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Toll-like receptors and airway disease
Drug Discovery Today: Therapeutic Strategies
Vol. 3, No. 3 2006
Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Respiratory diseases
Toll-like receptors and airway disease Sabine Hoffjan*, Jo¨rg T. Epplen Department of Human Genetics, Ruhr-University, Bochum, Germany
The increasing prevalence of asthma may be, at least in part, due to a lack of microbial contact and infections in early life. Toll-like receptors (TLRs) are innate immune receptors that recognize microbial patterns, inducing a proinflammatory immune response that may counter-
Section Editors: Roy Goldie – Faculty of Health Sciences, Flinders University, Adelaide, Australia Peter Henry – School of Medicine & Phamacology, The University of Western Australia, Nedlands, Australia
balance allergic diathesis. Thus, TLRs and downstream signalling molecules are promising drug targets for asthma. Novel TLR ligands are being developed that aim at inducing T helper cell (Th)1 cytokine production to counteract or prevent the Th2-dominated immune response in allergic diseases. Introduction The human immune system can be roughly divided into two major parts: innate and adaptive immunity. While the adaptive immune system works via clonal expansion of antibodies specifically directed against a certain pathogen, the innate immune system senses pathogens through a limited number of nonclonal pattern recognition receptors (PRRs). These PRRs recognize molecular structures shared by several pathogens instead of specific protein structures, thus inducing rapid but not pathogen-specific immune responses. At present, the best known PRRs are the TOLL-LIKE RECEPTORS (TLRs; see Glossary), mammalian homologues of the Drosophila protein Toll. Dysregulation of innate immunity may play an important role in susceptibility to airway diseases, especially allergic asthma [1]. Thus, TLRs and their downstream signalling molecules are promising drug targets for asthma. This review summarizes biological functions of TLRs, the role of TLRs in asthma pathogenesis as revealed by epidemiological and genetic studies, and recent advances for therapeutic targeting of TLRs. *Corresponding author: S. Hoffjan ([email protected]) 1740-6773/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddstr.2006.09.008
TLR structure and ligands The Toll protein was originally identified in Drosophila melanogaster as a protein important for embryogenesis and innate immune responses. Currently, ten different Toll-like receptors are known in humans (TLR1-10, Fig. 1). They are transmembrane receptors containing two important structural domains: the Toll/IL-1 receptor (TIR) domain, which is also a common structure in members of the interleukin (IL)-1 receptor family, and the leucin-rich repeat (LRR) domain. The TIR domain conveys intracellular signalling, whereas the extracellular LRR domain is involved in ligand recognition. TLRs bind phylogenetically conserved microbial structures, the so-called pathogen-associated molecular patterns (PAMPs; see Glossary). The PAMPs that are recognized by the different TLRs as well as several synthetic ligands are summarized in Table 1. TLRs can also influence adaptive immunity. Activation of TLRs on antigen-presenting cells leads to the expression of surface molecules and activation of T lymphocytes. T helper cells differentiate either into Th1 cells that predominantly produce interleukin 12 (IL12) and interferon-g (IFN-g) or into Th2 cells characterized by production of IL4, IL5 and IL13. Th1 and Th2 immune responses are balanced in healthy individuals, whereas Th2 immunity predominates in asthma and allergic diseases. Most TLR ligands induce a shift towards Th1-dominated immunity that may counterbalance the Th2 predominance in asthma [2]. Yet, recent studies suggest that a third Th class, the so-called T regulative cells, is also important for regulation of balanced immune responses [2]. 317
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Glossary Adjuvant for immunotherapy: immuno-stimulatory agent that is applied together with the specific allergen in order to enhance immune reactions and possibly reduce the number of required injections. Hygiene hypothesis: theory that suggests that the increase in allergic disorders over past decades may be due to a general lack of microbial contact in industrialized countries. PAMPs: pathogen-associated molecular patterns; conserved molecular structures shared by several pathogens that are recognized by innate immune receptors. Toll-like receptors: innate immune receptors that recognize phylogenetically conserved molecular patterns (so-called PAMPs).
Intracellular signalling pathways Signalling through TLRs leads to the activation of transcription factors, including nuclear factor kB (NFkB) and interferon regulatory factors (IRFs), eventually leading to strong induction of Th1-dominated immunity. Overall, the different TLRs activate similar pathways although some TLRs also
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trigger their own specific signalling cascades (summarized in Fig. 1). This differentiation in signalling is mediated by intracellular TIR domain-containing adapter molecules that interact with the intracellular TIR domain of the TLRs. These adaptor molecules include Myd88, TIR domain-containing adapter protein (TIRAP), TIR domain-containing adapter protein inducing IFN-b (TRIF), and TRIF-related adapter molecule (TRAM). Myd88 is the main adaptor molecule that is utilized by all TLRs except by TLR3 (Fig. 1). Current knowledge about TLR signalling has been summarized in [3], yet new signalling molecules and interactive pathways are being discovered, and more detailed knowledge about the complex structure of TLR signalling will be gained.
Microbial exposure and susceptibility to airway disease: the hygiene hypothesis The prevalence of allergic diseases, including asthma, has increased in industrialized countries during the recent decades
Figure 1. Signalling through TLRs. Recognition of endotoxin (lipopolysaccharide, LPS) via the receptor complex including TLR4, CD14 and MD2, is mainly mediated by Myd88 and leads through activation of IL-1 receptor-associated kinase 4 (IRAK4), IRAK1 and TNF receptor-associated factor 6 (TRAF6) to the nuclear translocation and activation of NFkB. This pathway eventually results in enhanced expression of inflammatory cytokines such as IL6 and IL12b. A second, Myd88-independent pathway for TLR4 signalling is mediated by TRIF in association with TRAM, eventually leading to the activation of both NFkB and IRF3, thus inducing both inflammatory cytokines and IFN-b. TLR3, on the contrary, can directly associate with TRIF, also triggering the described pathway. TLR7/8/9 also utilize Myd88; downstream signalling of these TLRs can either activate NFkB, IRF7 or IRF5, thus inducing both inflammatory cytokines and IFN-a and -b. TLR: Toll-like receptor; IRAK: IL-1 receptor-associated kinase; TNF: Tumour necrosis factor; TRAF: TNF receptor-associated factor; NFkB: Nuclear factor kappa b; IL: Interleukin; TRIF: TIR domain-containing adapter protein inducing interferon b; TRAM: TRIF-related adapter molecule; IRF: Interferon regulatory factor; IFN: Interferon.
