review

Genetic variation in Toll-like receptors and disease susceptibility

Mihai G Netea1, Cisca Wijmenga2 & Luke A J ONeill3
Toll-like receptors (TLRs) are key initiators of the innate immune response and promote adaptive immunity. Much has been learned about the role of TLRs in human immunity from studies linking TLR genetic variation with disease. First, monogenic disorders associated with complete deficiency in certain TLR pathways, such as MyD88-IRAK4 or TLR3-Unc93b-TRIF-TRAF3, have demonstrated the specific roles of these pathways in host defense against pyogenic bacteria and herpesviruses, respectively. Second, common polymorphisms in genes encoding several TLRs and associated genes have been associated with both infectious and autoimmune diseases. The study of genetic variation in TLRs in various populations combined with information on infection has demonstrated complex interaction between genetic variation in TLRs and environmental factors. This interaction explains the differences in the effect of TLR polymorphisms on susceptibility to infection and autoimmune disease in various populations. One of the most important concepts to revolutionize the understanding of host defense against pathogenic microorganisms that has emerged during the past 20 years is the recognition of patterns of microbial structures by dedicated germline-encoded receptors known as pattern-recognition receptors (PRRs). This was proposed by Charles Janeway in 1992 (ref. 1) and gained supporting experimental evidence from a seminal study by Lemaitre and colleagues, who showed that Drosophila fruit flies that lack the hematocyte receptor Toll, which indirectly recognizes pathogens through the cytokine-like protein Spaetzle, are highly susceptible to infection with fungi and Gram-positive bacteria2. That first report was followed shortly by the discovery of Tolllike receptors (TLRs) expressed on cells of the mammalian immune system. These receptors recognize evolutionarily conserved structures of microorganisms and activate an inflammatory response3. The homology between the intracellular domains of TLRs and that of the type 1 receptor for interleukin 1 (IL-1) has provided the key to understanding the function of TLRs in the activation of innate hostdefense mechanisms4. During infection, the host inflammatory reaction is initiated by the recognition by PRRs of conserved structures of the pathogenic microorganisms known as pathogen-associated molecular patterns (PAMPs). Five major classes of PRR have been described: the TLRs, the CLRs (C-type lectin receptors), the NLRs (nucleotide-binding domain, leucine-rich repeatcontaining receptors), the RLRs (RNA helicase RIG-Ilike receptors) and the ALRs (cytoplasmic DNA receptor
1Department of Internal Medicine and Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands. 2Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. 3Trinity Biomedical Sciences Institute, School of Biochemistry and Immunology, Trinity College Dublin, The University of Dublin, Dublin, Ireland. Correspondence should be addressed to L.A.J.O. (laoneill@tcd.ie).

2012 Nature America, Inc. All rights reserved.

Published online 18 May 2012; doi:10.1038/ni.2284

AIM2like receptors)3,5. Among the families of PRRs, TLRs were the first to be described and have been studied most intensively. TLRs activate an acute inflammatory reaction after engaging with PAMPs from all the major classes of microorganisms, a reaction that represents the first line of innate host defense. Subsequently, stimulation via the TLR initiates and modulates the adaptive cellular and humoral immune responses6. More than 10 years of effort has identified the main signaling pathways activated by TLRs (Fig. 1). Signaling is initiated by adaptors that contain TollIL-1 receptor (TIR) domains. MyD88 is the universal adaptor, as it interacts with all the TLRs except TLR3, the receptor for double-stranded RNA. MyD88 also has a death domain, which recruits members of the IRAK (IL-1 receptorassociated kinase) family of serine-threonine kinases; this launches signaling pathways that culminate in the activation of transcription factors, most notably NF-B7. The structure of the multiprotein complex of the MyD88IRAK family has been solved, and this complex has been called the Myddosome8,9. Two Myddosomes have been characterized, defined by the presence of IRAK1 or IRAK2. In each, six MyD88 molecules assemble and interact with four IRAK4 molecules, which in turn interact with four IRAK1 molecules or four IRAK2 molecules. The interfaces between the components have all been solved structurally and thus detailed knowledge of the amino acids involved in the interactions is available. Notably, the Myddosome is also used by the receptors for IL-1, IL-18 and IL-33, which makes MyD88 especially important for inflammation and host defense. This is also true for the IRAK molecules, with IRAK4 being important for the activation of T cells by IL-1 (in cells of the TH17 subset of helper T cells) and most likely IL-18 (in cells of the TH1 subset of helper T cells)10. This broadens the role of this system into adaptive immunity. The other adaptors for the TLR system have more restricted uses. Mal (TIRAP) interacts with MyD88 and is required for signaling by the bacterial lipopeptide receptor TLR2 with a small amount of stimulation and is essential for signaling by the lipopolysaccharide
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TLR4, in which two amino acid changes (D299G and T399I) were reported to decrease the interaction of the receptor with lipopolysaccharide16 and to increase the susceptibility of patients to sepsis due to infection with Gram-negative bacteria 17. During the subsequent decade, a multitude of studies described genetic variation in practically all TLRs. The genetic variation in these receptors can be broadly described as resulting in complete functional deficiency that leads to primary immunodeficiency syndromes or having a variable effect on the function of the receptors; the latter has been studied in the context of diseases in case-control association studies.
