THE TOLL-LIKE RECEPTOR FAMILY

Contents

• TLR1
• TLR2
• TLR3
• TLR4
• TLR5
• TLR6
• TLR7
• TLR8
• TLR9
• TLR10
• References

Host defense against infectious disease is a harrowing task considering not only the vast diversity of pathogens, but also their rapid replication and mutation rates.

Front-line, anti-microbial defense is accomplished by the innate immune system with the help of pattern recognition receptors (PRRs), such as the Toll-like receptors (TLRs), in early detection of specific classes of pathogens.^1-4 The broad classes of pathogens (e.g. viruses, bacteria, and fungi) constitutively express a set of class-specific, mutation-resistant molecules called pathogen-associated molecular patterns (PAMPs). These microbial molecular markers may be composed of proteins, carbohydrates, lipids, nucleic acids and/or combinations thereof, and may be located internally or externally.^1-6

PRRs have evolved to take advantage of these three salient PAMP qualities. First, constitutive expression allows the host to detect the pathogen regardless of its life cycle stage. Second, class specificity allows the host to distinguish between pathogens and thereby tailor its response. Third, mutation resistance allows the host to recognize the pathogen regardless of its particular strain.^1-3,5 PRRs are also nonclonal, or expressed on all cells of a given type, and germ-line encoded, or independent of immunologic memory.^1,2,6 PRRs do more than merely recognize pathogens via their PAMPs. Once bound, PRRs tend to cluster, recruit other extracellular and intracellular proteins to the complex, and initiate signaling cascades that ultimately impact transcription.^1,3,4 Further, PRRs are involved in activation of complement, coagulation, phagocytosis, inflammation, and apoptosis functions in response to pathogen detection.^1-3 There are several types of PRRs including complement, glucan, mannose, scavenger, and toll-like receptors, each with specific PAMP ligands, expression patterns, signaling pathways, and anti-pathogen responses.^1,5,6

Toll, the founding member of the Toll/Toll-like receptor family, was initially described in Drosophila 15 years ago as a developmental protein essential for embryonic axis establishment.⁷ It has since been ascribed a role in fungal pathogen detection and response.⁸ More recently, the TLR family has been described as type I transmembrane (TM) PRRs that possess varying numbers of extracellular N-terminal leucine-rich repeat (LRR) motifs, followed by a cysteine-rich region, a TM domain, and an intracellular Toll/IL-1 R (TIR) motif.^7,9-14 The LLR domain is important for ligand binding and associated signaling and is a common feature of PRRs.^6,16 The TIR domain is important in protein-protein interactions and is typically associated with innate immunity.^17,18 Several lines of evidence argue that TLRs play an important role in innate immunity.^1,6 First, they are PRRs that recognize a spectrum of PAMPs. Second, TLR spatial expression is coincident with the host's environmental interface. Third, TLRs directly and indirectly induce innate and adaptive immune responses.^1,6

The TIR domain also unites a larger IL-1 R/TLR superfamily that is composed of three subgroups.¹⁸ Members of the first group possess immunoglobin domains in their extracellular regions and include IL-1 and IL-18 receptors and accessory proteins as well as ST2. The second group encompasses the TLRs. The third group includes intracellular adaptor proteins important for signaling.¹⁸ While only a few other Toll-like proteins have been cloned in Drosophila, the human TLR family is composed of at least 10 members, TLR1 through 10.^9-15 Each TLR is specific in its expression patterns and PAMP sensitivities.¹

[Note: due to space constraints, TLR ligands, or PAMPs, will not be covered extensively; where appropriate, reviews will be cited. Also, TLR signaling will not be covered within the scope of this minireview. For recent reviews on TLR-associated signal transduction cascades, see references 4, 18-20.]

Figure 1. Toll-like receptor (TLR) protein structure. All of the TLRs are Type I transmembrane proteins possessing a variable number of N-terminal leucine rich repeats (LRRs) followed by a cysteine rich domain, a transmembrane (TM) domain, and an intracellular Toll/IL-1 receptor (TIR) domain. The variation in amino acid number and molecular weight of the different TLRs is most significantly contributed by differences in the numbers of LRRs. Gene Bank Accession numbers and chromosomal locations for each of the TLRs are also given.

TLR1

TLR1 was cloned out of a human erythroleukemic (TF-1) cell line-derived cDNA library (GenBank Accession # U88540).¹⁰ TLR1 maps to chromosome 4p14 and its sequence encodes a putative 786 amino acid (aa) protein with 18 N-terminal LRRs and a calculated molecular weight of 84 kDa (Figure 1).^10,18 TLR1 is most closely related to TLR6 and TLR10 with 68% and 48% overall aa sequence identity, respectively (Figure 2).

