Review ArticleLPS/TLR4 signal transduction pathway
Introduction
The Toll protein first discovered in Drosophila, was shown to be essential for determining the dorsal–ventral patterning during embryogenesis [1], [2] and an early form of the innate immune system [3], [4]. The mammalian Toll-like receptors (TLRs) are germline-encoded receptors expressed by cells of the innate immune system that are stimulated by structural motifs characteristically expressed by bacteria, viruses and fungi known as pathogen-associated molecular patterns (PAMPs) [5], [6]. Importantly, TLR interactions trigger the expression of proinflammatory cytokines as well as the functional maturation of antigen presenting cells of the innate immune system [6], [7].
Many PAMPs have been defined that interact with particular TLRs. For example, the TLR2/TLR6 heterodimer can be stimulated by several bacterial components, such as lipoteichoic acid (LTA) and peptidoglycan (PG). Viral DNA is rich in unmethylated CpG motifs, which stimulates TLR9. While TLR3 interacts with viral double-stranded RNA, TLR7/8 can sense guanosine- or uridine-rich single-stranded RNA from viruses. Collectively, the innate immune system utilizes TLRs and cytosolic sensors (RIG-I, MDA5, ets.) to detect viruses [8]. Thus, using TLR as critical sensors, the innate immune system has devised a way to decode the type of invading pathogen and trigger an appropriate effective immune response.
Evidence suggests that several PAMPs can stimulate TLR4. These molecules include lipopolysaccharide (LPS) from Gram-negative bacteria, fusion (F) protein from respiratory syncytial virus (RSV) and the envelope protein from mouse mammary tumor virus (MMTV) [9], [10]. In addition, endogenous molecules can also interact directly or indirectly with TLR4, such as heat-shock proteins, hyaluronic acid and β-defensin 2 [11], [12], [13].
LPS is one of the best studied immunostimulatory components of bacteria and can induce systemic inflammation and sepsis if excessive signals occur [14]. LPS is an important structural component of the outer membrane of Gram-negative bacteria. LPS consists of three parts: lipid A, a core oligosaccharide, and an O side chain [15], [16]. Lipid A is the main PAMP of LPS. Using the C3H/HeJ mouse strain which is known to have a defective response to LPS, Beutler’s group demonstrated that TLR4 is an important sensor for LPS [17].
LPS stimulation of mammalian cells occurs through a series of interactions with several proteins including the LPS binding protein (LBP), CD14, MD-2 and TLR4 [18], [19]. LBP is a soluble shuttle protein which directly binds to LPS and facilitates the association between LPS and CD14 [20], [21]. CD14 is a glycosylphosphatidylinositol-anchored protein, which also exists in a soluble form. CD14 facilitates the transfer of LPS to the TLR4/MD-2 receptor complex and modulates LPS recognition [22]. MD-2 is a soluble protein that non-covalently associates with TLR4 but can directly form a complex with LPS in the absence TLR4 [23], [24], [25]. Although no evidence suggests that TLR4 can bind LPS directly, TLR4 can enhance the binding of LPS to MD-2 [26]. Therefore LPS stimulation of TLR4, includes the participation of several molecules, and the currently favoured model is outlined in Fig. 1 [19], [27].
Upon LPS recognition, TLR4 undergoes oligomerization and recruits its downstream adaptors through interactions with the TIR (Toll-interleukin-1 receptor) domains. TIR domains contain three highly conserved regions, which mediate protein–protein interactions between the TLRs and signal transduction adaptor proteins. The TIR domain of TLR4 is critical for signal transduction, because a single point mutation in the TIR domain can abolish the response to LPS [17]. There are five TIR domain-containing adaptor proteins: MyD88 (myeloid differentiation primary response gene 88), TIRAP (TIR domain-containing adaptor protein, also known as Mal, MyD88-adapter-like), TRIF (TIR domain-containing adaptor inducing IFN-β), TRAM (TRIF-related adaptor molecule), and SARM (sterile α and HEAT-Armadillo motifs-containing protein) [28]. Different TLRs use different combinations of adaptor proteins to determine downstream signaling. Interestingly, TLR4 is the only known TLR which utilizes all these adaptor proteins.
Studies using knockout mice have revealed important roles for these adaptors in TLR4 signaling. MyD88 was first described as a myeloid differentiation primary response gene [29]. It was later suggested to be the critical adaptor in the interleukin-1 receptor (IL-1R) signaling pathway [30], [31]. Because both the IL-1R family and the TLR family contained TIR domains, studies were also done to determine whether MyD88 was involved in TLR-mediated signaling pathways. MyD88-deficient mice were shown to be resistant to LPS-induced septic shock, and MyD88-deficient macrophages failed to produce proinflammatory cytokines after LPS stimulation, despite the ability to activate nuclear factor-κB (NF-κB) [32]. In addition, the expression of Type I interferons and interferon-inducible genes was not impaired in MyD88-deficient macrophages [33]. This demonstrated an important role for MyD88 downstream of IL-1R and TLR signaling, but also indicated that other molecules are involved in the induction of a subset of LPS induced responses.
TIRAP/Mal was cloned through a computer-based search for proteins containing TIR domains [34], [35]. TIRAP-deficient mice were generated in subsequent studies and had a phenotype similar to MyD88 knockout mice [36], [37]. TIRAP also contains a phosphatidylinositol 4,5-bisphosphate (PIP2) binding domain, which mediates TIRAP recruitment to the plasma membrane. TIRAP then facilitates the association between MyD88 and the TLR4 cytoplasmic domain to initiate MyD88-dependent downstream signaling [38].
TRAM was also cloned by homology of the TIR domain [39], while TRIF was cloned using multiple approaches [40], [41], [42]. Studies using knockout mice indicated that TRIF and TRAM mediate MyD88-independent signaling and will be discussed in detail later [39], [42], [43]. Studies suggest that TRAM associates with the plasma membrane through myristoylation, and is essential for TLR4 signal transduction [44] (Fig. 1). SARM was suggested to function as an inhibitor of TRIF-mediated signaling in the human HEK293 cell line [45]. However, the role of SARM in vivo is still unclear.
Section snippets
TLR4 signal transduction
TLR4 signaling has been divided into MyD88-dependent and MyD88-independent (TRIF-dependent) pathways. Based on studies using MyD88-deficient macrophages, the MyD88-dependent pathway was shown to be responsible for proinflammatory cytokine expression, while the MyD88-independent pathway mediates the induction of Type I interferons and interferon-inducible genes (Fig. 1).
Negative regulation of TLR4 signaling pathway
Because TLR4 stimulation can induce potent responses such as sepsis, inhibitory pathways are necessary to protect the host from inflammation-induced damage. TLR4 signaling can be regulated at multiple levels by many negative regulators. Typically mice lacking these key regulators exhibit enhanced TLR4 responses [74]. RP105 (radioprotective 105), ST2L (also known as IL1Rl) and SIGIRR (single immunoglobulin IL-1R-related molecule) are expressed on the cell surface and their inhibitory functions
Conclusion
Recent studies have provided tremendous insights into LPS/TLR4 signaling pathway. The knowledge from this pathway also provides models for how other TLR signaling pathways may be regulated. Because improper regulation of LPS/TLR4 signaling has the potential to induce massive inflammation and cause acute sepsis or chronic inflammatory disorders, it is important to further explore this pathway and evaluate novel targets to counteract these conditions [90], [91].
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