Genetic variations in toll-like receptor pathway genes influence asthma and atopy

Authors


  • Edited by: Hans-Uwe Simon

Prof. Michael Kabesch, Clinic for Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany.
Tel.: +49-511-532-3325
Fax: +49-511-532-9125
E-mail: kabesch.michael@mh-hannover.de

Abstract

To cite this article: Tesse R, Pandey RC, Kabesch M. Genetic variations in toll-like receptor pathway genes influence asthma and atopy. Allergy 2011; 66: 307–316.

Abstract

Innate immunity is a pivotal defence system of higher organisms. Based on a limited number of receptors, it is capable of recognizing pathogens and to initiate immune responses. Major components of these innate immunity pathogen recognition receptors are the toll-like receptors (TLRs), a family of 11 in humans. They are all membrane bound and through dimerization and complex downstream signaling, TLRs elicit a variety of specific and profound effects. In recent years, the role of TLRs signaling was not only investigated in infection and inflammation but also in allergy. Fuelled by the hygiene hypothesis, which suggests that allergies develop because of a change in microbial exposure and associated immune signals early in life, it had been speculated that alterations in TLRs signaling could influence allergy development. Thus, TLR genes, genetic variations of these genes, and their association with asthma and other atopic diseases were investigated in recent years. This review provides an overview of TLR genetics in allergic diseases.

Abbreviations
ATG16L1

autophagy-related 16-like 1

CARD

caspase-recruitment domain

CpG-DNA

cytidine-phosphate-guanosine (CpG) motifs containing DNA

dsRNA

double-stranded RNA

Foxp3

forkhead/winged helix transcription factor

GATA3

trans-acting T cell-specific transcription factor

GWAS

genome-wide association study

IFN

interferon

IKK complex

inhibitor of nuclear factor-B (IB)-kinase complex

IL1RL1 (or ST2)

interleukin-1 receptor family member

IRAKs

IL-1-receptor-associated kinases

IRFs

IFN-regulatory factors

LPS

lipopolysaccharide

LRR

leucine-rich repeat

LTA

lipoteichoic acid

MAPKs

mitogen-associated protein kinases

MD-2

myeloid differentiation protein-2

MyD88

myeloid differentiation primary-response protein 88

NFκB

nuclear factor-B (NF-B)

NOD

nucleotide-binding oligomerization domain

PAMPs

pathogen-associated molecular patterns

SHP-1

Src homology 2 domain containing phosphatases

SIGIRR

single immunoglobulin IL-1R-related molecule

SOCS1

suppressor of cytokine signaling 1

ssRNA

single-stranded RNA

TANK

TRAF family member-associated NFκB activator

T-bet

T-box expressed in T cells transcription factor

TBK1

TANK-binding kinase-1

TIRAP

TIR-domain-containing adaptor

TLRs

toll-like receptors

TOLLIP

toll-interacting protein

TRAF6

tumor-necrosis-factor-receptor-associated factor 6

TRAM

TRIF-related adaptor molecule

TRIF

TIR-domain-containing adaptor protein inducing IFN

In most Western countries, the prevalence of asthma increased over the last four decades of the 20th century. It has been estimated that 300 million individuals are affected all over the world (1). In addition to a strong genetic component in the pathogenesis of asthma (2), environmental factors may influence susceptibility for atopic diseases and the subsequent development of such diseases by affecting the activation of immune responses. More specifically, the maturation of immunity, as well as the timing and diversity of microbial exposure during the first year of life, are nowadays considered important factors, which may modify the risk of asthma in genetically susceptible individuals (3). Innate immunity is the pivotal system that facilitates the interaction of higher organisms with the microbial environment in the first pace and defends higher organisms against pathogens. Innate immunity is an evolutionary old defence system found in plants as well as insects and mammals. Its aim is the fast detection of pathogens from the environment when they get into contact with the organisms and to provide an efficient first line immune response. If not working properly, this may have profound effects.

