TLRs were first identified as receptors for the recognition of discrete molecules of microbial origin when one of them, TLR4, was shown to be absolutely required to detect LPS (7
). Gene targeting established the function of most of the other TLRs (8
). It now appears that each TLR responds to a limited repertoire of ligand molecules (Table 1
). In general, these molecules are represented by many microbial taxa.
In some cases, molecules unique to one microbe or at most restricted to a small clade of microbes have been observed to activate TLRs. For example, the F protein of RSV, or the G glycoprotein of VSV, or the Env protein of mouse mammary tumor virus (MMTV) can each activate TLR4 (although in each case in a qualitatively different manner).
Structural and genetic data suggest that TLRs exist as dimeric proteins (either heterodimers or homodimers), and it is widely held that activation entails a conformational change elicited by ligand binding. All of the TLRs are single-spanning type I transmembrane proteins. The ectodomains of TLRs are composed of leucine-rich repeat motifs and adopt an aesthetically pleasing curved solenoid shape, which is believed to be relatively rigid except where interrupted by irregular loops that may act as hinges, permitting some degree of flexibility. The ectodomains of TLRs are variably glycosylated, and glycosylation may restrict the types of interactions that can occur between subunits (12
On the cytoplasmic side, TLRs have a characteristic protein domain called a TIR [Toll/interleukin-1 (IL-1) receptor] domain. The TIR domain always accounts for the major portion of the cytoplasmic domain and serves two purposes. First, it contains an oligomerization site, maintaining dimeric interactions between TLR subunits. Second, it contains a site that recruits cytoplasmic adapter proteins that also contain TIR domains. The adapter proteins also have oligomerization sites and through ‘face to face’ and ‘back to back’ interactions can presumably form extended chains in the cytoplasm of the cell following activation (13
), as described in more detail below. TIR domains are found not only in Toll-like receptors but also in receptors of the IL-1/IL-18/IL-33 family, which do not have leucine-rich repeat motifs in their ectodomains but are built of immunoglobulin-type domain, represented in varying numbers of copies. TIR domains are also represented in plant disease resistance proteins and in some proteins encoded by bacteria and viruses. In microbes, some of the TIR domains almost certainly operate as molecular mimics to disrupt TLR signal transduction.
The IL-1, IL-18, and IL-33 receptors each bind well defined endogenous proteins and elicit inflammatory responses. IL-1, IL-18, and possibly IL-33 as well are each induced in response to TLR signal transduction events, which activate NF-κB. The strategy of cytokine production and subsequent activation of additional TIR domain-mediated signaling may be seen as a device for generalizing the inflammatory response and spreading a warning signal beyond the bounds of the initial inflammatory stimulus. Moreover, at times, signaling via IL-1 is clearly involved in autoinflammatory diseases. These may be seen as the consequence of continuous microbial stimulation or, perhaps at times, sterile inflammatory reactions that result from a failure in normal ‘braking’ mechanisms the terminate TIR domain signaling.
In some cases, it is clear that TLRs act as the membrane-spanning components of receptor complexes. For example, TLR4 is tightly associated with MD-2, a small molecule that binds to a hinge region of the TLR4 ectodomain and physically engages LPS, triggering activation of the complex (14
). CD14, a glycosylphosphoinositol-linked leucine-rich repeat protein, also assists in the activation of TLR4, specifically by highly glycosylated forms of LPS. It is not entirely clear how this occurs, but it is reasonable to hypothesize that CD14, too, is part of a multisubunit complex and contributes to receptor activation. Another example may be seen in the case of the TLR2/TLR6 complex, in which CD36 plays an important role in the recognition of some microbial components (15
), and CD14 plays an important role in the recognition of others (16
). While associated subunits have not been identified for TLR3, TLR5, TLR7, and TLR9, it is possible that these TLRs also behave as parts of complexes that have not yet been fully deciphered.
