Trends in Molecular Medicine
ReviewSignaling to NF-κB by Toll-like receptors
Introduction
The innate immune system is evolutionarily conserved and is the first line of defense in host protection against invading microbial pathogens [1]. In insects and mammals, innate immune responses are triggered by the detection of pathogen-associated molecular patterns (PAMPs). Drosophila Toll, which was initially identified as a receptor essential for dorso-ventral polarity during development, was first shown to participate in innate immune responses against fungal infections in 1996 [2]. Subsequently, numerous homologues of Toll, termed Toll-like receptors (TLRs), were identified in mammals and were demonstrated to recognize PAMPs and to elicit innate immune responses, such as the induction of inflammatory cytokines [1]. TLRs are single-pass transmembrane proteins composed of N-terminal extracellular leucine-rich repeats (LRRs) that are responsible for recognition of specific pathogen components, a membrane-spanning domain and a C-terminal cytoplasmic domain similar to the cytoplasmic region of the interleukin-1 (IL-1) receptor, known as the Toll/IL-1 receptor (TIR) domain, which is required for downstream signaling.
The TLR family now consists of more than 13 members, each detecting distinct PAMPs derived from various microbial pathogens, such as viruses, bacteria, protozoa and fungi [1]. These microbial components include bacterial lipopolysaccharide (LPS; TLR4 ligand), lipoproteins (TLR2 ligand), flagellin (TLR5 ligand), bacterial CpG DNA (TLR9 ligand), viral single-stranded RNA (TLR7 ligand) and viral double-stranded RNA (TLR3 ligand) (Figure 1). TLRs are expressed on various immune cells, such as macrophages, dendritic cells (DCs), B cells and neutrophils, as well as on non-immune cells, such as fibroblast cells, epithelial cells and keratinocytes. After recognition of PAMPs, TLRs activate the same signaling components as those used in IL-1 receptor (IL-1R) signaling, which results in appropriate immune responses required for host defense. TLRs recruit a set of adaptor proteins with TIR domains by homophilic interaction of their TIR domains. These interactions result in triggering of downstream signaling cascades leading to the activation of transcription factor nuclear factor-kappaB (NF-κB), which controls induction of proinflammatory cytokines and chemokines as well as the upregulation of co-stimulatory molecules on DCs that are essential for T-cell activation. TLR recognition of PAMPs is a key element for induction of the inflammatory response, as well as for instruction of the adaptive immune response against pathogens. Here, we review recent progress in our understanding of the role of NF-κB in TLR signaling pathways and the therapeutic implications of these pathways.
Section snippets
The NF-κB family
The NF-κB family comprises dimeric transcription factors that contain Rel-homology domains (RHDs) that bind to discrete DNA sequences known as κB sites (5′-GGGRNNYYCC-3′; R, purine; Y, pyrimidine; N, any nucleotide) present in promoter and enhancer regions of various genes [3]. In mammalian cells, there are five members of the NF-κB family: RelA (p65), RelB, C-Rel, p105 (NF-κB1; a precursor of p50) and p100 (NF-κB2; a precursor of p52) (Figure 2). NF-κB proteins form homo- or hetero-dimers and
TIR-domain-containing adaptors
Subsequent to recognition of a PAMP, TLRs recruit a specific combination of TIR-domain-containing adaptors, including myeloid differentiation primary response gene 88 (MyD88), TIR-containing adaptor protein/ MyD88-adaptor-like (TIRAP/MAL), TIR-containing adaptor inducing interferon-β (IFN-β)/TIR-domain-containing adaptor molecule 1 (TRIF/TICAM1) and TIR-domain-containing adaptor molecule/TRIF-related adaptor molecule 2 (TRAM/TICAM2) (Figure 1) 7, 8. In TLR5-, TLR7- and TLR9-signaling, the
NF-κB activation through a MyD88-dependent pathway