The toll-like receptor system

A limited number of receptors expressed on the surface of and within cells recognize pathogen-associated molecular patterns (PAMPs). Leucine-rich repeat (LRR) domains for pathogen recognition are typical for these receptors (4). The so far identified spectrum of PAMPs recognized by this system is broad. Toll-like receptors (TLRs, toll receptors were primarily identified in Drosophila) are the most prominent and most extensively studied group of such PAMP receptors. The Drosophila toll receptor was initially discovered in the early 1980s through a mutagenesis screen for genes involved in dorso-ventral patterning of the Drosophila embryo. ‘Toll’ is the German exclamation attributed to the later Noble price laureate Christiane Nüsslein-Volhard for ‘amazing’ or ‘great’ on its discovery. In humans, 11 TLRs have been identified (5, 6). With regard to their cellular localization, TLRs can be divided into two subgroups: TLR1, TLR2, TLR4, TLR5, TLR6, TLR10, and TLR11 are expressed on the cell surface, while TLR3, TLR7, TLR8, and TLR9 are localized in intracellular vesicles such as the endosome or lysosome (Fig. 1). TLR2 is involved in the recognition of a wide range of PAMPs derived from bacteria, fungi, parasites, and viruses. It generally forms a heterodimer with TLR1 and TLR6; TLR1-TLR2 responds to the bacterial triacylated lipopeptide, whereas TLR2-TLR6 recognizes the mycobacterial diacylated lipopeptide. TLR4 is essential for responses to lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria. Similarly, TLR5 recognizes flagellin, a protein component of bacterial flagella. TLR3 responds to viral double-stranded RNA, while TLR7 and TLR8 preferentially recognize single-stranded RNA. TLR9 was originally identified to respond to unmethylated 2′-deoxyribo-cytidine-phosphate-guanosine (CpG) DNA motifs that are frequently present in bacteria (7). The knowledge of specific ligands of TLR10 and 11 is still incomplete. Recent studies have demonstrated that TLR10 shares some common agonists with TLR1 (and TLR2) (8). TLR11 is activated by a profilin-like molecule from Toxoplasma gondii in mice, and in mice, it is also a receptor for uropathogenic Escherichia coli (E. coli) (9–11).

Figure 1.

 Schematic presentation of the associations of toll-like receptors (TLRs) and TLR signaling molecules with atopic disorders and other human diseases, as reported in study population with more than 200 children or adults. Associations of TLR polymorphisms and their signaling molecules, with asthma (blue), atopic dermatitis (yellow), atopic sensitization (green), and other disorders (orange) are indicated by color; gray shades indicate absence of reported association. Binding of indicated ligands to TLRs leads to interaction with adaptor proteins, initiation of respective signaling cascades. Finally, translocation of the transcription factor NF-κB (and others) to the nucleus occurs and pro-inflammatory genes are activated. See text for more details.

It is striking that such a limited number of pathogen recognition receptors should be capable of recognizing an abundant number of pathogens and to initiate complex immune responses. The first secret of success for the system is that rather than recognizing individual pathogens specifically, the innate immune system recognizes pathogen patterns common to different genera or metasignals of infection. Furthermore, dimerization of receptors and variability in downstream signaling as well as cell-specific regulation adds to the complexity of the system and allows for particular PAMP-specific immune responses and the activation of proinflammatory genes.

TLR-dependent downstream signaling is classified based on the recruitment of adapter molecules in a myeloid differentiation primary-response protein 88 (MyD88)-dependent and/or independent signaling cascades. TLR1-2, TLR2-6, TLR5, TLR7, 8, and 9 recruit MyD88-dependent pathway resulting in phosphorylation of interleukin (IL)-1-receptor-associated kinases (IRAKs) and the involvement of tumor-necrosis-factor-receptor-associated factor 6 (TRAF6) and IKK complex [(inhibitor of nuclear factor-B (IB)-kinase complex)], leading to downstream activation of NFκB [(nuclear factor-B (NF-B)] and mitogen-associated protein kinases (MAPK) pathways, which induce cytokines/chemokines production (Fig. 1) (7, 12). A tight regulation of these pathways also results from posttranslational modification processes such as phosphorylation and ubiquitination (13). TLR3 acts through a MyD88-independent pathway and recruits TRIF [toll-interleukin-receptor (TIR)-domain-containing adaptor protein inducing interferon (IFN)], to induce type I IFN production through IFN-regulatory factor 3 (IRF3) (Fig. 1), while TLR7 and TLR9 induce type I IFN expression through IRF7. TLR4 uses both the MyD88-dependent pathway as well as the independent one. TIR-domain-containing adaptor (TIRAP), TRIF, and TRIF-related adaptor molecule (TRAM) are adapter molecules that participate in TLR signaling. TIRAP is involved in TLR2 and TLR4-mediated activation of the MyD88-dependent pathway. TRAM is the specific adapter of TLR4-mediated MyD88-independent response, which bridges TLR4 and TRIF. TLR4 forms also a complex with another LRR adaptor protein known as myeloid differentiation protein-2 (MD-2) on the cell surface (Fig. 1) (7, 12).