Some of the TLRs appear to signal chiefly from the cell surface (TLR1/TLR2, TLR2/TLR6, TLR4, and TLR5). Others—those that detect nucleic acids—signal from internal compartments within the cell. These include TLR3, TLR7, and TLR9 (and TLR8 in humans). Because agents that block acidification of the endolysosomal compartment inhibit the perception of TLR3, TLR7, and TLR9 ligands, it is believed that nucleic acid sensing occurs strictly within these compartments. TLRs are expressed by a wide variety of cells, some of which are professional components of the immune system and some of which are not. Although considerable work has gone into the analysis of which cells respond to which TLR ligands, a complete summary of the response patterns would be premature and also beyond the scope of this review. It is interesting, however, to note that the induction of type I IFN responses in vivo seems to depend largely (though not entirely) upon activation of plasmacytoid dendritic cells, also known as interferon (IFN)-producing cells. These cells express TLR9 and other nucleic acid sensing TLRs, and probably most of their response to nucleic acids is TLR mediated (although an alternative pathway for nucleic acid perception exists as described below).
Very different modes of receptor binding have been proposed for different TLRs based on co-crystallization studies. The lipid A moiety of LPS, for example, is believed to engage MD-2, the small accessory subunit of the TLR complex described above, and to cause a conformational change sensed by the holoprotein complex. Double-stranded RNA is believed to span two subunits of TLR3, uniting them and causing activation (17
). Tri-acyl lipopeptides are believed to insert into the interstices of the leucine-rich repeat coils of TLR1 and TLR2, uniting them and causing activation (18
). It may be proposed that these essentially extracellular (or intravesicular) events are sensed across the lipid bilayer through torsion and cause a change in the spatial orientation of TIR domains on adjoining TLR subunits. This activity leads to the recruitment and organization of adapter proteins that carry the signal into the cytoplasm.
Four TIR adapter proteins [myeloid differentiation factor 88 (MyD88), MyD88-adapter-like protein (MAL), TIR-domain-containing adapter-inducing IFN-β (TRIF), and translocating chain-associating membrane protein (TRAM)] mediate virtually all TLR signals, and in the absence of two of the adapters (MyD88 and TRIF), no discernable signaling is known to arise from any of the TLRs or from the IL-1 or IL-18 receptors. MyD88 is believed to be capable of signaling by itself (from TLR5, TLR7, TLR8, and TLR9, as well as the IL-1 and IL-18 receptors, none of which is known to depend upon other adapters), but when signaling from the TLR1/TLR2 heterodimer or from the TLR2/6 heterodimer, it depends in part upon the presence of MAL, a second adapter protein, and the one with strongest primary sequence homology with MyD88. MyD88-dependent signaling from the TLR4 complex also depends upon MAL. Hence, MyD88 seems to operate in a functional pair with MAL, at least when signaling is nucleated by some of the TIR receptor proteins.
A number of interpretations may be attached to this observation. According to one basic model, MyD88 never engages the TLR1/TLR2, TLR2/TLR6, or TLR4 receptor TIR domains directly, but instead depends upon MAL to do so. Hence, MyD88 has contact only with MAL, which acts as a ‘bridge’ to the receptor TIR domain. Other possibilities also exist, and the bridge model is contested by specific genetic evidence (13
). This evidence comes in the form of receptor-selective mutations of MyD88, including the Pococurante
mutation of MyD88: I178N, and the classical P712H mutation first identified in the TLR4 TIR domain in C3H/HeJ mice, and localized to the so-called BB loop of the TIR domain. These mutations behave identically, and both reside on the surface of all TIR domains. Both mutations, when engrafted into MyD88, are capable of interrupting MyD88-dependent signaling from TLR4 and TLR9 but not from TLR2/TLR6. If a simple bridge was provided by MAL in both cases, it is difficult to understand how a mutation of the MyD88 TIR domain could discriminate between the two triggering receptors.
Other hypotheses may be advanced to explain the dependency of MyD88 on MAL for some of the TIR domain receptors but not others. For example, MyD88 may be altered post-translationally (e.g. by phosphorylation) after contact with the TLR1/TLR2 or TLR2/TLR6 heterodimers or the TLR4 homodimer. In this state, it may recruit MAL, and further ‘chaining’ between MyD88 and MAL may be nucleated.
No crystal structure of MyD88 exists, but in the absence of such data, the TIR domains of all of the adapter proteins can be modeled on the existing structures of the TLR1 and TLR2 TIR domains, derived from X-ray crystallography. Docking studies can then be performed to predict how MyD88 is most likely to interact with receptor TIR domains. The most favored mode of interaction between the MyD88 TIR domain and the TLR2 TIR domain (face-to-face interaction) entails reciprocal engagement of surfaces containing both the BB loop and the Poc site, described above. The second most favored mode of interaction (back-to-back interaction) entails the reciprocal engagement of αE helices from both the receptor and adapter TIR domains. This suggests that initial contact between receptor and adapter TIR domains is face-to-face, and back-to-back interaction may follow to allow the recruitment of more adapter molecules ().