In vivo, two different sized transcripts for TLR1 are observed suggesting that the mRNA is alternatively spliced to generate two different forms of the protein. TLR1 mRNA is ubiquitously expressed and found at higher levels than the other TLRs.¹⁰ Of the major leukocyte populations, TLR1 is most highly expressed by monocytes, but is also expressed by macrophages, dendritic cells (DCs), polymorphonuclear leukocytes, B, T, and NK cells (Figure 3).^21-24 In vitro, TLR1 mRNA and protein expression is upregulated in monocytic leukemic (THP-1) cells upon PMA-induced differentiation. While TLR1 expression is most significantly upregulated by autocrine IL-6, it is also elevated by IFN-γß, IL-10, and TNF-α. However, TLR1 level is unaffected by exposure to both Gram-positive and Gram-negative bacteria.²¹ Ex vivo, both monocyte and granulocyte TLR1 expression is downregulated after exposure to Gram-negative bacteria.²¹ TLR1 forms a heterodimer with TLR2 (as do other TLRs, see below). While the significance of TLR heterodimerization is not clearly understood, it is thought that some TLRs, such as TLR1, function to specify or enhance the PAMP sensitivity of TLR2.^25-27 However, there is evidence that TLR2 may not signal as a homodimer and that TLR1 in this case is required for downstream functionality.²⁸ TLR1 also heterodimerizes with TLR4, not to enhance its function, but rather to inhibit TLR4 activity.²⁹ [Note: see TLR2 for TLR1/TLR2 heterodimer PAMP sensitivities.]

A. 1 2 3 4 5 6 7 8 9 10 1 100 29 23 25 22 68 21 23 22 48 2 100 22 26 23 31 24 24 25 29 3 100 24 26 23 26 26 24 24 4 100 24 25 23 24 24 23 5 100 23 24 25 24 22 6 100 22 22 22 46 7 100 43 36 22 8 100 35 21 9 100 21 10 100

B.

Figure 2. A. Values result from whole-sequence pairwise comparisons of all 10 TLRs represented as percent amino acid sequence identity. B. Evolutionary relationships of TLRs represented by a phylogenetic tree derived from whole-sequence comparisons of all 10 TLRs. [Note: figure adapted from Chuang, T. & R.J. Ulevitch (2001) Biochim. Biophys. Acta 1518:157.]

TLR2

TLR2 was first cloned using a human lung-derived cDNA library (GenBank Accession # U88878).¹⁰ It was also cloned, a short time later, from human peripheral blood leukocyte (PBL)- and prostate-derived cDNA libraries by a different group, who called it Toll/IL-1-like 4 (TIL4; GenBank Accession # AF051152).¹¹ TLR2 maps to chromosome 4q31-32 and encodes a putative 784 aa protein with 19 N-terminal LLRs and a calculated molecular weight of 84 kDa (Figure 1).^10,11,18 TLR2 is most closely related to TLR6 with 31% overall aa sequence identity (Figure 2).

In vivo, two different sized transcripts for TLR2 are observed suggesting that the mRNA is alternatively spliced.⁹ TLR2 mRNA expression is observed in brain, heart, lung, and spleen tissues and is highest in PBLs, specifically those of myelomonocytic origin (Figure 3).^{10,11,21-24,30} In vitro, while PMA-differentiated THP-1 TLR2 is most significantly upregulated by autocrine IL-6 and TNF-α, IL-1β, and IL-10. Further, TLR2 mRNA expression is elevated after exposure to both Gram-positive and Gram-negative bacteria.²¹ Ex vivo, the increase in TLR2 expression in monocytes and granulocytes on exposure to Gram-negative bacteria is only very modest.²¹

TLR2 forms heterodimers with TLR1, TLR6,^1-4,18,28 and possibly TLR10,² where each complex is particularly sensitive to subsets of TLR2-associated PAMPs.^25-27 TLR2 complexes recognize a wide range of PAMPs, mostly from bacteria. These include lipoarabinomannan (LAM), lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), and other glycolipids, glycoproteins, and lipoproteins. TLR2 complexes are also capable of detecting viruses, including measles virus (MV), human cytomegalovirus (HCMV), and hepatitis C virus (HCV) and fungal PAMPs, including zymosan.^{1-4,6,18,31,32} More recent studies have suggested that, like TLR4, TLR2 complexes require CD14 for detection of PAMPs and/or signaling.^31,33 Aside from detection of non-self patterns, TLR2 complexes are also capable of detecting altered self patterns, such as those displayed by necrotic cells.⁴ Further, recent evidence indicates that TLR2 is recruited to phagosomes and may be directly involved in the internalization of microbial products by cells.³⁴

