TOLL-LIKE RECEPTORS REGULATION OF VIRAL INFECTION AND DISEASE

Abstract

In recent years, it has become increasingly evident that mammalian Toll-like receptors (TLRs) play a critical role in determining the outcome of virus infection. TLRs have evolved to recognize viral nucleic acids, and promote the stimulation of innate and adaptive immune responses. Interestingly, the study of mice harboring deficiencies in various TLR proteins and their adaptors suggests that TLR activation promotes protective anti-viral immunity in some cases, while exacerbating virus-induced disease in others. In this report we describe the interactions of viruses with both the TLR system and the intracellular recognition system and highlight the role of TLRs in shaping the outcome of virus infection in both a positive and negative manner.

1. Introduction

While viral innate immunity is regulated at multiples levels, evolutionary pressures have selected viral recognition as a key, if not the key, checkpoint in the cellular response to viral infection. It was known as early as the 1950s, through studies performed by Isaacs and Lindenmann, that virus infection leads to the release a soluble factor which “interfered” with the infection and replication in trans¹. This factor was subsequently identified as type I interferon (IFN) and the importance of IFN in innate immunity is evidenced by the observation that mice with a genetic deficiency in the IFN receptor (IFNα/βR^-/-) are exquisitely susceptible to multiple viral infections². At that time however, the cellular pathways responsible for the recognition of virus infection and the subsequent stimulation of an IFN response were less clear. Recent years have seen a dramatic increase in our understanding of how viruses are “sensed” by the innate immune system and the functional consequences of such virus-host interactions. The proposal of pattern-recognition receptors (PRRs) and the discovery of mammalian Toll-like receptors (TLRs) by Medzhitov and Janeway has opened a new chapter in the course of viral recognition and innate immunity³. Viral recognition by TLRs potently stimulates innate immune responses through the stimulation of IFNα/β-dependent and independent pathways.

The Toll pathway was originally discovered in Drosophila for its role in regulating dorsal-ventral polarity^{4, 5} and Hoffmann and colleagues subsequently demonstrated that Toll mutants failed to produce anti-fungal peptides and were hypersensitive to fungal infections⁶. Since then, up to 13 mammalian TLRs have been identified and much effort has recently focused on both identifying the precise structures recognized by each TLR, and the functional effects of those interactions on microbial pathogenesis^{7, 8}. TLRs are members of the PRR family, which recognize conserved microbial moieties termed, pathogen-associated molecular patterns (PAMPs), as originally proposed by Charles Janeway^{9, 10}. TLR triggering, following virus detection, catalyzes a complex signaling cascade initiated by the toll/interleukin-1 receptor (TIR) domain in the cytoplasmic tail of the TLR, which ultimately leads to expression of variety of genes^11-13. TIR domain-containing adaptor molecules, MyD88, which is utilized by all TLRs except for TLR3, as well as TIRAP, TRIF, and TRAM (for TLR4), are recruited to the receptor and activate a complex containing IRAKs and TRAFs which signal through NF-kB¹⁴. The molecular details of TLR signaling have been well described in other reviews^{11, 12, 14-16}.

Ultimately, TLR stimulation culminates in the synthesis of antiviral cytokines, such as type I IFNs, IL-1β, and IL-6, which may directly suppress viral replication. These signals efficiently recruit immune cells to sites of virus infection and activate dendritic cells (DCs) - effectively linking innate recognition to the induction of adaptive immunity^17-19. Many aspects of how TLR biology impact innate immunity have been described elsewhere. Therefore, here we highlight recent evidence revealing the means by which TLRs affect the outcome of viral infection, and discuss the growing complexity in the control of TLR-dependent immune induction.

