PRINCIPLES IN INNATE SIGNAL TRANSDUCTION
Upon engagement of TLRs by individual PAMPs, a number of different signaling pathways are triggered. Signal transduction is mediated initially by a family of adaptor molecules, which at least in part determines the specificity of the response (280). Recruitment of one or several adaptor molecules to a given TLR is followed by activation of downstream signal transduction pathways via phosphorylation, ubiquitination, or protein-protein interactions, ultimately culminating in activation of transcription factors that regulate the expression of genes involved in inflammation and antimicrobial host defenses (7) (Fig. 4).
FIG. 4. TLRs and TIR domain-containing adaptor molecules. TLR1/2 and TLR2/6 utilize MyD88 and Mal as adaptors. TLR3 is dependent on TRIF for signaling. In the case of TLR4, four different adaptors, i.e., MyD88, Mal, TRIF, and TRAM, are involved, whereas TLR5, -7, -8, and -9 utilize only MyD88. The fifth adaptor, SARM, negatively regulates TRIF-dependent signaling. Overall, MyD88-dependent signaling induces proinflammatory cytokine production, whereas TRIF-dependent signaling stimulates a type I IFN response. In pDCs, stimulation of TLR7 or TLR9 induces type I IFN production by a mechanism dependent on MyD88. See text for further details.
TLR-induced signaling pathways can be broadly classified on the basis of their utilization of different adaptor molecules, i.e., dependent on or independent of the adaptor MyD88 or TIR domain-containing adaptor inducing IFN- β (TRIF), and, additionally, their respective activation of individual kinases and transcription factors (6, 280). Three major signaling pathways responsible for mediating TLR-induced responses include (i) NF- κ B, (ii) mitogen-activated protein kinases (MAPKs), and (iii) IFN regulatory factors (IRFs) (6, 7, 177). Whereas NF- κ B and MAPKs play central roles in induction of a proinflammatory response, IRFs are essential for stimulation of IFN production (6, 177).
TLR Adaptor Molecules
Following ligand binding, TLRs dimerize and undergo conformational changes required for the subsequent recruitment of cytosolic TIR domain-containing adaptor molecules (280). MyD88, which was the first adaptor molecule to be identified (44, 381), is involved in signaling triggered by all TLRs, with the exception of TLR3, and plays a major role in TLR-induced signal transduction (176, 280).
Despite the prominent role of MyD88, studies with MyD88-deficient mice revealed the existence of both MyD88-dependent and -independent pathways (176, 180), resulting in subsequent identification of additional adaptor molecules, including MyD88 adaptor-like protein (Mal) (90, 135), TRIF (283, 387), TRIF-related adaptor molecule (TRAM) (91, 284, 386), and sterile alpha- and armadillo motif-containing protein (SARM) (51, 253).
Figure 4 illustrates the utilization of different adaptor molecules by individual TLRs. Based on findings that NF-B activation and the inflammatory cytokine response are abolished in mice deficient in MyD88 but normal in mice deficient in Mal, TRIF, or TRAM in response to agonists of TLR5, -7, and -9, it was inferred that MyD88 is used as the only adaptor by these TLRs (120, 135, 384–386). MyD88 is also utilized by TLR2, heterodimerizing with TLR1 or TLR6 (287), but in addition, Mal appears to be required for linking TLR2 and MyD88 together (135, 385). In the case of TLR4, four different adaptors, i.e., MyD88, Mal, TRIF, and TRAM, are involved in signal transduction (129, 135, 384–386), reflecting the complexity of signaling downstream of this receptor (Fig. 4).
Several attempts have been made to explain how TLR4 coordinates recruitment and signaling through two potentially competing adaptor systems. Recently, Kagan et al. presented data suggesting that TLR4 activates these two pathways in a sequential manner determined by endocytosis of the TLR4 complex (162). The authors proposed a scenario in which endocytosis of TLR4 terminates an initial phase of Mal-MyD88-dependent signaling and triggers a second phase of TRAM-TRIF-dependent signal transduction originating from TLR4 molecules localized in endosomes and aided by improved access to the adaptor protein tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) (162). Finally, TLR3 is the only TLR that does not use MyD88 but instead is dependent on TRIF for signaling (384). In contrast to these four adaptor molecules with activating potential, the fifth adaptor, SARM, appears to be a negative regulator of TRIF-dependent signaling in human cells (51), although this finding was not confirmed in studies of SARM-deficient mice (189). In summary, the existence of this family of adaptors has the important implication that the selective usage of individual adaptor molecules or combinations of molecules may explain, to some degree, the differential responses induced by different TLRs. Thus, TLR-induced signaling can be largely divided into MyD88-dependent and MyD88-independent, TRIF-dependent pathways, which are both capable of activating NF- κB, although each activates additional signaling components, including MAPKs and IRFs.
The MyD88-Dependent Signaling Pathway
MyD88-dependent signaling is activated downstream of all TLRs except TLR3 (7). In the pathways stimulated through TLR2, -4, and -5, MyD88 primarily drives inflammatory gene expression, whereas MyD88 relays signaling to type I IFN production in pathways triggered from TLR7 and TLR9, occurring primarily in pDCs. In this review, these two MyD88- dependent signaling pathways are described separately, with TLR4 signaling representing the prototypic MyD88-dependent proinflammatory pathway as illustrated in Fig. 5.
FIG. 5. Principles in TLR signaling. TLR4 activates both the MyD88-dependent and MyD88-independent, TRIF-dependent pathways. The MyD88-dependent pathway is responsible for early-phase NF-κB and MAPK activation, which control the induction of proinflammatory cytokines. The MyD88-independent, TRIF-dependent pathway activates IRF3, which is required for the induction of IFN-β- and IFN-inducible genes. In addition, this pathway mediates late-phase NF-κB as well as MAPK activation, also contributing to inflammatory responses.
In response to TLR4 stimulation by an appropriate PAMP, MyD88 associates with the cytoplasmic part of the receptor and subsequently recruits members of the IL-1 receptor (IL1R)-associated kinase (IRAK) family (44, 381). Following association with MyD88, IRAK4 and IRAK1/2 are sequentially phosphorylated, with IRAK4 being of particular importance, as it has been demonstrated to be indispensable for the response to IL-1 and various TLR ligands (348). IRAK1 was originally thought to play an essential role in TLR-induced NF- κB activation (164), but more recent data have emerged suggesting that instead IRAK2 may play a prominent role in NF- κB activation, particularly during the late phase of TLR signaling (175, 182). Further downstream, IRAK1, or alternatively IRAK2, associates with TRAF6, which acts as a ubiquitin protein ligase (E3) (214) that, together with the ubiquitination enzyme complex (E2), catalyzes the synthesis of K63-linked polyubiquitin chains on TRAF6 itself and other substrates, including transforming growth factor-activated protein kinase 1 (TAK1) and the IκB kinase (IKK) subunit NF- κB essential modifier (NEMO) (378). A central step in the downstream signaling events is the recruitment of TAK1-binding protein 2 (TAB2) and TAB3 to ubiquitinated TRAF6, which brings TAK1 into proximity to the signaling complex, leading to its activation (165). TAK1 then stimulates two distinct pathways involving the IKK complex and the MAPK pathway, respectively (378) (Fig. 5). In the first pathway, TAK1-mediated activation of the IKK complex results in site-specific phosphorylation of the inhibitory IκB protein. Being the point of convergence for multiple NF- κB -inducible stimuli, IKK represents an essential component in many inflammatory signaling pathways (112). This high-molecular-weight kinase is composed of two structurally related kinases, IKK α and IKK β , as well as the chaperone IKK complex-associated protein and the adaptor NEMO/IKK. NEMO appears to function as a signal integrator in the NF- κB pathway by receiving and transmitting converging signals from various stimuli and pathways to common downstream signaling events (112, 244, 311, 397). Despite early reports of IKK dependency upon ubiquitination for optimal kinase activity, it was only recently resolved that direct ubiquitination of NEMO is mediated by TRAF6 (346). Following phosphorylation, IκB undergoes proteasomal degradation to allow activation and translocation of NF- κB to the nucleus, where it binds to κB sites present in promoters and enhancers of a broad range of proinflammatory genes, which are then transcribed (112). In the second pathway, TAK1 phosphorylates members of the MAPK kinase (MKK) family, including MKK3, -4, -6, and -7 (54). MKK3/6 subsequently phosphorylate and activate p38, whereas MKK4/7 activate c-Jun N-terminal kinase (JNK). However, in studies analyzing T. gondii-activated signaling, an alternative mechanism involving p38 autophosphorylation proceeding in a TAK1-dependent and MKK3/6-independent pathway has been described (188). Ultimately, these signaling pathways lead to activation of the transcription factor activator protein 1 (AP1) (54). Moreover, other members of MKK kinases, most notably MKK kinase 3 and tumor-progression locus 2, have also been implicated in MAPK activation downstream of TLR4 (28, 142). The essential and nonredundant role played by TAK1 is strongly suggested by significantly reduced NF- κB , JNK, and p38 responses to various TLR ligands in cells derived from mice deficient in this kinase (317, 332).
