Toll-like receptor 4 (TLR4), also designated as CD284 (cluster of differentiation 284), is a key activator of the innate immune response and plays a central role in the fight against bacterial infections. TLR4 is a transmembrane protein of approximately 95 kDa that is encoded by the TLR4 gene.
TLR4 belongs to the toll-like receptor family which is representative of the pattern recognition receptors (PRR), so named for their ability to recognize evolutionarily conserved components of microorganisms (bacteria, viruses, fungi and parasites) called pathogen-associated molecular patterns (PAMPs). The recognition of a PAMP by a PRR triggers rapid activation of the innate immunity essential to fight infectious diseases.
TLR4 is expressed in immune cells mainly of myeloid origin, including monocytes, macrophages and dendritic cells (DC). It is also expressed at a lower level on some non-immune cells, including epithelium, endothelium, placental cells and beta cells in Langerhans islets. Most myeloid cells express also high amounts of plasma membrane-anchored CD14, which facilitates the activation of TLR4 by LPS and controls the subsequent internalization of the LPS-activated TLR4 important for receptor signaling and degradation.
The main ligands for TLR4 are lipopolysaccharides (LPS), the major components of the outer membrane of Gram-negative bacteria and some Gram-positive bacteria. TLR4 can also be activated by endogenous compounds called damage-associated molecular patterns (DAMPs), including high mobility group box protein 1 (HMGB1), S100 proteins, or histones. These compounds are released during tissue injury and by dying or necrotic cells.
Function
The first function described for TLR4 was the recognition of exogenous molecules from pathogens (PAMPs), in particular LPS molecules from gram-negative bacteria. As pattern recognition receptor, TLR4 plays a fundamental role in pathogen recognition and activation of innate immunity which is the first line of defense against invading micro-organisms. During infection, TLR4 responds to the LPS present in tissues and the bloodstream and triggers pro-inflammatory reactions facilitating eradication of the invading bacteria.
TLR4 is also involved in the recognition of endogenous DAMP molecules leading to different signaling outcomes than PAMPs, both quantitatively and qualitatively. DAMPs can activate TLR4 in non-infectious conditions to induce tissue repair and the activation of mainly proinflammatory responses. Generally, inflammation has a protective role. It is a complex and coordinated process followed by the induction of resolution pathways that restore tissue integrity and function. However, in some cases, an excessive and/or poorly regulated inflammatory response to DAMPs can be detrimental to the organism, accelerating the development or progression of pathologies such as a number of cancers and neurodegenerative diseases (as discussed below).
TLR4 binds LPS with the help of LPS-binding protein (LBP) and CD14, and an indispensable contribution of the MD-2 protein stably associated with the extracellular fragment of the receptor. TLR4 signaling responds to signals by forming a complex using an extracellular leucine-rich repeat domain (LRR) and an intracellular toll/interleukin-1 receptor (TIR) domain. LPS stimulation induces a series of interactions with several accessory proteins which form the TLR4 complex on the cell surface. LPS recognition is initiated by an LPS binding to an LBP. This LPS-LBP complex transfers the LPS to CD14 which is a glycosylphosphatidylinositol-anchored membrane protein that binds the LPS-LBP complex and facilitates the transfer of LPS to MD-2 protein, which is associated with the extracellular domain of TLR4. LPS binding promotes the dimerization of TLR4/MD-2 complex. The conformational changes of the TLR4 induce the recruitment of intracellular adaptor proteins containing the TIR domain which is necessary to activate the downstream signaling pathway.
The binding of an LPS molecule to the TLR4/MD-2 complex involves acyl chains and phosphate groups of lipid A, the conserved part of LPS and the main inducer of pro-inflammatory responses to LPS.
TLR4 activation and response to LPS is greatly influenced by the polysaccharide domain and the molecular structure of Lipid A moiety of the LPS molecules. Hexa-acylated and diphosphorylated LPS, like Escherichia coli LPS (O111:B4), is one of the most potent agonists of TLR4 whereas under-acylated LPS and dephosphorylated LPS species have a weaker pro-inflammatory activity especially in human cells. Structural determinants of this phenomenon are found in the TLR4/MD-2 complex and also in CD14 protein. The polysaccharide portion covalently bound to lipid A also plays an indispensable role in TLR4 activation through CD14/TLR4/MD-2. However, in addition to the lipid A domain, the polysaccharide moiety plays an important role in the binding and activation of the LPS molecules as the lipid A moiety alone was demonstrated to be significantly less active than the full LPS molecule.
Signaling
Unlike all the other TLRs, TLR4 stimulation triggers two signaling pathways called the MyD88-dependent and the TRIF-dependent one after the adaptor proteins involved in their induction. The MyD88-dependent signaling is triggered by TLR4 localized to the plasma membrane, while the TRIF-dependent one by the TLR4 internalized in endosomes.
These signaling pathways lead to the production of two sets of cytokines. The MyD88-dependent pathway induces the production of pro-inflammatory cytokines while TRIF-dependent pathway induces the production of type I interferons and chemokines. The molecular structure of TLR4 ligands (in particular LPS), as well as their complexation with proteins or lipids, greatly influence the action of these TLR4-related signaling pathways, leading to different cytokine balances.
