toll-like receptor

Toll-like receptors (TLRs) are type I transmembrane proteins that recognize microbes once they have breached physical barriers such as the skin or intestinal tract mucosa, and activate immune cell responses. They are believed to play a key role in the innate immune system. TLRs are a type of pattern recognition receptors (PRRs) and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs) . However, there are some exceptions to this general rule. TLRs are present in vertebrates (including fish, amphibians, reptiles and birds and mammals) as well as in invertebrates (such of the insect Drosophila where they have been extensively studied). 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 defense against infection. The TLRs thus appear to be one of the most ancient, conserved components of the immune system.

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 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 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 Hoffmann and his colleagues to have an essential role in the fly's immunity to fungal infection^[1], 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,^[2] mapped to a chromosome by Taguchi and colleagues in 1996,^[3]. 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.^[4]

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. ^[5]. 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.

The function of the TLRs was discovered by Beutler and colleagues^[6]. 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^[7][8][9]. 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 is 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), that are not subject to mutation, 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. This work has been pursued most actively in the laboratories of Shizuo Akira and Richard Flavell^[10][11]. 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

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, in the laboratory of Bruce Beutler, 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^[12][13][14]. 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 powerful and important gateways for gene modulation.

Activation and effects

Following activation by ligands of microbial origin, several reactions are possible. Immune cells can produce signalling factors called cytokines which trigger inflammation. In the case of a bacterial factor, the pathogen might be phagocytosed and digested, and its antigens presented to CD4+ T cells. In the case of a viral factor, the infected cell may shut off its protein synthesis and may undergo programmed cell death (apoptosis). Immune cells that have detected a virus may also release anti-viral factors such as interferons.

The discovery of the Toll-like receptors finally identified the innate immune receptors that were responsible for many of the innate immune functions that had been studied for many years. Interestingly, TLRs seem only to be involved in the cytokine production and cellular activation in response to microbes, and do not play a significant role in the adhesion and phagocytosis of microorganisms.

Drugs interactions

Imiquimod (cardinally used in dermatology), and its successor R848, are ligands for TLR7 and TLR8 ^[15].