The MyD88-Independent, TRIF-Dependent Signaling Pathway
The existence of a MyD88-independent pathway downstream of TLR3 and TLR4 was indicated by data from MyD88- deficient mice displaying normal IFN- production (176, 180). Extensive molecular studies by Akira and associates then led to the identification of TRIF as the adaptor responsible for signaling in the MyD88-independent pathway (384). Subsequently, the equally important discovery of two IKK-related kinases, TRAF family member-associated NF-B activator (TANK)-binding kinase 1 (TBK1) and IKKε, and their essential role in induction of type I IFN were reported (89, 122, 330). During TLR3- and TLR4-mediated signaling, TRIF (associated with TRAM in the case of TLR4) (91, 386) is responsible for initiating a signaling pathway in which TRAF3 and TANK serve to bridge to the IKK-related kinases TBK1 and IKKε (108, 113, 276, 318), which mediate direct phosphorylation of IRF3 and IRF7 (89, 330). Studies with cells lacking TBK1 or IKKε have revealed that TBK1 and, to a lesser extent, IKKε are responsible for TRIF-mediated IFN responses (122, 240, 292). It is notable, however, that whereas TBK1 and IKKε are essential for TRIF-dependent IRF3/7 phosphorylation, these kinases are not involved in TLR-mediated NF-B activation (122). As a consequence of phosphorylation, IRF3 and IRF7 form hetero- or homodimers, translocate to the nucleus, and, in association with transcriptional coactivators such as CBP and p300, bind to target sequences in DNA, such as IFN-stimulated response elements (89, 330). Importantly, IRFs together with NF-B and AP1 form a multiprotein complex termed the enhanceosome, which induces transcription of the IFN- gene (364). TRIF-dependent activation of NF-B occurs through binding of TRAF6 to TRIF and subsequent ubiquitination-dependent recruitment and activation of TAK1 (318). In order to obtain robust NF-B activation, a second molecule, receptorinteracting protein 1 (RIP1), involved in TNF-receptor mediated NF-B activation, is also recruited to TRIF (245). RIP1 is polyubiquitinated to form a complex with TRAF6, and these two molecules appear to cooperate in facilitating TAK1 activation, resulting in IKK-mediated activation of NF-B as well as activation of the MAPK pathway (66). Thus, molecular signaling mechanisms within the TRIF-dependent pathway illustrate how selective binding of different molecules, i.e., either TRAF3 or TRAF6, results in recruitment and downstream activation of TBK1-IRF versus TAK1-IKK-NF-B, respectively (Fig. 5). An example of differential pathogen-induced activation of diverse signaling pathways emanating from a given TLR has been reported for two strains of N. meningitidis with different abilities to stimulate TLR4 through the MyD88- independent pathway (263). In molecular terms, this observation may be explained by the ability of CD14 to distinguish between different carbohydrate chains of LPS/LOS, which governs the MyD88-independent signaling pathway of TLR4 (157).
MyD88-Dependent Signaling from TLR7 and TLR9
It has been well established for some time that in addition to TLR3 and TLR4 activation, TLR7 and TLR9 activation also triggers IFN production in pDCs, a subset of DCs specialized in producing large amounts of type I IFN in response to virus infection (59). Intriguingly, however, TLR7- and TLR9-mediated IFN production requires MyD88, in contrast to TLR3- and TLR4-mediated IFN production, which is dependent on TRIF (128, 178, 384). Another notable difference between these pathways is that TLR7- and TLR9-mediated IFN production does not depend on TBK1/IKKε but instead proceeds through a molecular network including IRAK1, IRAK4, TRAF6, TRAF3, and IKK (113, 132, 138, 178, 276, 370) (Fig. 5). The presence in this network of IKK, which otherwise mediates its primary function as part of IKK, is surprising and still not fully understood. In pDCs, which are characterized by constitutive expression of IRF7, TLR7 and TLR9 signaling results in IRAK1-mediated phosphorylation of IRF7 and production of type I IFN (370). Several reports analyzing this pathway in mice deficient in relevant components have demonstrated an important and nonredundant role of MyD88, IRAK1/4, and IRF7 (178, 370), thus emphasizing that IRF7 is an essential transcription factor regulating IFN production in pDCs (133). In addition, stimulation of TLR7 and TLR9 also triggers MyD88-dependent activation of NF-B and MAPKs in all cell types by mechanisms similar to the ones operating downstream of TLR3 and TLR4. Finally, IRF5 has been reported to interact with MyD88 in hematopoietic cells (358) and to play a role in induction of type I IFN production through TLR7 and TLR9 (63, 291, 324). IRF5 also participates in induction of inflammatory cytokines through TLR3, -4, -7, and -9 (291, 324, 358). In summary, TLRs signal through a proinflammatory MyD88-dependent pathway mainly responsible for cytokine expression induced by potent activation of NF-B and MAPKs. An additional MyD88-dependent pathway is triggered by TLR7 and TLR9 and induces type IFN production, particularly in pDCs. On the other hand, a MyD88-independent, TRIF-dependent pathway is essential for mounting a type I IFN response in non-pDCs as well as for contributing to activation of NF-B.
Signaling by Cytosolic PRRs
In addition to RNA detection in the endosomal compartment through TLR3- and TLR7/8-mediated recognition of dsRNA and ssRNA, respectively, several cytosolic RNA-sensing receptors have been identified, including RLRs and NLRs, as previously described (Fig. 6).
FIG. 6. Intracellular RNA recognition and signaling. Cytosolic dsRNA or 5-triphosphate ssRNA is recognized primarily by the cytoplasmic RNA helicases RIG-I and MDA5, which mediate interaction with the adaptor IPS-1, localized to mitochondria, and trigger signaling to NF-κB and IRF3 via IKK and TBK/IKKε, respectively. dsRNA can also be recognized by TLR3 localized in the endosomal compartment or by cytosolic PKR, but whereas TLR3 triggers signaling to NF-κB and IRF3, PKR instead activates NF-κBand MAPKs. Finally, ssRNA is recognized by TLR7/8 in endosomes and induces signaling to IRF7 as well as to NF-κB and MAPKs (not shown in the figure).