2. The Nature of a Viral PAMP

Viral recognition by the innate immune system relies upon a group of germline-encoded receptors to alert the host to the presence of viral pathogens. Such a model has important implications for the molecular structures that are recognized by PRRs. First, the moieties selected for recognition must be absolutely distinct from natural cellular components in order to avoid the deleterious effects of self recognition²⁰. The activation of innate immunity directed against self antigens may directly catalyze autoimmune reactions; therefore, PRRs have evolved to discriminate signatures present in viruses, or virus-infected cells, which are absent from uninfected cells. Simultaneously, the ability to elude recognition by the innate immune system provides a distinct selective advantage on viral fitness. Given the high error rate of viral polymerase enzymes, especially those of viruses containing RNA genomes, PRRs must recognize invariant structures essential to virus survival, and thus not subject to mutagenesis as a viral evasion strategy^{9, 20, 21}. In general, innate PRRs have evolved to deal with this conundrum by targeting distinct features present in viral nucleic acids for recognition⁹. As these structures are generally sequence-independent, they are presented to the innate immune system by all viruses, even those which have evolved to produce a quasi-species, or swarm of viral genomes as a survival strategy. Ergo, while detection of viral proteins by TLRs clearly impinges upon virus-induced disease (see below), viral nucleic acids (either of the genome or produced during replication) better satisfy the Janeway definition of a viral PAMP.

3. Non-TLR-mediated Viral Recognition

Implicit in the existence of multiple PRRs is the assumption that each receptor recognizes a distinct molecular signature, such that cumulatively, the diversity of moieties recognized provides protection against all the major classes of infectious microbes in the relevant cellular compartments. However, which PRRs are utilized for the recognition of each specific viral pathogen represents an intense area of inquiry. While TLRs clearly play an important role in viral recognition (see below), many viruses induce type I IFN in a TLR-independent manner, suggesting the existence of additional sensing molecules. Indeed, recent work has uncovered new cytoplasmic PRRs which recognize viruses containing both DNA^{22, 23} and RNA^24-26 genomes.

RNA viruses are efficiently recognized by the cytoplasmic RNA helicases, retinoic acid-inducible gene-I (RIG-I)²⁷ and melanoma differentiation-associated gene 5 (MDA5)²⁸. Both of these proteins recognize viral RNA through their helicase domains. Binding to viral RNA catalyzes a conformational rearrangement which exposes a caspase activation and recruitment domain (CARD) to initiate antiviral signaling^24-26. Both RIG-I and MDA5 utilize a common adaptor molecule termed MAVS²⁹, IPS-1³⁰, Cardif³¹, or VISA³², which was independently identified by multiple groups and localizes to the mitochondrial membrane²⁹. The third member of the family, lgp2, displays sequence homology to the helicase domains of RIG-I and MDA5, however lacks a CARD, domain and therefore serves as a negative regulator of both sensors^33-35. Interestingly, a recent report suggests that lgp2 may play both a positive and negative regulatory role in RIG-I and MDA5-mediated innate immunity³⁶.

Analysis of RIG-I and MDA5 deficient animals has revealed important and distinct roles for these sensing molecules in innate immunity. RIG-I functions as a critical PRR for a number of viruses including Sendai virus (SeV), Vesicular stomatitis virus (VSV), Influenza virus (Flu), Hepatitis C virus (HCV), Japanese encephalitis virus (JEV), as well as small RNAs encoded by the DNA virus, Epstein-Barr virus (EBV)^{37, 38}. In contrast, MDA5 serves as a sensor for synthetic dsRNA and picornaviruses^{37, 39}. These molecules cumulatively provide the host with the ability to recognize a large group of viral pathogens in infected cells and mediate a critical aspect of innate antiviral defense. RIG-I/MDA5 are the critical sensors of viral infection in most cell types including fibroblasts and conventional dendritic cells (DCs), while plasmacytoid DCs rely upon the TLR pathway for RNA virus recognition⁴⁰ (see below).

An intracellular PRR responsible for sensing viruses with DNA genomes has also recently been identified. The existence of a TLR-independent cytoplasmic DNA sensing molecule was suggested from a number of studies utilizing DNA viruses and bacteria^{41, 42}. Recently, Takaoka et al. identified the DAI protein (also known as DLM-1/ZBP1) as a candidate intracellular DNA sensing molecule⁴³. DAI physically interacts with the B form of DNA in the cytosol and recruits an anti-viral signaling complex that results in the activation of NF-kB and the production of type I IFNs. IFN induction following B-DNA treatment was abrogated by a specific interfering RNA directed against DAI, suggesting that DAI is a critical cytoplasmic DNA sensor⁴³. Examination of DAI deficient animals should provide important insights into the role of this protein in antiviral defense in vivo.