Thus, the distinct use of adapter molecules in the TLR signaling leads to a variety of TLR responses against different pathogens. Downstream, the signaling cascade activates specific transcription factors to induce cytokines production. Following the MyD88-independent cascade, transcription factor IRF3 is activated and translocated into the nucleus, increasing IFN-β production. Similarly, the activation of MyD88-dependent pathway results in NFκB and MAPK signaling, which leads to an increased production of pro-inflammatory cytokines such as tumor necrosis factor and interleukins (10, 12).

Also, the regulation of complex TLR pathway signaling is important to prevent hyperresponsiveness and adverse effects (Fig. 1). Therefore, TLR signals are controlled by negative regulators such as IL1RL1 (also known as ST2, IL-1 receptor family member), single immunoglobulin IL-1R-related molecule (SIGIRR), suppressor of cytokine signaling 1 (SOCS1) and IRAK-M (also known as IRAK3, IL-1 receptor-associated kinase-3). SIGIRR and IL1RL1 have TIR domains that interrupt TLR signaling by sequestering MyD88 and TIRAP transiently. IRAK-M prevents dissociation of IRAK1–IRAK4 complex, averting their association with TRAF6, and induces LPS tolerance (12). In addition, intracellular signaling molecules also include toll-interacting protein (TOLLIP) as one of the regulator of TLR signaling (12). Multiple other mechanisms mediated by several molecules [i.e., src homology 2 domain-containing phosphatases (SHP-1), TRAF family member-associated NFκB (TANK), A20, autophagy-related 16-like 1 (ATG16L1)] also suppress harmful induction of cytokines by limiting pattern recognition receptors responses (14).

Other innate immunity receptors

As outlined, the knowledge on the innate immune system is continuously expanding, and recent reviews on the currents status of understanding are published regularly. It becomes increasingly clear that innate immunity not only plays a crucial and primary role in pathogen defence but that alterations within the PAMP recognition system can set the stage for immune disorders. Compelling evidence also suggests that concomitant recognition of PAMPs and damage-associated molecular patterns, such as uric acid and adenosine triphosphate, is needed to start an immune response to allergens (15). Genetic variation in these systems may not only change host susceptibility to pathogens and lead to severe immune deficiency syndromes, but may affect common inflammatory diseases such as autoimmune disorders and allergy (16, 17).

Further groups of innate immunity-associated pathogen receptors comprise nucleotide-binding oligomerization/caspase-recruitment (NOD/CARD) proteins, which are located also in intracellular compartments (14, 18). Genetic variation within these intracellular pathogen receptors may also be associated with asthma and allergy development (19–22). However, this review will focus on the association between genetic variation in TLR pathway genes and their effect on allergy and asthma.

Innate immunity pathways and allergies

While the susceptibility for asthma and atopy is genetic, the decision if and when atopic disease develops is a multifactorial event to which environmental factors contribute significantly.

These external factors interact with genetically determined individual structures at interfaces of the human organism with the environment, such as the skin, the gut, and the lung mucosa. Environmental factors can increase the risk for asthma as shown for the exposure to environmental tobacco smoke prenatally or during childhood (23, 24), or decrease the asthma risk, as it has been observed in numerous studies for children raised on traditional farms (25, 26). Living on a farm is associated with a traditional lifestyle, close and early contact with livestock, a high and diverse burden of microbial exposure, and the intake of unprocessed food. While the exact mechanisms by which farm life protects from asthma have not yet been identified, it seems to be the exposure to microbes that decrease the risk for atopy and atopic asthma in a dose-dependent manner (27). On the other hand, the prevalence of nonatopic wheeze rises with increased exposure to microbes in the same population. As expected, there is no ‘one environment protection fits all’ (28). Protection from one disease comes with the drawback of an increased risk for other conditions.