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Figure 1 TLRs, their main ligands and the intracellular pathways that lead to the stimulation of proinflammatory cytokines. ssRNA, single-stranded RNA; dsRNA, double-stranded RNA.

receptor TLR4 (ref. 11). Mal is also required for signaling by the receptor RAGE (receptor for advanced glycation end products), which has been linked to several inflammatory, degenerative and hyperproliferative diseases12. The adaptor TRAM is used only by TLR4 and interacts with the adaptor TRIF at endosomes, which leads to activation of the transcription factor IRF3. TRIF is also the sole adaptor used by TLR3. Finally, the fifth TIR domaincontaining adaptor, SARM, is inhibitory for TRIF-dependent signaling and thus limits signaling by TLR3 and TLR4 (ref. 13). Through these complex mechanisms, TLRs serve an important role in host defense against infection. In addition, as a result of their interactions with endogenous ligands, they are also involved in the pathophysiology of inflammatory and autoimmune diseases14. Genetic variation in TLRs Soon after the first description of TLRs, genetic variability in these molecules was proposed to result in differences in susceptibility to infectious and inflammatory diseases15. The first genetic variation to be described in TLRs (in the year 2000) were polymorphisms in
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Primary immunodeficiency in the TLR pathways Complete defects in two main TLR-dependent pathways have been described so far. One defect leads to greater susceptibility to pyogenic bacteria (MyD88-IRAK4 deficiency) and the other results in greater susceptibility to herpesviruses (TLR3-Unc93bTRAF3 deficiency). Studies have identified patients with homozygous or compound heterozygous mutations in IRAK4 (refs. 1825) or MYD88 (refs. 26,27) that abolish protein production and result in a primary immunodeficiency syndrome characterized by greater susceptibility to pyogenic bacteria. Deficiency in MyD88-IRAK4 results in defective cell stimulation after engagement of TLRs and IL-1 receptors with subsequently impaired production of proinflammatory cytokines, followed by invasive and localized bacterial diseases. The invasive infections, such as meningitis, sepsis, arthritis or osteomyelitis, are caused by Streptococcus pneumoniae and Staphylococcus aureus and, less frequently, by Pseudomonas aeruginosa and Salmonella species. Localized bacterial diseases such as cellulitis, furunculosis and folliculitis are caused mainly by S. aureus, followed by P. aeruginosa and S. pneumoniae1828. So far, 15 different mutations have been identified in IRAK4, including insertions and deletions and missense, nonsense and splice-site mutations, and three different autosomal recessive mutations have been described in MYD88 deficiency in patients suffering from recurrent infection with pyogenic bacteria. Two of the mutations that lead to defective MyD88 (deletion of Glu52 or the substitution L93P) affect amino acids in key positions for the interaction between MyD88 and IRAK4 in the Myddosome8. A third mutation results in the substitution R98C, which is also in a key position for the so-called type 3 death domaindeath domain interactions found in the Myddosome structure29. Interestingly, the life-threatening infections in people with defective MyD88 first occur during in early infancy, as expected in a primary immunodeficiency, but they become less frequent and less severe during early adolescence, after which no life-threatening infections have been documented. Although the invasive infections in early childhood account for a cumulative mortality of 3040% (ref. 24), all adult patients have had a favorable clinical course without major infections and apparently with no prophylaxis needed28. This may indicate that the development of proficient adaptive immune responses (mediated by either T cells or B cells) later in life may compensate for the defects in the inflammatory reaction24, and if that phenomenon could be demonstrated, it would represent a major shift in the understanding of immune host defense. However, it should also be acknowledged that although MyD88-IRAK4 deficiency results in mortality of only 3040% in childhood, this prognosis is true only in advanced countries with well-developed intensive-care treatment facilities and in patients under protection with antibiotic treatment; the natural course of the disease would otherwise most likely be fully lethal. Although they are not related to primary immunodeficiency, striking examples of how somatic mutations in MYD88 can contribute to