4. TLRs as Sensors of Virus infection

As mentioned above, PRRs in general, and the TLRs specifically have evolved to recognize unique signatures overrepresented in viral nucleic acids. The first TLR implicated in viral nucleic acid recognition was TLR3⁴⁴. TLRs 3, 7, 8, and 9 typically reside in the endosome as opposed to at the plasma membrane, where they can gain access to viral nucleic acids. This appears to be a well-suited strategy as most viruses, or viral signatures, localize to the endosome either during entry and uncoating, or during assembly and budding^{45, 46}. Double stranded RNA intermediates are produced during the replication cycle of most viruses, including viruses with DNA genomes⁴⁷, and these RNA secondary structures are thought to be recognized by TLR3. It is clear that TLR3 responds to the artificial dsRNA mimic, polyiosinic-polycytidylic acid (polyIC), when it is provided extracellularly^{37, 39}. However, evidence supporting TLR3-mediated recognition of authentic viral replication intermediates remains elusive. Indeed, much speculation exists regarding the role of TLR3 in viral infections in vivo^{48, 49}. It has been reported that TLR3 is involved in recognizing virus-produced dsRNA in the context of phagocytized apoptotic cells⁵⁰. In fact, it has been suggested that TLR3 has evolved specifically to detect the presence of virus-infected dead, or dying cells⁵¹. Further studies will be required to clearly elucidate the role of TLR3 in viral recognition and pathogenesis in vivo.

TLR7 was originally shown to be responsible for mediating the antiviral effects induced following delivery of imidazoquinoline compounds⁵². It is now clear that TLR7 and TLR8 recognize single stranded RNA (ssRNA) and induce innate immune responses to ssRNA-viruses⁵³. Infection of TLR7^-/- mice in vivo, or of immune cells, especially plasmacytoid DCs (pDCs) in vitro, with Flu, SeV, and VSV resulted in abrogated IFN and cytokine responses^{54, 55}. TLR7-dependent signaling was also induced following delivery single stranded RNA oligonucleotides derived from human immunodeficiency virus (HIV) RNA⁵⁶. Furthermore, Diebold et al. demonstrated that uridine and ribose, the defining signatures of RNA, are both necessary and sufficient for TLR7 stimulation⁵⁷. Furthermore, viral fusion and/or uncoating⁵⁸ and endosomal acidification^{54, 59} are required for TLR7-dependent immunity. TLR7 recognition of viral RNA, synthetic polyU RNA, and even non-viral, cellular RNA in the endosome is sufficient to stimulate TLR7-dependent cytokine production^55-57. Together these results suggest a key regulatory role for endosomal nucleic acid localization in TLR7-dependent antiviral immune induction; however, the precise structural requirements that govern TLR7-mediated RNA recognition remain elusive. A more detailed analysis of the contribution of endosomal targeting in TLR-mediated immune induction is described below.

TLR9 is also located in the endosome and likewise mediates recognition of viral nucleic acids. In this case, it is the recognition of viral DNA that stimulates TLR9. Bacterial DNA sequences containing hypomethylated CpG motifs were first shown to activate TLR9⁶⁰. It is now clear that TLR9 is the principal means by which HSV-1 and HSV-2 stimulate type I IFNs in pDCs in vivo^{61, 62}. As predicted, this response was dependent upon acidification of the endosome, as treatment of with chloroquine or bafilomycin⁶¹ abrogated recognition. Furthermore, Honda et al. demonstrated that the ability of pDCs to retain the TLR9 ligand, CpG DNA, in the endosomal vesicle for long periods of time is critical to recruit a signaling complex containing MyD88 and the transcription factor IRF-7⁶³. In particular, CpG localization to the early endosomes (transferrin receptor positive compartment) correlated with the ability of a cell to signal through TLR9⁶³. Interestingly, a TLR9 molecule “retargeted” to the plasma membrane was unable to respond to viral nucleic acids, however now responded to self DNA which did not stimulate wild type TLR9⁶⁴. These results suggest that not only is endosomal localization important to trigger TLR-viral nucleic acid interactions, but that endosomal TLRs may limit access to non-viral nucleic acids via an active “sequestering” mechanism^{64, 65} in the endosome. Such a mechanism may provide protection from the deleterious effects of “inappropriate” nucleic acid recognition^{51, 64, 65}.

