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* From the Academic Unit of Respiratory Medicine, University of Sheffield, Sheffield, UK
Correspondence to: Ian Sabroe, PhD, Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, L Floor, Royal Hallamshire Hospital, Sheffield, S10 2JF, UK; e-mail: i.sabroe{at}sheffield.ac.uk
Abstract
Toll-like receptors (TLRs) are pivotal in human response to microbial stimuli. Their activation and signaling underpin much of the observed epidemiologic data generated by the hygiene hypothesis, and their contribution to infectious exacerbations of airways disease is likely to be highly important. Our growing knowledge in this field will have a significant impact on the understanding of the pathogenesis of inflammatory diseases, and TLR-based therapies are already in early clinical trials to modify atopic disease severity.
Key Words: hygiene • infection • Toll-like receptor • treatment
The innate immune system is crucial to our day-to-day survival and defense against infection. The recent identification of the Toll-like receptor (TLR) family has unlocked some of the most long-lasting and greatest secrets of the innate immune system, and has triggered a resurgent interest in the role of innate immunity in human disease. Therapeutics targeting TLRs (as either antagonists or agonists) have enormous potential to prevent inflammation, change the pattern of allergic disease, or improve responses to infection. Early clinical trials of such therapeutics are already showing real promise. Much more importantly, the discovery of TLRs has also triggered a reappraisal of disease pathogenesis in chronic lung disease, the benefits of which may be substantial and result not just in new therapies, but entire therapeutic approaches.
The primary function of TLRs is as sensors of infective stimuli. They are therefore placed to regulate the interactions that underpin sensitization to allergens (ie, the protective effect of "dirt" defined by the hygiene hypothesis). In chronic disease, activation of innate immunity can often result in excessive inflammation and can have a deleterious effect on the host, and exacerbations of many lung diseases have infection at their root. Examples include COPD and asthma, for which infections are the principal cause of acute exacerbations. This role of microbial stimuli, regulating both sensitization and exacerbations in diseases such as asthma, immediately underscores the central role that TLRs must play in respiratory disease.
TLRs: Origins, Structure, and Function
Detection of bacteria and responses to microbes is one of the most important functions of the immune system. Astonishingly therefore, it was only in the late 1990s that the receptors allowing our immune system to sample and respond to microbial stimuli were identified. Lipopolysaccharide (LPS) is the major septic shock-inducing component of Gram-negative bacterial cell walls; and while work in the early 1990s identified CD14 as a key receptor facilitating LPS recognition, the lack of an intracellular signaling face of CD14 demonstrated that this molecule could only be part of the story of endotoxin responses.1 To reveal the mechanisms resulting in mammalian recognition of LPS, we had to return to a much more primitive species: the fruit fly.
The toll gene was initially identified in Drosophila species, in which it is essential for normal development of the fly.2 Meaning "great," "crazy," or "weird" in German, toll-deficient flies failed to develop past the embryo stage. A clever strategy using temperature-sensitive gene expression alleles allowed expression of the Toll protein in larvae but its suppression in adult flies. These studies revealed that flies deficient in Toll were highly vulnerable to fungal infections.3
The realization that proteins with homology to Toll (TLRs) were encoded by the mammalian genome sparked considerable interest.4 Two strands of evidence revealed the importance of TLRs in humans. Firstly, it was shown that activation of TLRs stimulated signaling pathways associated with immunity.5 Most importantly, however, positional cloning research published in 1998 and 1999 on two naturally occurring mouse strains that are susceptible to Gram-negative infection, and unable to respond normally to LPS, revealed that these mice were also deficient in one of these TLRs, TLR-4.67 Subsequently in humans, TLR-4 missense mutations affecting the extracellular domain have also been associated with endotoxin hyporesponsiveness,8 thus highlighting the critical role of TLRs in immune defense.