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Table 1. Natural and synthetic ligands of human TLRs Receptor
Dimerization
Natural ligands
Synthetic ligands
TLR 1
Heterodimer with TLR2
?
Triacyl lipopeptides
TLR2
Heterodimer with TLR1 Heterodimer with TLR6
Peptidoglycan
Triacyl lipopeptides Diacyl lipopeptides
TLR3
–
Double-stranded RNAb
a
c
d
Poly I:C
TLR4
Homodimer
Endotoxin (LPS ) HSP 60
Monophosphoryl lipid A Aminoalcylglucosaminide-4-phosphates
TLR5
–
Bacterial flagellin
?
TLR6
Heterodimer with TLR2
?
Diacyl lipopeptides
TLR7
–
Single-stranded RNA
Imidazole quinolines, Guanosine nucleotides
TLR8
–
Single-stranded RNA
Imidazole quinolines
TLR9
–
Bacterial DNAe, Viral DNA
CpGf oligodeoxynucleotides
TLR10
–
?
?
a
Toll-like receptor. b Ribonucleic acid. c Lipopolysaccharide. d Heat shock protein. e Deoxyribonucleic acid. f Cytosine–guanine dinucleotide.
[2]. An attempt to explain this phenomenon is the so-called HYGIENE HYPOTHESIS (see Glossary). According to this theory, the general lack of microbial stimuli in early childhood in industrialized countries influences the developing immune system towards allergic diseases [4]. In other words, activation of innate immune responses early in life seems to be associated with protection from allergic diseases such as asthma. Epidemiological studies have shown that children living on farms and being exposed to many different microbial structures have a lower prevalence of atopy and asthma than children raised in urban areas [5]. A reduced risk of atopy has further been associated with increased childhood infections, higher number of siblings, early day-care entry and reduced use of antibiotics, among others. TLRs are involved in the first immune response to both acute infections and noninvasive microbial products. TLR4, for example, represents the receptor for endotoxin (lipopolysaccharide, LPS), a part of the cell wall of Gram-negative bacteria that is found ubiquitously in the environment. Endotoxin has been suggested as one of the major factors mediating the protection from allergic diseases (reviewed in [6]). Yet, endotoxin exposure can also aggravate asthma symptoms in patients and even induce airway hyperresponsiveness and an inflammatory response in healthy probands [6]. One explanation for this paradoxical observation is that the effect of endotoxin on asthma symptoms seems to depend critically on the timing of exposure: although environmental exposure to endotoxin early in life (before sensitization has occurred) has been associated with reduced asthma risk, endotoxin exposure later in life may exacerbate preexisting asthma [6]. Additionally, genetic differences in TLRs or downstream signalling molecules potentially influ-
ence the host’s ability to recognize and react to environmental factors such as endotoxin. Therefore, the effects of TLR polymorphisms have come into focus recently.
Genetic association studies for TLR and related genes Two coding variations were discovered in the TLR4 gene (GenBank accession no. NM_138554), Asp299Gly and Thr399Ile that were associated with hyporesponsiveness to inhaled endotoxin in humans [7]. Yet, only one study found a direct association of the TLR4 Asp299Gly polymorphism with asthma in Swedish school children [8], whereas three other studies showed no differences in the overall risk for asthma between carriers of the wild type and the less frequent genotypes [9–11] (Table 2). On the other hand, the Asp299Gly polymorphism was associated with a modified response to endotoxin [11], indicating gene–environment interaction. Similarly, a polymorphism at position 16934 in the TLR2 gene (GenBank accession no. NM_003264) was significantly associated with asthma and atopy only in children who grew up on farms [12]. Additional evidence for the importance of gene-environment interplay comes from the studies of CD14 (GenBank accession no. NM_000591), which associates with TLR4 to form the receptor complex for endotoxin. The T allele of the CD14 159 (C/T) promoter polymorphism was associated with a decreased total serum IgE in a cohort of children from Tucson (Arizona), whereas no association of this SNP with allergy or IgE levels was evident in a large German cohort. In the Hutterites, an isolated population from South Dakota, the -159T allele was instead associated with an increased risk for atopy [13]. Recently, an intriguing explanation for this phenomenon suggested that the level of endotoxin exposure may influence the switch over from the www.drugdiscoverytoday.com
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Table 2. Association of variations in genes encoding TLRs and related molecules with airway diseases in humans Gene a
TLR 1 TLR2
Gene name
Location
Variant
Phenotype
Population
Association?
Refs
Toll-like receptor 1
4p14
–
–
–
–
–
Toll-like receptor 2
4q31
16934 A/T
Asthma in children of farmers Asthma
Austrian/German
Yes
[12]
Japanese
No
[32]
7 A/C, IVS3+71C/A, Leu412Phe, 1377 C/T
Asthma
Japanese
No
[32]
Asp299Gly
Asthma
No
[9]
Yes
[10]
Thr399Ile
Severity of atopy in asthmatics Asthma Asthma Severe RSVc infection COPDd Asthma
North American, Canadian Caucasian
196147del 191 G/A, 597 T/C, 1350 T/C TLR3
Toll-like receptor 3
TLR4
Toll-like receptor 4
9q33
b
Nob Yes Yes Yes No
[11] [8] [15] [14] [9]
Asthma Severe RSV infection
German Swedish Israeli German North American, Canadian German Israeli
Nob Yes
[11] [15]
TLR5
Toll-like receptor 5
1q41
–
–
–
–
–
TLR6
Toll-like receptor 6
4p13
Ser249Pro
Asthma Childhood asthma
African American German
Yes Yes
[33] [34]
TLR7
Toll-like receptor 7
Xp22
–
–
–
–
–
TLR8
Toll-like receptor 8
Xp22
–
–
–
–
–
TLR9
Toll-like receptor 9
1237 C/T
Asthma
European American African American
Yes No
[35]
TLR10
Toll-like receptor 10
4p14
1635 G/A 1031 G/A 2322 A/G
Asthma Asthma Asthma
Japanese European American North American
No Yes Yes
[32] [36]
CD14
Monocyte differentiation antigen CD14
5q31
159 C/T
IgEe
North American
Yes
[37]
SPTf IgE, SPT Atopic asthma Asthma, IgE Asthma IgE in asthmatics BHRg, atopy Asthma Asthma Asthma, asthma severity Asthma, asthma severity Asthma Severe RSV infection
Hutterites Dutch Icelandic German Caucasian Chinese Australian Finnish Indian Barbados Australian Mexican, Puerto Rican Israeli
Yes No No No No Yes Yes No Yes Yesb No Nob No
[38] [39] [40] [41] [42] [43] [44] [8] [45] [46] [47] [48] [15]
a
Toll-like receptor. Article includes evidence for gene-environment interaction. c Respiratory syncytial virus infection. d Chronic obstructive pulmonary disease. e Immunoglobulin E. f Skin prick test. g Bronchial hyperresponsiveness. b
Th2-biased cytokine profile at birth to a Th1-biased cytokine profile in early childhood, and that endotoxin levels might interact with the CD14 genotype to confer either risk to or protection from atopic phenotypes later in life [13]. 320
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Associations of genetic variations with asthma have further been reported for the TLR3 (GenBank accession no. NM_003265), TLR6 (GenBank accession no. NM_006068), TLR9 (GenBank accession no. NM_017442) and TLR10
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(GenBank accession no. NM_030956) genes (Table 2). The TLR4 coding variations have further been associated with two other airway diseases: the Asp299Gly polymorphism showed an association with chronic obstructive pulmonary disease (COPD) in one study [14], and both coding variations were associated with severe respiratory syncytial virus infection [15]. Yet, for most of these associations, the results still await confirmation in large-scale studies and additional populations. In conclusion, there is convincing evidence that variation in genes encoding TLRs may play a role in the pathogenesis of airway diseases, especially asthma. Yet, a complex interplay between genetic variation and environmental factors obviously needs to be taken into account.