The adaptive immune system, represented by T and B cells, has a well-known, direct impact on allergies. B cells, under the direction and tight control of T cells, produce immunoglobulin (Ig)E, a prerequisite for an allergic immune response. While these mechanisms and their role in allergy had long been known, increasing doubt exists, that the adaptive immune system is responsible for the initiation of the immune deviations that lead to the development of asthma (28). Rather, the adaptive immune system may be triggered to amplify immune signals toward an allergic response. Novel research findings suggest that first triggers for such an immune deviation may arise from the innate immune system at barrier interfaces.

Thus, a competent innate immune system is the first prerequisite for the immunological integrity of the surface–environment interface of higher organisms; a competent and interactive adaptive immune system is the second. Only then, the surface barrier function is effective. Recent hypotheses suggest that in allergies, this barrier between the organism and the environment may be disturbed: either by a shift in challenges from the environment or by (genetic) alterations of the barrier itself, e.g., by mutations of pathogen recognition receptors, co-molecules, or downstream signaling components of this delicate immune network.

Genetic alterations in the toll-like receptor system and allergy development

A special program of the American National Institute of Health (NIH) was devoted to identify genetic variation in components of the innate immune system, with a special focus on TLR genes (http://www.pharmgat.org/IIPGA2/). Indeed, a number of these TLR genes showed genetic variation, and the cluster where many of these TLR genes are located on chromosomes 4 (TLR1, 6, 10), was identified to be one of the most variable regions of the human genome (29). Allele frequencies correlate with a North–South gradient in the United Kingdom. Variation in these receptors reflects the potential for adaptation to different environment and may allow for the recognition of a wider variety of pathogens in the polymorphic population. More adaptation to changes in the (microbial) environment may increase the chances for survival of the species in different environments. However, genetic variation in TLRs introduced by mutation may also predispose to the development of immune deviations and disease. Because the airways are regularly exposed to inhaled suspended environmental allergens and PAMPs, asthma susceptibility may be at least partially associated with disordered immune responses to common environmental inhalants (30). Thus, asthma susceptibility might also be associated with variation in genes encoding components of innate immunity, making them biologically plausible candidate genes for asthma (31).

TLR4 and CD14

TLR4 is a major signaling receptor for LPS, the major component of endotoxins, which are released from the membrane of Gram-negative bacteria present in the environment. TLR4 is not only expressed by specialized immune cells but also by airway epithelial cells, which make up the first line of defence against inhaled antigens (32).

Variation in the TLR4 gene may modulate responsiveness to LPS and thus could play a role in the development of asthma. Several groups have studied the effects of the D299G (rs4986790) exonic TLR4 polymorphism on asthma with conflicting results. Reports on asthma specifically associated with LPS showed that D299G, which interrupts TLR4-mediated LPS signaling, was correlated with hyporesponsiveness to endotoxin in humans (33–35). Fagerås Böttcher et al. (36) observed that the same TLR4 polymorphism was associated with high prevalence of asthma and reduced LPS-induced IL12 (p70) responses in school-aged Swedish children; Saçkesen et al. (37) also showed that the heterozygosity for the same TLR polymorphism was associated with mild forms of asthma in Turkish children (Table 1). However, other studies have not demonstrated such effect of the D299G polymorphism on the overall incidence of asthma (38–41). Interestingly, because of ethnic variation in TLR4 allele distribution, the D299G polymorphism was not found in Singaporean Chinese by DNA sequencing (42). More recently, Penders et al. (43) examined within the KOALA birth cohort study (Child, Parent, Health, Focus on Lifestyle and Predisposition) a gene–environment interaction between gut microbiota, genetic variation, and the development of atopy. Specifically, the interaction between intestinal E. coli colonization at 1 month of age and TLR4/CD14 haplotype tagging and functional polymorphisms in association with subsequent development of eczema and allergic sensitization at age 2 years were studied. The authors found that intestinal colonization by E. coli was associated with decreased risk of sensitization in children with the TT genotype for a promoter TLR4 polymorphism (rs10759932).

