Science Magazine TLR Connections Map

Toll like receptors (TLRs) are transmembrane proteins that localize either on plasma membrane or in intracellular vesicles. They are an evolutionarily conserved family of cell surface proteins that recognize pathogen-associated molecular patterns (PAMPs). To date, there are at least 13 TLRs discovered in mammals that can detect PAMPs. These PAMPS include 1) LPS-lipopolysaccharide on ,(via TLR4) 2) mannose, fucose and other sugar residues, 3) teichoic acid which is part of the peptidoglycan cell wall of  and 4) N-formyl peptides (all prokaryotic proteins begin with a formyl-methionine).

Once engaged, TLRs prompt the cells to unleash particular types of cytokines. These protein messengers then recruit additional macrophages, DCs and other immune cells to wall off and nonspecifically attack the microbe.

TLRs of mammalian cells were discovered relatively recently and have provided a unified view of the innate immune response and inflammation. Induction of IL-1 and TNF? is an early event in the process. Because these  share intracellular signalling pathways with the TLRs, they serve to amplify the innate immune response.

It is conceivable that microbial adjuvants might one day be obsolete, replaced by TLR agonists or perhaps by TLR mimetics. Many of the factors that mediate adjuvant responses, including upregulation of costimulatory molecules that drive the adaptive immune response are cytokines. IFN? has a particular important part to play, although specific NFkB regulated cytokines contribute as well. It is conceivable that TLR pathways could be bypassed entirely. 

Synthetic DNA oligonucleotides bearing unmethylated CpG motifs (TLR9 agonists) have been used for their adjuvant properties. So, too have LPS partial structures (TLR4 agonists) with diminished toxicity. DNA oligonucleotides ahve also been coupled to rpotein antigens, with striking effects on the adaptive immune response.

 In addition to the conserved structural features among the TLRs, there seems to be another level of organization. The TLRs that are involved in the recognition of microbial products (TLR1, TLR2, TLR4, TLR5 and TLR6 and TLR11) are displayed on the cell surface. By contrast, TLR7, TLR8 and TLR9 are localized intracellularly and their natural ligands might only be found within acidic compartments such as phagolysosomes. TLRs that recognize nucleic acids signal from endosomes, whereas cell-surface TLRs sense lipids and proteins. 

 Interaction with different sets of signaling proteins allows TLRs to activate different sets of genes that hone the cell’s response to better match the type of pathogen being encoutered. For example, TLR3 and TLR7 sense the presence of viruses. They then trigger a string of molecular interactions that induce the production and release of interferon, the major antiviral cytokine. TLR2, which is activated by bacteria, stimulates the release of a blend of cytokines that does not include interferon but is more suited to activating an effectiv antibacterial response by the body.

 Structure of TLRs

 All TLRs are type I transmembrane receptors characterized by a highly variable extracellular region containing a leucine-rich repeat domain (LRR) involved in ligand binding and an intracellular tail containing a highly conserved region, the TIR homologydomain, which mediates interaction between TLRs and downstream signaling molecules.

 Functions of TLRs

Interaction with different sets of signalling proteins allows TLRs to activate different sets of genes that hone the cell’s response to better match the type of pathogen being encountered. Among the primary cytokines produced in response to TLR activation, TNF, IL12 and the type I interferons are of key importance for the induction of further innate immune processes and also for activation of adaptive immunity. 

• recognition of viruses: Among 11 reported TLRs, TLR3, TLR4, TLR7, TLR8 and TLR9 are involved in the recognition of viral components. They then trigger a string of molecular interactions that induce the production and release of interferon, the major antiviral cytokine. TLR2, which is activated by bacteria, stimulates the release of a blend of cytokines that is more suited to activating an effective antibacterial response.
• upregulation co-stimulatory molecules: Ligand binding of TLRs up-regulates CD83, costimulatory molecules, and CCR7, which drives DC migration to T cell areas of draining lymph nodes.
• IFNy release: TLR activation also leads to the secretion of huge amounts of IFN? by plasmacytoid DCs and IL-6, IL-10, TNF, and or IL-12 by conventional DCs, depending on the subset and particular stimulus.