5. Endosomal Targeting and Autophagy in TLR-mediated Viral Immunity

As described above, endosomal targeting of TLRs is an important regulatory control mechanism; however the precise role of endosomal localization of TLRs in the generation of anti-viral immunity is not completely understood. Recent studies performed by Lee et al. shed new light in this regard by identifying autophagy as a critical mediator of endosomal TLR-mediated virus recognition⁶⁶ (reviewed in^{67, 68}). Recognition of dsDNA viruses and ssRNA viruses in pDCs is mediated by endosomal TLR9 and TLR7, respectively⁴⁰. It has been proposed that viral nucleic acid recognition by TLR7 requires only the presence of viral RNA in the endosomal/lysosomal compartment⁵³, suggesting that RNA replication is not required. However, this interpretation is blurred by the observation that UV-inactivated VSV⁶⁶, SeV⁶⁶, and RSV⁶⁹ failed to induce IFN production in pDCs. This, combined with the fact that VSV and SeV recognition in pDCs is TLR7-dependent⁶⁶, and that both viruses replicate in the cytoplasm, suggest that cytoplasmic replication intermediates are required for viral recognition by TLR7.

The study by Lee et al. demonstrated that this cytoplasmic sampling by endosomal TLRs occurs via autophagy. Autophagy represents an ancient, conserved pathway responsible for the recycling of long lived proteins and organelles⁷⁰. Viral recognition by endosomal TLRs was dependent upon autophagy, as pDCs derived from animals lacking a critical autophagy protein (atg-5) failed to induce a type I IFN response following VSV infection⁶⁶. Indeed recent studies suggest that autophagy is utilized by the immune systems for the generation of both innate and adaptive immune responses to viral and non-viral pathogens^71-74. Interestingly, while IFNα responses were abrogated in atg-5-deficient mice in response to a TRL9 ligand, the IL-12 response was unaffected⁶⁶. This observation suggests that cytoplasmic sampling via autophagy, instead of simply delivering cytoplasmic contents to the endosomal TLR, may instead play a role in regulating the trafficking of TLRs in intracellular compartments, or the recruitment of a TLR signaling complex to the correct intracellular location^66-68. These findings further underscore the role of the endosome in TLR biology and predict the existence of a family of adaptor proteins that direct TLR to the appropriate intracellular compartments.

In this regard, the endoplasmic reticulum membrane protein, UNC-93B^75-78, has been shown to be required for signaling through intracellular TLRs. This protein was originally identified by Tabeta et al. as a single N-ethyl-N-nitrosourea-induced mutation in the Unc93b1 gene, which markedly reduced both endosomal TLR signaling and MHC class I and class II presentation^{76, 78}. Mice harboring this mutated UNC-93B allele failed to respond to endosomal TLR ligands and were dramatically more susceptible to murine CMV infection. UNC-93B was reported to exert its effects independent of endosomal acidification or TLR intracellular localization⁷⁶. Recently, Brinkmann et al. demonstrated a physical interaction between wild type, but not mutated, UNC-93B and all three endosomal TLRs (TLR3, 7, and 9)⁷⁵. Moreover, the interaction of UNC-93B and TLRs occurred via the TLR transmembrane domain⁷⁵, which has previously been demonstrated to harbor the sorting signal required for TLR7⁷⁹ and TLR9^{64, 80} intracellular localization. Genetic evidence further implicates UNC-93B as a critical regulator of endosomal TLR function as Casrouge et al. identified deleterious mutations in the Unc93b1 gene in two patients with severe HSV encephalitis⁷⁷. The mechanism by which an ER-resident protein UNC-93B orchestrates endosomal TLR trafficking and signaling awaits further investigation.

