The curved leucine-rich repeat region of Toll-like receptors, represented here by TLR3
Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single membrane-spanning non-catalytic receptors that recognize structurally conserved molecules derived from microbes. Once these microbes have breached physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs which activates immune cell responses.
They receive their name from their similarity to the protein coded by the Toll gene identified in Drosophila in 1985 by Christiane Nüsslein-Volhard.
Diversity
TLRs are a type of pattern recognition receptor (PRR) and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). TLRs together with the Interleukin-1 receptors form a receptor superfamily, known as the “Interleukin-1 Receptor/Toll-Like Receptor Superfamily”; all members of this family have in common a so-called TIR (Toll-IL-1 receptor) domain.
Three subgroups of TIR domains exist. Proteins with subgroup 1 TIR domains are receptors for interleukins that are produced by macrophages, monocytes and dendritic cells and all have extracellular Immunoglobulin (Ig) domains. Proteins with subgroup 2 TIR domains are classical TLRs, and bind directly or indirectly to molecules of microbial origin. A third subgroup of proteins containing TIR domains consists of adaptor proteins that are exclusively cytosolic and mediate signaling from proteins of subgroups 1 and 2.
TLRs are present in vertebrates, as well as in invertebrates. Molecular building blocks of the TLRs are represented in bacteria and in plants, and in the latter kingdom, are well known to be required for host defence against infection. The TLRs thus appear to be one of the most ancient, conserved components of the immune system.
[edit] Discovery
When microbes were first recognized as the cause of infectious diseases, it was immediately clear that multicellular organisms must be capable of recognizing them when infected, and hence, capable of recognizing molecules unique to microbes. A large body of literature, spanning most of the last century, attests to the search for the key molecules and their receptors. More than 100 years ago, Richard Pfeiffer, a student of Robert Koch, coined the term “endotoxin” to describe a substance produced by Gram-negative bacteria that could provoke fever and shock in experimental animals. In the decades that followed, endotoxin was chemically characterized and identified as a lipopolysaccharide (LPS) produced by most Gram-negative bacteria. Other molecules (bacterial lipopeptides, flagellin, and unmethylated DNA) were shown in turn to provoke host responses that are normally protective. However, these responses can be detrimental if they are excessively prolonged or intense. It followed logically that there must be receptors for such molecules, capable of alerting the host to the presence of infection, but these remained elusive for many years.
Toll-like receptors are now counted among the key molecules that alert the immune system to the presence of microbial infections. They are named for their similarity to Toll, a receptor first identified in the fruit fly Drosophila melanogaster, and originally known for its developmental function in that organism. In 1996, Toll was found by Jules A. Hoffmann and his colleagues to have an essential role in the fly’s immunity to fungal infection, which it achieved by activating the synthesis of antimicrobial peptides.
The first reported human Toll-like receptor was described by Nomura and colleagues in 1994,mapped to a chromosome by Taguchi and colleagues in 1996.Because the immune function of Toll in Drosophila was not then known, it was assumed that TIL (now known as TLR1) might participate in mammalian development. However, in 1991 (prior to the discovery of TIL) it was observed that a molecule with a clear role in immune function in mammals, the interleukin-1 (IL-1) receptor, also had homology to drosophila Toll; the cytoplasmic portions of both molecules were similar.
In 1997, Charles Janeway and Ruslan Medzhitov showed that a Toll-like receptor now known as TLR4 could, when artificially ligated using antibodies, induce the activation of certain genes necessary for initiating an adaptive immune response.However, the function of the TLRs remained unknown in the wake of this work, and in particular, no ligand had been identified for any mammalian TLR.
TLR function was discovered by Bruce A. Beutler and colleagues. These workers used positional cloning to prove that mice that could not respond to LPS had mutations that abolished the function of TLR4. This identified TLR4 as a key component of the receptor for LPS, and strongly suggested that other Toll-like receptors might detect other signature molecules of microbes, such as those mentioned above.
In turn, the other TLR genes were ablated in mice by gene targeting, largely in the laboratory of Shizuo Akira and colleagues. Each TLR is now believed to detect a discrete collection of molecules of microbial origin, and to signal the presence of infections.
Extended family
It has been estimated that most mammalian species have between ten and fifteen types of Toll-like receptors. Thirteen TLRs (named simply TLR1 to TLR13) have been identified in humans and mice together, and equivalent forms of many of these have been found in other mammalian species.[8][9][10] However, equivalents of certain TLR found in humans are not present in all mammals. For example, a gene coding for a protein analogous to TLR10 in humans is present in mice, but appears to have been damaged at some point in the past by a retrovirus. On the other hand, mice express TLRs 11, 12, and 13, none of which are represented in humans. Other mammals may express TLRs which are not found in humans. This may complicate the process of using experimental animals as models of human innate immunity.