Binding of dsRNA or 5- triphosphate RNA to RLRs triggers signaling via CARDCARD interactions between the helicase RIG-I/MDA5 and the adaptor protein IPS-1 (179). This adaptor protein, which has been independently characterized by several groups and also named, in abbreviated form, CARDIF (246), MAVS (329), or VISA (383), appears to be associated with the outer mitochondrial membrane (329), and this association is essential for the function of IPS-1 (246). Yet another adaptor protein, stimulator of IFN genes (STING, also termed MITA), was recently described (150, 402). Although also present in the mitochondrial membrane, STING resides predominantly in the endoplasmic reticulum (ER) and interacts with RIG-I and IPS-1, which are linked to the mitochondrial membrane, thus potentially allowing cross talk between these two organelles, in terms of both viral recognition and signaling (150, 402). Indeed, a central position of mitochondria in virus-induced NF-B activation was already reported prior to the identification of RLRs, IPS-1, and STING (259). In this early study, NF-B activation in HSV-infected murine macrophages was demonstrated to be associated with generation of mitochondrial oxidative stress and calcium release and to be dependent on activation of TAK1, MKK kinase 1, and IKK (259). Since many viruses replicate in the membranous web connecting the ER to mitochondria, the localization of dsRNA sensing and signaling pathways to these organelles may provide the host with an opportunity to sense an incoming viral challenge and subsequently coordinate an immune or apoptotic response (125). However, the precise connection of mitochondrial oxidative stress and calcium-dependent signaling to mitochondrionassociated adaptor proteins such as IPS-1 remains to be fully understood. In the RLR pathway, IPS-1 appears to be the point of divergence of two different signaling pathways, involving either IRFs or NF-B, as illustrated in Fig. 6. A central role of TNF receptor-associated death domain (TRADD) in signal transduction from IPS-1 to both of these pathways was reported by Michallet et al. (251). TRADD, which is also an essential adaptor for the TNF receptor, was demonstrated to be recruited to IPS-1 and to orchestrate complex formation with downstream effector molecules (251). Thus, the IPS-1– TRADD complex recruits the E3 ubiquitin ligase TRAF3 and the adaptor protein TANK (108, 251, 313), which subsequently activate TBK1/IKKε to phosphorylate IRF3 and IRF7, ultimately resulting in an antiviral response mediated by IFN- production (172, 174, 393). Alternatively, the IPS-1–TRADD complex propagates the signal to NF-B by a mechanism involving interaction with Fas-associated death domain, RIP1, and caspase-8/10, which were also originally identified in the signaling pathway downstream of the TNF receptor (27, 355). These molecules transduce the signal to IKK, eventually mediating NF-B activation. NEMO has been demonstrated to participate not only in NF-B activation but also in IRF3 activation (401), although the mechanism, by which NEMO activates IRF3 remains to be clarified. In similarity with most TLR signaling pathways, RLRs also propagate activating signals to MAPKs (329). Intracellular sensing of RNA has also been attributed to PKR, which plays a role in IFN and stress responses (270, 312, 389). In studies of PKR signal transduction pathways, it was demonstrated that PKR-mediated NF-B activation in response to poly(I-C) is transduced through NF-B-inducing kinase and IKK (396). Moreover, a number of reports have described the involvement of PKR in signaling induced by diverse proinflammatory stimuli, including TNF- (74, 396), IFN- (68), and LPS (109). It remains unsettled, however, whether PKR kinase activity is a prerequisite for signal transduction or whether PKR mediates its functions as a structural protein through interaction with other kinases and adaptor proteins. For instance, it has been suggested that PKR may participate in TLR3-dependent NF-B activation by functioning as a scaffold protein as part of an intracellular complex containing TRAF6, TAK1, and TAB2 (158), and others have demonstrated that interaction of PKR with TRAF2 and TRAF5 leads to activation of IKK (100). Furthermore, one study with human keratinocytes has suggested that PKR may be involved in the dsRNA response through both TLR3 and RLR signaling pathways (163). This was supported by findings of PKR facilitating IPS-1-dependent signaling to induce IFN- and apoptosis in response to vaccinia virus infection and 5- triphosphate RNA (237, 399). Alternatively, PKR may be associated with the IKK complex under certain conditions or in response to specific stimuli (312, 396), thus explaining the importance of PKR in obtaining a full response under some experimental conditions, whereas in other situations PKR seems to be redundant. Such models may be a way to reconcile the apparent involvement of PKR in several proinflammatory pathways with recent data on more prominent dsRNA-sensing systems, such as TLR3 and RIG-I. Finally, several pieces of evidence suggest that PKR activates p38 in response to dsRNA by a mechanism that may include PKR-mediated phosphorylation of the p38 upstream kinase MKK6 (334). Regarding cytosolic DNA sensors, including DAI, strong evidence suggests that these receptors signal via TBK1/IKKε to IRFs, whereas the pathway for NF-B activation remains unknown (149, 344, 345, 357). Considerably more data describing signaling mechanisms have been collected in the case of NOD proteins. Signaling by these cytosolic PRRs involves recruitment of the kinase RIP2, which promotes polyubiquitination of NEMO and TAK1 (1, 382), a prerequisite for activation of NF-B. Likewise, the adaptor molecule CARD9 is directly recruited to NOD proteins, thus allowing downstream activation of MAPK signaling (169).
INNATE AND ADAPTIVE IMMUNE RESPONSES
Proinflammatory signaling pathways induced by PRRs activate the innate immune response and also play a role in the activation, maturation, and shaping of the adaptive immune response (151). To initiate these responses, the transcription factors NF-B, AP1, and IRF3/7 play pivotal roles (54, 177) due to their capacity to stimulate the production of proinflammatory mediators, including cytokines and IFNs. However, tight regulation is essential to ensure the strong, albeit transient, nature of these responses, and this is achieved via amplification early during infection, as well as restriction and downregulation when needed at later stages. It is notable that regulation of gene expression at the transcriptional level plays an important, although not exclusive, role in the generation of an adequate immune response; posttranscriptional mechanisms regulating the decay of mRNAs encoding various proand anti-inflammatory molecules also contribute to achieve this goal.
NF-B-Inducible Proinflammatory Mediators
The central role played by NF-B in both innate and adaptive inflammation and immunity is mediated by the coordinate expression of multiple genes essential for the immune response. The importance of NF-B is revealed by the extensive list of NF-B-inducible genes, including those for proinflammatory cytokines such as IL-1, IL-6, and TNF-, as well as chemokines, including IL-8 and RANTES (99). Moreover, the expression of cell adhesion molecules, such as intercellular adhesion molecule-1 and E-selectin, is upregulated. A major class of molecules involved in many aspects of the inflammatory response and upregulated in response to cellular pathogen recognition consists of immunoreceptors, including cytokine and chemokine receptors, immunoglobulins, TLRs, major histocompatibility complex (MHC) molecules, and costimulatory molecules (99, 151, 252). Together, these NF-B-inducible proteins participate in the activation and recruitment of leukocytes to sites of inflammation, in enhanced phagocytosis of microbes, in complement- or NK cell-mediated cellular lysis, and in enhanced antigen presentation. Finally, in some instances, it appears to be advantageous for the host to stimulate cell division and inhibit apoptosis, and in order to achieve these effects, NF-B induces a number of growth factors and antiapoptotic proteins (99, 171).
Posttranscriptional Regulatory Mechanisms
Posttranscriptional regulation, particularly control of mRNA stability, also represents an important level of regulation of the proinflammatory response (184). In this respect, two prominent mechanisms are micro-RNAs and AU-rich element (ARE)-mediated mRNA degradation (184). RNA degradation proceeds through specific, inducible ARE-binding proteins that determine the fate of a given mRNA, i.e., degradation versus stabilization, through binding to the 3 untranslated region (29). Many gene products involved in immunity and inflammation, especially cytokines and chemokines, harbor such destabilization sequence elements (92). ARE-binding proteins function by targeting AU-rich RNA to the exosome for degradation or by recruitment of alternative degradation factors (87, 139). Several signaling pathways with impact on mRNA stability have been described, with the p38 MAPK pathway being one of the most prominent (92). The importance of ARE-mediated mRNA regulation in generation of an inflammatory response was described in a study of live S. pneumoniae-induced TNF- production, which was demonstrated to be highly dependent on p38 and AREs in the 3 untranslated region of TNF- mRNA (257). However, despite previous findings that many purified and synthetic TLR ligands can stabilize RNA (114, 302), in this study, TNF- mRNA stabilization was found to be independent of MyD88. An interpretation of these results is that other members of the PRR family, such as NLRs or DNA receptors, may be activated by S. pneumoniae and mediate the effect (257). The close relationship between PRRs and the mRNA-regulating machinery is suggested by other examples illustrating both cooperation and interplay between these evolutionary ancient systems. For instance, several micro-RNAs which have the ability to bind and induce degradation of specific mRNA transcripts through the enzyme Dicer (145) are encoded by LPS-responsive genes (275, 354), and likewise, specific micro-RNAs are involved in regulation of TRAF6 and IRAK1, prominent members of TLR-induced signaling pathways (354).
Leukocyte Recruitment and Activation
Overall, pathogen recognition through PRRs regulates leukocyte recruitment to sites of infection by activating several cell type subsets, including tissue stromal cells, tissue-resident innate cells (most notably DCs and macrophages), and circulating leukocytes. Leukocyte recruitment is a complex process involving a series of interactions with integrins expressed on vascular endothelium. The sequential rolling, adhesion, and migration of leukocytes into the inflamed tissue is tightly regulated through induced expression of cell adhesion molecules and integrins in response to PRR activation (141, 151, 204). The acute inflammatory cellular infiltrate consists of monocytes, DCs, neutrophils, and NK cells. Among these, neutrophils and NK cells are critical innate effector cells protecting the host by killing pathogenic microbes and infected cells, respectively (31, 151). Whereas neutrophils express mRNA for all TLRs and can be activated by most PAMPs, NK cells seem to be activated mainly by type I IFN produced by pDCs in a TLR-dependent manner. These activated NK cells in turn secrete IFN-, hence augmenting pathogen clearance by macrophages (229, 315). Despite the central role played by these innate immune cells, recognition of PAMPs by epithelial, endothelial, and hematopoietic cells in different tissues through TLRs is also an integral part of innate immune defenses at sites of infection (151, 236).
IFN Responses
The ability of IFNs to confer an antiviral state on cells is their defining activity and at the same time one of the fundamental properties that allowed their discovery (148). Type I IFN, which is induced by PRR signaling and plays a major role in innate antiviral defenses, consists of IFN-α and IFN-β. IFN-γ, on the other hand, belongs to type II IFN, is produced by T lymphocytes and NK cells, and plays a central role in activation of macrophages (143).