Therapeutic targeting of TLRs Because the activation of innate immune responses may be protective against the development of allergy by inducing Th1 cytokine production, therapeutic activation of TLRs is a promising strategy to counterbalance the Th2-dominated immune response in allergic diseases such as asthma. Natural as well as synthetic ligands of several TLRs have been tested for therapeutic use over the past few years, and the first clinical trials are under way (Table 3).
Targeting TLR9: cytosine–guanine dinucleotide DNA Unmethylated cytosine–guanine dinucleotide (CpG) motifs are common in prokaryotic (e.g. bacterial) DNA but less abundant in eukaryotic DNA. CpG DNA is detected via TLR9 expressed on immune cells and induces strong Th1dominated immune responses. In murine models of asthma, administration of CpG-containing oligodeoxynucleotides (CpG ODNs) successfully prevented the development of airway inflammation and hyperresponsiveness as well as
reduced chronic asthma symptoms [16]. Initial clinical trials with CpG ODNs as ADJUVANTS FOR IMMUNOTHERAPY (see Glossary) in allergic patients have already shown promising results. For allergen-specific immunotherapy, increasing doses of a specific allergen extract are administered weekly or biweekly over a long period to reduce the level of sensitivity to the allergen in question. Immunostimulators as adjuvants are thought to augment immune responses and thus, may reduce the number of required injections. For targeting TLR9, purified short ragweed pollen antigen Amb a 1 was conjugated to a 22-base-long immunostimulatory oligodeoxynucleotide (Amb a 1-immunostimulatory DNA sequence conjugate [AIC]). In one clinical trial, treatment of ragweed-sensitive allergic rhinitis patients with six escalating doses of AIC or placebo showed that Th1 cytokine production in the nasal mucosa was increased and Th2 cytokine production was decreased in AIC-treated patients [17]. A reduction in chest and nasal symptoms was observed for the second ragweed season after immunotherapy [17]. In another study, administration of six doses of AIC likewise led to a shift form the Th2-dominated immunity towards the Th1-dominated immunity [18]. No adverse side effects have been reported in humans so far. Yet, in mice, chronic CpG ODN administration led to structural as well as functional defects in the lymphoid organs and ultimately to liver necrosis [19]. Taken together, these studies – although very preliminary – show that targeting TLR9 via CpG ODNs is a promising approach for asthma and allergic diseases, but long-term adverse effects still have to be excluded.
Targeting TLR2: mycobacteria Mycobacterial lipoproteins bind to TLR2, subsequently leading to IL-12 production and a switch towards Th1-dominated
Table 3. Therapeutic targeting of TLRs Pros
Cons
Latest developments
Who is working on this strategy?
Refs
Targeting TLRa9: CpG ODNb
Potent adjuvant for immunotherapy
Long-term effects unknown; damage to lymphoid organs in mouse model
Clinical testing of Amb a 1 conjugated to AIC for immunotherapy
Targeting TLR4: MPLc1
Potent adjuvant for immunotherapy
Long-term effects unknown
In clinical use as adjuvant (Pollinex Quattro1)
Targeting TLR2: BCGd vaccination
Improvement of asthma symptoms
Adverse local reactions
Initial clinical trials
[20]
Targeting TLR2: M. vaccae
More effective than BCG in mouse models
Long-term effects unknown
Initial clinical trials
[24]
Targeting TLR7/8: imiquimod/ resiqimod
Potent local immunomodulatory drugs for several skin diseases Mouse model for asthma
Long-term effects unknown
In clinical use for dermatological diseases (Aldara1)
[17,18]
Corixa Corporation (http://www.corixa.com/)
3M Laboratories (http://www.3m.com/)
[27]
[29] [30]
a
Toll-like receptor. Cytosine–guanine dinucleotide-containing oligodeoxynucleotides. Monophosphoryl lipid A. d Bacillus Calmette-Gue´rin. b c
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immunity. Epidemiological studies suggest that the prevalences of tuberculosis and allergic disorders in a given area are negatively correlated (reviewed in [20]). Thus, vaccination with bacillus Calmette-Gue´rin (BCG) was evaluated as a therapeutic strategy for asthma. Studies in mice showed that BCG could reverse Th2-dominated immune responses and could positively influence asthma symptoms [20]. One clinical study in humans showed that administration of BCG to allergic children led to a decline in IgE levels but no obvious change in cytokine production from peripheral blood mononuclear cells [21]. In another randomized control trial, significant improvement in pulmonary functions and reduced need for asthma medication was obvious after BCG vaccination in asthmatic patients [22]. Yet, a clinical trial with repeated intradermal injections of heat-killed BCG had to be stopped because of excessive local reactions to BCG in some individuals [23]. Recent studies suggested that immunization with nonpathogenic Mycobacterium vaccae may be more effective for the treatment or prevention of allergic symptoms than BCG in murine models [20]. An initial clinical trial with a single intradermal injection of heat-killed M. vaccae (SRL172) showed no change in the early asthmatic response, whereas 3 weeks after treatment pulmonary functions were slightly increased and IgE levels and IL-5 production were decreased [24]. More comprehensive approaches are clearly needed to evaluate the therapeutic potential of SRL172 for asthma.