6. Identification of Authentic Viral PAMPs

Both the TLR and intracellular pathways play a pivotal role in sensing virus infection, and these pathways are differentially utilized for the recognition of distinct viruses in a cell type-dependent manner. However, little evidence exists regarding the precise viral structures that serve as the authentic PAMP in vivo in either system. For example, the induction of IFNα by VSV, SeV, and Flu in pDCs is completely dependent upon TLR7. However, neither the particular viral ligand, which binds to TLR7, nor the stage in the viral life cycle in which such interactions occur, are understood. Presumably TLR7 senses a specific invariant feature present in the genomic RNA or viral replication intermediates of all these viruses. The genomes of RNA viruses do not exist as naked RNA, but instead are efficiently sequestered as a ribonucleoprotein complex. How and where the genomic RNA is accessed by TLR7 remains unclear.

In the case of RIG-I, Hornung et al. showed that the 5’ triphosphate structure of single stranded RNA is necessary and sufficient to serve as the viral PAMP⁸¹. This observation is likewise supported by the studies performed by Pichlmair et al. who also concluded that uncapped, 5’ triphosphate structures serve as the ligand for RIG-I⁸². Moreover, a recent report from Gale and colleagues demonstrated that RIG-I is activated by both the 5’ and 3’ untranslated regions of HCV, suggesting an authentic PAMP is present in the conserved secondary structure of the non-translated regions of the genome⁸³. In addition, Silverman and colleagues have recently unveiled a novel regulatory element in the production of viral PAMPs⁸⁴. The authors demonstrated that cellular RNAs cleaved by the antiviral endoribonuclease, RNaseL, which contain 3’ monophosphoryl group, efficiently serve as a ligand for both RIG-I and MDA5⁸⁴. These results suggest that following initiation of an antiviral cascade, even RNAs of cellular origin can serve as a viral PAMP to amplify the RNA helicase-induced pathways, and highlight the complexities involved in the identification of viral PAMPs.

7. TLRs Shape the Outcome of Viral Infection

Up to this point we have limited our analysis to what is known regarding TLR-mediated recognition of viral signatures without consideration to how such recognition impinges upon viral pathogenesis. In general terms, virus-induced disease can be characterized as direct, owing to cellular destruction as a result of viral replication; or indirect, in which the cellular response to the virus itself is the etiological cause of clinical disease. Consequent to TLR ligation, a plethora of cellular pathways are simultaneously launched, which may affect virus-induced disease in an unexpected manner. Studies in TLR knockout (KO) animals have demonstrated that TLRs either: 1) play no role, or a limited role; 2) play a dominant role in protection; or 3) exacerbate clinical manifestations, depending upon the specific viral system (Fig. 1). These studies suggest that TLRs affect virus outcome by a variety of mechanisms⁸⁵ including: directly modulating the magnitude and/or duration of viral replication, catalyzing or dampening the virus-triggered inflammatory response, and/or the activation of virus-specific adaptive immunity. In this section we highlight reports that demonstrate instances in which virus-induced TLR stimulation positively or negatively regulate clinical disease in mice.

Figure 1. Positive and negative consequences of TLR-virus interaction.

Viral sensing, following either direct fusion with the plasma membrane, or receptor-mediated endocytosis, initiates innate immunity and regulates virus-induced disease. Depending upon the viral genome content and entry mechanism, viral nucleic acids, or replicative intermediates, are recognized by one of the cellular sensing molecules such as DAI, RIG-I/MDA5, or one of the endosomal TLRs (3, 7, 9), and an innate anti-viral program is activated. Recognition of RNA viruses (RIG-I/MDA5) and DNA viruses (DAI) by the intracellular sensors stimulates innate immunity that limits replication and promotes protection. In the case of the TLR pathway, viral sensing by endosomal TLRs may lead to a protective innate and adaptive immune response. For certain ssRNA viruses, the delivery of viral PAMP to the late endosome/lysosome compartment is mediated by autophagy. Conversely, these interactions may exacerbate virus-induced disease, leading to a poor outcome for the host. Likewise, plasma membrane TLRs may also promote virus-induced inflammation. Together, these examples highlight how viral-TLR interactions affect virus-induced disease both in a positive and negative fashion.

