Toll-like receptors (TLRs) are an evolutionarily conserved family of pattern recognition receptors that are expressed by most cell types and recognize products of a wide variety of microbes, as well as molecules expressed or released by stressed and dying cells. Toll was discovered as a Drosophila gene involved in establishing the dorsal-ventral axis during development of the fruit fly, but subsequently it was discovered that the Toll protein also mediated antimicrobial responses in these organisms. Furthermore, the cytoplasmic domain of Toll was found to be similar to the cytoplasmic region of the receptor for the innate immune cytokine interleukin-1 (IL-1). These discoveries led to the identification of mammalian homologs of Toll, which were named Toll-like receptors (TLRs). There are 10 different functional TLRs in humans, named TLR1 through TLR10 (Fig. 1). Mice express TLRs homologous to human TLRs 1 through 9, plus three more (TLRs 11 through 13), but they do not express TLR10. The function of TLR10 remains poorly understood compared to that of other TLRs and there is some evidence that it may have anti-inflammatory functions, unlike all the other TLRs.

Fig1. Structure, location, and specificities of mammalian Toll-like receptors (TLRs). (A) Model of TLR3 with bound double-stranded RNA (dsRNA). The intraendosomal structure of the ligand-binding domains with bound dsRNA was determined by x-ray crystallography, and the structure of the transmembrane and cytoplasmic domains was modeled based on the crystal structure of TLR10. The binding of the ligand allows for dimerization and signaling by the Toll/interleukin-1 receptor (TIR) domains. (B) Note that some TLRs are expressed on the cell surface and others in endosomes. TLRs may form homodimers or heterodimers. CpG, Cytosine guanine–rich oligonucleotide; LPS, lipopolysaccharide; ssRNA, single-stranded RNA. A, Modified from Liu L, Botos I, Wang Y, Leonard JN, Shiloach J, Segal DM, Davies DR. Structural basis of Toll-like receptor 3 signaling with double-stranded RNA. Science. 2008;320(5874):379–381.

The TLRs are dimeric integral membrane glycoproteins that contain leucine-rich repeats flanked by characteristic cysteine rich motifs in their extracellular regions, which are involved in ligand binding, and a Toll/IL-1 receptor (TIR) domain in their cytoplasmic tails, which is essential for signaling. TIR domains are also found in the cytoplasmic tails of the receptors for the cytokines IL-1, IL-18, and IL-33, and similar signaling pathways are engaged by TLRs and these cytokines.

TLRs are involved in responses to a wide variety of molecules that are expressed by microbes but not by healthy mammalian cells. The ligands that the different TLRs recognize are structurally diverse and include products of all classes of microorganisms (see Fig. 1), as in the following examples:

• Bacterial cell wall constituents: LPS of gram-negative bacteria, which binds TLR4; and peptidoglycan and lipoteichoic acid of gram-positive bacteria, which bind TLR2.

• Bacterial surface proteins: flagellin, a protein subunit component of the flagella of motile bacteria, which binds to TLR5.

• Viral and bacterial nucleic acids: double-stranded RNAs, which are present in viruses but not mammalian cells, bind to TLR3; single-stranded RNAs in viruses, which are distinguished from cellular cytoplasmic single-stranded RNA transcripts by their location within endosomes and by their high guanosine and uridine content, bind to TLR7 and TLR8; and unmethylated CpG nucleotide repeats, which are common in prokaryotic DNA but rare in vertebrate genomes, bind to TLR9.

TLRs are also involved in responses to endogenous molecules whose expression or location indicates cell damage. Examples of host molecules that engage TLRs include heat shock proteins (HSPs), which are chaperones induced in response to various cell stresses and bind to TLR4, and high mobility group box 1 (HMGB1), an abundant DNA-binding protein involved in transcription and DNA repair, which binds to TLR2. Both HSPs and HMGB1 are normally intracellular proteins but may become extracellular when released from injured or dying cells. From their extracellular location, they activate TLR2 and TLR4 signaling in DCs, macrophages, and other cell types.

The structural basis of TLR specificities resides in the multiple leucine-rich modules of these receptors, which are located outside the plasma membrane or inside the lumen of endosomes and bind directly to PAMPs or to adaptor molecules that bind the PAMPs. There are between 16 and 28 leucine-rich repeats in TLRs. Each of these modules is composed of 20 to 30 amino acids that include conserved LxxLxLxxN motifs (where L is leucine, x is any amino acid, and N is asparagine) and amino acid residues that vary among TLRs. The ligand-binding variable residues of the modules are on the convex surface formed by α helices and β turns or loops. These repeats contribute to the ability of some TLRs to bind hydrophobic molecules, such as bacterial LPS. Ligand binding to the leucine-rich domains induces physical interactions between TLR molecules that lead to the formation of TLR dimers. The homodimers or heterodimers have different specificities, which increases the number of PAMPs that can be recognized by the small number of TLRs expressed. For example, TLR1/TLR2 dimers recognize different lipopeptides than TLR2/TLR6 dimers, and TLR2/TLR2 homodimers recognize peptidoglycans.

The binding of TLRs to their ligands is also influenced by various non-TLR accessory molecules. This is best defined for TLR4 recognition of LPS. LPS first binds to soluble LPS-binding protein in the blood or extracellular fluid, and this complex facilitates delivery of the LPS to the surface of the responding cell. An extracellular protein called MD2 (myeloid differentiation protein 2) binds to the lipid A component of LPS, forming a complex that then interacts with TLR4 and initiates signaling. Another protein, called CD14, is also required for efficient LPS-induced signaling. CD14 is expressed by most cells (except endothelial cells) as a soluble protein or as a glycophosphatidylinositol-linked membrane protein.