For TLR3 and TLR4, different adapters are also involved in ‘MyD88-independent signaling’ (19
). One of these adapters, shared by both TLR3 and TLR4, is called TRIF [or TIR-containing adapter molecule-1 (TICAM1)]. TRIF is the sole adapter used by TLR3 and activates both NF-κB and IFN-regulatory factor 3 (IRF3) signal transduction. To activate NF-κB, TRIF interacts with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), which is a key signaling intermediate of the MyD88-dependent pathway as well. To activate IRF3, TRIF interacts with one of two protein kinases: TANK-binding kinase 1 (TBK1) or inhibitor of NF-κB kinase i (IKKι), which in turn phosphorylate IRF3, permitting its translocation to the nucleus (22
TLR4 activates not only TRIF but also TRAM (also called TICAM2), its closest protein homologue, and an adapter protein that exists only to serve TLR4 signaling (19
). Like MAL, TRAM has been proposed as a bridging adapter protein. As with MAL, there are few direct data to support this hypothesis, and other possibilities exist.
Some TLR4 stimuli (for example, VSV infection) cause TRAM-dependent (and TRIF-independent) signal transduction, leading to the activation of IRF7 and type I IFN production (23
). There is thus considerable flexibility in signal transduction downstream of TLR4. Depending upon the ligand involved, it may be strictly MyD88/MAL dependent, strictly MyD88 independent, or TRAM dependent. In the absence of MyD88 and TRIF (in mice with compound homozygous mutations), no TLR signaling can occur. This would imply that neither MAL nor TRAM is by itself capable of propagating a signal.
MyD88 contains not only a TIR domain but also an N-terminal death domain and is known to recruit protein kinases of the IL-1 receptor-associated kinase (IRAK) family to the activation complex via a death domain interaction. IRAK4 is believed to be activated and to phosphorylate IRAK1 and/or IRAK2, which serve a positive role in signal transduction (24
). IRAK-M, the remaining member of this protein family, serves an inhibitory role in signaling (25
). The activated IRAK proteins somehow recruit TRAF6, a more distal component of the signaling chain, to the activation complex.
TRAF6, which as noted above is also activated by TRIF (or TRIF/TRAM) recruitment, is a protein with multiple ring-finger motifs that complex zinc and is endowed with catalytic activity, serving as an E3 ubiquitin ligase (26
). The intrinsic E3 ligase activity of TRAF6 acts in concert with an E1 ligase as well as two other protein complexes, known as TRAF6-regulated IKK activators (TRIKAs). TRIKA1 is an E2 ubiquitin ligase complex consisting of Ubc13 and a Ubc-like protein Uev1A. Together with TRAF6, TRIKA1 attaches a chain of K63-linked ubiquitin subunits on NF-κB essential modulator (NEMO) (the IKKγ subunit) and on TRAF6 itself. TRIKA2 is a complex of proteins including the transforming growth factor-β-activated kinase 1 (TAK1) and the adapter proteins TAK1-binding protein 1 (TAB1) and TAB2. These proteins act together to permit the recruitment of IKKβ and its activation. TAB2 and TAB3 both bind to the K63-linked ubiquitin chain, which serves as an anchorage site for IKK activation. Once activated, the IKK complex phosphorylates IκB, which is subsequently degraded in K48-ubiquitination dependent process. The role of TRAF6 as a central way-station in signaling via both MyD88-dependent and MyD88-independent pathways makes it the focus of both activating and inactivating signals. The polyubiquitin chains that are attached by TRIKA1 complex proteins can be removed by the action of de-ubiquitination proteins such as CYLD and A20 ().