TLR3 and TLR4 activate TRIF to induce type I IFNs and TLR7 and TLR9 utilize MyD88 to activate type I IFNs

• Tolerance: In addition to controlling the expression of costimulatory molecules on DCs, TLRs induce DCs to relieve suppression of effector T cells by regulatory T cells. Regulatory or suppressor T cells (T regs) are CD4+CD25+ cells that suppress the activity of self-reactive T cells in the periphery. Supression can be relieved, however, by the production by mature DCs of soluble factors, in particular, IL-6. Thus by restricting IL-6 production to TLR-stimulated DCs, suppression is only relieved in the presence of infection. Pathogen-specific T cells may then be activated by these same TLR-induced DCs expressing co-stimulatory molecules in the context of foreign presentation.
• Negative signalling: TLR signaling also initiates negative regulatory signals that are thought to help resolve the host inflammatory resposnes. Such regulatory molecules include IRAK-M, SOCS1, A20, MyD88 short, which appear to negatively regulate TLR immune responses.

Types of TLRs

TLR3, TLR7, TLR8, TLR9 and TLR13 are endosomal receptors that sense nucleic acids that have been endocytosed or phagocytosed. The crystal structures of most TLRs bound to theri respective ligands have been diciphered. TLRs have a ligand-recognition domain facing the lumen of the endosome, a transmembrane domain, and a signlaing domain facing the cytosol. (Greenbaum, “Sequence-specific sensing of nucleic acids” Trends Immunol 2017, 38(1); 53-65)

• TLR1 senses bacterial produces of Mycobacteria and Borrelia burgdorferi.
• TLR2:

• TLR3 recognizes the double-stranded RNA (dsRNA) that appears after infection by RNA viruses. dsRNA is also recognized by dsRNA-activated kinase. TLR3 activation results in increased IL-12, IFN-alpha and IFN-beta expression.

TLR uses TRIF but not MyD88 in signalling. recognizes LPS.

• TLR5 recognizes flagellin, the major structural component of bacterial flagella. People who die from Legionnaire’s diases often harbor a mutation in TLR5 that disables the protein, compromising their innate immune response.
• TLR7 is thought to recognize influenza viral compounds or endogenous tumor molecules released from virus infected cells or tumors and interacts with bacterial DNA motif CpG. TLR7 binds single stranded RNA (ssRNA), the genomic material of many viruses, such as influenza virus and Sendai virus (SeV). TLR7 is also expressed on both  DCs, and activation of these cells through TLR7 induces the production of IFNalpha. in pDC and IL-12 in myDC.
• TLR8: as well as TLR7 recognize ssRNA and share a general specificity for U rich ssRNA. (Greenbaum, “Sequence-specific sensing of nucleic acids” Trends Immunol 2017, 38(1); 53-65)
• TLR9 recognizes and initiates a response to cytosine-phosphate-guanine (CpG) (unmethylated cytosine linked to guanine) sequences. These sequences are represented in abundance in microbial sequences but are much less common in mammalian sequences. CpG ODNs are arpidly internalized by immune cells, perhaps involving phosphatidylinositol 3-kinases (PI3Ks), and they interact with TLR9 that is present in endocytic vesicles. This is a highly specific interaction, with the triggering of cells being abrogated by elimination of the CpG motif through either inversion or methylation. B cells and pDCs are the main human cell types that express TLR9 and respond directly to CpG stimulation. TLR9 signals exclusively via MyD88, which is not known to activate IRF3. Nevertheless, TLR9 signaling can also induce IFN? via an unknown pathway.
• TLR13: is an endosomal TLR expressed in mice but not humans. TLR13 recognizes its ligand in a stringently sequence-specific manner, sensing a highly conserved bacterail 23S rRNA sequence that contains 5′ -GAAGACC-3′. Notably, this sequence is found within a region of RNA targeted by certain antibiotics and clincial isolates of Staphylococcus aureus resistant to these antibiotics are unable to stimulate mouse TLR13. Overall, TLR13 functions as a sequence and conformation specific PRR. (Greenbaum, “Sequence-specific sensing of nucleic acids” Trends Immunol 2017, 38(1); 53-65)

Many companies are pursuing immunomodulator drugs targeting toll-like receptors, including the following:

Dynavax Technologies Coley 3M Idera Pharmaceuticals