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human malignancies for both chronic lymphocytic leukemia (2.9%) and diffuse large B cell lymphoma (29%) have been reported in two studies30,31. Both identified the same amino acid substitution, L265P, in the TIR domain of MyD88. The L265P mutant spontaneously assembled and activated the Myddosome complex, leading to the constitutive production of many cytokines, including IL-6, that are integral to the disease pathogenesis. An IRAK4 inhibitor was selectively lethal for B cell lymphoma cells, presumably acting by inactivating the constitutively active Myddosome complex, which suggests that an inhibitor of IRAK4 could have utility in treatment of lymphoma. The recognition of viral pathogens by the innate immune system is mediated by receptors from the following two classes of intracellular PRRs: the RLR family, and several members of TLRs that recognize nucleic acids (TLR9, which recognizes unmethylated bacterial or fungal DNA; TLR7 and TLR8, which recognize single-stranded RNA; and TLR3, which recognizes double-stranded RNA) 32. Although no defects in receptors of the RLR family have been reported so far, patients with autosomal dominant33 or recessive34 missense mutations in TLR3, patients with autosomal dominant mutation of TRAF3 (ref. 35), patients autosomal recessive mutations in UNC93B1, which encodes a molecule in the TLR3 pathway36, and patients with autosomal recessive or dominant mutation of TRIF37 all present with a clinical syndrome characterized by recurrent herpesvirus encephalitis. This disease occurs mainly in early childhood, in children 3 months to 6 years of age, during primary infection with herpes simplex virus type 1 (refs. 28,38). Interestingly, this immunodeficiency syndrome leads to greater susceptibility only to herpesvirus encephalitis and to no diseases caused by other pathogens. The functional defect in patients with such deficiency in TLR3-UNC93B1-TRIF-TRAF3 is probably the result of less capacity to release type I interferons. Type I interferons have a crucial role in antiviral host defense39,40, and in vitro experiments have shown loss of TLR3-dependent induction of interferons in these patients33. Why patients with this immunodeficiency are susceptible only to herpesvirus remains a mystery. TLR polymorphisms: individual and population levels Although the complete deficiencies in the TLR pathways described above have a strong, and sometimes devastating, effect at the level of the individual people affected (with the notable exception of TLR5 deficiency, discussed below), they are generally rare events with a limited effect on the scale of an entire population. Consequently, those mutations with severely deleterious effects are under strong purifying selection and will never increase in frequency. In contrast, the genes encoding TLRs are extremely polymorphic and encode many variant amino acid sites. The underlying nucleotide variation within a species is compatible with purifying selection driven by pathogens41. Before genome-wide association studies, TLRs were considered excellent functional candidates for involvement in enhanced susceptibility to and severity of both infections15 and autoimmune and inflammatory diseases4244. Polymorphisms in all TLRs have been described, and a wealth of studies have reported their association with enhanced susceptibility to and severity of infections. Several excellent reviews have already described those association studies in detail 15,4547; therefore, we will focus mainly on discussing the functional and evolutionary implications of these polymorphisms. A key obstacle, however, to understanding the true role of TLR polymorphisms in enhanced susceptibility to infectious disease has been the lack of availability of large infection cohorts for genetic-associations studies and the lack of replication of associations among different studies and across populations. Several suggestive examples emphasize this point. For example, the TLR4 haplotype that results in glycine at position 299
