Toll-like receptors: very clever molecules

By Elizabeth J. Hennessy and Luke A.J. O’Neill *

A review of the history of toll-like receptors and their role in innate immunity

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  1. Introduction
  2. Discovery of TLRs
  3. Specificity of TLRs
  4. TLR signaling pathways
  5. The cleverness of TLRs and their role in disease
  6. Reference list

Introduction

Activation of the host innate immune response depends on specific recognition of conserved microbial signature molecules called pathogen-associated molecular patterns (PAMPs) or the now more widely accepted microbe-associated molecular patterns (MAMPs) because they recognize any microbe regardless of its degree of pathogenicity1. These molecular patterns are highly conserved throughout evolution and are not likely to be mutated because they are essential for the survival of the microbe. Pattern recognition receptors (PRRs) like Toll-like receptors (TLRs) act by specifically recognizing PAMPs. These transmembrane receptors form early barriers against infectious disease and are the first line of innate immune defense. The recognition of a PAMP leads to the binding, engulfment and subsequent destruction of the invading microbe as well as the activation of cell signaling cascades that lead to the inflammatory response and adaptive immunity.

Discovery of TLRs

TLRs are expressed at high levels on immune phagocytic cells such as macrophages and dendritic cells. Phagocytic cells were first identified by the Russian scientist Ilya Mechnikov in 1883 when he observed the ability of starfish larvae to engulf particles introduced to their bodies2. His theory that white blood cells in the human body also had the ability to phagocytose microbes was initially ridiculed by other researchers but was later proven correct. Aspects of innate immunity are found in all multi-cellular organisms including plants and insects3-5 (See Figure 1 for timeline of TLR research).

Figure 1. Timeline of the history of Toll-like receptors (TLRs).

Figure 1. Timeline of the history of Toll-like receptors (TLRs).
The history of TLRs began with the discovery of phagocytic cells by Mechnikov in 1883, followed by the first description of what is now known as IL-1 in 1940, and the IL-1RI TIR domain. TLR adaptor molecules MyD88 and IRAK were first discovered in IL-1 signaling. Homology between IL-1RI and drosophila Toll was found in 1991 and this led to the discovery of human Toll in 1997. In 1998, Toll was discovered to be a receptor for LPS.

Interleukin-1 (IL-1) was one of the first cytokines to be described and was discovered by researchers trying to find the proteins responsible for the macrophage response to lipopolysaccharide (LPS) found on gram-negative bacteria6,7. IL-1 is one of the most effective pro-inflammatory cytokines. The transcription factor NF-κB is activated in response to IL-1 and this activation is followed by the transcription of inflammatory genes.

Researchers studying development of the fruit fly Drosophila melanogaster first discovered the Toll receptor when they found that a mutation in the Toll gene resulted in abnormal development. The mutated flies were termed Toll, the German for “wow” after fly embryos carrying the mutation were viewed under the microscope and appeared so different to wild-type flies. Adult fruit flies with the mutation were more susceptible to fungal infection and the activation of Toll resulted in the production of anti-fungal peptides5. The Drosophila Toll protein was shown to activate a transcription factor termed Dorsal which is a fly homologue of the transcription factor NF-κB8. It was also found that the cytosolic signaling domain of Drosophila Toll shares homology with the previously identified type I IL-1 receptor (IL-1RI).

A more closely related mammalian homologue to Drosophila Toll was identified called hToll, which was subsequently renamed TLR4 because it was “Toll-like”. This receptor was shown to induce the expression of the NF-κB signaling pathway and inflammatory genes like IL-1RI. Mice that were resistant to the effects of LPS were found to have a mutation in their TLR4 gene9. Thirteen TLRs have been identified in mouse and ten in human, each being responsible for the specific recognition of different PAMPs10-12. In 1994, researchers discovered a plant protein that conveyed resistance to tobacco mosaic virus called N protein. This protein had homology to IL1-RI and Toll, the conserved domain was named the Toll-IL-1 receptor resistance (TIR) domain. The homology between the plant N protein, IL-1RI and the Toll protein gave researchers reason to believe that proteins this similar must be very important in immune defense13.

Specificity of TLRs

The specificity of the TLRs has remained largely unchanged through the course of evolution allowing for an immediate response to invading threats and the ability for the host cell to distinguish between self and non-self and prevent auto-immune disorders from developing14. TLRs recognize conserved structures of microbes or ligands of exogenous and endogenous (host-derived) sources. These can include bacterial or viral nucleic acids, the unmethylated CpG islands of pathogen DNA or proteins unique to microbes such as flagellin15. Other TLR ligands include lipids and carbohydrates synthesized by bacteria such as LPS and lipoteichoic acid (LTA).