Targeting TLR4: endotoxin (LPS) The dual role of endotoxin (LPS) exposure in the development of asthma has already been discussed above. In mouse models, endotoxin exposure both in infancy and in prenatal period decreased susceptibility for sensitization to common allergens although airway hyperresponsiveness was not affected [25,26]. Furthermore, the immunostimulatory adjuvant effect of LPS has long been evident. Although LPS itself is too toxic for clinical use, derivatives of LPS have already been widely used as adjuvants for vaccines (summarized in [27]). Monophosphoryl lipid A is less toxic than LPS but has similar immunostimulatory activity. MPL1 (Corixa Corporation; http://www.corixa.com/) has been used in more than 120,000 vaccine doses to date and has so far proven to be safe and effective (for allergen-specific immunotherapy: Pollinex Quattro1 vaccine; Bencard Corporation; http://www. bencard.com/). Additionally, a new class of lipid A mimetics, aminoalcylglucosaminide-4-phosphates, has been developed that equally activates TLR4 and constitutes promising adjuvants for future therapy [28].
Targeting other TLRs The synthetic TLR7 or TLR8 agonists, imiquimod and resiquimod (imidazole quinolines), are the first TLR agonists that were used for clinical therapy in humans [29]. They 322
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are applied topically (imiqimod: 5% cream, Aldara1; 3M; http://www.3m.com/) and are used routinely for treatment of a variety of skin disorders including warts and skin cancer. A recent study suggested that resiquimod may also inhibit allergen-induced Th2 responses and airway hyperresponsiveness in a mouse model of asthma [30], offering interesting new perspectives for these drugs. Additional TLR ligands are currently being evaluated as potential therapeutics, including dsRNA motifs (TLR3) and flagellin (TLR5), but have not been used in clinical trials yet.
Probiotics A key player in the normal development of tolerance towards ubiquitous microbial products is the intestinal microflora. Because the hygiene hypothesis suggested that a general lack of microbial stimuli for the developing immune system may lead to allergic diseases, the use of live non-pathogenic microorganisms (e.g. Lactobacilli and Bifidobacteria) in the so-called probiotic foods has been postulated to be protective against allergies and asthma. Yet, the results of clinical studies are still somewhat contradictory to date (reviewed in [31]).
Conclusions Rapidly growing knowledge about TLR function and signalling has led to a variety of promising novel drugs for asthma and allergic diseases that are currently under basic research, tested preclinically or in clinical trials. Additionally, there is initial evidence that TLRs also play a role in the pathogenesis of other chronic airway diseases such as COPD. Yet, the longterm effects are unknown for all potential new therapies so far, and genetic differences in TLRs or downstream signalling molecules may influence the host’s response to these therapeutics. Thus, controlled clinical trials are warranted, and pharmacogenetic aspects should also be taken into consideration.
Acknowledgement This work was supported by a ‘Lise-Meitner’ grant 2006 of the Ministry for Innovation, Science and Technology NorthRhine Westfalia.
Related articles Yang, I.A. et al. (2006) The role of Toll-like receptors and related receptors of the innate immune system in asthma. Curr. Opin. Allergy Clin. Immunol. 6, 23–28 Renz, H. et al. (2006) The immunological basis of the hygiene hypothesis. Chem. Immunol. Allergy 91, 30–48 Kaisho, T. and Akira, S. (2006) Toll-like receptor function and signaling. J. Allergy Clin. Immunol. 117, 979–987 Ulevitch, R.J. (2004) Therapeutics targeting the innate immune system. Nat. Rev. Immunol. 4, 512–520
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Outstanding issues Why can therapeutic targeting of TLRs possibly counterbalance or prevent allergic diseases such as asthma? Is variation in genes encoding TLRs and related molecules associated with susceptibility to airway diseases? Which of the novel therapeutic strategies targeting TLRs have already been introduced into clinical trials?
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Choi, I.S. and Koh, Y.I. (2002) Therapeutic effects of BCG vaccination in adult asthmatic patients: a randomized, controlled trial. Ann. Allergy Asthma Immunol. 88, 584–591 Shirtcliffe, P.M. et al. (2004) Effect of repeated intradermal injections of heat-inactivated Mycobacterium bovis bacillus Calmette-Guerin in adult asthma. Clin. Exp. Allergy 34, 207–212 Camporota, L. et al. (2003) The effects of Mycobacterium vaccae on allergen-induced airway responses in atopic asthma. Eur. Respir. J. 21, 287–293 Gerhold, K. et al. (2002) Endotoxins prevent murine IgE production, T(H)2 immune responses, and development of airway eosinophilia but not airway hyperreactivity. J. Allergy Clin. Immunol. 110, 110–116 Blumer, N. et al. (2005) Prenatal lipopolysaccharide-exposure prevents allergic sensitization and airway inflammation, but not airway responsiveness in a murine model of experimental asthma. Clin. Exp. Allergy 35, 397–402 Baldridge, J.R. et al. (2004) Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents. Expert Opin. Biol. Ther. 4, 1129–1138 Evans, J.T. et al. (2003) Enhancement of antigen-specific immunity via the TLR4 ligands MPL adjuvant and Ribi.529. Expert. Rev. Vaccines 2, 219–229 Dockrell, D.H. and Kinghorn, G.R. (2001) Imiquimod and resiquimod as novel immunomodulators. J. Antimicrob. Chemother. 48, 751–755 Quarcoo, D. et al. (2004) Resiquimod, a new immune response modifier from the family of imidazoquinolinamines, inhibits allergen-induced Th2 responses, airway inflammation and airway hyper-reactivity in mice. Clin. Exp. Allergy 34, 1314–1320 Bjorksten, B. (2005) Evidence of probiotics in prevention of allergy and asthma. Curr. Drug. Targets Inflamm. Allergy 4, 599–604 Noguchi, E. et al. (2004) An association study of asthma and total serum immunoglobin E levels for Toll-like receptor polymorphisms in a Japanese population. Clin. Exp. Allergy 34, 177–183 Tantisira, K. et al. (2004) Toll-like receptor 6 gene (TLR6): single-nucleotide polymorphism frequencies and preliminary association with the diagnosis of asthma. Genes Immun. 5, 343–346 Hoffjan, S. et al. (2005) Evaluation of the toll-like receptor 6 Ser249Pro polymorphism in patients with asthma, atopic dermatitis and chronic obstructive pulmonary disease. BMC Med. Genet. 