TLR3

TLR3 was cloned from a human placenta-derived cDNA library (GenBank Accession # U88879).¹⁰ It maps to chromosome 4q35 and its sequence encodes a putative 904 aa protein with 24 N-terminal LRRs and a calculated molecular weight of 97 kDa (Figure 1).^10,18 TLR3 is most closely related to TLR5, TLR7, and TLR8, each with 26% overall aa sequence identity (Figure 2).

In vivo, two different sized transcripts for TLR3 are observed suggesting that the mRNA is alternatively spliced to generate two different forms of the protein. TLR3 mRNA is expressed at highest levels in placenta and pancreas.¹⁰ There are conflicting reports regarding the expression of TLR3 in particular leukocyte populations. Some suggest that TLR3 is only expressed by DCs,^22,30 while others find that TLR3 is expressed by T or NK cells (Figure 3).^21,23 In vitro, PMA-differentiated THP-1 TLR3 is moderately upregulated by autocrine IFN-γ, IL-1ß, IL-6, IL-10, and TNF-α. Further, TLR3 mRNA is elevated after exposure to Gram-negative bacteria and to an even greater extent in response to Gram-positive bacteria.²¹ Ex vivo, TLR3 expression is elevated in both monocytes and granulocytes upon exposure to Gram-negative bacteria.²¹ TLR3 forms a homodimer and recognizes viral double stranded RNA (dsRNA).^2-4,6,18 While it is generally assumed that TLRs are expressed on the cell surface, it now appears that some, particularly those TLRs sensitive to internal PAMPs such as dsRNA in the case of TLR3, may be localized intracellularly, perhaps to the lysosomal compartment.²⁸

Figure 3. Toll-like receptor (TLR) leukocyte expression patterns and PAMP specificities. Each of the TLRs is expressed on a different subset of leukocytes and each of the TLRs detects different subsets of pathogens allowing vigilant surveillance by the immune system.

TLR4

TLR4 was initially cloned as the human homolog of Drosophila Toll (dToll) and thus was first named hToll (GenBank Accession # H48602).⁹ A human fetal liver/spleen EST database was screened for sequences similar to that of the TIR domain, yielding the hToll sequence.⁹ Shortly thereafter, TLR4 was cloned by another group from a human fetal liver-derived cDNA library (GenBank Accession # U88880).¹⁰ TLR4 maps to chromosome 9q32-33. It shows a high degree of similarity to dToll over the entire aa sequence. The TLR4 sequence encodes an 839 aa protein with 22 N-terminal LRR regions and a calculated molecular weight of 90 kDa (Figure 1).^9,10,18 TLR4 is most closely related to TLR1 and TLR6 each with 25% overall aa sequence identity (Figure 2).

In vivo, TLR4 mRNA is expressed as a single transcript, and found at highest levels in spleen and PBLs.^9,10 Of the PBL populations, TLR4 is expressed by B cells, DCs, monocytes, macrophages, granulocytes, and T cells.^9,10,30 Other reports suggest that TLR4 is only expressed in myelomonocytic cells and is highest in mononuclear cells (Figure 3).^21-23 In vitro, TLR4 mRNA and protein expression is upregulated in THP-1 cells upon PMA-induced differentiation. TLR4 is moderately upregulated by autocrine IFN-γ, IL-1β. TLR4 mRNA expression in THP-1 cells is unaffected by exposure to both Gram-positive and Gram-negative bacteria.²¹ Ex vivo, granulocyte, and especially monocyte, TLR4 expression is upregulated upon exposure to Gram-negative bacteria.²¹