The ability of TLRs to confer innate protection against virus infection and disease has been well documented. Hardarson et al. recently reported a direct role for TLR3 in protection from virus-induced disease⁸⁶. Encephalomycoarditis virus (EMCV)-induced mortality was significantly higher in TLR3 KOs. This was directly proportional to the increase in viral titers both in the heart and the liver in TLR3KO mice, suggesting that the ability of TLR3 to limit viral replication is responsible for alleviating the downstream disease manifestations in the EMCV model⁸⁶. A protective role for TLR3 in viral pathogenesis is also suggested by the observation that abrogating mutations in the TLR3 protein are associated with severe influenza⁸⁷ and HSV-1⁸⁸ disease in human patients. Further, TLR9 plays a protective role in response to murine CMV, evidenced by the observation that animals deficient in either TLR9 or MyD88 are hypersensitive to murine CMV-induced mortality⁸⁹. Additionally, in the murine CMV model, both TLR2⁹⁰ and TLR9⁹¹ appear to regulate the activation of a protective NK cell response. Additionally, Lund et al. demonstrated that TLR9 confers protection against mucosal HSV-2 infection in a pDC-dependent manner⁹². However, TLR9 deficiency did not affect HSV-1 titers in a cutaneous infection model⁶², suggesting the potential for route-specific, TLR-mediated protective effects.

In other viral models, not only do TLRs fail to limit viral replication, but dramatically promote aberrant inflammatory responses, leading to increased mortality^{48, 93-95}. RSV-infected TLR3 KO mice display increased pulmonary inflammation and eosinophil recruitment to the lung even though viral titers are unaffected⁹³. Likewise, TLR3 KO mice are largely resistant to Punta Toro virus (PTV) infection, whereas approximately 90% of wild type mice succumb to a lethal liver inflammatory disease. Again, viral titers were largely unaffected in TLR3 KOs. Further support for TLR3-mediated inflammation stems from studies of West Nile virus (WNV) and influenza (Flu) virus infections of TLR3 KO animals^{96, 97}. In the case of WNV, TLR3 KO animals exhibited decreased susceptibility to peripheral infection, decreased systemic IL-6 and TNFα responses, and increased permeability of the blood brain barrier (BBB)⁹⁶. The authors went on to show that increased BBB permeability was dependent upon TLR3-induced TNFα production, suggesting that, during natural infections, TLR3-dependent systemic inflammation promotes virus entry into the brain by modulating the integrity of the BBB⁹⁸. Moreover, while viral titers of Flu virus are significantly higher in the lungs of TLR3 KO mice as compared to wild type mice, the production of proinflammatory cytokines and subsequent Flu-induced mortality is dramatically reduced in the absence of TLR3⁹⁷. Taken together, these results suggest the dual role of TLR3 in viral pathogenesis, leading to diminished disease by directly inhibiting viral replication in one model, and promoting virus-induced inflammation in others.

Analysis of the TLR system in the pathogenesis of HSV infection further supports the notion that TLR interactions catalyze virus-induced inflammation. Kurt-Jones and colleagues demonstrated that both adult, and especially neonatal, TLR2 KO mice produce a diminished proinflammatory cytokine response and are significantly more resistant to mortality caused by HSV-1^{99, 100}. Interestingly, this effect was not due to a viral replication defect, as brain titers in both wild type and TLR2^-/- animals were essentially equivalent¹⁰⁰. Of note, an additional report demonstrated that TLR2 induces apoptosis of HSV-infected microgial cells, potentially implicating microglial cells in the regulation of the inflammatory response following HSV infection¹⁰¹. The regulation of HSV-induced disease by TLR2 is not limited to models of HSV encephalitis, as TLR2 KO mice were also considerably resistant to ocular lesions associated with stromal keratitis caused by HSV-1¹⁰². Interestingly, a recent report identified an association between naturally occurring polymorphisms in TLR2 and the severity and recurrence of HSV-2 infections in humans¹⁰³, suggesting a role for TLR2 in the regulation of HSV-induced disease in the human population.