Finally, type III IFN is represented by IFN- λ and shares many properties with type I IFN (18). As previously described, type I IFN can be induced through different pathways depending on the cell type involved. In pDCs, which are responsible for the majority of IFN produced during a viral infection, stimulation of TLR7 or TLR9 with relevant ligands induces type I IFN in a MyD88- dependent manner (132, 178). Conversely, in macrophages, conventional DCs, and fibroblasts, type I IFN is induced by cytoplasmic RNA primarily via RLR-mediated signaling or, alternatively, by TLR3 or TLR4 stimulation by a mechanism dependent on TRIF (172, 384). An important aspect adding to the complexity of IFN production and regulation is the existence of a positive feedback loop, which represents a way to significantly enhance the IFN response (227). Whereas IRF3 is constitutively expressed, IRF7 expression is weak in unstimulated non-pDCs but is dramatically upregulated in response to virus infection, LPS, or type I IFN (59, 227). Initial expression of IFN- β is therefore largely dependent on phosphorylation and activation of IRF3, but the secreted IFN- β subsequently acts on neighboring cells to induce expression of IRF7. Finally, IRF7 phosphorylated during virus infection together with phosphorylated IRF3 induces the production of both IFN- α and IFN- β subtypes, thereby amplifying the response (133, 227, 316). IFNs mediate their responses by signaling through distinct but related pathways via specific type 1 or 2 receptors that bind to Janus kinases (JAKs) and subsequently activate signal transducers and activators of transcription (STATs), resulting in expression of a broad range of IFN-stimulated genes (74, 342). Classical functions of type I IFN comprise antiviral activities mediated by PKR, 2-5-oligoadenylate synthase/2-5A-dependent RNase L, and Mx proteins, which together inhibit protein synthesis, induce RNA cleavage, and interfere with viral replication (342). Type I IFN also plays a prominent role in inhibition of cellular growth and control of apoptosis, although the effect may be either pro- or antiapoptotic depending on the cellular context (342). The importance of IFN in innate immunity can be extended to include a role in adaptive immune defenses as well (151). For instance, cross-presentation of viral antigens occurs via a mechanism dependent on type I IFN (209a), and B-lymphocyte isotype switching and differentiation into plasma cells have also been attributed to IFN- α / β as well as to IFN- γ (209). Finally, DC maturation is induced by IFN- α / β following stimulation with unmethylated CpG DNA, poly(I-C), LPS, or viral infection (129, 131, 137). It is notable that only a subset of TLRs, including TLR3, -4, -7, and -9, have the ability to induce IFN, although through different signaling pathways. However, since many pathogens activate several TLRs or additional PRRs, most antimicrobial responses seem to include some degree of IFN production. In regard to type II IFN, IFN- γ participates in many aspects of innate and adaptive immunity in cooperation with type I IFN, but IFN- γ also has unique roles, of which activation of macrophages and intracellular killing of microbial pathogens are among the most prominent (342). Despite the IFN system being known for decades, novel functions of these molecules have been described in recent years. For instance, accumulating evidence suggests that type I IFN may not only be involved in antiviral defenses but also may play a role in antibacterial defenses (71), representing a subject of current interest in the field.
Bridging Innate and Adaptive Immune Defenses
A number of studies have established the important link between innate and adaptive immunity provided by PRRs, particularly via TLR-mediated maturation of DCs and activation of pathogen-specific T lymphocytes (151, 303). Following antigen uptake by DCs, these cells become activated and migrate to regional lymph nodes to present antigenic peptides in the context of relevant MHC molecules. During this process, phagocytosis, upregulation of costimulatory molecules (including CD80, CD86, and CD40, and antigen-presenting MHC molecules), switches in chemokine receptor expression, and cytokine secretion are all events that are regulated through the recognition of pathogens by PRRs expressed on DCs (129, 131, 137, 151). Importantly, different subsets of DCs express different and nonoverlapping sets of TLRs, which together with the selective tissue distribution may explain some of the diverse functions carried out by these DC subsets (77). This subject has been excellently reviewed by Iwasaki and Medzhitov (151) but is not within the scope of the present review. After having undergone maturation, DCs are endowed with the ability to stimulate naïve CD4 T lymphocytes into different T helper (Th) subsets, the differentiation of which is controlled by a variety of factors, including TLR-induced cytokines. The Th1- Th2 paradigm introduced by Mosmann et al. in 1986 states that Th cells can be separated into two distinct subsets, depending on the cytokines they produce (265). In general, Th1 responses are important for protection against viruses and intracellular bacteria, whereas Th2 responses mediate immunity to extracellular protozoa at mucosal surfaces and are involved in allergic responses. More recently, a Th17 subset with important functions in protection against certain bacterial infections and with possible roles in the development of autoimmunity was discovered (286, 372). In the process of DC-mediated polarization of different Th subsets, IL-12 plays a critical role in inducing Th1 responses, most notably IFN- (368), whereas IL-23 and IL-1 in humans, and transforming growth factor and IL-6 in mice, participate in inducing Th17 responses with preferential secretion of IL17, IL-21, and IL-22 (225, 286, 372). In contrast, Th2 responses consisting of IL-4, IL-5, and IL-13 are largely dependent on IL-4 (265). The demonstration that mice deficient in MyD88 display severely compromised Th1 differentiation and instead undergo Th2 differentiation is consistent with the view that TLRs primarily control the induction of Th1 and Th17 responses as opposed to Th2 responses (323). The relationship between PRRs and the induction of Th17 responses has been further elaborated in a recent report demonstrating that bacteria prime DCs to stimulate IL-17 production in human memory T lymphocytes through the NOD2 ligand MDP (372). Also of interest is the demonstration of a critical role of NOD1 in synergizing with TLRs to prime Th1 and Th17 pathways in vivo (93). TLRs may also play a role in mechanisms of immune suppression by regulatory T lymphocytes and in humoral immune responses mediated by B lymphocytes (48, 289, 290). Collectively, the important coupling of pathogen recognition with the induction of costimulation and hence activation of pathogen-specific T lymphocytes, an idea originally introduced by Janeway (153), supports the view that innate immunity and adaptive immunity should be perceived as tightly connected and interacting mechanisms of defense, rather than as two separate systems.
INTERFERENCE WITH PATHOGEN-ACTIVATED SIGNALING
Although the generation of a potent immune response is of crucial importance for the containment and eradication of microbial infection, excessive or inappropriate inflammation may be harmful to the host and result in immunopathology or autoimmunity. The innate immune system therefore needs to be able to control inflammatory signaling during infection and, not least, to downregulate the inflammatory response once the infection has been resolved. Recent progress in understanding mechanisms of regulation of innate immunity has revealed a common theme, in which PRRs that detect microbial pathogens activate both a proinflammatory response directed against the pathogen and a specific inhibitory pathway to limit the duration and magnitude of inflammation. This implies that both activating and inhibitory signals are induced sequentially by a given PAMP during generation of the inflammatory response. Furthermore, pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses by interfering with molecules involved in pathogen recognition and signaling.
Endogenous Mechanisms
Several cellular molecules that negatively regulate PRR signaling, particularly targeting the NF-B pathway, have been identified (Fig. 7).
FIG. 7. Negative regulation of PRR signaling. Signaling through PRRs is negatively regulated by endogenous and microbial proteins. Endogenous negative control of PRR signaling (molecules depicted in red) serves the purpose of mediating negative feedback on the inflammatory response, whereas microbial interference with PRRs (viral molecules shown in green) constitutes a means to evade the host antimicrobial response. See text for further details.
First, the fifth TLR adaptor molecule SARM acts as a negative regulator of TRIF-dependent signaling from TLR3 and TLR4 (51). TLR adaptor molecules are also the target of another inhibitory molecule, MyD88s, an alternatively spliced variant of MyD88, which does not bind to IRAK4 and hence does not promote IRAK1/2 phosphorylation and NF-B activation in response to LPS (43). Similar principles are employed by TIR domain-containing orphan receptors, such as single immunoglobulin IL-1R-related molecule and ST2, which have been demonstrated to negatively regulate TLR signaling by sequestration of MyD88 and Mal (41, 377). In addition, a molecule belonging to the family of IRAKs but deficient in kinase activity, and therefore abrogating further downstream signaling to NF-B, has been identified and termed IRAK-M (155). Importantly, IRAK-M-deficient mice secrete elevated levels of proinflammatory cytokines and are defective in induction of LPS tolerance (191). Another family of proteins with negative regulatory function is the suppressor of cytokine signaling (SOCS) proteins. These proteins, originally defined by their ability to interfere with cytokineinduced JAK-STAT signaling, are inducible by both cytokines and TLR ligands, although their action appears to be mediated indirectly by negative regulation of paracrine IFN- signaling rather than by direct inhibition of TLR-induced signaling pathways (25, 67). A key role in terminating TLR responses has been attributed to the deubiquitinating enzymes A20 and CYLD, which regulate TLR- and TNF--induced NF-B activation by a mechanism involving deubiquitination of TRAF6 (38, 193, 394). Likewise, recent data suggest that A20 restricts NOD-mediated signaling to NF-B by interfering with NOD2-induced ubiquitination of RIP2 (126). More recently, it was demonstrated that the tripartite-motif protein family member TRIM30 negatively regulates TLR4-mediated NF-B activation by specifically inducing degradation of TAB2/3 required for TAK1 activation and function, thus preventing downstream signaling to NF-B (331). Since TRIM30 gene expression is itself stimulated by NF-B, this represents a negative feedback loop with potentially important implications due to the central position of TAK1 in TLR- as well as in IL-1- and TNF-- induced signaling (331). At a level further downstream, another example of an elegant autoregulatory circuit is provided by the NF-B-mediated induction of IB gene expression (56). Newly synthesized IB protein in the cytoplasm or nucleus binds to free activated NF-B, thereby preventing NF-B from inducing further inflammatory mediators and in this way contributing to downregulation of the inflammatory response (347). A direct role of IKK in the degradation of the NF-B subunits p65 and c-Rel bound to the promoter has also been suggested as a means of terminating the response and suppressing DNA-bound NF-B (205). Within the IFN pathway, a negative regulatory feedback circuit is provided by the ring finger protein RNF125, which induces ubiquitination and proteasomal degradation of RIG-I, MDA5, and IPS-1, resulting in termination of IFN responses (21). Since RNF125 is itself induced by IFN, this again illustrates a manner, in which the initial stimulus activating RLR signaling subsequently triggers production of a factor capable of terminating the response. Furthermore, the RNA helicase LGP2 devoid of CARDs prevents activation of antiviral responses, most likely through sequestration of dsRNA from RIG-I and by prevention of RIG-I multimerization (310, 314). In addition, CYLD inhibits the ubiquitination essential for signaling through RIG-I, and accordingly, loss of CYLD causes constitutive activation of TBK1/IKKε (94, 398). Finally, an important level of negative regulation in TLR-induced signaling appears to be RNA-destabilizing proteins, the absence of which leads to exaggerated inflammation in experimental animals due to overproduction of TNF- and IL-1 as a result of impaired degradation of mRNA encoding these cytokines (219).