6, 34 Lazarus, R. et al. (2003) Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium, and haplotypes in three U.S. ethnic groups and exploratory case-control disease association studies (small star, filled). Genomics 81, 85–91 Lazarus, R. et al. (2004) Toll-like receptor 10 genetic variation is associated with asthma in two independent samples. Am. J. Respir. Crit. Care Med. 170, 594–600 Baldini, M. et al. (1999) A Polymorphism in the 50 flanking region of the CD14 gene is associated with circulating soluble CD14 levels and with total serum immunoglobulin E. Am. J. Respir. Cell Mol. Biol. 20, 976–983 Ober, C. et al. (2000) A second-generation genomewide screen for asthmasusceptibility alleles in a founder population. Am. J. Hum. Genet. 67, 1154–1162 Koppelman, G.H. et al. (2001) Association of a promoter polymorphism of the CD14 gene and atopy. Am. J. Respir. Crit. Care Med. 163, 965–969 Hakonarson, H. et al. (2001) Allelic frequencies and patterns of singlenucleotide polymorphisms in candidate genes for asthma and atopy in Iceland. Am. J. Respir. Crit. Care Med. 164, 2036–2044 Sengler, C. et al. (2003) Evaluation of the CD14 C159 T polymorphism in the German multicenter allergy study cohort. Clin. Exp. Allergy 33, 166– 169 Heinzmann, A. et al. (2003) Promoter polymorphisms of the CD14 gene are not associated with bronchial asthma in Caucasian children. Eur. J. Immunogenet. 30, 345–348 Leung, T.F. et al. (2003) The C159T polymorphism in the CD14 promoter is associated with serum total IgE concentration in atopic Chinese children. Pediatr. Allergy Immunol. 14, 255–260 O’Donnell, A.R. et al. (2004) Age-specific relationship between CD14 and atopy in a cohort assessed from age 8 to 25 years. Am. J. Respir. Crit. Care Med. 169, 615–622
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Sharma, M. et al. (2004) Suggestive evidence of association of C159T functional polymorphism of the CD14 gene with atopic asthma in northern and northwestern Indian populations. Immunogenetics 56, 544–547 Zambelli-Weiner, A. et al. (2005) Evaluation of the CD14/260 polymorphism and house dust endotoxin exposure in the Barbados Asthma Genetics Study. J. Allergy Clin. Immunol. 115, 1203–1209
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Kedda, M.A. et al. (2005) The CD14 C159T polymorphism is not associated with asthma or asthma severity in an Australian adult population. Thorax 60, 211–214 Choudry, S. et al. (2005) CD14 tobacco gene-environment interaction modifies asthma severity and immunoglobulin E levels in Latinos with asthma. Am. J. Respir. Crit. Care Med. 172, 173–182
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Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Respiratory diseases
Toll-like receptors and airway disease Sabine Hoffjan*, Jo¨rg T. Epplen Department of Human Genetics, Ruhr-University, Bochum, Germany
The increasing prevalence of asthma may be, at least in part, due to a lack of microbial contact and infections in early life. Toll-like receptors (TLRs) are innate immune receptors that recognize microbial patterns, inducing a proinflammatory immune response that may counter-
Section Editors: Roy Goldie – Faculty of Health Sciences, Flinders University, Adelaide, Australia Peter Henry – School of Medicine & Phamacology, The University of Western Australia, Nedlands, Australia
balance allergic diathesis. Thus, TLRs and downstream signalling molecules are promising drug targets for asthma. Novel TLR ligands are being developed that aim at inducing T helper cell (Th)1 cytokine production to counteract or prevent the Th2-dominated immune response in allergic diseases. Introduction The human immune system can be roughly divided into two major parts: innate and adaptive immunity. While the adaptive immune system works via clonal expansion of antibodies specifically directed against a certain pathogen, the innate immune system senses pathogens through a limited number of nonclonal pattern recognition receptors (PRRs). These PRRs recognize molecular structures shared by several pathogens instead of specific protein structures, thus inducing rapid but not pathogen-specific immune responses. At present, the best known PRRs are the TOLL-LIKE RECEPTORS (TLRs; see Glossary), mammalian homologues of the Drosophila protein Toll. Dysregulation of innate immunity may play an important role in susceptibility to airway diseases, especially allergic asthma [1]. Thus, TLRs and their downstream signalling molecules are promising drug targets for asthma. This review summarizes biological functions of TLRs, the role of TLRs in asthma pathogenesis as revealed by epidemiological and genetic studies, and recent advances for therapeutic targeting of TLRs. *Corresponding author: S. Hoffjan ([email protected]) 1740-6773/$ ß 2006 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddstr.2006.09.008
TLR structure and ligands The Toll protein was originally identified in Drosophila melanogaster as a protein important for embryogenesis and innate immune responses. Currently, ten different Toll-like receptors are known in humans (TLR1-10, Fig. 1). They are transmembrane receptors containing two important structural domains: the Toll/IL-1 receptor (TIR) domain, which is also a common structure in members of the interleukin (IL)-1 receptor family, and the leucin-rich repeat (LRR) domain. The TIR domain conveys intracellular signalling, whereas the extracellular LRR domain is involved in ligand recognition. TLRs bind phylogenetically conserved microbial structures, the so-called pathogen-associated molecular patterns (PAMPs; see Glossary). The PAMPs that are recognized by the different TLRs as well as several synthetic ligands are summarized in Table 1. TLRs can also influence adaptive immunity. Activation of TLRs on antigen-presenting cells leads to the expression of surface molecules and activation of T lymphocytes. T helper cells differentiate either into Th1 cells that predominantly produce interleukin 12 (IL12) and interferon-g (IFN-g) or into Th2 cells characterized by production of IL4, IL5 and IL13. Th1 and Th2 immune responses are balanced in healthy individuals, whereas Th2 immunity predominates in asthma and allergic diseases. Most TLR ligands induce a shift towards Th1-dominated immunity that may counterbalance the Th2 predominance in asthma [2]. Yet, recent studies suggest that a third Th class, the so-called T regulative cells, is also important for regulation of balanced immune responses [2]. 317
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Glossary Adjuvant for immunotherapy: immuno-stimulatory agent that is applied together with the specific allergen in order to enhance immune reactions and possibly reduce the number of required injections. Hygiene hypothesis: theory that suggests that the increase in allergic disorders over past decades may be due to a general lack of microbial contact in industrialized countries. PAMPs: pathogen-associated molecular patterns; conserved molecular structures shared by several pathogens that are recognized by innate immune receptors. Toll-like receptors: innate immune receptors that recognize phylogenetically conserved molecular patterns (so-called PAMPs).