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(Gly299) and isoleucine at position 399 (Ile399) has been shown in two of four studies to be associated with a greater risk of sepsis48 and is suggested to result in a defective response to lipopolysaccharide16. However, various studies have failed to replicate those data at both genetic and functional levels49. The lack of replication might due to the small sample sizes, population stratification or the fact that the effect of this haplotype is restricted to certain subgroups of patients defined by criteria such as severity of disease or age of onset. Other polymorphisms associated with enhanced susceptibility to disease are those in the adaptor Mal (encoded by TIRAP), which is part of the TLR2 and TLR4 pathways. Single-nucleotide polymorphisms (SNPs) in TIRAP (resulting in the substitutions C558T and S180L) were initially shown to be associated with protection against tuberculosis50,51. However, meta-analysis of 6,584 patients with tuberculosis and 7,294 uninfected control subjects has not demonstrated any evidence of involvement of the S180L substitution in protection against susceptibility to tuberculosis52. Still, reports suggesting an association, particularly in South Indian and South African populations with tuberculosis meningitis, continue to appear53,54. A published review has nicely highlighted the problems faced in attempting to ascribe host genetics to enhanced susceptibility to tuberculosis, including differences in phenotype definition in both patients with tuberculosis and uninfected control subjects, consideration of latent versus active tuberculosis disease, population substructures and subsequent differences in linkage-disequilibrium patterns, and differences in the Mycobacterium tuberculosis strains causing the disease55. The S180L form of Mal has also been associated with malaria, pneumococcal disease and bacteraemia50, severe sepsis5658, Chagas cardiomyopathy59, systemic lupus erythematosus60, failure of the vaccine against Haemophilus influenza serotype b61 and Behets disease62. For unambiguous establishment of such associations, replication studies of larger populations are needed, given the relatively low odds ratios for most of them. The evidence of an association between the S180L variant of Mal and the failure of the vaccine seems to indicate a rather strong effect (odds ratio, 5.6; P = 1.2 107), possibly because of the tightly controlled nature of the study, which involved administering a vaccine to a defined population with careful followup. However, this study might also have been confounded by the typing of an indirect SNP and the low frequency of the risk allele. Clearly, many challenges remain in the analysis of genetic susceptibility to infectious diseases. The structure of Mal has been solved 63, and the Leu180 form has differences in structure compared with that of the Ser180 form. The amino acid at position 180 is very close to the aspartic acid at position 96 in Mal in the crystal structure, and substitution with asparagine at position 96 impairs the function of Mal29,64, which suggests that this region of Mal may be important for the functioning of Mal protein (Fig. 2). A final case that exemplifies the difficulties encountered in assessing the function of TLR polymorphisms is TLR5 deficiency. TLR5 recognizes flagellin, an important PAMP of flagellated bacteria65. A loss-of-function mutation in TLR5 (replacement of sequence encoding Arg392 with a stop codon) that results in a total defect in flagellin recognition has been described66. Interestingly, the mutated allele is present in approximately 10% of European populations and as much as 23% of other populations. People homozygous for this mutation are not characterized by severe primary immunodeficiency, although there are unconfirmed studies suggesting enhanced susceptibility to infections caused by Legionella pneumophila66 and to recurrent cystitis67. A protective effect of this mutation on systemic lupus erythematosus and Crohns disease has also been suggested68,69. The moderate to high frequency of the mutation resulting in a complete TLR5 defect
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Figure 2 The structure of the Myddosome and Mal provide information on the molecular basis of why variants in MyD88 and Mal are associated with disease. (a) The Myddosome has a stoichiometry of 14 and is composed of six MyD88, four IRAK4 and four IRAK2 molecules7. (b) Glu52 of MyD88 is at a key interface between MyD88 and IRAK4. Deletion of this amino acid in human leads to a greater risk of pyogenic infection and death in childhood and would disrupt the MyD88-IRAK4 complex. (c) The structure of Mal shows that Asp96 and Ser180 are in close proximity 63. The variant Leu180 is associated with many diseases, and Asp96 of Mal cannot associate with MyD88. Changes in these amino acids alter the structure of Mal in this region. Leucine at position 180 of Mal would cause steric occlusion of a cavity in Mal that could alter signaling, whereas asparagine at position 96 would alter the distribution of negative charge (red), which is probably key for the interaction with MyD88.