It transpires that the identification of TLRs as critical to recognition of, and responses to, Gram-negative bacteria is merely one function of this vital family of pattern-recognition receptors. To date, 10 human TLRs have been described, each responding to distinct and varied pathogen associated molecular products (mice also possess an eleventh functional TLR). TLRs can be broadly classified as having antibacterial or antiviral responses. TLR-1, TLR-2, TLR-4, TLR-5, and TLR-6 principally (although not exclusively) serve antibacterial roles, through the detection of Gram-negative LPS (TLR-4) and Gram-positive lipoproteins (TLR-2, acting as a heterodimer with TLR-1 or TLR-6).9101112 TLR-5 recognizes flagellin (the key protein in bacterial flagellae). TLR-3, TLR-7, and TLR-8 respond to forms of RNA (double stranded for TLR-3, single stranded for TLR-7 and TLR-8) and have been implicated in antiviral responses.1314 TLR-9 distinguishes between human and nonhuman DNA through its ability to recognize so-called "CpG" (cytosine-guanine dinucleotide) motifs15: motifs that are less represented in human DNA (and further obscured in the human by cytosine methylation). The roles of TLR-9 are principally antibacterial, although it has clearly some ability to also recognize viral DNA. Interestingly, TLRs (particularly TLR-4) have also been implicated in the response to a number of endogenous molecules that are released at sites of cell damage and tissue injury. These endogenous agonists include heat shock protein 601617 and heat shock protein 70,18 fragments of fibronectin, and oligosaccharides of hyaluronic acid.19 This detection of danger signals from cell damage suggests additional roles for TLRs in the pathogenesis of sterile inflammation, and a study20 suggests that TLR signaling via hyaluronan is important in the maintenance of lung epithelial integrity and recovery from injury.
LPS and lipoprotein molecules may differ widely between bacterial species, but almost are all recognized by the TLRs. We understand only poorly how a restricted family of receptors can respond to such a diversity of exogenous and endogenous agonists, especially as these agonists often have very little structural similarity. This has given rise to the classing of the TLRs as pattern recognition receptors, and their agonists as pathogen-associated molecular patterns. There is still a paucity of data demonstrating direct physical association of many of these agonists with their receptors. It is however becoming increasingly appreciated that TLRs can at times utilize a number of accessory proteins to facilitate recognition. This is particularly true for TLR4, which requires myeloid differentiation-2,21 and CD14,1 these proteins helping to overcome the hydrophobicity and facilitate the recognition of LPS. Deployment of a limited number of TLRs in a combinational pattern may also diversify the repertoire of Toll-mediated responses, as exemplified by TLR-2 heterodimerization with TLR-1 and TLR-6 in response to lipoproteins from Gram-positive organisms.222324
There are a number of excellent reviews on TLR structure and signaling to which the reader is directed should they wish to pursue this interesting area.2526 In brief, the TLRs are comprised of an extra cellular domain containing blocks of leucine-rich repeats, which are thought to be primarily involved in ligand recognition. The cytoplasmic domains share sequence and functional homology with interleukin (IL)-1 receptors, and are thus named Toll/IL-1R domains. Toll/IL-1R domains serve as a scaffold for a complex series of protein-protein interactions, ultimately leading to the downstream activation of effector proteins and transcription factors such as mitogen-activated protein kinase (MAPK), phosphoinositide 3 kinase, nuclear factor (NF)-
B, and the interferon (IFN) regulatory factors (these latter transcription factors are important in type I IFN generation, particularly in the case of antiviral responses) [Fig 1
].