Viral Immune Evasion Strategies
Given the fact that mounting an inflammatory response through PRRs is a prerequisite for containment and eradication of invading pathogens, it is not surprising that most pathogens have developed mechanisms for modulating or interfering with PRR-mediated responses. Viruses are the class of pathogens that have evolved the most diverse and sophisticated molecular mechanisms for interfering with antimicrobial and proinflammatory responses. Due to the ability of viruses to exploit the cellular machinery during their replication cycle, an intricate virus-host relationship has developed throughout evolution, and this allows many types of viruses to interfere profoundly with host signaling (125) (Fig. 7). Overall, viruses can interfere in multiple ways with NF-B and IRF pathways to inhibit induction of proinflammatory molecules and IFN. Paradoxically, however, viruses can also stimulate NF-B activation in order to induce viral replication due to the presence of NF-B DNA-binding sites in many viral promoters, including the human immunodeficiency virus (HIV) long terminal repeat (23, 125). Viruses with oncogenic potential can induce constitutive NF-B activation and modulate cellular growth pathways and apoptosis, hence profoundly influencing cell cycle regulation and promoting malignant transformation, as in the case of Epstein-Barr virus (lymphoma), human herpesvirus 8 (Kaposi sarcoma), and human T-cell leukemia/lymphotropic virus type I (adult T-cell leukemia) (125). One of the first examples of viral interference with TLRinduced signaling to be described was the identification of the vaccinia virus proteins A46R and A52R, with putative TIR domains targeting host MyD88 and TRIF and suppressing TLR- and IL-1R-induced NF-B activation (39, 341). However, despite its sequence similarities with TIR domains, A52R appears to fold like a Bcl-2-like domain and hence is not a bona fide viral TIR family member (106). In addition, the HSV-1 immediate-early protein infected-cell protein 0 (ICP0) inhibits IRF3- and IRF7-mediated activation of IFN-stimulated genes (215). Likewise, RSV NS1/2 interferes with TBK1- mediated phosphorylation of IRF3 (339), whereas measles virus prevents phosphorylation of IRF7 (322). Moreover, as might be expected based on recent knowledge on the central role played by dsRNA-sensing systems, RLR-mediated signaling pathways are also the target of several viral components, of which the most extensively studied is the hepatitis C virus (HCV) NS3/4 serine protease. In addition to cleaving TRIF and thereby abrogating TLR3 responses to extracellular and endosomal dsRNA (212), NS3/4 also targets IPS-1 and prevents cytosolic dsRNA-induced production of type I IFN (212, 246). In this manner, HCV targets both TLR-dependent and -independent arms of the host antiviral response. The induction of IFN- by viral dsRNA can also be inhibited by paramyxoviruses, such as parainfluenza virus type 2, mumps virus, and Hendra virus, through association between the viral V protein and MDA5 (17). Similarly, RIG-I signaling is targeted by the influenza A virus NS1 and the Ebola virus VP35 proteins through sequestration of viral RNA and direct interaction with RIG-I/IPS-1 (49, 250, 295), the result being inhibition of downstream signaling to IRF3. Recently, a novel mechanism for prevention of RIG-I activation was described, consisting of virally induced posttranscriptional removal of 5- triphosphate RNA from genomes of negative-strand RNA viruses, including Hantaan virus, Crimean-Congo hemorrhagic fever virus, and Borna disease virus (111). By cleavage of triphosphates from their RNA, these viruses impede RIG-Ibinding and activation (268). Additionally, PKR appears to be the target of several viral proteins, including the E2 and NS5A proteins of HCV, which bind to PKR to inhibit IRF1 activation (95, 293), and the influenza A NS1 protein, which functions as an IFN antagonist in part through inhibition of PKR (213), although the precise mechanism of action is controversial (125, 250). Finally, proinflammatory cytokine and IFN expression can also be manipulated by viral interference with the posttranscriptional machinery of the host. For instance, vesicular stomatitis virus M protein inhibits nuclear-to-cytoplasmic export of mRNAs, including those encoding IFN-/ (124). Moreover, in studies of virus-host interactions, HSV-1 has been reported to suppress proinflammatory cytokine production by a mechanism involving destabilization of mRNA encoding proinflammatory cytokines, in a process dependent on the viral immediate-early proteins ICP4 and ICP27, thus impeding the antiviral host response to infection (260).
Bacterial, Protozoan, and Fungal Immune Evasion Strategies
Although viral evasion strategies may be the most elaborate and have been studied most extensively, other classes of pathogens have also developed several elegant evasion strategies, either by altering their PAMPs to be less immunogenic or through activation of anti-inflammatory responses induced by different virulence factors. Certain bacteria, such as Helicobacter pylori, Porphyromonas gingivalis, and L. pneumophila, synthesize modified forms of LPS, which are not recognized by TLR4 (134), and similarly, Campylobacter jejuni, H. pylori, and Bartonella bacilliformis produce subclasses of flagellin that do not activate TLR5 (16). Recently, a novel family of virulence factors was identified in Escherichia coli and Brucella melitensis (57). These proteins, which have been named TIR domaincontaining proteins, impede TLR signaling through direct binding to MyD88, thus suppressing innate immunity and increasing bacterial virulence (57). Important enteric pathogens, including Shigella, Salmonella, and Yersinia species, are endowed with a type III secretion system mediating the delivery of effector proteins into host cells, thus representing bacterial virulence factors (9). One such type III effector protein is the Yop protein of Yersinia pestis, which has been demonstrated to act as a deubiquitinating protease acting on TRAF6 and TRAF3 to inhibit NF-B/ MAPK and IRF signaling, respectively (350). Other data suggest that Yop may also act as an acetyltransferase that modifies critical residues in the activation loop of MAPK6 and IKK, thereby preventing their phosphorylation and further downstream signaling (267). A similar type III effector is represented by the Shigella OspG protein kinase, which appears to bind a subset of ubiquitin-conjugating enzymes, including those mediating degradation of IB, thereby preventing nuclear translocation of NF-B (186). Finally, another class of virulence factors is the V antigens, which in the case of Yersinia species induce an anti-inflammatory response by stimulating TLR2-mediated IL-10 production, ultimately resulting in TLR2-deficient mice being less susceptible to yersinia infection (336). Likewise, the Vi antigen of S. enterica serotype Typhi exerts an immunosuppressive effect in the intestinal mucosa that promotes bacterial dissemination and invasive disease (298). The Vi antigen is therefore considered to play a role in the differences in pathogenesis between S. enterica serotype Typhi and S. enterica serotype Typhimurium, the latter of which does not express the Vi antigen and causes only local inflammatory disease (298). At present, much less is known about protozoan and fungal immune evasion strategies, although in particular in the case of protozoans, such strategies are likely to be of major importance for the establishment of long-term residence within their hosts (98). For instance, T. gondii induces LPS unresponsiveness mediated at least in part by activation of STAT3, a transcription factor also used by IL-10 to exert anti-inflammatory activities (45). T. gondii also negatively regulates LPS-induced MAPK activation and IL-12 production, although it remains unresolved whether the target is a p38-activating kinase or induction of a p38 MAPK phosphatase (188). Suppression of macrophage IL-12 production is also the final outcome of infection with Leishmania mexicana due to expression of amastigote-specific cysteine peptidases that proteolytically degrade IB and NF-B (47). Finally, a number of fungal immune evasion strategies have been described, exemplified by IL-10- mediated immunosuppression induced through TLR2 by C. albicans and A. fumigatus (272).
