User:Kinkreet/Immunology/Innate recognition

The innate immune system was once thought to recognise non-specifically to general patterns, but from the discovery of Toll-like receptors (TLRs) in the 1990s, it was revised to a model in which the innate system have germline-encoded pattern-recognition receptors (PRRs) that recognises specifically four different types of signals: general patterns on pathogens which is not found on the self (most of these patterns are lipopeptides, glucans, glycans and DNA and proteins epxressed on many microbial classes); host proteins modified by microbial enzymes (such as fungal proteases cleaving host proteins, Staphylococcus coagulase, and diptheria toxin); danger signals produced by the host (uric acid, heat shock proteins, chitin oligomers, DAMPs etc.) and finally it recognises any cell which do not express self-antigens (MHC molecules), as a sign of cell death.

There are many types of PRRs: Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and Nod-like receptors (NLRs); but of all these TLRs are the most studied and thought to be the most important. RLRs are cytosolic receptors which includes RIG-I, Mda5 and LGP2, which binds to RNA viruses; NLRs contain more than 20 members and binds to some PAMPs, non-PAMP particles and response to cellular stresses. There are other uncharacterised PRRs that binds to dsDNA and induce the upregulation of type 1 interferon. Many of these PRRs are expressed on non-immune cells.

The immune system is more susceptible to infection if the PRRs no longer recognize its ligands, or the downstream signalling cascade or effector product has been compromised; autoimmune diseases, acute and chronic inflammation can occur when the PRRs recognises self-antigens as foreign; dysregulation of cytokines will lead to acute and chronic inflammation; and so the study of PRRs are essential to understand many immunodiseases.

Toll-like Receptors

Toll-like receptors (TLRs) are a group of 10 (human)- 12 (mouse) molecules expressed by macrophages, DCs, B and specific T lymphocytes, fibroblasts and epithelial cells, to recognise specific conserved molecular patterns on pathogens. Different patterns stimulates different TLRs and induce the expression of different genes, as well as to initiate the adaptive immunity. Whereas the adaptive immunity is antigen-specific, TLRs only recognises patterns. Out of the 10/12 TLR identified in humans and mouse, TLR-1 to TLR-9 are conserved in both species. "Mouse TLR10 is not functional because of a retrovirus insertion, and TLR11, TLR12 and TLR13 have been lost from the human genome."

It was found by studying mouse with defects in each of these TLRs, that each TLR have a distinct ligand, and produce distinct effects. Some cells also have cell-specific pathways, which produce different effects through the binding of the same ligand than in other cell types.

TLRs can be localised on the plasma membrane (TLR-1,2,4,5,6,11) but also intracellularly on endosomes (TLR-3,7,8,9), and endoplasmic reticulum (ER), lysosomes and endolysosomes. Their location depends on the location of their intended ligand; surface TLRs recognizes mainly microbial membrane components, cytosolic TLRs recognizes mainly microbial nucleic acids.

Structure

TLRs form a superfamily of proteins, first of which is the Toll receptor, which controls the pathway controlling the dorsoventral (back and front) polarity pattern in insect embryogenesis. But it was also found that defects in the Toll receptor and pathway led to susceptibility to fungal infections, now known to be because the fungus have proteases which cleave persephone (in Drosophila) and become activated to cleave SPE, which interns cleaves Spatzle, which is recognised by the Toll receptor. Failure to recognise fungal infections lead to the believe that Toll receptor is involved in the innate immune response. Insects, like all invertebrates, have no adaptive immune response, and so is more prone to these infections.

The cytoplasmic region of the TLRs have stark homology to the interleukin-1 receptors (IL-1Rs), a very common domain on pattern recognition molecules in both the innate and adaptive immune system; and so they can be thought of as part of a larger Toll–interleukin 1 (IL-1) receptor (TIR) superfamily. Within the cytoplasmic domain, there is a region of ～200 amino acid residues which are conserved, and is known as the TIR domain. The TIR domain is characterised by 3 high-homology regions (Box 1,2 and 3) and consists of 5 parallel β-sheets linked by loops to, and surrounded by, 5 α-helices.

However, the extracellular region differs greatly - TLRs have variable numbers of tandem leucine-rich repeat (LRRs) motifs, whereas IL-1Rs contains 3 immunoglobulin-like domains. The LRR motifs is made up of 19-25 tandem repeats of a 24-29 residue conserved sequence of XLXXLXLXX and X○XX○X4FXXLX, where ○ denotes a hydrophobic residue, and X denotes any amino acid. The extracellular domain of all TLRs form a horseshoe shape, and the concave structure is thought to be involved in recognition; however, different TLRs can recognise different structurally unrelated ligands, so it might be true that ligand recognition do not depend simply on the shape of the receptor.