While viral nucleic acids serve as the true viral PAMP, interactions with TLRs and viral proteins can also provide a protective role in viral pathogenesis. For example, Kurt-Jones et al. demonstrated that RSV peak titers and the longevity of persistence were significantly increased in mice harboring a natural deletion in TLR4 (C57BL10/ScCr mice) compared to wild type mice¹⁰⁴ . Speculation exists as to the physiological role of TLR4 in RSV pathogenesis, as CF7BL10/ScCr mice are known also to harbor a defect in the gene encoding IL-12Rβ2¹⁰⁵, and an additional study suggested that the cytokine secretion defect in CF7BL10/ScCr mice is not due to the TLR mutation, but instead explained by theIL-12 deficiency¹⁰⁶. However, Tal et al. demonstrated that abrogating mutations in TLR4 are significantly overrepresented in children who experience severe RSV disease compared to children with mild RSV symptoms, suggesting a potential role for TLR4 in mediating the clinical outcome of RSV disease in its natural population¹⁰⁶. These results exemplify the importance of evaluating TLRs in natural models of virus infection.

Viral TLR systems provide a unique opportunity to study host pathogen interactions. In addition to playing both protective and pathogenic roles in viral pathogenesis, two studies suggested that viruses rely upon TLR signaling as a survival mechanism^{107, 108}. As mentioned above, the HA protein of Measles virus induces cytokine secretion in a TLR2-dependent manner¹⁰⁷. Interestingly, this interaction also results in upregulated expression of the MV receptor, CD150, suggesting that HA-TLR2 interactions in fact benefit the virus at the expense of the host¹⁰⁷, unless increased infection can be viewed as an effective antiviral strategy. Likewise, the interaction of the MMTV env protein with TLR4 provides a selective advantage to the virus by promoting infection of target cells, transcription of the viral genome, and release of the immunoregulatory cytokine, IL-10¹⁰⁸. It has been suggested that IL-10 may serve as a master regulator of chronic vs. acute viral infections¹⁰⁹, and thus it warrants analyzing whether TLR-dependent IL-10 production plays an important role in the pathogenesis of other chronic viral systems, such as HIV and HCV.

Taken together, the examples described here provide strong evidence that TLRs dramatically affect the outcome of viral infection. This complex regulation may protect the host from virus-induced disease either by limiting viral replication, or by tempering virus-induced immune pathology (Figure 1). Conversely, TLRs may predispose the host to the deleterious manifestations associated with virus infection. Future studies will be required to define the precise mechanisms by which TLRs modulate viral pathogenesis in each specific system and what factors delineate the balance between protective and pathogenic immunity.

8. Perspectives and Concluding Remarks

Defining the pathways that regulate viral pathogenesis is an important endeavor, with serious ramifications. Viral-induced disease represents a significant threat to human health and the world economy. Our understanding of the role of TLRs in viral pathogenesis has expanded significantly over the last few years and it is clear that TLRs provide protective anti-viral immunity in some cases, while clearly exacerbating virus-induced disease in others. To date, a systematic understanding of the factors that regulate this balance is lacking. This, in turn, provides us with an opportunity to establish if such a hierarchy does in fact exist, and if so, the potential to lessen the burden of virus-induced disease.