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in human populations, without a severe primary immunodeficiency phenotype, suggests a redundant role for TLR5 in host defense70. In conclusion, although many studies have suggested associations of TLR polymorphisms with disease processes, such conclusions should be interpreted with caution. Any of several factors may provide the basis for variation and discrepancies, such as the small sample sizes of many studies, population stratification (in particular in admixture populations) and restriction of the effect to certain subgroups of patients. In addition, all of those studies were of cohorts of limited sample size, and positive findings were often not replicated. Now it is possible to perform much more powerful genetic association studies and to control for the confounding factors that might have inflated some of the published results (such as population stratification). For example, genome-wide association studies have linked TLR7 and TLR8 to the genetic susceptibility to celiac disease (which involves an abnormal intestinal immune response to dietary gluten), one of the most common autoimmune disorders71. The advance of whole-genome and whole-exome sequencing is expected to identify many more such cases. Finally, one important but underestimated aspect of the influence of TLR polymorphisms on human diseases is heterogeneity in the frequency of polymorphisms and haplotypes among populations, often the result of past and present pressures exerted by local infections. Evolutionary pressure of infection A crucial aspect of the prevalence of polymorphisms in genes encoding TLRs and other genes related to immunity and their effect on susceptibility to infectious and autoimmune diseases is represented by the evolutionary processes that have influenced and shaped their spread in modern human populations. As discussed in detail below, several studies have suggested that certain polymorphisms in genes encoding TLRs in modern populations have resulted from positive selection through protection from infection. Subsequently, these studies may explain, at least in part, the differences among various populations in disease susceptibility. Classic examples of such differences are the lower susceptibility to malaria of populations in sub-Saharan Africa due to variants of the gene encoding hemoglobin 72 and the greater prevalence of autoimmune diseases in European populations than in African populations73. Systematic analyses of the evolutionary dynamics of TLRs in humans have reported the presence of selective forces that have shaped the evolution of this class of PRRs74,75. A first important observation was that all TLRs have undergone processes of purifying selection (that is, the elimination of gene variants with deleterious effects), albeit of different intensities. Interestingly, it seems that the intracellular TLRs (TLR3, TLR7, TLR8 and TLR9), which recognize mainly (viral) nucleic acids, have been under stronger purifying selection than have TLRs associated with the cell membrane. That has
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led to speculation that either viral infections have exerted a stronger evolutionary pressure than have bacterial infections during the evolution of Homo sapiens or membrane TLRs are partially redundant74 and alternative recognition mechanisms are available to take over the activation of host defense when membrane TLRs are defective. Still, TLR4 and TLR1 are also under clear evolutionary pressure, and strong evidence of recent selection in the TLR1-TLR6-TLR10 cluster has become apparent, especially in European populations74,76, which indicates that membrane TLRs also have nonredundant roles in host defense. Those data are supported by a study identifying positive selection for several TLRs, as well as IRAK4, in both African and European populations77. Notably, members of the gene family encoding IL-1 cytokines (related to TLRs through the TIR domain) mostly show signs of balancing selection in the same populations77, and this is in line with a study linking the IL-1 family of cytokines to an abundance of pathogen species and a greater likelihood of developing a disease caused by parasites78. Studies suggesting an effect of selection pressures acting at the level of TLRs are also supported by a report investigating the evolution of TLRs in several primate species41. The authors concluded that distinct signatures of positive selection are present in most TLRs, more so in virus-sensing TLRs than in those that do not sense viruses. Moreover, the strongest evidence of positive selection is found in TLR1 and TLR4 (ref. 41), two of the genes identified as target of selection by the earlier studies74,76. A similar analysis has been made of the evolution of the TIR domaincontaining