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The ability to develop knock-out mice deficient in specific components of TLR signaling has emphasized the important role of TLRs in infection control. For example, TLR-2–deficient mice have been shown to be susceptible to Staphylococcus aureus,12 pneumococcal,27 and mycobacterial infections; TLR-4–deficient mice are susceptible to Gram-negative infections28; and TLR-3–deficient mice are susceptible to viruses such as cytomegalovirus.29 Completely nonfunctional variants are extremely rare in humans, presumably as they would carry little chance of survival of individuals to reproductive age. Recently, however, individuals have been identified with recurrent life-threatening bacterial infections who have mutations in IL-receptor–associated kinase 4, a key component of TLR and IL-1 signaling pathways.3031 Several studies have begun to examine the impact of TLR deficiency (ie, knock-out mice) on survival in pneumonia models. Such studies3233 are generating data that clearly indicate a role for TLRs in the inflammatory response to microbes; although depending on the microbe and model, contributions of individual TLRs to bacterial clearance are still variable. Archer and Roy34 showed a role for TLR-2 in responses to Legionella pneumophila, and TLR-2 and TLR-4 both seem important in responses to Chlamydia pneumoniae.35 Gene deficiencies do not always correlate with impaired microbe clearance, as illustrated by a study36 showing no effect of TLR-2 deficiency on survival in response to pneumococcal infections, although TLR-4 deficiency prevents recognition of pneumococcal pneumolysin, and does convey increased risk of death from pneumonia in mouse models.37 TLR-5 also contributes importantly to responses to bacteria such as Pseudomonas aeruginosa.3238 These data make it clear that, in man, microbial recognition, and probably effective clearance, will heavily involve TLR-mediated recognition.
There has also been a growing interest in the identification of TLR genetic variations that can predict disease susceptibility. CD14 polymorphisms, a key accessory molecule for TLR signaling, have been associated with increased prevalence of positive bacterial culture findings and sepsis attributed to Gram-negative infections in a critically ill population,39 as well as the susceptibility to chronic C pneumoniae infection in patients with coronary artery disease.40 Arbour et al8 also showed that two polymorphisms of the TLR-4 gene were present in a higher proportion of individuals who are hyporesponsive to inhaled LPS. This report was followed by a number of studies investigating the potential impact of these TLR-4 polymorphisms on the course of infectious diseases. Two groups4142 demonstrated the association between the development of septic shock and TLR-4 polymorphisms. There have also been correlations between TLR-4 polymorphisms and severity of malaria and Candida infection.4344 These polymorphisms do not confer susceptibility to all Gram-negative infections, since other groups4546 have shown no correlation in other infectious diseases such as meningococcal disease, and again one should be mindful that very significant hypofunctioning TLR-4 alleles are likely to have a very strong negative selection pressure across the generations. In keeping with a potential role for TLR-4 in the detection of tissue damage, there is some evidence that polymorphisms in this gene may influence risks of transplant rejection,47 and may be important in risks of atherosclerotic disease.48 TLR-2 polymorphisms have also been linked to susceptibility to staphylococcal infection,49 lepromatous leprosy,50 and have been attributed as a risk factor for coronary restenosis after stenting.51 Although some of these studies are still relatively small in scale, they reinforce the important role of TLRs in pathogen recognition and immune response in humans.
TLRs in Disease
The role of TLRs in the detection of and response to microbial stimulation and tissue damage suggests that these receptors will play integral roles in many lung diseases (Fig 2
). The reader will immediately be able to see that neutralization of excessive inflammation may prevent tissue damage in COPD or cystic fibrosis, while enhancement of antimicrobial potential could equally perhaps be of use in the same conditions. While the manner of exploitation of TLRs in these diseases remains to be determined, for asthma and allergic disease TLR-based treatments are already in clinical trials.