Table 1.   Overview of the associations of CD14 and toll-like receptors (TLRs) polymorphisms (rs numbers) with atopic disorders and other human diseases, restricted to studies with more than 200 probands
GENESNP rsAssociation with diseases (reference number)
AsthmaAtopic eczemaAtopic sensitizationInfectious inflammation autoimmunity
  1. SNP, single nucleotide polymorphism; NR, not reported; TB, tuberculosis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus.

CD14rs2569190No (48, 79)Yes (50)Yes (47, 51, 82, 83)Sepsis (87, 88)
TLR1rs5743595Yes (55)NRNRNR
rs4833095Yes (55)NRNRNR
TLR2rs4696480Yes (38)NRYes (38)Sepsis (87)
rs3804099Yes (55)NR TB Meningitis (89)
rs5743708No (40)No (56)Yes (60)NR
TLR4rs4986790Yes (37)No 56)No (90)Sepsis (88); RA (91)
rs4986791Yes (55)NRNRNR
TLR6rs5743789Yes (55)NoNRNR
rs5743810Yes (55)No (92)NRNR
TLR7rs5743781Yes (66)NRNRNR
TLR8rs5744077Yes (66)NRNRNR
TLR9rs5743836No (55)Yes (64)No (65)SLE (93)
rs187084Yes (55)NRNo (65)NR
TLR10rs11096956Yes (68)NRNRNR
rs4129009Yes (55, 68)NRNRNR

The first hints that genetic alterations in the innate immunity’s pathogen recognition system may modify allergy risk actually came from CD14, a co-molecule in TLR4 signaling. CD14 is a glycosylphosphatidylinositol (GPI)-linked protein containing LRRs, which is involved, together with an LPS-binding protein (LBP), in response to LPS. CD14 is expressed on the surface of macrophages and monocytes and is also present in soluble form. It binds LBP and delivers LPS-LBP to the TLR4-MD-2 complex. CD14 may be considered a crucial link between nonadaptive and adaptive immune responses to environmental antigens. In fact, the engagement of CD14 by LPS and other bacterial cell wall-derived molecules enhances IL12/IL18 expression from antigen presenting cells, including dendritic cells and could favor T helper (Th)1 rather than Th2-type responses, which in turn may result in inhibition of IgE responses to environmental allergens (44). On the other hand, reduced exposure to LPS may be responsible for the delayed development of the mature, Th1 immune response, and this could be one of the many effects that LPS exposure in early life may have on the development of Th2-dominated immune disorders such as allergies. Fuelled by the hygiene hypothesis and the epidemiologic data showing that the presence of LPS in house dust is inversely correlated with atopy (45), and numerous genetic studies of atopic populations have been performed looking at specific CD14 variants.

A promoter polymorphism (rs2569190) was associated with levels of soluble CD14 protein in the serum in many studies (46–48) and phenotypes of allergy in some others (49–51). This CD14 polymorphism (rs2569190) leading to a C→T nucleotide substitution, located 260 bp from translation start site and 159 bp from transcription start site, alters CD14 promoter activity in vitro by decreasing the affinity of Sp protein binding and thus enhancing transcriptional activity (52).

TLR2 system

The TLR2 receptor is a major mammalian pathogen pattern receptor, which can form dimers with TLR1 or TLR6. The TLR1–TLR2 dimer recognizes triacylated lipopeptides, while the TLR2–TLR6 dimer recognizes diacylated lipopeptides. TLR2 responds to a wide variety of microbes, such as Gram-positive bacteria, Gram-negative bacteria, mycobacteria, mycoplasma (7), that are capable to induce severe pulmonary infections. Mice deficient in TLR2 and its downstream effector, MyD88, were found to be highly susceptible to an intravenous inoculation with Staphylococcus aureus (S. aureus) (53). Of all the TLRs, much attention has been paid to TLR2 in terms of immune-modulate disease pathogenesis. In particular, a role for TLR2 in such diseases as rheumatoid arthritis, type I diabetes, inflammatory bowel disease, and psoriasis, has been indicated (54).