adaptors79. MyD88 and TRIF have been shown to have evolved under purifying selection, which indicates their role is essential and nonredundant for host survival. MyD88 is the least polymorphic adaptor, with the genes encoding Mal and TRIF having the greatest nucleotide diversity. Nonsynonymous polymorphisms in Mal are common, whereas they are present at low frequency in the other adaptors. Overall, however, there is evidence of more constraints on the adaptors than on the TLRs, presumably because there are fewer adaptors and therefore less redundancy. The stronger selecting constraint on MyD88 and TRIF than on plasma-membrane TLRs indicates that the integrity of these adaptors is very important for host defense. This is probably due to the nonredundant roles of all of the TLRs and receptors for IL-1 (with TLR4 using both, and TLR3 using TRIF). Adaptations of Mal in Europe, of TRIF in Europe and of TRAM in Asia, possibly as a result of infectious diseases, have been reported. The determinants of TLR selection are most probably infections that cause high morbidity and mortality in people before they reach reproductive age. It is beyond the scope of this review to discuss all
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Figure 3 The major routes of migration of modern humans after the movement out of Africa 100,000 years ago, and the worldwide geographical distribution of haplotypes of TLR4 and TIRAP (which encodes Mal). Average allele frequencies of the African, European, Asian and New World continents were adapted from refs. 56 and 80. Migration routes and dates are adapted from ref. 90. The TLR4 allele encoding Gly299 (299 allele) was selected in Africa because it provided protection against malaria but was negatively selected in Eurasia because it resulted in greater susceptibility to septic shock. Constraints imposed by the migration routes of Homo sapiens have influenced the absence of this allele in the Americas, despite a high prevalence of malaria. In contrast, the allele encoding Gly299 and Thr399 (399 allele) has most probably followed a variable distribution influenced mainly by genetic drift80. The distribution of the S180L polymorphism of Mal (180 allele) shows balanced evolution: the moderately greater cytokine production in people heterozygous for this allele protects them from susceptibility to several major diseases such as tuberculosis, malaria and pneumococcal pneumonia, whereas an overshoot in cytokine release in people homozygous for the allele encoding Leu180 is deleterious in septic shock. The distribution of the Mal S180L polymorphism is heavily skewed toward the Indo-European populations in which it most probably occurred.

Wild-type TIRAP TLR4 180 allele Wild-type 299 allele 399 allele 299 & 399 allele Europe Asia

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the studies and data that have accumulated to support this hypothesis, but we will put forward a few powerful examples in support of this proposition. In addition, we should add that immunological research is now at a crossroads in the study of selection pressures on TLRs, as new databases such as the 1000 Genomes Project will prove to be an invaluable resource for such studies in the coming years, as this project will identify most genetic variants with a frequency of at least 1% in the 25 different and very diverse populations studied. TLR4 is one of the best-studied PRRs, and the distribution of TLR4 polymorphisms, as well as of the SNP that results in the S180L substitution of Mal, varies among different populations (Fig. 3). This is most probably because TLR4 with glycine at position 299 has protective effects against mortality due to Plasmodium falciparum cerebral malaria in Africa, where the TLR4 allele encoding Gly299 is highly prevalent (up to 15% in some populations)80. However, the deleterious effects of the product of this allele on the severity of Gram-negative sepsis prevented its fixation (that is, an increase in allele frequency to 100%) in Africa and may have been the selective force that led to nearly complete loss of this allele in Europe and Asia80. In addition, the SNP encoding the S180L substitution of Mal that results in an enhanced cytokine response is proposed to have been under balancing selection by protection against tuberculosis, malaria, pneumococcal infection50 and septic shock56 in heterozygous carriers. Similarly, it has been suggested that the positive selection of certain TLR1 alleles in Europeans is due to an attenuated inflammatory response, with potential beneficial effects during sepsis 74. Many other studies have reported important effects of polymorphisms of genes encoding receptors of the other PRR classes on susceptibility to infections, and comprehensive reviews have been published on this subject15,4547.