Intracellular signalling pathways Signalling through TLRs leads to the activation of transcription factors, including nuclear factor kB (NFkB) and interferon regulatory factors (IRFs), eventually leading to strong induction of Th1-dominated immunity. Overall, the different TLRs activate similar pathways although some TLRs also
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trigger their own specific signalling cascades (summarized in Fig. 1). This differentiation in signalling is mediated by intracellular TIR domain-containing adapter molecules that interact with the intracellular TIR domain of the TLRs. These adaptor molecules include Myd88, TIR domain-containing adapter protein (TIRAP), TIR domain-containing adapter protein inducing IFN-b (TRIF), and TRIF-related adapter molecule (TRAM). Myd88 is the main adaptor molecule that is utilized by all TLRs except by TLR3 (Fig. 1). Current knowledge about TLR signalling has been summarized in [3], yet new signalling molecules and interactive pathways are being discovered, and more detailed knowledge about the complex structure of TLR signalling will be gained.
Microbial exposure and susceptibility to airway disease: the hygiene hypothesis The prevalence of allergic diseases, including asthma, has increased in industrialized countries during the recent decades
Figure 1. Signalling through TLRs. Recognition of endotoxin (lipopolysaccharide, LPS) via the receptor complex including TLR4, CD14 and MD2, is mainly mediated by Myd88 and leads through activation of IL-1 receptor-associated kinase 4 (IRAK4), IRAK1 and TNF receptor-associated factor 6 (TRAF6) to the nuclear translocation and activation of NFkB. This pathway eventually results in enhanced expression of inflammatory cytokines such as IL6 and IL12b. A second, Myd88-independent pathway for TLR4 signalling is mediated by TRIF in association with TRAM, eventually leading to the activation of both NFkB and IRF3, thus inducing both inflammatory cytokines and IFN-b. TLR3, on the contrary, can directly associate with TRIF, also triggering the described pathway. TLR7/8/9 also utilize Myd88; downstream signalling of these TLRs can either activate NFkB, IRF7 or IRF5, thus inducing both inflammatory cytokines and IFN-a and -b. TLR: Toll-like receptor; IRAK: IL-1 receptor-associated kinase; TNF: Tumour necrosis factor; TRAF: TNF receptor-associated factor; NFkB: Nuclear factor kappa b; IL: Interleukin; TRIF: TIR domain-containing adapter protein inducing interferon b; TRAM: TRIF-related adapter molecule; IRF: Interferon regulatory factor; IFN: Interferon.
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Table 1. Natural and synthetic ligands of human TLRs Receptor
Dimerization
Natural ligands
Synthetic ligands
TLR 1
Heterodimer with TLR2
?
Triacyl lipopeptides
TLR2
Heterodimer with TLR1 Heterodimer with TLR6
Peptidoglycan
Triacyl lipopeptides Diacyl lipopeptides
TLR3
–
Double-stranded RNAb
a
c
d
Poly I:C
TLR4
Homodimer
Endotoxin (LPS ) HSP 60
Monophosphoryl lipid A Aminoalcylglucosaminide-4-phosphates
TLR5
–
Bacterial flagellin
?
TLR6
Heterodimer with TLR2
?
Diacyl lipopeptides
TLR7
–
Single-stranded RNA
Imidazole quinolines, Guanosine nucleotides
TLR8
–
Single-stranded RNA
Imidazole quinolines
TLR9
–
Bacterial DNAe, Viral DNA
CpGf oligodeoxynucleotides
TLR10
–
?
?
a
Toll-like receptor. b Ribonucleic acid. c Lipopolysaccharide. d Heat shock protein. e Deoxyribonucleic acid. f Cytosine–guanine dinucleotide.
[2]. An attempt to explain this phenomenon is the so-called HYGIENE HYPOTHESIS (see Glossary). According to this theory, the general lack of microbial stimuli in early childhood in industrialized countries influences the developing immune system towards allergic diseases [4]. In other words, activation of innate immune responses early in life seems to be associated with protection from allergic diseases such as asthma. Epidemiological studies have shown that children living on farms and being exposed to many different microbial structures have a lower prevalence of atopy and asthma than children raised in urban areas [5]. A reduced risk of atopy has further been associated with increased childhood infections, higher number of siblings, early day-care entry and reduced use of antibiotics, among others. TLRs are involved in the first immune response to both acute infections and noninvasive microbial products. TLR4, for example, represents the receptor for endotoxin (lipopolysaccharide, LPS), a part of the cell wall of Gram-negative bacteria that is found ubiquitously in the environment. Endotoxin has been suggested as one of the major factors mediating the protection from allergic diseases (reviewed in [6]). Yet, endotoxin exposure can also aggravate asthma symptoms in patients and even induce airway hyperresponsiveness and an inflammatory response in healthy probands [6]. One explanation for this paradoxical observation is that the effect of endotoxin on asthma symptoms seems to depend critically on the timing of exposure: although environmental exposure to endotoxin early in life (before sensitization has occurred) has been associated with reduced asthma risk, endotoxin exposure later in life may exacerbate preexisting asthma [6]. Additionally, genetic differences in TLRs or downstream signalling molecules potentially influ-
ence the host’s ability to recognize and react to environmental factors such as endotoxin. Therefore, the effects of TLR polymorphisms have come into focus recently.
Genetic association studies for TLR and related genes Two coding variations were discovered in the TLR4 gene (GenBank accession no. NM_138554), Asp299Gly and Thr399Ile that were associated with hyporesponsiveness to inhaled endotoxin in humans [7]. Yet, only one study found a direct association of the TLR4 Asp299Gly polymorphism with asthma in Swedish school children [8], whereas three other studies showed no differences in the overall risk for asthma between carriers of the wild type and the less frequent genotypes [9–11] (Table 2). On the other hand, the Asp299Gly polymorphism was associated with a modified response to endotoxin [11], indicating gene–environment interaction. Similarly, a polymorphism at position 16934 in the TLR2 gene (GenBank accession no. NM_003264) was significantly associated with asthma and atopy only in children who grew up on farms [12]. Additional evidence for the importance of gene-environment interplay comes from the studies of CD14 (GenBank accession no. NM_000591), which associates with TLR4 to form the receptor complex for endotoxin. The T allele of the CD14 159 (C/T) promoter polymorphism was associated with a decreased total serum IgE in a cohort of children from Tucson (Arizona), whereas no association of this SNP with allergy or IgE levels was evident in a large German cohort. In the Hutterites, an isolated population from South Dakota, the -159T allele was instead associated with an increased risk for atopy [13]. Recently, an intriguing explanation for this phenomenon suggested that the level of endotoxin exposure may influence the switch over from the www.drugdiscoverytoday.com
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Table 2. Association of variations in genes encoding TLRs and related molecules with airway diseases in humans Gene a
TLR 1 TLR2
Gene name
Location
Variant
Phenotype
Population
Association?
