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:firstname.lastname@example.org