, and that the frequency of TLR-8-129C/+1A was significantly higher in male patients with HCV infection compared with healthy controls, as well as that variations in TLR-7 and TLR-8 genes might impair immune responses during HCV infection 90. The same group reported recently that these variations affect NF-kB signalling and cytokine production upon stimulation of human monocytes with specific TLR-7/TLR-8 agonists, thus modulating immune responses during HCV infection 91. Finally, the TLR-8 SNPs Met1Val and −129G/C polymorphisms render individuals more susceptible to Crimean–Congo haemorrhagic fever 92 and tuberculosis 93. The association of TLR-7/TLR-8 SNPs and infection are presented in Table 5.

TLR-9

Little is known about the clinical relevance of TLR-9 gene polymorphisms in critical illness. It was concluded that the TLR-9 polymorphisms rs187084 C>T transition (occurring in the 5′ near gene of TLR-9) and rs352162 T>G (located in the 3-flanking region of TLR-9) were useful to provide relevant risk estimates for the development of sepsis and multiple organ dysfunction in patients with major blunt trauma 94. Data from a Swiss HIV cohort in 2007 implicated two TLR-9 polymorphisms, A1635G (rs352140, Pro545Pro) and G1174A (rs352139, intron near 5′ UTR) with the rapid progression of HIV type 1 infection, with patients displaying a rapid CD4 cell decline 95. SNP A1635G occurs in exon2 of the TLR-9 gene but does not lead to any amino acid change (Pro545Pro), while G1174A is located in intron 1. Two more recent studies, however, showed that the A1635G allele was associated with a lower viral load set-point and slower progression 76,96. This discrepancy may be attributed to the fact that the Swiss 2007 cohort included subjects enrolled before the application of highly active anti-retroviral therapy, therefore not including rapid progressors who may have deceased and thus posing a bias in the study.

Finally, a link between allergic bronchopulmonary aspergillosis (ABPA) was demonstrated by Carvalho and co-workers 73, where patients with ABPA had a significantly higher frequency of allele C for the T-1237C SNP in TLR-9 (rs5743836) than control patients (20·5 versus 9·4%). This polymorphism is located within the putative promoter of the TLR-9 gene and has been implicated in chronic inflammatory diseases, including asthma. The same SNP was tested for a possible role in infection risk by human papilloma virus (HPV), but found not to be associated significantly with either HPV clearance or persistence 97. Table 6 presents TLR-9 SNPs and their association with human infectious diseases.

TLR-10

No ligand has been described for TLR-10. Due to its phylogenetic and physical proximity of TLR-10 to TLR-1 and TLR-6, several groups have looked for associations between this receptor and human diseases, with indications that TLR-10 SNPs may be linked to increased risk of certain cancers (to be described elsewhere) 36.