MEDICAL IMPLICATIONS
The pivotal role of TLRs in innate host defenses was first indicated by the finding that C3H/HeJ mice with nonfunctional TLR4 are resistant to LPS-mediated shock (296), and valuable insights into TLR-mediated pathogen recognition and signaling has since been gained from the use of knockout mice challenged with individual purified PAMPs or entire microbial pathogens. However, data from in vitro studies or mouse models may not be entirely representative of the physiological situation in the human organism with respect to understanding natural conditions of infection and immunity, primary immunodeficiencies, altered disease susceptibility, and development of autoimmunity. Therefore, it is important to translate findings from cell culture and animal studies to the human organism, where important lessons may be learned from the identification and description of associations between alterations in the function of PRRs and development of human diseases.
Primary Immunodeficiencies and Susceptibility to Infectious Diseases
To date, six Mendelian primary immunodeficiencies associated with impaired TLR signaling and NF-B activation have been reported (52, 62, 78, 294, 376, 400) (Table 2). These genetic disorders are excessively rare, and progress has been made primarily through international collaborations directed by Casanova (196). First, anhydrotic ectodermal dysplasia with immunodeficiency (EDA-ID) is an X-linked recessive immunodeficiency caused by mutations in NEMO, a critical component of the IKK complex, and resulting in impaired NF-B signaling (78). Affected patients display abnormalities in ectodermal development, including absent or diminished dental growth and hair, as well as various degrees of immunodeficiency. The infectious phenotype is dominated by invasive infections by encapsulated pyogenic bacteria, such as Haemophilus influenzae, S. pyogenes, and S. aureus (195). These patients can also be affected by weakly pathogenic mycobacteria, viral illnesses with herpesviruses (282), and fungal diseases, particularly P. jirovecii pneumonitis (78, 195). Laboratory investigations reveal various degrees of hyper-immunoglobulin M syndrome (152), NK cell abnormalities (282), and a poor response to LPS, IL-1, and TNF- (196). Moreover, a second form of EDA-ID inherited in a dominant way has been reported in a patient with a mutation in the ikba gene (62), which prevents IB phosphorylation and NF-B activation. The third primary immunodeficiency, involving IRAK4, is inherited as an autosomal recessive disorder (294). As previously described, IRAK4 plays an important role in signal propagation to NF-B and MAPKs downstream of certain TLRs (294, 348). Accordingly, poor inflammatory responses and recurrent pyogenic bacterial infections, particularly with S. pneumoniae, have been reported in patients with this disorder (294). Blood cells from these patients fail to produce IL-1, IL-6, IL-8, IL-12, and TNF- in response to various TLR agonists (196), and fibroblasts are unable to activate both NF-B and MAPK pathways following IL-1 stimulation (196). Given the central position of IRAK4 in TLR and IL-1R signaling, the relatively mild phenotype of IRAK4-deficient patients may seem surprising. However, a similar immunological and infectious phenotype was recently described in the case of MyD88-deficient patients, who are vulnerable to recurrent pyogenic infections early in life (376), supporting a narrow but nonredundant role of both IRAK4 and MyD88 in protective immunity against certain bacterial infections. The observation that both IRAK4- and MyD88-deficient patients are affected mostly in the neonatal and childhood period and then appear to improve with age may also suggest that maturation of TLR-independent innate immunity or T and B lymphocytespecific adaptive responses progressively compensate for the poor innate immune response early in life. The first immunodeficiency originating from a TLR mutation was identified in a recent report describing increased susceptibility to HSV encephalitis in patients with rare mutations in TLR3, indicating a possible role for this receptor in central nervous system infections with HSV and possibly other neurotropic viruses (400). Finally, a genetic predisposition to HSV encephalitis was identified in two children lacking functional UNC-93B (52), an ER protein required for delivering nucleotide-sensing TLRs (TLR3, -7, -8, and -9) from the ER to endosomes (190, 351). The list of associations between PRR single-nucleotide polymorphisms (SNPs) and altered susceptibility to different pathogens is rapidly expanding; some examples are given below, and a more extensive overview is presented in Table 2. However, interpretation of the causal association between an SNP and alteration in disease susceptibility at the mechanistic level is frequently hampered by inadequate knowledge of the molecular consequences of the polymorphism, i.e., whether any given SNP results in diminished or increased inflammation.
Two SNPs in TLR2 have been linked to reduced NF-B activation and human diseases (363). First, the Arg677Trp polymorphism has been associated with leprosy in a Korean population (167) and with susceptibility to tuberculosis in a Tunisian population (33). At the mechanistic level, this may be explained by the mutation causing reduced IL-12 levels in response to mycobacteria, thereby resulting in diminished IFN--mediated Th1 responses. The second functional TLR2 variant, Arg753Gln, is overrepresented in patients with staphylococcal septic shock (217) and associated with an increased risk of developing tuberculosis (278), in agreement with TLR2 recognizing PAMPs from these two pathogens. The central position of TLR4 as the receptor for LPS of gram-negative bacteria has inspired several studies on the role of this receptor in human diseases. Two relatively frequent cosegregating polymorphisms in TLR4, Asp299Gly and Thr399Ile, have been linked to an increased risk of infection and septic shock with gram-negative bacteria as well as to an increased risk of severe malaria in African children (4, 218, 255). Furthermore, rare TLR4 mutations were associated with increased meningococcal susceptibility in a study involving a large collection of patients with meningococcal sepsis (337). In a case-control study, other TLR4 polymorphisms have been associated with resistance to Legionnaires’ disease, suggesting a protective association (116). In contrast, TLR5 has a stop codon variant (TLR5 392STOP) that is associated with increased susceptibility to pneumonia caused by L. pneumophila (117). Regarding TLR9, data from the Swiss HIV cohort have suggested that TLR9 polymorphisms may influence the clinical course of HIV type 1 infection, based on findings that two SNPs in TLR9 were associated with rapid progression of HIV type 1 infection (36). Although the cellular mechanism behind this association remains to be elucidated, a possible explanation may be that TLR9-mediated NF-B activation enhances HIV replication by binding to HIV long terminal repeats or, alternatively, that nonspecific immunostimulation participates in CD4 T-lymphocyte depletion and exhaustion of cellular immunity (75, 83). A negative influence of excessive immunostimulation was also reported by Arcaroli et al., who identified a variant IRAK1 haplotype (Leu522Ser) associated with increased NF-B activation and increased severity of sepsis (20). In a large epidemiological study, heterozygosity of a Mal Ser180Leu functional variant has been associated with protection from invasive pneumococcal disease, bacteremia, malaria, and tuberculosis (185). The authors investigated the functional consequences of this amino acid substitution and concluded that the Mal variant attenuated TLR2 signal transduction, thus implying that strong TLR2 activation may be disadvantageous in these common infectious diseases (185). Collectively, the majority of studies strongly suggest that in humans as well as in mice, TLRs play an important role in inflammatory immune responses. However, genetic population studies seem to indicate that no strong selective pressure operates on TLRs, and thus it has been speculated that TLRs may be redundant and that other sensors of innate immunity can substitute for their functions (196). Alternatively, it may be that a more profound defect in pan-TLR signaling has not been identified in humans, simply because it would be incompatible with survival, thus supporting the current idea that TLRs are key sensors of invading pathogens in innate immune defenses.
Hereditary Periodic Fever Syndromes and Other Disorders of NLRs
Alterations in the function of NLR proteins can also cause human disease. Mutations in NALP3/cryopyrin or other subunits of the inflammasome have been linked to relatively rare hereditary periodic fever syndromes as a consequence of constitutive caspase activity and excessive production of IL-1 (233). Patients with the syndromes neonatal-onset multisystem inflammatory disease, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and familiary Mediterranean fever suffer from periodic attacks of fever and inflammation (86, 233). Fever episodes seem to be triggered more or less spontaneously, possibly by subclinical viral infections or cellular stress activating the inflammasome, and the resultant IL-1 hyperproduction results in a clinical picture resembling a microbial infection (169, 233). Accordingly, some of the periodic fever syndromes have been successfully treated with the specific IL-1R antagonist Anakinra (130). Other members of the family of NLRs have been implicated in aberrant NF-B regulation, although mutations can result in either inhibition or activation of NF-B. For instance, a missense point mutation in the human NOD2 gene resulting in defective NF-B activation has been correlated to increased susceptibility to Crohn’s disease (144, 277). In contrast, constitutive NF-B activation is responsible for development of early-onset sarcoidosis and Blau syndrome, two closely related juvenile systemic granulomatosis syndromes, originating from other mutations in NOD2 (166). Finally, SNPs in NALP1 were demonstrated to contribute to the risk of vitiligo-associated multiple autoimmune diseases (160).