Ligands
Because the specificity of Toll-like receptors (and other innate immune receptors) cannot easily be changed in the course of evolution, these receptors recognize molecules that are constantly associated with threats (i.e. pathogen or cell stress) and are highly specific to these threats (i.e. cannot be mistaken for self molecules). Pathogen-associated molecules that meet this requirement are usually critical to the pathogen’s function and cannot be eliminated or changed through mutation; they are said to be evolutionarily conserved. Well conserved features in pathogens include bacterial cell-surface lipopolysaccharides (LPS), lipoproteins, lipopeptides and lipoarabinomannan; proteins such as flagellin from bacterial flagella; double-stranded RNA of viruses or the unmethylated CpG islands of bacterial and viral DNA; and certain other RNA and DNA. For most of the TLRs, ligand recognition specificity has now been established by gene targeting (also known as “gene knockout”): a technique by which individual genes may be selectively deleted in mice. see the table below for a summary of known TLR ligands.
Endogenous ligands
The stereotypic inflammatory response provoked by TLR activation has prompted speculation that endogenous activators of TLRs might participate in autoimmune diseases. TLRs have been suspected of binding to host molecules including fibrinogen (involved in blood clotting) and heat shock proteins (HSPs)and host DNA.
Signaling
Signaling pathway of Toll-like receptors. Dashed grey lines represent unknown associations.
TLRs are believed to function as dimers. Though most TLRs appear to function as homodimers, TLR2 forms heterodimers with TLR1 or TLR6, each dimer having a different ligand specificity. TLRs may also depend on other co-receptors for full ligand sensitivity, such as in the case of TLR4′s recognition of LPS, which requires MD-2. CD14 and LPS Binding Protein (LBP) are known to facilitate the presentation of LPS to MD-2.
The adapter proteins and kinases that mediate TLR signaling have also been targeted. In addition, random germline mutagenesis with ENU has been used to decipher the TLR signaling pathways. When activated, TLRs recruit adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adapter molecules are known to be involved in signaling. These proteins are known as MyD88, Tirap (also called Mal), Trif, and Tram.[13][14][15] The adapters activate other molecules within the cell, including certain protein kinases (IRAK1, IRAK4, TBK1, and IKKi) that amplify the signal, and ultimately lead to the induction or suppression of genes that orchestrate the inflammatory response. In all, thousands of genes are activated by TLR signaling, and collectively, the TLRs constitutes one of the most pleiotropic yet tightly regulated gateways for gene modulation.
The Role of Toll-like Receptors in Host Innate Immune Defense
The ability of host cells to sense and respond to non-self is dependent upon a number of secreted and cell-associated molecules of the innate immune system. In 1996, a receptor known as Toll was found to protect fruit flies from fungal infection by activating the production of antifungal peptides. Homologues of this receptor, called Toll-like receptors (TLRs), were subsequently uncovered in vertebrates and are the focus of our research.
Humans possess ten TLR family members subsets of which are expressed in epithelial cells, endothelial cells as well as leukocyte subtypes found in tissue and blood. TLRs are transmembrane receptors whose extracellular domain discriminates self from nonself through recognition of conserved structural components of viruses, bacteria, fungi, and protozoans. These include cell wall or membrane components of bacteria and fungi as well as modified nucleic acids of certain bacteria and viruses (see figure). Upon engaging a cognate microbial agonist, TLRs activate intracellular signals which induce the expression and cellular release of cytokines, chemokines and other mediators that facilitate local inflammation. Viral sensing TLRs trigger the production of Type I interferons which are essential for early antiviral defense. In addition to providing immediate protection for the host, the engagement of TLRs drives antigen presentation leading to the T and B cell responses associated with adaptive immunity. In this context, it is not surprising that inappropriate TLR activation is directly associated with a variety of inflammatory disorders ranging from sepsis, atherosclerosis, asthma, and certain autoimmune disorders.