Refs
Toll-like receptor 1
4p14
–
–
–
–
–
Toll-like receptor 2
4q31
16934 A/T
Asthma in children of farmers Asthma
Austrian/German
Yes
[12]
Japanese
No
[32]
7 A/C, IVS3+71C/A, Leu412Phe, 1377 C/T
Asthma
Japanese
No
[32]
Asp299Gly
Asthma
No
[9]
Yes
[10]
Thr399Ile
Severity of atopy in asthmatics Asthma Asthma Severe RSVc infection COPDd Asthma
North American, Canadian Caucasian
196147del 191 G/A, 597 T/C, 1350 T/C TLR3
Toll-like receptor 3
TLR4
Toll-like receptor 4
9q33
b
Nob Yes Yes Yes No
[11] [8] [15] [14] [9]
Asthma Severe RSV infection
German Swedish Israeli German North American, Canadian German Israeli
Nob Yes
[11] [15]
TLR5
Toll-like receptor 5
1q41
–
–
–
–
–
TLR6
Toll-like receptor 6
4p13
Ser249Pro
Asthma Childhood asthma
African American German
Yes Yes
[33] [34]
TLR7
Toll-like receptor 7
Xp22
–
–
–
–
–
TLR8
Toll-like receptor 8
Xp22
–
–
–
–
–
TLR9
Toll-like receptor 9
1237 C/T
Asthma
European American African American
Yes No
[35]
TLR10
Toll-like receptor 10
4p14
1635 G/A 1031 G/A 2322 A/G
Asthma Asthma Asthma
Japanese European American North American
No Yes Yes
[32] [36]
CD14
Monocyte differentiation antigen CD14
5q31
159 C/T
IgEe
North American
Yes
[37]
SPTf IgE, SPT Atopic asthma Asthma, IgE Asthma IgE in asthmatics BHRg, atopy Asthma Asthma Asthma, asthma severity Asthma, asthma severity Asthma Severe RSV infection
Hutterites Dutch Icelandic German Caucasian Chinese Australian Finnish Indian Barbados Australian Mexican, Puerto Rican Israeli
Yes No No No No Yes Yes No Yes Yesb No Nob No
[38] [39] [40] [41] [42] [43] [44] [8] [45] [46] [47] [48] [15]
a
Toll-like receptor. Article includes evidence for gene-environment interaction. c Respiratory syncytial virus infection. d Chronic obstructive pulmonary disease. e Immunoglobulin E. f Skin prick test. g Bronchial hyperresponsiveness. b
Th2-biased cytokine profile at birth to a Th1-biased cytokine profile in early childhood, and that endotoxin levels might interact with the CD14 genotype to confer either risk to or protection from atopic phenotypes later in life [13]. 320
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Associations of genetic variations with asthma have further been reported for the TLR3 (GenBank accession no. NM_003265), TLR6 (GenBank accession no. NM_006068), TLR9 (GenBank accession no. NM_017442) and TLR10
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(GenBank accession no. NM_030956) genes (Table 2). The TLR4 coding variations have further been associated with two other airway diseases: the Asp299Gly polymorphism showed an association with chronic obstructive pulmonary disease (COPD) in one study [14], and both coding variations were associated with severe respiratory syncytial virus infection [15]. Yet, for most of these associations, the results still await confirmation in large-scale studies and additional populations. In conclusion, there is convincing evidence that variation in genes encoding TLRs may play a role in the pathogenesis of airway diseases, especially asthma. Yet, a complex interplay between genetic variation and environmental factors obviously needs to be taken into account.
Therapeutic targeting of TLRs Because the activation of innate immune responses may be protective against the development of allergy by inducing Th1 cytokine production, therapeutic activation of TLRs is a promising strategy to counterbalance the Th2-dominated immune response in allergic diseases such as asthma. Natural as well as synthetic ligands of several TLRs have been tested for therapeutic use over the past few years, and the first clinical trials are under way (Table 3).
Targeting TLR9: cytosine–guanine dinucleotide DNA Unmethylated cytosine–guanine dinucleotide (CpG) motifs are common in prokaryotic (e.g. bacterial) DNA but less abundant in eukaryotic DNA. CpG DNA is detected via TLR9 expressed on immune cells and induces strong Th1dominated immune responses. In murine models of asthma, administration of CpG-containing oligodeoxynucleotides (CpG ODNs) successfully prevented the development of airway inflammation and hyperresponsiveness as well as
reduced chronic asthma symptoms [16]. Initial clinical trials with CpG ODNs as ADJUVANTS FOR IMMUNOTHERAPY (see Glossary) in allergic patients have already shown promising results. For allergen-specific immunotherapy, increasing doses of a specific allergen extract are administered weekly or biweekly over a long period to reduce the level of sensitivity to the allergen in question. Immunostimulators as adjuvants are thought to augment immune responses and thus, may reduce the number of required injections. For targeting TLR9, purified short ragweed pollen antigen Amb a 1 was conjugated to a 22-base-long immunostimulatory oligodeoxynucleotide (Amb a 1-immunostimulatory DNA sequence conjugate [AIC]). In one clinical trial, treatment of ragweed-sensitive allergic rhinitis patients with six escalating doses of AIC or placebo showed that Th1 cytokine production in the nasal mucosa was increased and Th2 cytokine production was decreased in AIC-treated patients [17]. A reduction in chest and nasal symptoms was observed for the second ragweed season after immunotherapy [17]. In another study, administration of six doses of AIC likewise led to a shift form the Th2-dominated immunity towards the Th1-dominated immunity [18]. No adverse side effects have been reported in humans so far. Yet, in mice, chronic CpG ODN administration led to structural as well as functional defects in the lymphoid organs and ultimately to liver necrosis [19]. Taken together, these studies – although very preliminary – show that targeting TLR9 via CpG ODNs is a promising approach for asthma and allergic diseases, but long-term adverse effects still have to be excluded.