TLR4 forms a homodimer and requires the extracellular association of an additional component, MD-2. Although TLR2 complexes are capable of recognizing LPS, TLR4 is generally considered the LPS receptor. MD-2-associated TLR4 homodimers do not bind LPS directly, however. LPS must first be bound by the soluble LPS binding protein (LBP). LBP is then bound by either soluble or GPI-linked CD14. While the exact mechanism is still unclear, it is thought that LBP transfers LPS to CD14 thereby activating TLR4.^1-4,6,18 Recently a new model for TLR4 detection of LPS has emerged. It suggests that additional cell type-dependent components are required for LPS detection by TLR4 including CXCR4, GDF-5, CD55, various heat shock proteins (HSPs), and complement receptors (CRs).³⁵ The TLR4 complex also recognizes a few other bacterial PAMPs including LTA. Further, the TLR4 complex recognizes viruses including respiratory syncytial virus (RSV), hepatitis C virus (HCV), and mouse mammary tumor virus (MMTV). The TLR4 complex can also recognize endogenous ligands, for example, heat shock proteins, fibrinogen, fibronectin, surfactant protein A (SP-A), and β-defensins.^1-4,18,32,36 TLR4 also forms heterodimers both with TLR5, which presumably enhances its activity, and also with TLR1, which inhibits its activity.^29,37

TLR5

A partial TLR5 sequence was cloned out of a human multiple sclerosis plaque-derived cDNA library (GenBank Accession # U88881) and mapped to chromosome 1q33.3.¹⁰ Full-length TLR5 was cloned a short time later from human PBL- and prostate-derived cDNA libraries and called TIL3 (GenBank accession number AF051151).¹¹ This group mapped TLR5 to chromosome 1q41-42. The gene encodes a putative 858 aa protein with a calculated molecular weight of 91 kDa (Figure 1).^11,18 It is most closely related to TLR3 with 26% overall aa sequence identity (Figure 2).

In vivo, TLR5 mRNA is expressed as a single transcript in ovary, prostate, and PBLs.^10,11 Initially thought to be restricted to myelomonocytic cells, TLR5 now appears to be expressed by several PBL populations with the highest expression found in monocytes (Figure 3).^21,22 In vitro, TLR5 is most significantly upregulated in PMA-differentiated THP-1 cells by autocrine IL-6, IL-10, and TNF-α, but is also elevated by IFN-γβ. Further, TLR5 mRNA expression is elevated after exposure to both Gram-positive and Gram-negative bacteria.²¹ Ex vivo, however, granulocyte and in particular monocyte TLR5 expression is downregulated upon exposure to Gram-negative bacteria.²¹ TLR5 forms a homodimer as well as a heterodimer with TLR4. Both complexes function to recognize the Flagellin protein of flagellated bacteria.^1-4,6,18,37

TLR6

TLR6 was cloned using a human placenta-derived cDNA library (GenBank accession number AB020807) and the gene mapped to chromosome 4p14.¹² The TLR6 sequence encodes a 796 aa protein containing 20 N-terminal LRR motifs with a calculated molecular weight of 91 kDa (Figure 1).^12,18 TLR6 is most closely related to TLR1, TLR10, and TLR2 with 68%, 46%, and 31% overall aa sequence identity, respectively (Figure 2).

In vivo, TLR6 transcript is observed in thymus, spleen, and lung.¹² TLR6 mRNA expression is highest in B cells and monocytes (Figure 3).^21,23 In vitro, TLR6 mRNA expression is upregulated in THP-1 cells upon PMA-induced differentiation. Further, TLR6 is moderately upregulated by autocrine IFN-γ, IL-1β. However, TLR6 mRNA expression in THP-1 cells is unaffected by exposure to both Gram-positive and Gram-negative bacteria.²¹ Ex vivo, monocyte and, in particular, granulocyte TLR6 expression is downregulated upon exposure to Gram-negative bacteria.²¹ TLR6 forms a heterodimer with TLR2. Like TLR1, TLR6 is thought to specify or enhance the PAMP sensitivity of TLR2 and contribute to its signaling capabilities through heterodimerization.^25-28 [Note: see TLR2 for TLR6/TLR2 heterodimer PAMP sensitivities.]

TLR7

TLR7 was cloned from a human placenta cDNA library (GenBank Accession # AF240467, AF245702) and mapped to human chromosome Xp22.^13,14 The TLR7 sequence encodes a 1049 aa protein containing 27 N-terminal LRRs with a calculated molecular weight of 121 kDa (Figure 1).^13,18 TLR7 is most closely related to TLR8 and TLR9 with 43% and 36% overall aa sequence identity, respectively (Figure 2).

