Toll-like receptors (“TLRs”) are a class of proteins that play a role in the innate immune system. They are single membrane-spanning non-catalytic receptors that recognize structurally conserved molecules derived from microbes. Once these microbes have breached physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs that then activate the immune cell responses.
TLRs are a type of pattern recognition receptor (“PRR”) and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). TLRs, together with the Interleukin-1 receptors, form a receptor superfamily known as the “Interleukin-1 Receptor/Toll-Like Receptor Superfamily”; all members of this family have in common a so-called TIR (Toll-IL-1 receptor) domain.
Three subgroups of TIR domains exist. Proteins with subgroup 1 TIR domains are receptors for interleukins that are produced by macrophages, monocytes and dendritic cells and all have extracellular Immunoglobulin (Ig) domains. Proteins with subgroup 2 TIR domains are classical TLRs, and bind directly or indirectly to molecules of microbial origin. A third subgroup of proteins containing TIR domains consists of adaptor proteins that are exclusively cytosolic and mediate signaling from proteins of subgroups 1 and 2.
TLRs are present in vertebrates, as well as in invertebrates. It is generally believed 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. It is generally believed that the endogenous activators of TLRs may participate in autoimmune diseases due to the sterotypic inflammatory response provoked by TLR activation. TLRs may be involved in the binding to host molecules including fibrinogen (involved in blood clotting) and heat shock proteins (HSPs) and host DNA.
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 different ligand specificity. TLRs also may 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 Lipopolysaccharide 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 studied. Random germline mutagenesis with ENU has been used to study the TLR signaling pathways. When activated, it is believed that 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. The adapters may 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.
Toll-like receptors bind and become activated by different ligands, which, in turn are located on different types of organisms or structures. They also have different adapters to respond to activation and are located sometimes at the cell surface and sometimes to internal cell compartments. Furthermore, they are expressed by different types of leucocytes or other cell types (as shown in Table 1 below).
TABLE 1LigandReceptorLigand(s)LocationAdaptorsLocationCell TypesTLR1multiple triacylbacteriaMyD88/cell surfacemonocytes/macrophages;lipopeptidesMALa subset of dendriticcells; B lymphocytesTLR2multiple glycolipids;bacteria;MyD88/cell surfacemonocytes/macrophages;multiple lipopeptides;host cells;MALmyeloid dendritic cells;multiple lipoproteins;fungimast cellslipteichoic acid;HSP70; zymosanTLR3dsRNA; poly I:CvirusesTRIFcell compartmentdendritic cells; BlymphocytesTLR4lipopolysaccharide;Gram-MyD88;cell surfacemonocytes/macrophages;heat shock proteins;negativeMAL;myeloid dendritic cells;fibrinogen; heparanbacteria;TRIF;mast cells; intestinalsulfate fragments;host cellsTRAMepitheliumhyaluronic acidfragmentsTLR5flagellinbacteriaMyD88cell surfacemonocytes/macrophages;a subset of dendriticcells; intestinalepitheliumTLR6multiple diacylmycoplasmaMyD88;cell surfacemonocytes/macrophages;lipopeptidesMALmast cells; BlymphocytesTLR7imidazoquinoline;smallMyD88cell surfacemonocytes/macrophages;loxoribine;syntheticplasmacytoid dendriticbropirimine; ssRNAcompoundscells; B lymphocytesTLR8small syntheticMyD88cell compartmentmonocytes/macrophages;compounds; ssRNAa subset of dendriticcells; mast cellsTLR9unmethylated CpGbacteriaMyD88cell compartmentmonocytes/macrophages;DNAplasmacytoid dendriticcells; B lymphocytesTLR10*********cell surfacemonocytes/macrophages;B lymphocytesTLR11profilmToxoplasmaMyD88cell surfacemonocytes/macrophages;gondiiliver cells; kidney;bladder epitheliumTLR12***************TLR13***************
Several reactions are possible following activation by ligands of microbial origin. Immune cells may produce signaling factors (cytokines) that trigger inflammation. For example, with a bacterial factor, the pathogen may be phagocytosed and digested, and its antigens presented to CD4+ T cells; with 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 also may release anti-viral factors such as interferons.
There is a need for a cell system that can express TLRs and, therefore, be used to study gastrointestinal disorders in a gastrointestinal segment-specific manner.
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