In farmer’s children but not in other children growing up in rural environments without farm exposure, the A to T nucleotide substitution at site −16934 bp from the transcription start site, in TLR2 gene promoter region (rs4696480), was reported to significantly reduce the risk to develop allergic sensitization, hay fever, and asthma (Table 1) (38). However, as analyses were performed here in very small subgroups and the prevalence of asthma and other atopic diseases is extremely low in farmer’s children, these data must be viewed as preliminary until replicated in independent populations. No conclusions for children living in urban environments can be drawn from that study at this point.

When our research group recently screened genetic variants in the human TLR genes 1–10 systematically in large urban populations, we found associations between TLR polymorphisms and asthma development in children (55). Specifically, genetic variants in TLR receptors 1 and 6, forming complexes with TLR2, showed strong and replicable associations with atopic asthma in large populations of European children. Most polymorphisms significantly associated with atopic asthma were found in TLRs 1, 2, 4, 6, and 10 that predominantly recognize bacterial-derived ligands. Considering the broad spectrum of signals that are recognized by the TLR system, this is intriguing. These polymorphisms do not seem to lead to a loss of function of the respective TLR (thus, no severe effect on infection susceptibility is likely) but to a modification of effects associated with the TLR2 heterodimer network.

The role of TLR2 mutations has also been investigated in the pathogenesis of atopic dermatitis (55, 56). One hallmark of this skin disorder is the susceptibility to colonization with S. aureus (57). Enhanced susceptibility toward these bacteria may result from an insufficient host defence. TLR2 has emerged as a principal receptor in the defence against S. aureus (58), in that respect the role of the TLR2 polymorphism R753Q (rs5743708), which leads to an amino acid change, in atopic phenotypes has been studied. Of particular interest, Ahmad-Nejad et al. (59) documented that German patients with atopic dermatitis carrying this mutation had severe eczema, very high total, allergen-specific, and superantigen-specific IgE titers compared to those bearing the wild-type TLR2. However, other authors could not replicate the findings in a larger cohort of Germans (56). We observed that the same mutation R753Q resulted in reduced TLR2 activity in vitro experiments, assessed by NF-κB-driven reporter gene activation and IL-8 release assays, and also influenced the risk to develop atopic sensitization. Carriers of the 753Q allele in our population showed higher risk to develop atopy, as measured by skin prick test, elevated total serum IgE levels, and increased allergen-specific IgE (60).

The fact that TLR2 and associated TLRs are involved in the recognition of certain classes of bacterial components may not be arbitrary. Rather, these components recognized by the TLR2-associated system may share certain properties. The TLR2 pathway with the associated components TLR1 and TLR6 may by far not be the only factors in the innate immune system that contribute to the genesis of asthma and allergy, but the acquired data may help to pin down the mechanisms of disease development and protection in asthma. Signals by the TLR2 network, which are modified by the genetic makeup of the TLR2 receptor system and dependent on the dose of microbial exposure, seem to trigger effects by the adaptive immune system, resulting in elevated total and specific IgE levels, asthma, and atopic diseases (60). However, between pathogen recognition and asthma development, numerous intermediate regulatory effects may take place, such as IFN and cytokine production and transcription factors directing cell development (e.g., T-box expressed in T cells (T-bet), forkhead/winged helix transcriptor factor (Foxp3), and trans-acting T cell-specific transcription factor GATA3 for T-cells). Alterations and feed back events in these intermediate steps may modify or even counterbalance genetic susceptibility for disease development.

Other toll-like receptors

TLR9 is a receptor for bacterial CpG-DNA motifs, which have been found in increased levels in dust from rural homes (61). Only few studies have investigated TLR9 polymorphisms in relation to allergy or asthma, showing inconsistent results. A possible association of the promoter polymorphism C-1237T (rs5743836) with asthma has been reported by Lazarus et al. (62) in 67 European American asthmatics. However, other authors failed in reproducing such results testing more TLR9 polymorphisms in a bigger population (63). The same polymorphism in TLR9 was reported to be associated with atopic eczema in two German family cohorts (64), but not with total and specific IgE against common aeroallergens (65).

Polymorphisms with putative functional significance of TLR7 and TLR8, on the X chromosome, were recently found significantly associated to asthma and related disorders in two wide and independently ascertained Danish family samples (Table 1) (66).