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It is increasingly appreciated that infections often trigger and perpetuate inflammation in patients with asthma, and that TLRs are likely to play an important role in this response. However, with the emergence of the hygiene hypothesis it is also apparent that early exposure to a variety of microbial products and specific infections can confer a protection against the development of atopy.5556 Evidence for a modifying role of genetic variations in TLRs (and other proteins involved in TLR signaling) in asthma susceptibility reinforce this potential. CD14 polymorphisms are associated with reduced IgE levels and atopic phenotype in children.57 Polymorphisms in TLR-2 also influence atopic disease risk in children of farmers in particular.58 Although the function of TLR-10 is still unknown, single-nucleotide polymorphisms in this gene are associated with the asthma phenotype in two independent samples.59 The mechanism of the protective effect of TLR signaling is still under investigation. While it is clear that there is redirection away from a T-helper type 2 (Th2) response to allergens to a T-helper type 1 (Th1) pattern, it is also likely that microbial agonists regulate T-regulatory cell responses either directly or via the dendritic cell.6061 In mouse models of allergic sensitization, low levels of LPS inhalation via TLR4 signaling could drive a Th2 phenotype response, while in contrast, high levels induced Th1 responses.62 This action of endotoxin (and other microbial agonists) again may be mediated at the level of the dendritic cell/T cell interface since TLR agonists can clearly modify this interaction in potentially complicated patterns.6364 An exciting aspect of TLR biology is the emergence of evidence that administration of CpG DNA motifs (that signal via TLR-9) have profound therapeutic potential in modulating the Th1/Th2 response. CpG-mediated activation of TLR-9 administered at the same time as a potential allergen strongly favors a Th1 phenotype response and can mitigate established Th2 patterns of inflammation. CpG motifs can be conjugated to allergen, ensuring their direct action at the site of allergen processing and presentation, and reducing potential for side effects.65 A recent phase 2 trial66 has shown that immunotherapy with a ragweed-CpG conjugate represented a safe and very effective treatment for human allergic rhinitis. Though these therapies are still in their early days, the potential to use TLR-based approaches to treat allergic disease, or enhance antitumor immunity, or potentiate antimicrobial responses, using such strategies are clearly very exciting.
The pathology of chronic asthma clearly has multiple components. Repeated rounds of acute inflammation link into the establishment of chronic, perhaps self-perpetuating, inflammatory processes. Airway remodeling and chronic inflammation alters the local tissue phenotype and cellular makeup, resulting in a disease whose pathology changes over time. Continual dialogue between the innate and adaptive responses can influence sensitization and drive allergic and innate immune inflammation, the pattern of which can vary between individuals, and over time and with acute and chronic disease phases within a single patient. Throughout the lifetime of this network, the innate immune system remains crucial to disease pathogenesis. Our traditional terminologies of innate and adaptive, or Th1 and Th2 type immunity, describe this pathology poorly. We have recently proposed that the term contiguous immunity for the temporally and spatially linked inflammatory networks that regulate this disease is a more appropriate term that opens up the way to future research to define and intervene in these pathways.67
Conclusion
The identification of TLRs and the elaboration of their roles in the human innate immune system have provided us with a vital opportunity to reassess the pathology of inflammatory lung disease, a window into the biology of innate immunity, and the opportunity to develop badly needed new treatments for lung diseases such as asthma. While the field is still young, the early move into clinical trials for some therapies is deeply encouraging, and suggests that understanding these pathways will transform our treatment of some very troublesome diseases. TLR-directed therapies can be classified into three broad areas. Firstly, drugs are in trial that exploit the adjuvant potential of TLR agonists to drive effective Th1 responses. These may provide effective treatments for allergic disease (by immunotherapeutic strategies to reduce Th2 disease), or generate effective adjuvants that may be useful in conventional vaccination or antitumor vaccines. Secondly, TLR agonists might in theory boost immunity when it is deficient, enhancing local immunity.68 Finally, TLR antagonists may be useful to reduce excessive inflammation triggered at sites of sterile or infective inflammation. The complex nature of the interaction of specific microbes with the immune system, and the huge importance of TLRs to immune function, suggest that each of these approaches will present challenges, but it seems likely that TLR-targeted treatments will become familiar over the years to come.
Footnotes
Abbreviations: CpG = cytosine-guanine dinucleotide; IFN = interferon; IL = interleukin; LPS = lipopolysaccharide; MAPK = mitogen-activated protein kinase; NF = nuclear factor; Th1 = T-helper type 1; Th2 = T helper type 2; TLR = Toll-like receptor
Professor Sabroe is supported by a Medical Research Council Senior Clinical Fellowship. The authors have no conflicts of interest to disclose.
Received for publication November 27, 2006. Accepted for publication January 2, 2007.
References
B by Toll-like receptor 3. Nature 2001;413,732-738[CrossRef][Medline]B signaling. J Cell Biol 2003;162,1099-1110
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