MyD88 – dependent pathway
The MyD88-dependent pathway is regulated by two adaptor-associated proteins: Myeloid Differentiation Primary Response Gene 88 (MyD88) and TIR Domain-Containing Adaptor Protein (TIRAP). It also involves the activation of IL-1 Receptor-Associated Kinases (IRAKs) and the adaptor molecules TNF Receptor-Associated Factor 6 (TRAF6). TRAF6 induces the activation of TAK1 (Transforming growth factor-β-Activated Kinase 1) that leads to the activation of MAPK cascades (Mitogen-Activated Protein Kinase) and the IκB Kinases (IKK), called IKKα and IKKβ. IKKs' signaling pathway leads to the induction of the transcription factor NF-κB, while activation of MAPK cascades lead to the activation of another transcription factor AP-1. These two transcription factors induce the expression of genes encoding pro-inflammatory mediators, such as tumor necrosis factor α (TNF-α), interleukin (IL)-6, and type III interferons (IFNλ1/2).
TRIF – dependent pathway
The TRIF-dependent pathway involves the internalization of TLR4 in endosomes and the recruitment of the adaptor proteins TIR-domain-containing adaptor inducing interferon-β (TRIF) and TRIF-related Adaptor Molecule (TRAM). TRAM-TRIF signals activate the ubiquitin ligase TRAF3 followed by the activation of non-canonical IKK kinases: TANK binding kinase 1 (TBK1) and IKKε. TBK1 phosphorylates the pLxIS consensus motif of TRIF that is necessary to recruit interferon regulatory factor (IRF) 3. IRF3 is also phosphorylated by TBK1 and then dissociates from TRIF, dimerizes and translocates to the nucleus. Finally, IRF3 induces the expression of genes encoding type I IFN such as interferon beta (IFN-β), the chemokine CCL5/ RANTES and interferon-regulated genes as that encoding the chemokine CXCL10/IP-10. TRIF-dependent signaling pathway of TLR4 is known to play a central role in the stimulation of innate immune cells such as macrophages, the maturation of DCs and the induction and recruitment of Th1 adaptive immune responses.
Immune cell activation TLR4 activation by LPS enables a rapid stimulation of a wide range of innate immune cells such as macrophages and DCs. This leads to the secretion of pro-inflammatory and type I interferons cytokines, chemokines. Production levels of these cytokines/chemokines vary according to the degree of activation of the MyD88 and TRIF signaling pathways by TLR4 agonist molecules. TLR4 activation also induces the stimulation of antigen presentation and upregulation of costimulatory molecules (such as CD40, CD80 and CD86) on innate immune cells which are required for antigen presentation for T lymphocytes. This explains why TLR4 activation by LPS is also known to stimulate the generation of effective adaptive immune responses and to induce their recruitment, polarization and maintenance via the panel of cytokines and chemokines produced.
The TRIF and MyD88 signaling pathways have a different but complementary impact on immune cell activation. Macrophage stimulation has been shown to be strictly dependent on TRIF pathway activation whereas DC activation and maturation depend on both the MyD88 and TRIF pathways. The increased expression of costimulatory and MHC molecules is a hallmark of DC maturation required for antigen presentation by these cells. However, significant differences were found in the signaling pathways leading to this phenomenon. In macrophages, the upregulation of costimulatory molecules depends strictly on the TRIF-dependent pathway, whereas in DC both the MyD88- and TRIF-dependent ones are involved. The increased cell surface presence of the costimulatory molecules and also of MHC II is a hallmark of DC maturation required for antigen presentation by these cells.
The activation of MyD88 and TRIF signaling pathways were also found to induce Th1 polarization of the T cells responses through DC maturation and the panel of cytokines produced. Low activation of MYD88 pathway is however important for effective cytotoxic T-cell differentiation by facilitating fusion of MHC I-bearing recycling endosomes with phagosomes allowing cross-presentation of antigens. In contrast, robust activation of MYD88 pathway induces excessive production of pro-inflammatory cytokines leading to life-threatening pathological consequences such as cytokine storms.
The impact of TLR4 activation on the innate and adaptive immune system explains why TLR4 agonists, such as LPS derivatives, have been developed as vaccine adjuvants. Among them is GSK's Monophosphorylated Lipid A (MPL), a detoxified Lipid A derived from Salmonella LPS, which is the first and only natural immunostimulant to have been approved as adjuvant in five human vaccines.
Evolutionary history and polymorphism TLR4 originated when TLR2 and TLR4 diverged about 500 million years ago near the beginning of vertebrate evolution. Sequence alignments of human and great ape TLR4 exons have demonstrated that not much evolution has occurred in human TLR4 since our divergence from our last common ancestor with chimpanzees; human and chimp TLR4 exons only differ by three substitutions while humans and baboons are 93.5% similar in the extracellular domain. Notably, humans possess a greater number of early stop codons in TLR4 than great apes; in a study of 158 humans worldwide, 0.6% had a nonsense mutation. This suggests that there are weaker evolutionary pressures on the human TLR4 than on our primate relatives. The distribution of human TLR4 polymorphisms matches the out-of-Africa migration, and it is likely that the polymorphisms were generated in Africa before migration to other continents.