Two polymorphisms of the TLR10 gene (rs11096956 and rs4129009) have been also associated with risk of asthma in two independent samples, although the ligands for TLR10 are not yet well characterized (Table 1) (67, 68). Recently, it has been reported that TLR10 can bind to some shared common ligands with TLR1 and 2, but its signaling pathways seem to differ from all other TLRs and is not yet understood. In particular, TLR10 fails to activate NF-κB IL8 and IFN-driven reporters (8). Furthermore, genetic polymorphisms of the TLR6-TLR1-TLR10 gene cluster are in tight linkage disequilibrium, and it may be difficult to dissect the effect of one polymorphism in the locus from others using population genetics alone (55). These novel findings make TLR10 an attractive candidate to deliver nonclassical TLR effects, which may be connected to asthma and allergy.

Zhang et al. (9) have reported that TLR11 sequences are present in the genomes of many mammalian species, including humans. TLR11 is expressed in macrophages, and liver, kidney, and bladder epithelial cells, in a distinct pattern from that of the other known TLRs. Cells expressing TLR11 respond specifically to uropathogenic bacteria. No genetic studies in the relationship of TLR11 variants and allergy-related phenotype have yet been carried out. However, it has been demonstrated that TLR11 expression on epithelial cells in murine models is regulated (among other factors) by an epithelium-specific transcription factor, ESE3 (69), which was proposed as a candidate molecule related to asthma susceptibility in another context (69, 70). However, it is not yet clear if and what role TLR11 is playing in humans, especially as its expression in humans is hampered by the presence of stop codons in the putative open reading frame of the gene (9). Also, systematic expression studies of TLR11 in humans are still lacking.

Genetic variations in toll-like receptor pathway genes

There is growing evidence that also polymorphisms in genes coding for proteins involved in the TLR pathway are correlated with asthma and allergy. A recent study revealed that several genes in the TLR-related MyD88-dependent pathway such as IL1RL1, MD2, MAP3K7IP1, and MAP3K7IP1 were associated with atopy and/or asthma in a Dutch population (21). The same study also described evidences of gene–gene interaction among these TLR-related pathway candidate genes. On the contrary, Hoffjan et al. (71) failed to find a significant association between polymorphisms in the NFKB1 gene and atopic dermatitis in Dutch children.

With regards to regulatory molecules of the TLR pathway, Balaci et al. (72) demonstrated an association of polymorphisms in IRAK-M gene with early-onset persistent asthma in an Italian population. Furthermore, Gudbjartsson et al. (73) demonstrated that one polymorphism in the IL1RL1 gene (rs1420101) was significantly related with eosinophil’s count and asthma in a large study population in a genome-wide association study (GWAS) approach. Finally, IL1RL1-IL18R1-IL18RAP gene cluster polymorphisms showed an association with asthma and IgE levels in two independent Dutch populations (74). Other authors have also suggested an association of TOLLIP gene polymorphisms with atopic dermatitis (75). No evidences on functional effects of these polymorphisms in TLR pathway genes have been reported so far.

Gene–environment interactions in TLR genes and TLR pathway

Most complex human diseases, such as asthma, are considered the result of interactions between genetic variations and environmental exposures. Recently, gene–environment interactions were found between 10 polymorphisms in candidate genes CD14, TLR2, TLR4, and TLR9 and living in the country during childhood, in adults enrolled in the EGEA (French Epidemiological study on the Genetics and Environment of Asthma, Bronchial Hyperresponsiveness, and Atopy) study (76). Similarly, as previously described, Eder et al. (38) have found that a polymorphism in the TLR2 gene (rs4696480) was strongly associated with frequency of asthma and allergies in children of European farmers. Such interaction studies are so far limited to TLR genes and do not include TLRs pathway genes.

In the past, the focus for gene-by-environment studies was mainly on CD14, and most published data on gene–environment interactions in atopic diseases deal with the promoter polymorphism C159T (rs2569190) in the CD14 gene and microbial exposure. Conflicting results of the association between this polymorphism and atopic sensitization could be because of differences in environmental exposures in the population studied or differences in exposure assessments (48, 77–80). Indeed, CD14 effect seems to be dependent on the level of microbial exposure. This was first suggested by Donata Vercelli in her ‘endotoxin-switch theory’ (81) and later shown by association studies (44, 80, 82, 83). These data indicated that the polymorphism in the CD14 promoter modified IgE levels according to the level of endotoxin exposure (as measured in the mattress of children). Depending on microbial exposure, the polymorphism effect may switch. In farmers (40, 83) and even nonfarmers populations (82), children exposed to high levels of endotoxin, the polymorphic C allele is associated with less IgE (83) and less allergy (82) and the opposite effect was observed with the same allele in the presence of low endotoxin levels.