MyD88 is composed of a TIR domain and a death domain. Upon TLR activation, through its death domain, MyD88 interacts with the death domains of members of the IRAK (IL-1 receptor-associated kinase) family of protein kinases, including IRAK1, IRAK2, IRAK4 and IRAK-M, 7, 8. IRAK4 is initially activated, which in turn phosphorylates and activates IRAK1. It has been suggested that IRAK-M, which lacks intrinsic kinase activity, can negatively regulate TLR signaling by preventing the dissociation of
NF-κB activation through the TRIF-dependent pathway
In addition to the MyD88-dependent pathway, NF-κB is activated by the TRIF-dependent pathway. The downstream pathway of TRIF, which is used by TLR4 and TLR3, leads to NF-κB activation and induction of inflammatory cytokines (Figure 3). Activation of NF-κB by TRIF is initiated through two distinct regions [25]. TRIF directly binds TRAF6 via its TRAF6-binding motifs in the N-terminal region 26, 27. TRIF with mutations of the TRAF6-binding motifs is unable to activate NF-κB when overexpressed,
IKK-related kinases: TBK1 and IKKi
Two additional members of the IKK family, IKKi (also known as IKKɛ) and TBK1 [TRAF family member-associated NF-κB activator (TANK) binding kinase-1, also known as T2K or NAK] [32], which were initially implicated in NF-κB activation, have been identified (Figure 2). IKKi was originally isolated as an LPS-inducible protein in mouse macrophages and was shown to exhibit sequence similarity to canonical IKKs [33]. Its overexpression in cultured cells markedly activates NF-κB. IKKɛ was subsequently
Activation of IKK-related kinases through a TRIF-dependent pathway
Two groups reported the important finding that TBK1 and IKKi have essential roles in the induction of type I IFN (single IFN-β and multiple IFN-α) through phosphorylation and activation of the transcription factors IRF (interferon regulatory factor)3 and IRF7 41, 42. In unstimulated cells, IRF3 is primarily present in the cytoplasm in an inactive form. However, stimulation with TLR3 and TLR4 ligands or by viral infection causes TBK1- and IKKi-mediated phosphorylation of the C-terminal regions
Early and late phase NF-κB activation through TLR4
Cells derived from MyD88-deficient mice fail to activate NF-κB and fail to produce inflammatory cytokines in response to various TLR ligands, including TLR2, TLR5, TLR7 and TLR9. MyD88-deficient cells also fail to produce inflammatory cytokines in response to LPS. However, LPS activates NF-κB in MyD88-deficient mice [17]. Notably, NF-κB activation in MyD88-deficient cells is delayed in reaching a peak compared with wild-type cells [17]. By contrast, LPS-induced IRF3 activation and IFN-β
TLR7 and TLR9 signaling to NF-κB and IRF7
Plasmacytoid DCs (pDCs; also known as IFN-producing cells) are a subset of DCs that rapidly secrete large amounts of type I IFN, typically IFN-α, during virus infection 51, 52. pDCs preferentially survey virus infections through the TLR system [53]. pDCs express TLR7 and TLR9 and can produce type I IFN and inflammatory cytokines in response to their cognate ligands, viral ssRNA and DNA [54]. Notably, type I IFN induction through TLR7 and TLR9 absolutely depends on MyD88, unlike TLR3 and TLR4
Roles of IκBζ and IκBNS in TLR signaling
Among the IκB family members, IκBζ, IκBNS and Bcl-3 are nuclear proteins bound to a NF-κB subunit. IκBζ is weakly expressed but markedly induced in response to TLR ligands and IL-1β, but not to TNF-α, via the MyD88-dependent pathway [62]. IκBζ-deficient macrophages produce no detectable level of IL-6 but produce normal amounts of TNF-α in response to LPS. In vitro studies show that overexpression of IκBζ enhanced LPS-stimulated production of IL-6 and that IκBζ protein preferentially binds p50.
Concluding remarks
All TLRs activate NF-κB through the MyD88- or TRIF-dependent pathway or both. MyD88 activates NF-κB through the IRAKs–TRAF6–TAK1–IKKα/β pathway whereas TRIF activates NF-κB through the RIP1/TRAF6–TAK1–IKKα/β pathway. The activation of NF-κB is important for eliciting innate immune responses as well as for the subsequent development of adaptive immune responses. Thus, agonists that activate TLR signaling pathways will probably be useful as adjuvants to treat infectious diseases as well as
Acknowledgements
We thank the members of our laboratory for helpful discussions.
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