Our knowledge of the TLR system in viral disease stems mostly from studies of TLR KO mice. However, additional support comes from the observation that viruses have evolved molecular mechanisms to interfere with the TLR pathway, which would not have occurred if such strategies did not provide a selective advantage. For example, VV encodes two proteins, A46¹¹⁰ and A52¹¹¹, which antagonize the TLR system independently. Deletion of either gene from the viral genome resulted in an attenuated phenotype, suggesting that the virus has evolved TLR inhibition as a survival strategy^{110, 111}. Likewise, inhibition of TLR-dependent signaling by several HCV proteins has also been documented^{112, 113}, further validating the importance of TLR evasion. As more TLR regulatory pathways are discovered, no doubt this list will likewise expand, and may provide insights into virus maintenance and evolution.

Challenge 1

In this review, the role of a single TLR in mediating viral outcome was considered for simplicity; however, this is not the full picture. In the real world, such interactions are complex, often encompassing multiple TLRs that interact with distinct signatures in a single virus. This is particularly true in the case of the Herpes viruses where HSV interacts with TLRs 2, 3, and 9^{88, 114, 115}, and CMV interacts with TLRs 2, 3, and 9¹¹⁶. Defining the exact contribution of each TLR alone and in combination with the other activated TLRs is crucial to assess the extent to which TLRs modulate viral pathogenesis. This analysis is confounded by that fact that specific TLR signaling occurs in distinct immune compartments¹¹⁷, and is dependent upon viral genetics^{107, 115}, inoculation route¹¹⁸, and virus dose¹¹⁸. The issue of virus dose is interesting as most in vivo infection models rely upon delivery of relatively large doses of virus, which may mask subtle effects caused by virus TLR interactions under more physiological conditions, as suggested by Gowen et al⁹⁴. Likewise, it will be important to clarify whether the phenotype observed by Bieback et al., that TLR interaction is limited to wild type MV, and not attenuated strains, also holds true for other viral systems¹⁰⁷. Accordingly, analysis of clinical viral isolates with both mouse and natural human TLR proteins may identify novel roles for the TLR family in viral pathogenesis. It is possible that a reevaluation of these issues may uncover a previously unappreciated role of TLRs in shaping the outcome of viral disease.

Challenge 2

In analyzing the response to virus infection in genetically diverse hosts, such as in the human population, heterogeneity is the rule. Undoubtedly, this response is governed by the actions of numerous gene products to facilitate anti-viral immunity. As new TLR regulatory pathways continue to be discovered, it will be interesting to determine if polymorphisms in either TLRs, or TLR regulatory proteins correlate positively or negatively with disease severity in humans. In fact, polymorphisms in TLR3⁸⁷, TLR4¹⁰⁶, and the ER protein, UNC-93B⁷⁷ have all been shown to correlate with poor outcome following viral infection. Based upon the idea that TLRs negatively impact viral pathogenesis, it is also possible that a correlation exists between TLR polymorphisms and decreased susceptibility to viral infection. However, whether such mutations would be selected for if they abrogate TLR activity directed against other pathogens is unclear. It is possible that a molecular genetic analysis of TLR alleles in natural populations may provided a means to predict how an individual may respond to a TLR-containing virus or vaccine, and provide an opportunity for alternative intervention⁸⁵.

Challenge 3

Finally, it will be important to clarify the cellular mechanisms by which TLRs regulate the balance between protective and pathogenic immunity. In the case of WNV, TLR signaling promotes viral entry into the CNS by modulating the integrity of the blood brain barrier⁹⁶, however whether BBB effects are responsible for exacerbated disease in other models, such as for TLR2 and HSV infections¹⁰⁰, remains to be determined. Likewise, it has been suggested that TLR signaling in pDCs can function to downregulate aberrant virus-induced inflammation, at least in part, in a type I IFN-independent manner¹¹⁹. Identification of the regulatory programs initiated by TLR signaling will provide important new insights into the role of TLRs in viral pathogenesis.

In summary, the work outlined here provides clear evidence that TLRs play a pivotal role in viral pathogenesis on several levels. Furthermore, despite the identification of how TLR biology impacts virus-induced disease, many unanswered questions remain. Investigations over the next few years should begin to unravel the specific mechanics of TLR-mediated immune regulation and may provide novel antiviral intervention strategies.