Mechanism

TLR	Localization	Ligand	Effector Molecule	Pathway	Cellular Effect
TLR4	Plasma membrane and endosome	LPS	Early- and late-phase NF-κB, IRF3	MyD88 and TRIF	Inflammatory Cytokine and Type 1 Interferon production
TLR2-TLR1	Plasma membrane	triacyl lipopeptide (from Gram- bacteria)	NF-κB	MyD88	Inflammatory cytokine production
TLR2-TLR6	Plasma membrane	diacyl lipopeptide (from Gram+ bacteria and mycoplasma)	NF-κB	MyD88	Inflammatory cytokine production
TLR5	Plasma membrane	Flagellin	NF-κB	MyD88	Inflammatory cytokine production
TLR3	Endosome	dsRNA	NF-κB and IRF3	TRIF	Inflammatory Cytokine and Type 1 Interferon production
TLR7	Endosome	ssRNA	NF-κB and IRF7	MyD88	Inflammatory Cytokine and Type 1 Interferon production
TLR9	Endosome	DNA	NF-κB and IRF7	MyD88	Inflammatory Cytokine and Type 1 Interferon production

Recognition

Different TLRs are known to recognise different ligands, and are also found to recruit different adaptor molecules, and so able to tailor each response specifically to the pathogen.

Cell surface recognition

TLR-4 was the first TLR to be identified, and it is the best characterized; it is a plasma membrane-associated receptor known to function by forming a complex with MD-2, which is required for TLR-4 (and TLR-2 for that matter) to function. As mentioned below, lipopolysaccharide (LPS) on Gram- bacteria is a PAMP which should be recognised. In the blood, there exists LPS binding proteins (LBPs), an acute phase protein which binds to LPS and delivers it to CD14, a glycosylphosphatidylinositol (GPI) anchored leucine-rich repeat-containing protein on the macrophage surface. The LPS:CD14 complex on the surface of the macrophage can then interact laterally with (TLR4)₂:MD-2 complex on the same macrophage's cell surface. 5 out of 6 of the LPS chains associate with het hydrophobic pocket of MD-2, while the remaining chain associates at the interface between MD-2 and TLR-4; the negatively-charged phosphate groups can interact favourable with the positively charged residues on TLR4; this may explain the requirement of TLR-4 to complex with MD-2. Binding leads to a conformational change of TLR-4 that allows TIR-domain-containing adaptor proteins to bind to the TIR domain at the cytosolic tail, that triggers the downstream signalling cascade.

Cytosolic recognition

There are three different TLRs identified that exists in the cytosol, most commonly on the endosome membrane, they are all, at least partly, involved in recognising abnormal nucleic acids. TLR3 was found to produce a response to polyinosinic-polycytidylic acid (poly(I:C)), an analog of dsRNA. Because dsRNA is found in viral genomes, during replication of ssRNA, and in certain siRNA, they are thought to be key in recognising viral infections. Its role as a anti-viral receptor was confirmed when the structure was confirmed to bind to its ligand; it has a large horseshoe structure in the ectodomain, which is thought to increase its surface area to bind to dsRNA. This view is further solidified when mice deficient in TLR3 shows high susceptibility to murine cytomegalovirus; and humans deficient in TLR3 shows susceptibility to herpes simplex virus type 1 (HSV-1). TLR3 binding leads to the increased secretion of inflammatory cytokines and type 1 interferon by the TRIF-dependent pathway.

Downstream signalling

TLR-4's downstream signalling uses both of these pathways. Initially, the bound complex recruits TIR domain-containing adaptors TIRAP which allows MyD88 to bind; this binding leads to the activation of early-phase nuclear factor kappa B (NF-κB), which moves into the nucleus and induce transcription. Soon, the LPS:LBP:TLR-4₂:MD-2 complex is internalized to an endosome, where it recruits TRAM and subsequently TRIF, to activate late-phase NF-κB and, togetheer with activated early-phase NF-κB, leads to secretion of inflammatory cytokines; TRIF also activate IRF-3 that induce type 1 interferon production. TLR-4 and TLR-3 are the only two TLR that seems to be able to generate responses from the two pathways.

MyD88-dependent pathway

Adaptor protein myeloid differentiation primary-response protein 88 (MyD88) was first isolated in M1 myeloleukaemic cells as having increased expression after the cells were being induced with IL-6 to promote differentiation into macrophages. The structure of MyD88 contains an N-terminal death domain (DD), a linker region, and a C-terminal TIR domain. They exist as dimers where the DD domains, as well as the TIR domains interact with each other. When a ligand, such as IL-1, binds to the TLR/IL-1R complex, the MyD88 dimer binds to it and recruits and binds IRAK through the DD, which is present in both proteins.

IL-1R-associated kinases (IRAKs)’s role is to activate transmit and amplify the signal from MyD88 to downstream signalling molecules; all IRAKs have an N-terminal domain with a central serine/threonine-kinase domain. In mammals, four IRAKs have been identified - IRAK1, IRAK2, IRAK4 and IRAK-M - of which only IRAK1 and IRAK4 have intrinsic kinase activity. IRAK1 shows a marked increase kinase activity upon TLR/IL-1R stimulation, although a lack of kinase activity on IRAK1 can often be compensated by an increase concentration of IRAK1mut. In contrast, IRAK4 with its kinase activity removed led to no NB-κB (a downstream effector) activation; this suggests that IRAK4 is essential for TLR/IL-1R response, and is likely to act upstream of IRAK1.

PAMPs

The TLRs bind to pathogen-associated molecular patterns (PAMPs) as part of its recognition of molecules that are non-self. Common bacterial PAMPs include lipoteichoic acid (LTA), lipipolysaccharide (LPS, which contains Lipid A, a non-self antigen), peptidoglycan, lipoarabinomannan (LAM, a glycolipid), galactan (polymerized galactose) and mycolic acid.