In vivo, TLR7 mRNA is expressed in lung, placenta, spleen, lymph node, and tonsil.¹³ TLR7 mRNA expression is highest in monocytes, B cells, and DCs (Figure 3).^21,23 In vitro, TLR7 mRNA expression is upregulated in THP-1 cells upon PMA-induced differentiation. TLR7 is highly upregulated by exposure to IL-6 and to a slightly lesser extent by autocrine IFN-γ, IL-1β. TLR7 mRNA expression in THP-1 cells is elevated after exposure to both Gram-positive and Gram-negative bacteria.²¹ Ex vivo, expression of TLR7 is elevated after exposure to both Gram-positive and Gram-negative bacteria in monocytes and to a greater degree in granulocytes.²¹ Like TLR3, it appears that TLR7 may be localized intracellularly.²⁸ While natural ligands are still unknown, TLR7 does bind a group of small synthetic antiviral compounds called imidazoquinolines.^4,18

TLR8

TLR8 was cloned from a human placenta cDNA library (GenBank Accession # AF245703, AF246971) and mapped to chromosome Xp22.^13,14 The TLR8 sequence encodes a 1041 aa protein containing 26 N-terminal LRRs with a calculated molecular weight of 120 kDa (Figure 1).^13,18 TLR8 is most closely related to TLR7 and TLR9 with 43% and 35% overall aa sequence identity, respectively (Figure 2).

In vivo, TLR8 mRNA is expressed in lung, placenta, spleen, lymph node, bone marrow, and PBLs, with highest expression found in monocytes (Figure 3).^13,21,23 In vitro, TLR8 mRNA expression is upregulated in THP-1 cells upon PMA-induced differentiation. TLR8 is highly upregulated by autocrine IL-1β, IL-6, IL-10, and TNF-α, and is even more enhanced by exposure to IFN-γ. TLR8 mRNA expression in THP-1 cells is elevated after exposure to both Gram-positive and Gram-negative bacteria.²¹ Ex vivo, monocyte TLR8 expression increases while granulocyte expression decreases on exposure to Gram-negative bacteria.²¹ TLR8 may also be localized intracellularly and, like TLR7, binds imidazoquinolines while the natural ligands are still unknown.^4,18,28

TLR9

TLR9 was cloned from a human placenta cDNA library (GenBank Accession # AF245704, AF259262, AF259263) and mapped to chromosome 3p21.^13,14 The TLR9 sequence encodes a 1032 aa protein containing 27 N-terminal LRRs with a calculated molecular weight of 116 kDa (Figure 1).^13,18 TLR9 is most closely related to TLR7 and TLR8 with 36% and 35% overall aa sequence identity, respectively (Figure 2).

In vivo, TLR9 mRNA is expressed in spleen, lymph node, bone marrow, and PBLs.¹³ Specifically, TLR9 mRNA is expressed at the highest levels in B cells and DCs (Figure 3).^21,23,30 In vitro, TLR9 is moderately upregulated by autocrine IFN-γ, IL-1ß, IL-6, IL-10, and TNF-α in PMA-differentiated THP-1 cells. TLR9 mRNA expression in THP-1 cells is unaffected by exposure to both Gram-positive and Gram-negative bacteria.²¹ Ex vivo, TLR9 expression in monocytes and particularly in granulocytes is downregulated in response to Gram-negative bacteria.²¹ TLR9 forms a homodimer and recognizes unmethylated bacterial DNA.^1-4,6,18 TLR9 also appears to be localized internally, perhaps in lysosomic or endocytic compartments where it would more likely encounter PAMPs including unmethylated DNA.^1,28

TLR10

TLR10 was cloned from a human spleen-derived cDNA library (GenBank accession number AF296673)¹⁵ and has not yet been mapped. The TLR10 sequence encodes a putative 811 aa protein with molecular weight of 95 kDa (Figure 1).^15,18 TLR10 is most closely related to TLR1 and TLR6 with 48% and 46% overall aa identity, respectively (Figure 2).

In vivo, TLR10 mRNA expression is highest in immune system-related tissues including spleen, lymph node, thymus, tonsil.¹⁵ TLR10 mRNA is most highly expressed on B cells (Figure 3).^21,23 In vitro, TLR10 is moderately upregulated by autocrine IFN-γ, IL-1β, IL-6, IL-10, and TNF-α in PMA-differentiated THP-1 cells. TLR10 mRNA expression in THP-1 cells is elevated after exposure to both Gram-positive and Gram-negative bacteria.²¹ Ex vivo, monocyte TLR10 expression increases while granulocyte expression decreases on exposure to Gram-negative bacteria.²¹ While still little is known about TLR10 associations or ligands, some have speculated that it, like TLR1 and TLR6, may form a heterodimer with TLR2 and thereby be sensitive to similar PAMPs.^2,25-27 [Note: see TLR2 for possible TLR10/TLR2 heterodimer PAMP sensitivities.]

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