Each TLR has demonstrated its specificity for particular microbial components. TLRs that recognize bacterial and fungal components are localized on the cell surface whereas TLRs that recognize viral or microbial nucleic acids are localized to intracellular membranes and encounter ligands in endosomal or phagosomal compartments (Figure 2). As mentioned earlier, TLR4 recognizes LPS on gram-negative bacteria and with the assistance of LPS-binding protein (LBP) this recognition is enhanced. LBP functions to carry LPS to the CD14 molecule where it is then presented to the MD2/TLR4 complex16.

Figure 2. TLRs, their ligands and subsequent signaling pathways.

TLR signaling pathways
TLRs are involved in the recognition of microbial molecular patterns. Following the specific recognition of a microbial ligand by TLRs, various adaptor molecules are recruited to the TLR. This leads to the activation of signaling pathways, the transcription of inflammatory genes and the regulation of innate and adaptive immune responses.

TLR2 specifically recognizes components from gram-positive bacteria including LTA and MALP2 with the assistance of the scavenger receptor CD36. TLR2 can form a heterodimer with either TLR1 to recognize triacylated lipopeptides or TLR6 to recognize diacylated lipopeptides. TLR1, 2 and 6 are highly similar and were formed through an evolutionary gene duplication event17. Through this collaboration of TLRs, a more specific and wider array of microbial components can be recognized18. TLR5 recognizes flagellin, a constituent of bacterial flagella4. TLR7 and TLR8 are found in endosomes of cells and recognize single-stranded RNA from viruses. TLR9 is also found in endosomes and acts as a receptor for unmethylated CpG islands found in bacterial and viral DNA. TLR3 recognizes double-stranded RNA which is produced by replicating viruses as well as Polyriboinosinic polyribocytidylic acid (Poly I:C). TLR3 is essential in inducing a protective effect against West Nile Virus by restricting its replication19.

TLRs are only one set of receptors involved in the innate immune response, others include mannose-binding lectin (MBL), the nucleotide oligomerization domain family and complement components, but TLRs are of particular interest because their evolutionary conservation and their activation leads to the induction of many essential host defense genes via specific signaling pathways2.

TLR signaling pathways

All mammalian TLRs share the TIR domains followed by a transmembrane domain and multiple leucine rich repeats (LRR)20,21. Because these receptors are found across species, the importance of them in immunity became a focus for researchers and particularly the signaling cascades activated came into the spotlight. Some TLRs require additional molecules to facilitate the recognition of a ligand as well as the assistance of adaptor molecules.

When TLRs are activated, adaptor molecules are recruited from within the cytoplasm of the cell and lead to the activation of signaling cascades22. Adaptor molecules contain TIR domains like TLRs and it has been demonstrated that for signaling pathways to be initiated there needs to be an interaction between TIR domains. MyD88 was the first identified adaptor molecule. It was first shown to associate with IL-1RI. Its activation leads to the synthesis of pro-inflammatory cytokines such as TNF-α and IL-1. The importance of MyD88 was demonstrated through the use of MyD88 knock-out mice. These mice are resistant to the effects of LPS, peptidoglycan and lipopeptides and also have defective T-cell proliferation. The knock-out mice exhibited no impairment of IFN-regulatory factor 3 (IRF3) activation in response to LPS, leading to the idea that there are MyD88-independent TLR pathways where IFN-β is induced23.

Other adaptor molecules have been identified that enhance the specificity of individual TLR signaling. MyD88 adaptor-like (Mal), also known as Toll-IL-1 receptor domain containing adaptor protein (TIRAP) is used in TLR2 and TLR4 signaling24. The MyD88-independent pathway is mediated by Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF). TRIF is used by TLR3 and TLR4 and activates IRF3 which leads to the production of IFN-β. Lastly there is TRIF-related adaptor molecule (TRAM). The activation of TRAM is TLR4 induced and leads TRIF recruitment25. A splice variant of TRAM, TAG (TRAM-adaptor with a GOLD domain) was recently identified to have an inhibitory effect on MyD88-independent TLR4 signaling26. Following cell stimulation with LPS, both TRAM and TAG localize to late endosomes where TAG inhibits IRF3 activation.