Systemic Autoimmune Disorders
Since nucleic acids of the host are generally inaccessible to RNA- and DNA-sensing receptors present in specific compartments, they do not trigger PRR signaling. However, under certain pathological conditions, such as incomplete clearance of apoptotic cells, host-derived nucleic acids may become available for TLRs, an event that may break tolerance and result in autoimmunity (228, 236). Despite TLR3, -7, -8, and -9 being originally identified as receptors specific for microbial nucleic acids, more recently some of these receptors have been linked to the detection of endogenous host RNA and DNA in systemic autoimmune diseases (228). Fundamental progress in the understanding of the pathogenesis of systemic autoimmune diseases was made when the need for dual engagement of the B-cell receptor (BCR) and TLRs by autoreactive B lymphocytes was described (207). These self-reactive B lymphocytes are responsible for the formation of immune complexes by producing antibodies recognizing a limited range of nuclear self-autoantigens, including DNA, histones, RNA, and RNAbinding proteins (207). Binding of the immune complex by the BCR, or by the FcR in the case of pDCs, is followed by BCR-mediated endocytosis and delivery of the immune complex to an endosomal compartment, which allows ssRNA and CpG DNA motifs within the autoantigen to activate the cell through TLR7 and TLR9, respectively (203, 207, 241). Based on findings that immune complexes associated with self-DNA and -RNA can activate pDCs to produce large amounts of IFN-, coupled with clinical data revealing that IFN-producing pDCs accumulate in cutaneous systemic lupus erythematosus (SLE) lesions (84, 241), the common mechanism, by which TLRs play a role in SLE pathogenesis, is believed to be via production of IFN-. This is supported by epidemiological studies, which have demonstrated an association between a TLR9 polymorphism and susceptibility to SLE in a Japanese population (361). Furthermore, an association between genetic variants of IRF5, which is involved in regulating IFN- production, and SLE in multiple ethnic groups has now been established (105, 183, 358). Mechanistic insights into the process of immune complex formation and uptake, TLR activation, and type I IFN production may explain some fundamental observations on the nature of systemic autoimmune diseases, such as the association between infections and a triggering event in disease development, and the subsequent association between infection and disease flares once an autoimmune condition has manifested. However, despite recent progress in the understanding of autoimmune diseases such as SLE, the precise mechanism behind loss of peripheral tolerance remains unknown (228). Finally, uncontrolled excessive or constitutive NF-B activation has been associated with oncogenesis, particularly in the lymphoid system, and has been implicated in the development of multiple hematological malignancies as well as in several solid tumors. This subject is not within the scope of the present review but has been excellently reviewed by Courtois and Gilmore (61) and by Karin (171).
Therapeutic Implications
Since excessive inflammation plays an important role in the pathogenesis of many infectious diseases and autoimmune conditions as well as in the development of malignancy, targeting innate immune activation and signaling potentially holds great medical promise.
Glucocorticoids. Glucocorticoids have been used extensively in the treatment of various medical conditions, where their potent immunosuppressive and anti-inflammatory effects may be desirable. However, whereas the use of adjunctive dexamethasone for the treatment of bacterial meningitis, at least in an adult population in the developed world, is currently well documented from large clinical trials (72, 273, 320), the use of high-dose glucocorticoids in patients with septic shock remains controversial and may even be harmful (340). Several targets of glucocorticoids have been identified, but the molecular mechanisms behind these effects are very complex and remain incompletely understood. Since glucocorticoids are known to interfere with various signaling pathways and molecules involved in TLR signaling, it has been hypothesized that these pathways may be important targets of glucocorticoid action and hence may explain many of their effects (13, 266). A number of levels at which glucocorticoids can exert their effects have been defined, including interference with upstream signal transduction, direct interaction with the transcriptional machinery, and modulation of RNA stability (13, 266). Classically, glucocorticoids bind to the intracellular glucocorticoid receptor and subsequently translocate to the nucleus, where they bind to glucocorticoid response elements to activate expression of anti-inflammatory genes (3, 13, 258). Another major mechanism of action of glucocorticoids is repression of proinflammatory genes, which is achieved mainly through direct protein-protein interactions between the glucocorticoid ligand-receptor complex and the transcription factors NF-B and AP1 (266), thereby preventing interaction with the essential transcriptional coactivator CBP/p300 (266). Further upstream, glucocorticoids can inhibit TLR-induced proinflammatory signaling through MAPK, NF-B, and IRF pathways (3, 239, 321). The glucocorticoid dexamethasone has been demonstrated to inhibit the MAPKs extracellular signal-regulated kinases 1/2, JNK, and p38 by a mechanism involving upregulation and decreased degradation of MAPK phosphatase 1 (34, 46, 103, 200, 349). Further insight into the mechanisms of glucocorticoid-mediated interference with the inflammatory response during microbial infection was provided by a study demonstrating that dexamethasone inhibits TLR signaling induced by N. meningitidis and S. pneumoniae by targeting the NF-B pathway at several levels, including inhibition of IKK-mediated IB phosphorylation and NF-B DNA-binding activity as well as upregulation of IB resynthesis (258). Additionally, dexamethasone has been reported to inhibit IRF3 and the IFN response by targeting the IKKrelated kinase TBK1 (239). Given the central position of the molecules targeted by glucocorticoids (258, 266), their potent and broad range of effects are understandable, although their use may often seem like a double-edged sword. The long list of adverse effects of glucocorticoids and their profound interference with almost any physiological system in the organism is the rationale behind the effort to identify and develop therapeutic molecules with more specific cellular targets and thus theoretically a more narrow profile of adverse effects.
NF-B and p38 inhibitors. Specific inhibitors of the NF-B pathway have been the focus of interest for a number of years (388). More than a decade ago, nonsteroidal anti-inflammatory drugs, such as aspirin and sodium salicylate, were reported to prevent IB phosphorylation and NF-B nuclear translocation by direct inhibition of IKK kinase activity (388, 391). Novel specific inhibitors of IKK or inhibitors of the ubiquitinproteasome pathway that interferes with IB degradation and NF-B activation are being developed and tested in clinical trials (254). However, significant safety problems related to total inhibition of all NF-B-mediated functions, including regulation of inflammation and apoptosis, remain important matters of concern and the subject of ongoing studies. Turning to the MAPK pathway, p38 inhibitors have received much attention. Several chemically diverse compounds potently inhibit p38/ in an ATP-competitive manner, and specific and selective p38/ inhibitors have been demonstrated to block the production of IL-1, TNF-, and IL-6 both in vitro and in vivo, with potential implications for the treatment of inflammatory diseases (197). A number of p38 inhibitors have reached clinical trials in humans, but adverse effects, including liver toxicity and neurological manifestations, have been reported. One of the reasons for these undesirable side effects may be cross-reactivity against other cellular kinases, implying that the development of inhibitors that are non-ATP competitive may be advantageous.
TLR agonists. Triggering TLR activation during vaccination, either as part of the immunogen or as an adjuvant, is the most extensively explored application of TLR agonists (373). Direct TLR activation has been demonstrated in the case of the bacillus Calmette-Gue´rin (BCG) vaccine for the prevention of tuberculosis, in which cell wall components of BCG, including arabinogalactomannan and peptidoglycan, activate TLR2 and TLR4 in a MyD88-dependent manner (369). In contrast, an adjuvant effect of TLR activation has been demonstrated in an H. influenzae type B vaccine conjugated with a meningococcal protein (201), consistent with the activation of TLR2 by Neisseria outer membrane proteins (8, 234, 262). Furthermore, two hepatitis B virus vaccines using TLR4 agonists as adjuvants have been approved (79). One concern is whether a TLR4 agonist may mimic the effects of LPS and induce endotoxic shock (177). However, the TLR4 agonist monophosphoryl lipid A exhibits low toxicity compared to LPS and possess only weak ability to induce proinflammatory cytokines, which may be explained by its selective activation of the TRAM-TRIF pathway as opposed to the Mal-MyD88 pathway (235). Allergy and asthma, both of which are characterized by inappropriate Th2 responses to environmental allergens, have also been the targets of TLR agonist therapies. Given their ability to induce strong Th1 responses, TLR4 and TLR9 agonists are being developed for the treatment of allergic diseases, and such agonists have been administered along with an allergen (170). A successful example is the report of immunotherapy with a ragweed-TLR9 agonist vaccine for allergic rhinitis, with an observed shift in the Th2/Th1 ratio together with significant clinical benefits (64). In the treatment of infectious diseases, agonists of TLR3, -7, -8, and -9 have shown some promise, particularly against chronic viral infections, where the mechanism seem to involve production of type I IFN and generation of IFN-dependent antiviral responses (170). For example, the TLR7 agonist imiquimod has been approved for topical use against genital warts caused by human papillomavirus, whereas other TLR7 and TLR9 agonists are undergoing clinical trials for potential use during chronic infection with HCV and genital HSV infection (170). Finally, a potentially very important use of TLR agonists is in the treatment of cancer, which has been reviewed elsewhere (170, 373).