Targeting TLR2: mycobacteria Mycobacterial lipoproteins bind to TLR2, subsequently leading to IL-12 production and a switch towards Th1-dominated
Table 3. Therapeutic targeting of TLRs Pros
Cons
Latest developments
Who is working on this strategy?
Refs
Targeting TLRa9: CpG ODNb
Potent adjuvant for immunotherapy
Long-term effects unknown; damage to lymphoid organs in mouse model
Clinical testing of Amb a 1 conjugated to AIC for immunotherapy
Targeting TLR4: MPLc1
Potent adjuvant for immunotherapy
Long-term effects unknown
In clinical use as adjuvant (Pollinex Quattro1)
Targeting TLR2: BCGd vaccination
Improvement of asthma symptoms
Adverse local reactions
Initial clinical trials
[20]
Targeting TLR2: M. vaccae
More effective than BCG in mouse models
Long-term effects unknown
Initial clinical trials
[24]
Targeting TLR7/8: imiquimod/ resiqimod
Potent local immunomodulatory drugs for several skin diseases Mouse model for asthma
Long-term effects unknown
In clinical use for dermatological diseases (Aldara1)
[17,18]
Corixa Corporation (http://www.corixa.com/)
3M Laboratories (http://www.3m.com/)
[27]
[29] [30]
a
Toll-like receptor. Cytosine–guanine dinucleotide-containing oligodeoxynucleotides. Monophosphoryl lipid A. d Bacillus Calmette-Gue´rin. b c
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immunity. Epidemiological studies suggest that the prevalences of tuberculosis and allergic disorders in a given area are negatively correlated (reviewed in [20]). Thus, vaccination with bacillus Calmette-Gue´rin (BCG) was evaluated as a therapeutic strategy for asthma. Studies in mice showed that BCG could reverse Th2-dominated immune responses and could positively influence asthma symptoms [20]. One clinical study in humans showed that administration of BCG to allergic children led to a decline in IgE levels but no obvious change in cytokine production from peripheral blood mononuclear cells [21]. In another randomized control trial, significant improvement in pulmonary functions and reduced need for asthma medication was obvious after BCG vaccination in asthmatic patients [22]. Yet, a clinical trial with repeated intradermal injections of heat-killed BCG had to be stopped because of excessive local reactions to BCG in some individuals [23]. Recent studies suggested that immunization with nonpathogenic Mycobacterium vaccae may be more effective for the treatment or prevention of allergic symptoms than BCG in murine models [20]. An initial clinical trial with a single intradermal injection of heat-killed M. vaccae (SRL172) showed no change in the early asthmatic response, whereas 3 weeks after treatment pulmonary functions were slightly increased and IgE levels and IL-5 production were decreased [24]. More comprehensive approaches are clearly needed to evaluate the therapeutic potential of SRL172 for asthma.
Targeting TLR4: endotoxin (LPS) The dual role of endotoxin (LPS) exposure in the development of asthma has already been discussed above. In mouse models, endotoxin exposure both in infancy and in prenatal period decreased susceptibility for sensitization to common allergens although airway hyperresponsiveness was not affected [25,26]. Furthermore, the immunostimulatory adjuvant effect of LPS has long been evident. Although LPS itself is too toxic for clinical use, derivatives of LPS have already been widely used as adjuvants for vaccines (summarized in [27]). Monophosphoryl lipid A is less toxic than LPS but has similar immunostimulatory activity. MPL1 (Corixa Corporation; http://www.corixa.com/) has been used in more than 120,000 vaccine doses to date and has so far proven to be safe and effective (for allergen-specific immunotherapy: Pollinex Quattro1 vaccine; Bencard Corporation; http://www. bencard.com/). Additionally, a new class of lipid A mimetics, aminoalcylglucosaminide-4-phosphates, has been developed that equally activates TLR4 and constitutes promising adjuvants for future therapy [28].
Targeting other TLRs The synthetic TLR7 or TLR8 agonists, imiquimod and resiquimod (imidazole quinolines), are the first TLR agonists that were used for clinical therapy in humans [29]. They 322
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are applied topically (imiqimod: 5% cream, Aldara1; 3M; http://www.3m.com/) and are used routinely for treatment of a variety of skin disorders including warts and skin cancer. A recent study suggested that resiquimod may also inhibit allergen-induced Th2 responses and airway hyperresponsiveness in a mouse model of asthma [30], offering interesting new perspectives for these drugs. Additional TLR ligands are currently being evaluated as potential therapeutics, including dsRNA motifs (TLR3) and flagellin (TLR5), but have not been used in clinical trials yet.
Probiotics A key player in the normal development of tolerance towards ubiquitous microbial products is the intestinal microflora. Because the hygiene hypothesis suggested that a general lack of microbial stimuli for the developing immune system may lead to allergic diseases, the use of live non-pathogenic microorganisms (e.g. Lactobacilli and Bifidobacteria) in the so-called probiotic foods has been postulated to be protective against allergies and asthma. Yet, the results of clinical studies are still somewhat contradictory to date (reviewed in [31]).
Conclusions Rapidly growing knowledge about TLR function and signalling has led to a variety of promising novel drugs for asthma and allergic diseases that are currently under basic research, tested preclinically or in clinical trials. Additionally, there is initial evidence that TLRs also play a role in the pathogenesis of other chronic airway diseases such as COPD. Yet, the longterm effects are unknown for all potential new therapies so far, and genetic differences in TLRs or downstream signalling molecules may influence the host’s response to these therapeutics. Thus, controlled clinical trials are warranted, and pharmacogenetic aspects should also be taken into consideration.
Acknowledgement This work was supported by a ‘Lise-Meitner’ grant 2006 of the Ministry for Innovation, Science and Technology NorthRhine Westfalia.
Related articles Yang, I.A. et al. (2006) The role of Toll-like receptors and related receptors of the innate immune system in asthma. Curr. Opin. Allergy Clin. Immunol. 6, 23–28 Renz, H. et al. (2006) The immunological basis of the hygiene hypothesis. Chem. Immunol. Allergy 91, 30–48 Kaisho, T. and Akira, S. (2006) Toll-like receptor function and signaling. J. Allergy Clin. Immunol. 117, 979–987 Ulevitch, R.J. (2004) Therapeutics targeting the innate immune system. Nat. Rev. Immunol. 4, 512–520
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Outstanding issues Why can therapeutic targeting of TLRs possibly counterbalance or prevent allergic diseases such as asthma? Is variation in genes encoding TLRs and related molecules associated with susceptibility to airway diseases? Which of the novel therapeutic strategies targeting TLRs have already been introduced into clinical trials?
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