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Therefore, it is likely that selective forces exerted by pathogens have had an effect on the genetic makeup of genes encoding PRRs. However, it is also important to note that several studies published in the past few years have argued that nonadaptive mechanisms, such as genetic drift and geographical constraints, have also had an important role in the evolution of TLRs. Genetic drift and historical constraints In addition to selection, genetic drift is an important genetic process by which the frequencies of genetic variants both within and across populations change by chance alone. Processes such as population expansion and population bottlenecks can strongly influence the frequency of neutral variation, and it is very likely that such drift has influenced the frequency of at least some TLR polymorphisms (Fig. 3). For example, the frequency of the nonsense TLR5 polymorphism that results in replacement of the sequence encoding Arg392 with a stop codon ranges from 10% in Europeans to 23% in other populations70,74. The absence of major infectious complications in people who lack a functional TLR5 suggests that genetic drift, rather than adaptive processes, is the main mechanism behind the spread of this TLR5 variant, and this suggestion is supported by genetic studies74. Genetic drift is also the most likely cause of the variation of the TLR4 haplotype that encodes the D299G and T399I substitutions, which shows considerable variation in frequency among populations80. An obvious example of the role of geographical and historical constraints in influencing the genetic makeup of populations is the dependence of the genetics of one population on the genetic makeup of the population from which it originated. For example, the
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indigenous populations of South America, who have been living in warm climates for a relatively short period of time (the past 18,000 years at most), originate from human populations who lived in the cold climates of North-East Asia for much longer before that (estimated at 35,000 years). The effects of human-migration routes on the TLR4 and TIRAP polymorphisms have been studied56,80. Nonadaptive constraints such as population history explain why the TLR4 allele encoding Gly299 has been lost in Trio Indians from the Amazon jungle, where malaria is highly prevalent, despite the protective effect of the product of this allele against malaria in sub-Saharan African. The protective phenotype has probably been lost during the 25,000-year immigration of Homo sapiens to the Americas across the cold climates of northeast Asia, where the TLR4 allele encoding Gly299 was deleterious because of greater mortality from septic shock and possibly respiratory syncytial virus infection (the travel of these changes is presented in Fig. 3). Thus, the TLR4 haplotype encoding Gly299 that may prove protective against malaria in the Amazon tropical forest is paradoxically missing because of selective pressures in the opposite direction undergone by the ancestors of the target population during their migration through cold climates. An additional substantial geographical constraint was encountered by European populations during the final glacial maximum 20,000 years ago, a period in which the advancement of the ice cap up to Central Europe forced the remaining scattered populations to retreat to isolated regions of the Iberian, Italian and Balkan peninsulas81. This period, characterized by geographical constraints due to the isolation of populations in southern refuges, may have contributed to the spread of the TLR4 haplotype encoding Gly299 and Ile399 in European populations from the Iberian refuge82. In conclusion, all of the data discussed above suggest that in addition to adaptation through natural selection, the role of genetic drift and geographical factors should also be considered in the genetic history of TLRs. Consequences of TLR genetic variability for modern societies There is a substantial body of evidence indicating that variation in genes encoding TLRs and molecules involved in the cellular pathways associated with these receptors has evolved under selection driven by pathogens and infections83. However, a multitude of studies that accumulated during the past decade also support the proposal of the involvement of TLRs in enhanced susceptibility to severe immunodeficiency disorders. There is not yet overwhelming evidence to link TLRs to widespread susceptibility to infectious diseases, inflammatory disorders and autoimmune processes, although studies in this area may also have been hampered by power issues. TLRs can bind endogenous ligands (such as heat-shock proteins and HMGB1) that function as danger-associated molecular patterns and can thus initiate sterile inflammation14. In this manner, the host-defense mechanisms that have beneficial effects during infection can exert the deleterious effects of exaggerated inflammatory or autoimmune reactions (Fig. 4). The observation that celiac disease is associated with a locus containing both TLR7 and TLR8 is notable in this context71. This is particularly important, as both TLR7 and TLR8 are the target of strong purifying selection, possibly induced by resistance to viral infections74, and a viral trigger has long been proposed for celiac disease and other autoimmune disorders (such as infection with rotavirus84). Infectious pressures tend to select genetic variants of PRRs that result in strong immune responses required for the elimination of pathogens, but such selection would also favor genetic variants that enhance the inflammatory response and hence may lead to greater susceptibility to inflammatory or autoimmune diseases. It is important
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Figure 4 Adaptive evolution caused by infections, combined with nonadaptive evolution caused by genetic drift, population bottlenecks and migration routes, all contribute to PRR polymorphisms in various populations. These polymorphisms can give rise to resistance to infection via the sensing of PAMPs but also to a greater risk of autoimmunity, in which endogenous ligands acting via PRRs cause tissue damage and inflammation.