TLR antagonists. Modulation of the inflammatory response by TLR antagonists may be of clinical benefit in order to dampen exaggerated responses observed during some infectious diseases, thus avoiding immunopathology. Indeed, this is the strategy behind the development of TLR4 antagonists to prevent LPS-induced endotoxic shock during gram-negative bacterial sepsis. Two lipid A analogues, TAK-242 and Eritoran, have been approved for clinical trials involving patients with sepsis (308), and their potential benefits are awaited with interest, since previous efforts to modulate the pathophysiology of sepsis by inhibiting TNF- or LPS have been disappointing (107). The small molecule TAK-242 selectively inhibits TLR4-activated cytokine production by targeting the intracellular domain of the receptor, thus preventing signaling to NF-B through both the MyD88-dependent and -independent pathways (181). Finally, TLR antagonists have recently been suggested to possess desirable properties in the treatment of autoimmune diseases, most notably SLE (170, 373). Given that TLR7 and TLR9 have been implicated in the generation of autoantibodies directed against endogenous TLR ligands (228) and to be essential for pDC-mediated production of type I IFN, (84, 241), targeting of these TLRs may theoretically prevent the development and progression of autoimmune pathophysiology. In conclusion, as knowledge on the role of inflammation in the pathogenesis of infectious, allergic, autoimmune, and malignant conditions increases, the list of targets for therapeutic interference with TLRs or TLR signaling components expands accordingly. The vast number of preclinical and clinical approaches, of which only the most prominent examples are described above, seem to indicate that it is possible to translate the current understanding of TLRs and innate immunity into clinically relevant therapies. However, much experimental animal work, and not least clinical studies involving patients, is needed to evaluate possible advantageous effects as well as toxicity and side effects, such as the consequences of TLR inhibition in terms of enhanced susceptibility to infections or, alternatively, the consequences of TLR activation with regard to the development of malignancy. Some of these scenarios cannot be predicted at present due to our inadequate insight into the complexities and full biological perspectives of the TLR system and proinflammatory signaling in innate immune defenses. The great challenge remains to inhibit the detrimental components of the immune response while leaving untouched an appropriate and beneficial immune response essential for the resolution of infection and elimination of the invading pathogen.
CONCLUDING REMARKS AND PERSPECTIVES
In this review, important aspects of pathogen recognition and activation of signaling pathways ultimately resulting in a proinflammatory innate immune response have been presented. Rapid and efficient mechanisms for detection of invading pathogens and triggering of proinflammatory signaling are of pivotal importance for the initial containment and control of infection. To achieve this, the organism is in possession of PRRs, which recognize evolutionarily conserved structures on pathogens, PAMPs, and trigger innate immune responses, including production of proinflammatory cytokines and type I IFN (7). In addition, pathogen recognition by PRRs is a prerequisite for the activation and shaping of adaptive immune responses through maturation of antigen-presenting DCs and activation of antigen-specific T lymphocytes to resolve the infection and induce immunological memory (151). The development of the concept of pattern recognition and the identification of a large family of receptors with this ability has significantly changed the entire perception of the innate immune system, which is now recognized to have a high degree of specificity and complexity. Moreover, current insight into common principles of cell-cell interactions and intracellular signaling suggests that innate immunity and adaptive immunity are much more intimately connected than previously believed (242). Despite enormous progress in this field of research during recent years, many areas of uncertainty remain. While TLRs represent the most extensively studied group of innate immune receptors and have been assigned essential roles in the induction of proinflammatory and antimicrobial responses, accumulating evidence has revealed that they are part of a large and diverse family of PRRs. Thus, in addition to TLRs, cytosolic PRRs with the ability to sense intracellular PAMPs also seem to be important for both immediate innate protection and bridging to the adaptive immune response (93, 174, 372). Recent insight into the structure and function of these PRRs highlights the fascinating complexity of cellular pattern recognition. During a microbial infection, several different families of PRRs are activated in a cell type- and time-dependent manner by a given pathogen, and the PRR-derived signals appear to cooperate in the generation of a proinflammatory response. The relationship between TLRs and cytosolic PRRs and the principles after which they interact during infection are important issues that remain to be further elucidated. Although the primary function of PRRs is to sense foreign microbial material in the form of “infectious nonself,” in certain cases of “danger,” such as cellular injury or stress, PRRs stimulate inflammation in response to recognition of self molecules. These danger signals originate from host molecules with aberrant locations, structures, or interactions induced by, e.g., necrosis or oxidative damage of cells or tissues, as exemplified by uric acid, ATP, potassium, hyaluronan fragments, and HSPs (146, 236, 247, 362, 365). In this respect, HSPs, which are highly conserved immunodominant proteins with central roles in protein folding and transport, illustrate the intimate relationship between danger and pathogen recognition, which is also underscored by the fact that these proteins are shared between microbial pathogens and eukaryotes. Indeed, endogenous HSPs are upregulated and released by stressed, infected, necrotic, or malignant cells and mediate activation of APCs through TLR2 and TLR4/CD14 either directly or in association with PAMPs (22, 110, 279). Additionally, a role for HSPs as endogenous danger signals recognized by APCs independently of TLRs has been proposed (285). Recognition of endogenous danger signals is particularly evident in the case of the inflammasome, but it also seems to be a common theme for TLRs. Therefore, PRRs may be perceived as evolutionarily ancient detectors of cellular danger invoked not only by pathogens but also by other types of cellular stress. In line with this idea, it has been proposed that PRRs be regarded as sensors of both “microbe-associated molecular patterns” and “danger-associated molecular patterns” (236, 247). This concept places PRRs in a central position in the regulation of immunity and other aspects of cellular physiology. At present, the study of innate immune recognition and signaling remains an area of intensive investigation, with new ideas and concepts being proposed and challenged at an impressing pace. Entirely new and unpredicted functions of PRRs are emerging. For instance, work by Medzhitov and associates has suggested that TLRs are also involved in central aspects of cellular physiology, as demonstrated by the requirement for TLR recognition of commensal microflora in intestinal homeostasis (300). The great versatility of PRRs is also apparent when considering the variability in the microbial PAMPs recognized by these receptors. Indeed, pathogens with quite different biochemical compositions and with entirely different life cycles are recognized by slightly different yet surprisingly similar and overlapping mechanisms (7). Likewise, the signaling pathways they activate are shared to a great extent and regulated by the same overall molecular mechanisms. In the future, it may become apparent that PRR-activated signaling is far more tightly connected to other cellular signaling pathways than is currently realized. For instance, recent studies have demonstrated that RLR signaling utilizes some of the same molecules as the TNF- signaling pathway (251), which may seem surprising given the diverse functions mediated by these receptors. Describing these signaling pathways in more depth, including aspects concerning PRR expression and signaling in different cell types and in different anatomical locations within the organism, may hold the key to a more profound understanding of some of the central questions as to how specificity, complexity, and regulation of both innate and adaptive immunity is achieved. Due to the potency of the inflammatory response, tight regulation to limit the magnitude and duration of this response is crucial in order to avoid the development of immunopathology. Recent progress in understanding mechanisms of regulation of innate immunity has revealed a common theme, in which PRRs detecting microbial pathogens activate both a proinflammatory response directed against the pathogen and a specific inhibitory pathway to downregulate the inflammatory response once the infection has been resolved. The emerging picture reveals intricate molecular mechanisms by which innate inflammatory immune responses are regulated by tightly connected activating and inhibitory circuits to fine-tune and modulate an appropriate response over time. Given the extreme complexity of this system, it may seem surprising that it does not fail more frequently. Moreover, many pathogens, and viruses in particular, have developed sophisticated molecular strategies to subvert host defenses by targeting individual components of PRR-mediated signaling. In this way, interactions between host and pathogen have developed throughout evolution and continue to fascinate by their great ingenuity. Importantly, the molecular mechanisms of signal transduction pathways elicited by a given pathogen and the interactions between host and pathogen serve to illustrate and reveal important aspects of how the immune system functions in defense and disease. The importance of PRRs in regulating inflammatory responses is underscored by their involvement in a wide range of medical conditions. Primary immunodeficiencies as a consequence of impaired TLR signaling and NF-B activation have been identified (196), and accumulating evidence also links genetic polymorphisms in PRRs to altered susceptibility to infectious diseases. In addition, genetic polymorphisms resulting in increased IFN production have recently been associated with the development of systemic autoimmune diseases, particularly SLE (228), and rare mutations in NLR components of the inflammasome resulting in constitutive IL-1 production cause hereditary inflammatory fever syndromes (233). Thus, insight into pathogen recognition and proinflammatory signaling in infection and immunity is a prerequisite for understanding the nature of primary immunodeficiencies and the pathogenesis of infectious diseases and inflammatory disorders and hence for the development of novel therapeutic intervention strategies.