TLRs are found on the cell surface and on endosomal mem branes and are thus able to recognize microbes in different cellular locations (see Fig. 1). TLRs 1, 2, 4, 5, and 6 are expressed on the plasma membrane, where they recognize various bacterial and fungal PAMPs in the extracellular environment. Some of the most potent microbial stimuli for innate immune responses bind to these plasma membrane TLRs, such as bacterial LPS and lipoteichoic acid, which are recognized by TLR4 and TLR2, respectively. In contrast, TLRs 3, 7, 8, and 9 are mainly expressed inside cells on endosomal membranes, where TLR3 detects double-stranded RNA, TLR7 and TLR8 detect single-stranded RNA produced by viruses, and TLR9 detects unmethylated CpG motifs in bacterial or viral DNA. Single- and double-stranded RNA are not unique to microbes, but their location in endosomes reflects origin from microbes. This is because host cell RNA is not normally present in endosomes, but microbial RNA may end up in endosomes of neutrophils, macrophages, or plasmacytoid DCs when the microbes are phagocytosed by these cells. Enzymatic digestion of the microbes within endosomes will release their nucleic acids so these are able to bind TLRs in the endosomal membrane. Thus, the endosomal TLRs may distinguish nucleic acids of normal cells from microbial nucleic acids on the basis of the cellular location of these molecules. A protein called UNC93B is required for the transfer of TLRs synthesized in the endoplasmic reticulum to their endosomal localization. Genetic deficiency in TLR3 or UNC93B leads to susceptibility to certain viral infections, especially herpes simplex virus encephalitis, demonstrating the importance of the endosomal location of TLRs for innate defense against viruses.

TLR recognition of microbial ligands results in the activation of several signaling pathways and ultimately transcription factors, which induce the expression of genes whose products are important for inflammatory and antiviral responses (Fig. 2). The signaling pathways are initiated by ligand binding to the TLR at the cell surface or in endosomes, leading to dimerization of the TLR proteins. Ligand-induced TLR dimerization is predicted to bring the TIR domains of the cytoplasmic tails of each protein close to one another. This is followed by recruitment of TIR domain–containing adaptor proteins, which facilitate the recruitment and activation of various protein kinases, leading to the activation of different transcription factors. The major transcription factors that are activated by TLR signaling pathways are nuclear factor κB (NF-κB), interferon regulatory factor 3 (IRF3), and IRF7. NF-κB stimulates the expression of genes encoding many of the molecules required for inflammatory responses, including inflammatory cytokines (e.g., tumor necrosis factor [TNF] and IL-1), chemokines (e.g., CCL2 and CXCL8), and endothelial adhesion molecules (e.g., E-selectin). IRF3 and IRF7, together with NF-κB, promote the expression of genes encoding interferon (IFN)-α and IFN-β, which are both type I IFNs that are important for antiviral innate immune responses.

Fig2. Signaling pathways and functions of Toll-like receptors (TLRs). TLRs 1, 2, 5, and 6 use the adaptor protein MyD88 and activate the transcription factor nuclear factor kappa B (NF-κB), which induces inflammatory gene expression. TLR3 uses the adaptor protein TIR domain-containing adapter-inducing interferon-β (TRIF), which activates the interferon regulatory factor (IRF)-3 and IRF7 transcription factors and NF-κB that promote type I interferon (IFN) expression. TLR4 uses both MyD88 and TRIF, leading to activation of NF-κB and IRF3/7 pathways, respectively. TLRs 7, 8, and 9 in the endosome use MyD88, leading to activation of both NF-κB and -IRF3 and IRF7, promoting expression of inflammatory and antiviral defense genes. CpG, Cytosine guanine–rich oligonucleotide; dsRNA, double-stranded RNA; LPS, lipopolysaccharide; ssRNA, single-stranded DNA; TIR, Toll interleukin-1 receptor; TNF, tumor necrosis factor.

Different combinations of adaptors and signaling intermediates are used by different TLRs, accounting for the common and unique downstream effects of the TLRs. All TLRs except TLR3 engage the adaptor MyD88, while only TLR3 and TLR4 utilize the adaptor TRIF (TIR domain–containing adaptor inducing IFN-β). Broadly speaking, MyD88-dependent signaling down stream of some TLRs leads to proinflammatory gene expression that is dependent on TRAF (tumor necrosis factor receptor [TNF-R]-associated factor) family adaptor proteins that promote activation of IKK (inhibitor of NF-κB kinase), leading to NF-κB activation; the pathway of NF-κB activation is described in Chapter 7. MyD88-dependent signaling induces less robust IRF activation and type I IFN responses. This is the case for the cell surface TLRs 1, 5, and 6. It should be noted that additional transcription factors, such as AP1 (activator protein 1) and CREB, are also activated by MyD88-dependent TLR signaling, and work in concert with NF-κB to promote inflammatory gene expression.

The endosomal TLRs 7, 8, and 9 also engage MyD88, but in these cases, downstream signaling leads to TRAF-dependent activation of the kinases TBK1 (TANK-binding kinase 1) and IKK, which in turn activate IRFs and NF-κB respectively, inducing type I IFN–mediated antiviral responses and some degree of inflammation. Cell surface TLR4 engages both MyD88 and TRIF, leading to NF-κB, IRF3 and IRF7 activation, and both inflammatory and antiviral responses. Endosomal TLR3 signals only through TRIF, which activates NF-κB and IRF3. Thus, like TLR4, TLR3 signaling induces inflammatory and type I IFN mediated antiviral responses.

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