to realize that such responses may have beneficial effects early in life by protecting against childhood infections, whereas later in life, when autoimmune diseases become more prevalent, they may exert deleterious effects. Although this field of research is still in its infancy, important examples illustrate and support this proposition. A survey has shown that alleles and haplotypes related to innate immunity that are clearly compatible with positive selection, most probably due to a protective effect against infections, are in almost all cases associated with an greater risk of autoimmune disease85. That study is supported by data showing that pathogens lead to the selection of loci that confer both resistance to infection and, at the same time, susceptibility to autoimmune diseases78,83. For example, in celiac disease, recent positive selection has targeted several risk loci for celiac known to be associated with susceptibility to this disease83,86,87. One of these loci contains SH2B3, which encodes a protein with a structure reminiscent of that of cytokine-signalinginhibitory proteins (such as those of the SOCS family). The polymorphism of SH2B3 that results in the substitution R262W is associated with enhanced cytokine responses after stimulation of cells with bacterial peptidoglycans88. The SNP that encodes the R262W substitution is present at high prevalence in European populations but is almost absent in Asia and Africa. The sweep of the positive selection is estimated to have occurred around 1,2001,700 years ago88, coinciding with the collapse of the Roman Empire, during a period in which Europa was afflicted with the Justinian plague. Although the protein encoded by SH2B3 is not a member of the TLR family, its ability to modulate TLR signaling probably illustrates the general pattern of the effects exerted by genes encoding molecules of the immune system and most probably by TLRs as well. On the basis of the evidence presented above, it is rational to propose that the present high prevalence of certain alleles and haplotypes of genes encoding TLRs is the consequence not only of their protective effects against infections but also of nonadaptive genetic processes (for example, genetic drift and migration routes) of the past. In addition, extensive genetic variation is a driving force behind evolution and selection, as it allows modern humans to respond to a broad variety of infectious agents. In contrast, it might also predispose modern human populations to dysregulated immune responses that result in greater susceptibility to autoimmune and inflammatory diseases.
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Perspectives The two challenges that have emerged from the genetic analysis of the TLR system are the limited types of infections evident in primary immunodeficiencies and the low risk of infection in people bearing SNPs in a gene encoding one of the components of the TLR system, which in certain cases does not replicate across populations. Evolutionary analysis, however, strongly supports the proposal of a role for the TLR system in host defense, with redundancy in certain components possibly explaining the low risk of infection for particular single variants. However, such redundancy could be useful in the targeting of TLRs therapeutically for the treatment of autoimmune diseases or conditions, such as severe sepsis, or diseases of tissue injury, such as ischemia-reperfusion injury, conditions in which TLRs may have a less redundant role. For example, inhibiting TLR2 in heart ischemia has a clear benefit with no obvious signs of a greater risk of infection89. Similarly, inhibiting IRAK4 in B cell lymphomas that have mutant MyD88 could have considerable therapeutic benefits, possibly with a relatively limited risk of infection. Furthermore, if genetic variation can be used to predict severe sepsis or vaccine failure, testing for such variation could be useful for patient selection and personalized therapy or may help in the design of more efficient adjuvants. Further analysis of the functional consequences of genetic differences in the TLR system is needed to provide a better picture of what to target and for what indication.
ACkNOWLedGMeNts Supported by the Netherlands Organization for Scientific Research (M.G.N. and C.W.), Science Foundation Ireland (L.A.J.O.) and the European Research Council (L.A.J.O.). COMPetING FINANCIAL INteRests The authors declare no competing financial interests.
Published online at http://www.nature.com/doifinder/10.1038/ni.2284. reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.
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