Sensing Danger: The Biology of TLR-Mediated Inflammation

Toll-like receptors (TLRs) are renowned for their fundamental roles in innate sensing and initiating inflammatory responses. TLRs accomplish this remarkable task through interactions with conserved molecular structures known as pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides, that are expressed by microbial species. Once ligated, TLRs propagate stimuli via one of two intracellular signalling cascades culminating in the induced expression of pro-inflammatory genes needed for pathogen clearance and tissue remodelling.

TLRs are germline encoded type I transmembrane glycoproteins. The extracellular domain of a given TLRs contains a characteristic amount of contiguous Leucine-rich repeat (LRR) motifs that are flanked on either side by distinctive N-terminal and C-terminal cysteine-rich clusters (Bell et al., 2003; Matsushima et al., 2007). A single transmembrane (TM) helical domain connects the ectodomain with an intracellular cytoplasmic tail containing a Toll/IL-1R (TIR)-domain, that latter of which is needed for signal transduction (Matsushima et al., 2007). The LRR ectodomain adopts a horseshoe-like solenoid conformation that enables the binding of an extensive range of structurally unrelated PAMPs (Bell et al., 2003). This feature, in combination with the differential expression and distribution of TLRs, confers myeloid and lymphoid cells with the ability to recognize and respond to a vast array of pathogens (Hornung et al., 2002; Lu et al., 2008; Takeda and Akira, 2005).

Two distinct TLR signalling pathways are known, both of which have been characterized in detail over the past 15 years (O’Neill et al., 2013).

The MyD88-dependent signalling pathway is used by TLRs 1-2 and 4-10 (in humans) and is responsible for transducing stimuli that induce pro-inflammatory cytokines (as reviewed in Lu et al., 2008).

Activate TLRs rely on the TIR-domain containing adaptors Mal/TIRAP and MyD88 to signal via the universally conserved MyD88-Dependent Pathway (as reviewed in Lu et al., 2008). Active TLR complexes recruit these bridging adaptors to the plasma membrane, where homotypic interactions between TIR-domains facilitate association of the adaptors with the cytoplasmic tail of the receptor. In addition to its TIR-domain, MyD88 contains a death domain (DD) and, once activated, MyD88 transduces stimuli by recruiting the DD-containing adaptor IRAK-4, which is responsible for the recruitment, phosphorylation and subsequent activation of IRAK-1. Once phosphorylated, IRAK-1 recruits TRAF6, which is an essential component of the MyD88-dependent pathway. Phosphorylated IRAK-1 and TRAF6 dissociate from the receptor and join TAK1, TAB1, and TAB2 to form a complex at the plasma membrane, leading to phosphorylation of TAK1 and TAB2. Once this has occurred, TRAF6, TAK1, TAB1, and TAB2 translocate to the cytosol and interact with the cofactors UBC13 and UEV1A, both of which bind the RING-domain of TRAF6. This binding leads to ubiquitination of TRAF6, activation of TAK1, and subsequent activation of the MAPK and IKK Signalling Pathways. In IKK signalling, the active IKK complex phosphorylates IκB, which is then ubiquitinated and degraded by the proteasome. This degradation frees NF-κB family transcription factors from IκB-mediated repression. The now active transcription factors translocate to the nucleus, where they induce the expression of specific target genes, such as IL-6, IL-12, and TNF-alpha.

The TRIF-dependent signalling pathway is used by TLRs -3 and -4 (in humans) and is primarily responsible for transducing stimuli that induce Type I IFNs. This pathway also induces late-phase activation of the MAPK and IKK signalling pathways, however (as reviewed in Lu et al., 2008).

In the case of TLR4, the active TLR4/MD2/LPS complex is endocytosed in a clathrin-dependent manner (Felberbaum-Corti et al., 2003). Once in the endosome, TLR4’s intracellular cytoplasmic tail complexes with the TIR-domain containing adaptors TRAM and TRIF. Induction of Type I IFNs involves TRIF-mediated activation of TRAF3. TRAF3 associates with the downstream adaptors TBK1 and IKK-ε to activate IRF3. This association results in translocation of active IRF3 dimers into the nucleus, which drives the expression of Type I Interferons. By contrast, the activation of late-phase MAPK and IKK pathway-dependent responses involves TRIF-mediated recruited and activation of the downstream serine/threonine kinase RIP-1, which is necessary for the induction of late-phase MAPK and IKK pathway-dependent responses. A RIP-homotypic interaction motif (RHIM) within the C-terminal of the TRIF adaptor facilitates the TRIF-RIP1 interaction. RIP-1 then interacts with TRAF6 to promote TAK1 activation. Subsequent steps in the cascade are identical to those of the MyD88-dependent pathway and are as described above.

Lipopolysaccharide (LPS) is a fundamental component of the gram-negative bacterial cell wall and is known for being particularly immunogenic. The LPS-TLR4 interaction has been extensively characterised and is known to provoke inflammation readily. This TLR4-mediated response must be precisely regulated to prevent excessive and systemic inflammation that would otherwise comprise healthy tissues. This regulation of is made possible by negative regulators that target TLR4 signalling at the following levels:

TLR4 Activation: RP105 associates with MD1 to directly interact with the TLR4/MD2 signalling complex and prevent LPS binding (Divanovic et al., 2005).

Adaptor Recruitment: The IL-1R homologs ST2L and SIGIRR inhibit adaptor recruitment by the active TLR4 signalling complex (Brint et al., 2004; Qin et al., 2005). ST2L and SIGIRR exert their inhibitory effects by sequestering Mal and MyD88 in the cytoplasm. By contrast, the E3 ubiquitin ligases TRIAD3A (Fearns et al., 2006) and SOCS1 (Mansell et al., 2006) inhibit TLR signalling by targeting TIR-adaptors for proteasomal degradation.

Non-Functional Decoys: The presence of non-function isoforms of crucial signalling components also regulates TLR signalling. MyD88s and IRAK-2c, for example, are non-functional isoforms of the MyD88 and IRAK2 proteins, respectively (Burns et al., 2003; Hardy et al., 2004). The inclusion of one or both of these in signalling complexes inhibits signal transduction in a dominant-negative like manner.

Downstream Signalling Component Activation: IRAK-M prevents dissociation of the IRAKs from the MyD88 adaptor complex, thus preventing their translocation to the plasma membrane for association with other co-factors needed for signal transduction (Kobayashi et al., 2002; Wesche et al., 1999). Further, TLR signalling is inhibited by peptide fragments that form during proteolytic processing of specific signalling components, such as TRAF1. The proteolytic processing of TRAF1 by caspases produces peptides fragments that inhibit TRIF-induced NF-κB and IRF3 activation (Su et al., 2006). Lastly, the A20 de-ubiquitinating enzyme prevents proteasomal degradation of TRAF6 by removing ubiquitin moieties. As TRAF6 degradation is necessary for the activation of TAK1, inhibition of this process precludes TAK1 activation and blocks the activation of the IKK and MAPK pathways (Lu et al., 2008).

In the case of TLR4 signalling, LPS induces the expression of these negative regulators. In effect, this negative feedback loop down-regulates LPS induced inflammation and is critical for preventing overstimulation of TLR signalling. Failure to adequately regulate this process underlies several life-threating conditions, such as sepsis. Further, hypermorphic and hypomorphic mutations that affect signalling and inhibition of TLR4 have been identified and are associated with an increased risk of infection and chronic, systemic inflammation (Carpenter and O’Neill, 2007).

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