Not all mammalian TLRs are cell-surface receptors. The TLRs that recognize nucleic acids are located in the membranes of endosomes, to which they are transported via the endoplasmic reticulum. TLR-3 is expressed by macrophages, conventional dendritic cells, and intestinal epithelial cells; it recognizes double-stranded RNA (dsRNA), which is a replicative intermediate of many types of viruses, not only those with RNA genomes. dsRNA is internalized either by the direct endocytosis of viruses with double-stranded RNA genomes, such as rotavirus, or by the phagocytosis of dying cells in which viruses are replicating, and it encounters the TLRs when the incoming endocytic vesicle or phagosome fuses with the TLR-containing endosome. Crystallographic analysis shows that TLR-3 binds directly to dsRNA. The TLR-3 ectodomain (the ligand-binding domain) has two contact sites for dsRNA: one on the amino terminus and a second near the membrane-proximal carboxy terminus. The twofold symmetry of dsRNA allows it to bind simultaneously to two TLR-3 ectodomains, inducing a dimerization that brings the TIR domains of TLR-3 together and activates intracellular signaling. This can be verified by using poly I:C to artificially induce signaling. A synthetic polymer composed of inosinic and cytidylic acid, poly I:C binds to TLR-3 and functions as an analog of dsRNA; poly I:C is often used experimentally to activate this pathway. Mutations in the ectodomain of human TLR-3, which produce a dominantly acting loss-of-function mutant receptor, have been associated with encephalitis that is caused by a failure to control the herpes simplex virus.
TLR-7, TLR-8, and TLR-9, like TLR-3, are endosomal nucleotide sensors involved in the recognition of viruses. TLR-7 and TLR-9 are expressed by plasmacytoid dendritic cells, B cells, and eosinophils; TLR-8 is expressed primarily by monocytes and macrophages. TLR-7 and TLR-8 are activated by single-stranded RNA (ssRNA), which is a component of healthy mammalian cells, but it is normally confined to the nucleus and cytoplasm and is not present in endosomes. Many virus genomes, for example, those of orthomyxoviruses (such as influenza) and flaviviruses (such as West Nile virus), consist of ssRNA. When extracellular particles of these viruses are endocytosed by macrophages or dendritic cells, they are uncoated in the acidic environment of endosomes and lysosomes, exposing the ssRNA genome for recognition by TLR-7. Mice lacking TLR-7 have impaired immune responses to viruses such as influenza. In abnormal settings, TLR-7 may be activated by self-derived ssRNA. Normally, extracellular RNases degrade the ssRNA released from apoptotic cells during tissue injury. But in a mouse model of lupus nephritis, an inflammatory condition of the kidney, TLR-7 recognition of self ssRNA was observed to contribute to disease. Several studies have identified polymorphisms in the human TLR-7 gene that are associated with increased risk of the autoimmune disease systemic lupus erythematosus, suggesting a potential role in this disease. The role for TLR-8 has not been established as clearly from mouse model systems as for TLR-7. TLR-9 recognizes unmethylated CpG dinucleotides. In mammalian genomes, CpG dinucleotides in genomic DNA are heavily methylated on the cytosine by DNA methyltransferases. But in the genomes of bacteria and many viruses, CpG dinucleotides remain unmethylated and represent another pathogen-associated molecular pattern.