Microbes that penetrate an epithelial barrier and enter a tissue site are encountered by the three types of sentinel immune cells in the tissues: tissue macrophages, mast cells and immature dendritic cells. These sentinels must be able to distinguish between apoptotic particles generated by normal tissue turnover and particles that are indicative of infection. The molecules mainly responsible for making this pivotal distinction are those of the family of Toll-like receptors (TLRs). Stimulation of macrophages or mast cells through their Toll-like receptors leads to the synthesis and secretion of proinflammatory cytokines and lipid mediators, thereby initiating the inflammatory response that recruits both soluble immune components and immune cells from the blood, and which we describe in later sections. TLR stimulation of dendritic cells induces the initiation of an adaptive immune response, as we shall see in the next chapter. In this section and the next we shall focus on the structural and functional features of this family of receptors that enable it to detect the presence of infection and to signal an appropriate response.

The Toll-like receptors were named after the fruit-fly receptor Toll, which was first discovered because it has an important role in early fly development and was later recognized as contributing to innate immunity in adult flies. TLRs are characterized by an amino-terminal extracellular domain composed of repeated motifs high in leucine and known as leucine-rich repeats (LRRs), followed by a single transmembrane domain and a globular cytoplasmic domain called the Toll/interleukin 1 receptor (TIR) domain, or TIR domain that is also found in IL-1 receptors as well as in adaptors of the TLR signaling pathway, as we shall see in the next section. Thirteen TLRs have been identified in mammals so far. TLRs recognize constituents of microbial cell walls or pathogen-specific nucleic acids (Figure 3-30). Most of the determinants recognized by these receptors are molecules essential to the integrity, function or replication of microbes or viruses, and therefore the infectious agent cannot readily escape detection by changing them. For example, lipopolysaccharide (LPS), the major ligand for TLR4, is a central component of the outer membrane of Gram-negative bacteria (see Figure 3-3), and mutations that ablate the enzymes required for synthesis of LPS are lethal to most species of Gram-negative bacteria. Similarly, double-stranded RNA, the ligand for TLR3, is a central replication intermediate for all RNA viruses, so evasion of TLR3 recognition by these viruses is not easily achieved.

A striking feature of TLR ligands is their molecular diversity: they include nucleic acids, proteins, lipids and polysaccharides. How one family of receptors can recognize all of these different types of molecules is not well understood. Some mammalian TLRs are thought to bind directly to their innate immune ligands, but in other cases recognition is greatly facilitated by accessory proteins: one example of this is LPS binding by TLR4, to which two accessory proteins and a soluble lipid-transfer protein contribute.

The requirements for LPS recognition are illustrated in experiments in which the gene encoding TLR4 is introduced into cells that do not normally express it. TLR4 confers responsiveness to LPS on such cells only in the presence of a second polypeptide, called MD-2, which binds to the extracellular domain of TLR4 and enables it to bind to the relatively conserved inner lipid-containing region of the lipopolysaccharide (the outer polysaccharide region differs between different bacterial species).

Responses to forms of LPS from many but not all bacteria are also substantially increased by a second accessory molecule, CD14, a membrane protein expressed by monocytes, macrophages and neutrophils, that accepts LPS from a serum lipid transfer protein called LPS-binding protein (LBP). Like most lipids, LPS is released from cells only in very small amounts. Efficient recognition of LPS by innate immune system cells therefore requires a mechanism for extracting LPS monomers and making them available to cells. This is the function of LBP, which exchanges monomers of LPS for other lipids present in its lipid-binding site and then carries the LPS monomer to cells where it is transferred to CD14, which presents it to the TLR4–MD-2 complex (Figure 3-31). Genetic removal of CD14 or blocking its function with monoclonal antibodies greatly reduces the sensitivity of macrophages or neutrophils to LPS, although some responses still occur with LPS from some bacteria. CD14 also promotes responses to other bacterial cell wall components, including lipoteichoic acid of Gram-positive bacteria, and lipoarabinomannan of Mycobacterium tuberculosis.

In other cases, phagocytic receptors contribute to the recognition for TLR signaling. For example, dimers of TLR2 and TLR6 respond to lipid-containing ligands such as lipoproteins and lipoteichoic acid, which they recognize with the help of a scavenger receptor called CD36, whereas the recognition of fungal cell wall polysaccharides by these TLRs is facilitated by the transmembrane C-type lectin molecule dectin-1.

Whereas TLRs that recognize bacterial and fungal cell wall components are localized to the cell surface, TLRs that recognize viral or microbial nucleic acids are localized to intracellular membranes and are thought to encounter their ligands in phagosomes or endosomes (Figure 3-32). This localization is thought to be an adaptation ensuring that these receptors detect nucleic acids released from apoptotic host cells, microbial cells or virions only after phagocytosis and partial digestion of the ingested particles has released the nucleic acids. Cell-surface TLRs are also targeted to nascent phagosomes and upon ligand binding can signal there.

TLRs participating in innate immunity to viruses recognize a variety of nucleic-acid ligands produced by viruses, although the mechanism by which they distinguish cellular nucleic acids from viral nucleic acids is not always evident. The basis for discrimination is clearest for TLR3. This TLR recognizes double-stranded RNA (dsRNA), which is an obligatory replication intermediate for viruses with RNA genomes, whereas vertebrate cells express very little dsRNA. TLR7 and TLR8 recognize single-stranded RNA that is rich in guanosine, and it has not yet been established how viral RNAs are distinguished from cellular RNAs. TLR9 recognizes DNA motifs containing the dinucleotide CG where the C is unmethylated (CpG-containing DNA). This dinucleotide is about 10-fold under-represented in vertebrate DNA, and most such dinucleotides are methylated in vertebrate genomes where CpG methylation is a gene-silencing mechanism.

Definitions

CpG-containing DNA: DNA containing unmethylated C followed by G. Note that the sequence of bases adjacent to the CG motif also affects the stimulatory activity.

LBP: see LPS-binding protein.

leucine-rich repeat (LRR): unit of protein structure in which there are many repeats of a basic unit of approximately 25 amino acids.

lipoarabinomannan: a major immunostimulatory component of the lipid-rich mycobacterial cell wall, containing phosphatidylinositol linked to the carbohydrates mannose and arabinose.

LPS-binding protein (LBP): a lipid transfer protein of serum that can extract monomers of LPS from bacterial membranes and deliver them to the innate immune receptor CD14.

LRR: see leucine-rich repeat.

MD-2: a polypeptide that associates with the extracellular domain of the Toll-like receptor TLR4 and is required for LPS responsiveness.

TIR domain: see Toll/interleukin 1 receptor (TIR) domain.

TLR: see Toll-like receptor.

Toll/interleukin 1 receptor (TIR) domain: domain responsible for transmitting signals downstream of IL-1 receptors and Toll-like receptors.

Toll-like receptors (TLRs): family of receptors that have leucine-rich repeats in their extracellular domains and the TIR domain in their cytoplasmic domains.