Ubiquitin signaling in TLR-mediated activation of NF-κB
The activation of TAK1 (also known as MAP3K7) is a prerequisite not only for activation of IKKβ but also for the activation of mitogen-activated kinase (MAPK) kinases such as MKK6 (MAP2K6), which in turn phosphorylates Jun kinases (JNK), p38 kinase, and extracellular signal regulated kinase 1 (ERK1) and ERK2. Other protein kinases also play a key role in the downstream activation of signaling from all of the TLRs. Among these is Tpl2 (also known as MKK8, MAP3K8, or Cot), first shown to be important for LPS-induced responses not long after the TLRs were identified as signaling receptors (27
). Tpl2 is probably also activated by the TRAF6 complex and seems to be essential for the production of TNF, IL-6, and in some cells type I IFNs. Its key function lies in the activation of MAPKs including ERK1, ERK2, and p38.
Other proteins play supporting functions in TLR signaling. For example, UNC93B, a 12-spanning membrane protein located primarily in the endoplasmic reticulum, is required to allow TLR3, TLR7, and TLR9 to transit to the endosomes, where signaling actually occurs (28
). Similarly, gp96, an endoplasmic reticulum chaperone, is required for TLR4 to reach the cell surface (31
In the end, hundreds (and in some circumstances thousands) of genes are modulated by TLR activation: some upregulated and some suppressed. These events create the extremely complex changes that are known macroscopically as inflammation. TLR signaling also causes post-transcriptional changes that alter protein synthesis or trafficking within the cell. Most of these lie beyond the scope of discussion in this review, but where individual genes have been studied, it is seen that translational activation can result from TLR activation (in the case of the TNF mRNA, for example), and that degranulation of certain cells (such as mast cells) can be triggered by TLR signaling. While it has been proposed that the TLRs interpret the ‘molecular patterns’ present on microbes as a bar-code reader does and elicit a response finely attuned to the type of infection that is present, direct evidence of strong specificity is lacking. Some TLRs do indeed yield qualitatively different biological responses than others. However, the overlap in signaling is perhaps more striking, and the impression gained is one of a stereotypic response to infection.
Despite the seeming complexity of the TLR signaling pathways and the potential for redundancy in signaling in general, a number of mutations are capable of interrupting signal transduction completely or almost completely. Indeed, this is how the pathways have become clearly established as they are drawn today, and germline mutations remain the gold standard in pathway analysis. Random germline mutagenesis (forward genetics) and gene targeting (reverse genetics) have both played important roles in the dissection of these pathways. The current illustration based on forward genetic analysis (including both known and unknown genes) is illustrated in .
Overview of the TLR signaling pathways
Soon after Tlr4
was shown to encode the LPS receptor core by positional cloning, its mRNA was found to exist at low abundance: a fact that had undoubtedly frustrated efforts to identify the Lps
gene product through expression cDNA cloning. The protein itself also exists at remarkably low copy number. Titration experiments were performed by expressing epitope tagged versions of the wildtype (WT) (Lpsn
) and signaling defective, dominant inhibitory (Lpsd
) versions of the protein in RAW 264.7 cells: a mouse macrophage line that exhibits robust responses to LPS. Expression levels and LPS signal transduction were then measured in a large number of stable clones. Fewer than 1,000 molecules of the signaling defective isoform suppressed LPS signaling by 50%, suggesting that the endogenous WT protein was expressed at a similar level. Moreover, LPS signaling was dramatically augmented by overexpressing the WT isoform, which suggests that TLR4 itself is the limiting factor in LPS signaling to the level of TNF production (32
). This conclusion was later supported by transgenesis experiments performed in vivo
: it was shown that increasing the copy number of TLR4 increases sensitivity to the lethal effect of LPS (33
The lethal effect of LPS in an intact mouse is mediated principally by macrophages (34
) and is entirely dependent upon TLR4. One may estimate the quantity of TLR4 protein that delivers the lethal signal based on assumptions about the number of macrophages that exist in normal mice and on the added assumption that each macrophage expresses approximately 1,000 TLR4 monomers, as is the case for RAW 264.7 cells (32
). The most generous estimate concerning macrophage numbers (109
macrophages, or about 5% of the body weight of the mouse) leads to the conclusion that less than 0.15 μg of TLR4 mediates a lethal outcome when a large dose of LPS is administered. It may therefore be supposed that each TLR4 complex elicits the production of enormous numbers of cytokine molecules as a result of signal amplification within the TLR4-expressing cell and through secondary inducing effects of the cytokines themselves. Primary signal amplification probably occurs at catalytic steps in the signaling cascade: e.g. IRAK4, TAK1, and Tpl2 mediate phosphorylation of transcription factors, and at the levels of transcription and translation.