Summary of known mammalian TLRs
Toll-like receptors bind and become activated by different ligands, which, in turn are located on different types of organisms or structures. They also have different adapters to respond to activation and are located sometimes at the cell surface and sometimes to internal cell compartments. Furthermore, they are expressed by different types of leucocytes or other cell types:
| Receptor | Ligand(s) [16] | Ligand location [16] | Adapter(s) | Location | Cell types[16] |
|---|---|---|---|---|---|
| TLR 1 | multiple triacyl lipopeptides | Bacteria | MyD88/MAL | cell surface |
|
| TLR 2 | multiple glycolipids | Bacteria | MyD88/MAL | cell surface |
|
| multiple lipopeptides | Bacteria | ||||
| multiple lipoproteins | Bacteria | ||||
| lipoteichoic acid | Bacteria | ||||
| HSP70 | Host cells | ||||
| zymosan | Fungi | ||||
| Numerous others | |||||
| TLR 3 | double-stranded RNA, poly I:C | viruses | TRIF | cell compartment |
|
| TLR 4 | lipopolysaccharide | Gram-negative bacteria | MyD88/MAL/TRIF/TRAM | cell surface |
|
| several heat shock proteins | Bacteria and host cells | ||||
| fibrinogen | host cells | ||||
| heparan sulfate fragments | host cells | ||||
| hyaluronic acid fragments | host cells | ||||
| Numerous others | |||||
| TLR 5 | flagellin | Bacteria | MyD88 | cell surface |
|
| TLR 6 | multiple diacyl lipopeptides | Mycoplasma | MyD88/MAL | cell surface |
|
| TLR 7 | imidazoquinoline | small synthetic compounds | MyD88 | cell compartment |
|
| loxoribine (a guanosine analogue) | |||||
| bropirimine | |||||
| single-stranded RNA | |||||
| TLR 8 | small synthetic compounds; single-stranded RNA | MyD88 | cell compartment |
|
|
| TLR 9 | unmethylated CpG DNA | Bacteria | MyD88 | cell compartment |
|
| TLR 10 | unknown | unknown | unknown | cell surface |
|
| TLR 11 | Profilin | Toxoplasma gondii | MyD88 | cell surface |
|
| TLR 12 | unknown | unknown | ? | ||
| TLR 13 | unknown | unknown | ? | ||
| TLR 15 | unknown | unknown | ? | ||
|
|
|
The Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R)-family members share several signalling components, including the adaptor MyD88, Toll-interacting protein (TOLLIP), the protein kinase IRAK (IL-1R-associated kinase) and TRAF6 (TNF receptor-associated factor 6). TRAF6 can activate nuclear factor-B (NF-B) through TAK1 (TGF–activated kinase), and JNK (c-Jun N-terminal kinase) and p38 MAP kinases through MKK6 (mitogen-activated protein kinase kinase 6). TLR4 signals through another adaptor in addition to MyD88–TIRAP (Toll/interelukin-1 (IL-1) receptor domain-containing adaptor protein), which activates MyD88-independent signalling downstream of TLR4. The protein kinase PKR functions downstream of TIRAP, but its importance in this pathway has not yet been established.
Toll-like receptor (TLR)-signalling pathways are negatively regulated by several molecules that are induced by the stimulation of TLRs. IRAK-M (interleukin-1-receptor (IL-1R)-associated kinase M) inhibits the dissociation of the IRAK1–IRAK4 complex from the receptor. SOCS1 (suppressor of cytokine signalling 1) probably associates with IRAK1 and inhibits its activity. MyD88s (myeloid differentiation primary-response protein 88 short) blocks the association of IRAK4 with MyD88. The TIR (Toll/IL-1R)-domain-containing receptors SIGIRR (single immunoglobulin IL-1R-related molecule) and ST2 have also been shown to negatively modulate TLR signalling. IB, inhibitor of NF-B; IKK, IB kinase; NF-B, nuclear factor-B; TIRAP, TIR-domain-containing adaptor protein; TRAF6, tumour-necrosis-factor-receptor-associated factor 6.
Intracellular Proteins
| Gene Symbol | ||
|---|---|---|
| ATF1 | IRF8 (ISCBP) | MKK7 |
| ATF2 | JUN (AP-1) | MYD88 |
| BTK | MAP10K (JNK3) | NFKB1 |
| CD14 | MAP3K7 (TAK1) | NFKBIA |
| CHUK (IKKα) | MAP3K7IP1 (TAB1) | PIK3CA |
| CREB1 | MAPK1 (ERK) | PIK3CB |
| CREB3 | MAPK11 | PIK3R1 |
| FADD | MAPK12 | PIK3R3 (VPS34) |
| FOS | MAPK13 | RAC1 |
| IKBKB (IKKβ) | MAPK14 | RELA (NFκB) |
| IKBKG (IKKγ) | MAPK8 (JNK1) | RIPK2 |
| IRAK1 | MAPK9 (JNK2) | TBK1 |
| IRAK2 | MEK1 | TIRAP |
| IRAK3 (IRAKM) | MEKK3 | TRAF6 |
| IRAK4 | MKK3 (MEK3) | TRAM |
| IRF3 | MKK4 (MEK4) | UBE2N (UBC13) |
| IRF7 | MKK6 | |
 |
|
Transcribed Genes
| Gene Symbol | ||
|---|---|---|
| CCL3 (MIP-1α) | CD86 | IL8 |
| CCL4 (MIP-1β) | IFNA1 | IL12A |
| CCL5 (RANTES) | IFNB1 | TNF |
| CD40 | IL1B | |
| CD80 | IL6 | |
|
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