Simpson et al. (80) demonstrated that the effect of domestic endotoxin exposure on atopic diseases and sensitization differed based on CD14 genotype carriage status in a Manchester cohort. The same kind of effect has been reported by Eder et al. (77), in a small study of farmers’ and nonfarmers’ children from rural alpine areas. Moreover, these gene–environment studies focused on CD14 have raised some open question, as was previously pointed out in an editorial (78). Firstly, the C-159T polymorphism in the CD14 promoter affects the production of soluble CD14, but not always relates to IgE expression and atopic phenotypes (78, 80). Thus, the mechanism by which this polymorphism might influence IgE expression, which is critical to define a causal role of CD14 polymorphism in allergy pathogenesis, is still not well understood. Secondly, endotoxin may act as a surrogate for other related but yet unknown exposures, which may be an alternative explanation for the lack of consistency between studies. Thus, even if there are great expectations for potential applications of gene–environment interactions, they seem to be extremely complex, and the effectiveness of adoption of environmental control measures remains controversial.

Implications and future directions

There is evidence for a role of TLR polymorphisms in the pathogenesis of allergy-related disorders. Particularly, positive associations of polymorphisms in TLR2, TLR4, TLR1, TLR6 (and TLR10) have been found with asthma and atopy, although some results were not consistently replicated. Studies have been focused on examining polymorphisms in the endotoxin receptor (CD14) promoter region in respect to microbial exposure. These studies have become more sophisticated overtime, but the size and power of these studies is still insufficient for conclusive results. Large studies with good exposure data and genetic information will be available in the near future (such as the GABRIELA advanced study) to answer these questions. However, gene-by-environment studies on GWAS datasets are difficult and expensive because these kinds of studies require excessively large populations. For now, the coverage of polymorphisms in the TLR system in GWAS studies is also quite limited, and several methodological issues such as the problem of multiple comparisons need to be resolved.

Recently, it has been proposed that epigenetics may play a role in gene–environment interaction effects contributing to the development of asthma and allergies; however, convincing evidence for that is still lacking (84, 85). Epigenetic mechanisms mediate environmental effects on the human genome by controlling the transcriptional activity (and thus expression) of specific genes, at specific points in time, in specific organs. If epigenetic mechanisms are affecting asthma, then strong and consistent environmental factors (such as the protective effect of growing up on farms) are the most likely once to drive epigenetic effects. In the context of farm exposure, it could be possible that the TLR system is affected by epigenetic mechanisms. Indeed, TLR expression changes with farming exposure status (86), but the role of epigenetics in that has not been studied. Surely, genes of the innate immune system will be a target for future epigenetic studies.

With recent technological breakthroughs in genetics such as the 1000 genome sequencing project (http://1000genomes.org/), it is now possible to study individual genetic diversity on the genome level, assessing not only a few polymorphism at a time but more than a million per individual in one experiment. Sequencing the whole genome of thousands of patients and controls will be feasible in the near future. To properly investigate gene–environment interactions in asthma on a new level, novel tools and better studies to assess environmental exposures will be needed. Only then, we will be able to understand asthma as the complex interaction of genes and environments it truly is.

With a more comprehensive approach on how genetic factors influence interaction with the microbial environment, we may be able to identify pathways and mechanisms that are causal for asthma, but also may play a role in other diseases. With this novel insight, novel strategies to interfere with pathogenesis and prevent disease development may become available in the future.

Acknowledgments

All authors have contributed in writing this review. This work was supported by the National Genome Research Network (NGFN) research grant NGFN 01GS 0810, the GRK 1441 research grant to Ramesh Chandra Pandey, and the European Respiratory Society long term fellowship research program to Riccardina Tesse. Funding sources had no influence in the writing of the review and in the decision to submit the review for publication. We state that there are no conflicts of interest with this work for any author.

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