Following the stimulation of TLRs and the recruitment of adaptor molecules to the receptor complex, other molecules like IRAK1 (IL-1R-associated protein kinase), IRAK4 and TRAF6 (tumor necrosis factor receptor–associated factor) are also recruited27. This is followed by the disassociation of IRAK1 and TRAF6 from the receptor complex and their association with TAK1 (transforming growth factor-β-activated kinase). The activation of TAK1 leads to the activation of the IKK complex whose kinase activity is regulated by NEMO (NF-κB essential modulator). The activation of all of these molecules and complexes leads to the translocation of NF-κB into the nucleus of the cell, and the upregulation of pro-inflammatory cytokines28. Other crucial pathways are also activated in response to TLR ligands, these include MAPK, JNK, p38 and ERK, as well as the IRF pathway. Each of these pathways is responsible for the expression and activation of many immune regulatory genes.

The cleverness of TLRs and their role in disease

TLRs are essential regulators of innate and adaptive immune responses and their complexity continues to intrigue researchers29,30. Because of their importance, when they are mutated and not functioning as they should, auto-immune, inflammatory and infectious diseases can develop. Septic shock or excessive inflammation are possibly the most severe outcomes due to mutations of TLRs. They are thought to result from an inadequate negative regulation of TLR signaling leading to excessive pro-inflammatory cytokine production31.

Polymorphisms have been discovered in the genes encoding TLRs that are associated with disease progression and they demonstrate the importance of TLRs in a successful immune response. The best studied polymorphism is that of TLR4, D299G32-34. It was first identified in human patients with a decreased airway response to inhaled LPS. These patients had an increased risk of systemic inflammatory syndrome. The same polymorphism has also been associated with an increased risk of septic shock and decreased risk of atherosclerosis. A homozygous polymorphism in the Mal adaptor which is encoded by the TIRAP gene (S180L) has been linked with an increased susceptibility to invasive pneumococcal disease (IPD)35. A TLR2 polymorphism, identified as R753Q is associated with a predisposition to staphylococcal infections or tuberculosis in humans36. IRAK4 polymorphisms have also been identified. Children with homozygous polymorphisms have recurrent infections caused predominantly by gram-negative bacteria37,38. These infections seem to become less over time because the immune system has a mechanism of compensating for the clearance of gram-negative pathogens.

Recent work has been done showing the manipulation of TLRs by pathogens, particularly virsuses2,39. Mechanisms have been identified where pathogens are able to avoid detection by TLRs. Strains of bacteria, such as uropathogenic E. coli secrete proteins that are taken up by cells such as macrophages and disable adaptor molecules like MyD88 leading to a non-functioning TLR signaling pathway and the successful survival of the pathogen. The Vaccinia virus produces a protein A52R that sequesters IRAK2 and TRAF6 from the pathway. Some of the methods that bacteria and viruses use to manipulate TLR signaling are being used as therapies. Lipid A from Rhodobacter sphaeriodes and synthetic lipodisaccharide prevent activation of human TLR4 by LPS. This mechanism could possibly be used as a treatment for septic shock40. If more information can be uncovered regarding the methods bacteria use to manipulate TLRs in the host then more successful treatments can be used to eliminate infection.

Another area of research that has recently come into the spotlight and demonstrates the cleverness of TLRs is how they are needed to maintain homeostasis in the intestine41,42. Microbial ligands are not only produced by pathogenic bacteria they are also produced by the commensal microflora of the gut. TLRs on the surface of gastrointestinal cells are constantly being exposed to these ligands. It has been shown that commensal bacteria are recognized by TLRs under normal conditions and this recognition is essential for maintenance of homeostasis and a state of constant “controlled inflammation”. When there is an imbalance of gut microflora, inflammatory bowel diseases like colitis and Crohn’s Disease can develop.

As more and more genes in the TLR pathways are described it will become easier to manipulate the pathways to promote a defense against invading pathogens. In recent years molecules called microRNAs (miRNA) have been described that have a role in immune defense gene regulation43. Some miRNAs have been described as negative regulators of TLR signaling acting as a break on the pathway while others act as positive regulators and act as an accelerator. These molecules bind complementary to messenger RNA (mRNA) of a target gene and prevent translation of the protein. These small molecules could potentially be used as drug therapies. Details of TLR signaling pathways such as the discovery of the involvement of novel molecules like miRNA will lead to better and more effective therapies, and the ability to target specific immune processes, thus preventing uncontrolled infection and inflammation.

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Elizabeth J. Hennessy* and Luke A.J. O’Neill*#
Department of Biochemistry and Immunology, Trinity College Dublin, Ireland

# Corresponding author, E-mail:laoneill@tcd.ie