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- 2. Microbes induce abroad spectrum of cellular innate immune responses by a wide variety of cell types In addition to triggering their own uptake and killing by phagocytic cells. Several families of pattern recognition receptors (PRRs) play major roles in innate immunity. These PRRs bind to PAMPs as well as to some endogenous (self) DAMPs and trigger signal-transduction pathways that turn on expression of genes with important functions in innate immunity. Among the proteins encoded by these genes are antimicrobial molecules such as antimicrobial peptides and interferons, chemokines and cytokines that recruit and activate other cells, enzymes such as iNOS that generate antimicrobial molecules, and proinflammatory mediators (i.e., components that promote inflammation).
- 3. Toll-Like Receptors RecognizeMany Types of Pathogen Molecules Toll-like receptors (TLRs) were the first family of PRRs to be discovered and are still the best-characterized in terms of their structure, how they bind PAMPs and activate cells, and the extensive and varied set of innate immune responses that they induce. Discovery of Invertebrate Toll and Vertebrate Toll-like Receptors In the 1980s, in Germany it was discovered that Drosophila fruit fly embryos could not establish a proper dorsal-ventral (back to front) axis if the gene encoding the Toll membrane protein is mutated. (The name “Toll” comes from German slang meaning “weird,” referring to the mutant flies’ bizarrely scrambled anatomy.) For their subsequent characterization of the toll and related homeobox genes and their role in the regulation of embryonic development, Christiane Nusslein-Volhard, Eric Wieschaus, and Edward B. Lewis were awarded the Nobel Prize in Physiology and Medicine in 1995.
- 4. Many mutations ofthe toll gene were generated, and in 1996 Jules Hoff man and Bruno Lemaitre discovered that mutations in toll made flies highly susceptible to lethal infection with Aspergillus fumigatus, a fungus to which wild-type flies were immune . This studies showing that Toll and related proteins are involved in the activation of innate immune responses in invertebrates. For his pivotal contributions to the study of innate immunity in Drosophila, Jules Hoff man was a co-winner of the 2011 Nobel Prize in Physiology and Medicine.
- 5. Impaired innate immunityin fruit flies with a mutation in the Toll pathway. Severe infection with the fungus Aspergillus fumigatus (yellow) results from a mutation in the signaling pathway downstream of the Toll pathway in Drosophila that normally activates production of the antimicrobial peptide drosomycin.
- 6. Characterization of theToll protein surprisingly revealed that its cytoplasmic signaling domain was homologous to that of the vertebrate receptor for the cytokine IL-1 (IL-1R). Through a search for human proteins with cytoplasmic domains homologous to those of Toll and IL-1R, in 1997 Charles Janeway and Ruslan Medzhitov discovered a human gene for a protein similar to Toll that activated the expression of innate immunity genes in human cells. Appropriately, this and other vertebrate Toll relatives discovered soon thereafter were named Toll-like receptors (TLRs).
- 7. Through studies withmutant mice, in 1998 Bruce Beutler obtained the important proof that TLRs contribute to normal immune functions in mammals. Mice homozygous for a mutant form of a gene called lps were resistant to the harmful responses induced by lipolysaccharide (LPS; also known as endotoxin), a major component of the cell walls of Gram negative bacteria. In humans, a buildup of endotoxin from severe bacterial infection can induce too strong of an innate immune response, causing septic shock, a life-threatening condition in which vital organs such as the brain, heart, kidney, and liver may fail.
- 8. Beutler found thatthe defective mouse lps gene encoded a mutant form of one TLR, TLR4, which differed from the normal form by a single amino acid so that it no longer was activated by LPS. TLR4 is the cellular innate pattern recognition receptor that recognizes LPS and earned Beutler a share of the 2011 Nobel Prize. Invertebrates use receptors to respond to pathogens, also found in vertebrates, and that one of these receptors is responsible for LPS-induced innate immune responses.
- 9. TLRs and TheirLigands TLRs 1-10 are conserved between mice and humans, although TLR10 is not functional in mice, while TLRs 11-13 are expressed in mice but not in humans. Each TLR has a distinct repertoire of specificities for conserved PAMPs; the TLRs and some of their known PAMP ligands . Biochemical studies have revealed the structure of several TLRs and how they bind their specific ligands. TLRs are membrane-spanning proteins that share a common structural element in their extracellular region called leucine-rich repeats (LRRs); multiple LRRs make up the horseshoe-shaped extracellular ligand-binding domain of the TLR polypeptide chain .
- 11. When TLRs bindtheir PAMP or DAMP ligands via their extracellular LRR domains, they are induced to dimerize. In most cases each TLR dimerizes with itself, forming a homodimer, but TLR2 forms heterodimers by pairing with either TLR1 or TLR6.
- 12. Structures of TLR2/1with a bound lipopeptide and TLR3 with dsRNA are the characteristic “m”-shaped conformation of TLR dimers is apparent. TLRs exist both on the plasma membrane and in the membranes of endosomes and lysosome lysosomes; their cellular location is tailored to enable them to respond optimally to the particular microbial ligands they recognize. TLRs that recognize PAMPs on the outer surface of extracellular microbes are found on the plasma membrane, where they can bind these PAMPs and induce responses. TLRs that recognize internal microbial components that have to be exposed by the dismantling or degradation of endocytosed pathogens— nucleic acids in particular—are found in endosomes and lysosomes. Unique among the TLRs, TLR4 has been shown to move from the plasma membrane to endosomes/ lysosomes after binding LPS or other PAMPs. it activates different signaling pathways from the two locations.
- 13. In addition tomicrobial ligands, TLRs also recognize DAMPs, endogenous (self) components released by dead/ dying cells or damaged tissues. Among the DAMPS recognized by plasma membrane TLRs are heat shock and chromatin proteins, fragments of extracellular matrix components (such as fibronectin and hyaluronin), and oxidized low density lipoprotein and beta amyloid. While self nucleic acids usually do not activate the intracellular PRR, under certain circumstances (such as when bound by anti-DNA or antichromatin antibodies in individuals with the autoimmune disease lupus erythematosus) they can be endocytosed and activate endosomal/lysosomal TLRs, contributing to the disease .
- 14. Signaling Through TLRs:Overview Signaling through TLRs utilizes many of the principles and some of the signaling molecules, along with some unique to pathways activated by TLRs (and by other PRRs). An important example of a shared component is the transcription factor NF-kB. NF-kB is key for inducing many innate and inflammatory genes, including those encoding defensins; enzymes such as iNOS; chemokines; and cytokines such as the proinflammatory cytokines TNF-alpha, IL-1, and IL-6, produced by macrophages and dendritic cells. There are also TLR-specific signaling pathways and components that induce the expression of subsets of proteins, some of which are particularly effective in combating the type of pathogen recognized by the particular TLR(s).
- 15. The generation ofRNS requires the transcriptional activation of the gene for the enzyme inducible nitric oxide synthase (iNOS, or NOS2. Expression of iNOS is activated by microbial components binding to various PRRs. iNOS oxidizes L-arginine to yield L-citrulline and nitric oxide (NO), a potent antimicrobial agent. Collectively the ROS and RNS are highly toxic to phagocytosed microbes due to the alteration of microbial molecules through oxidation, hydroxylation, chlorination, nitration, and S-nitrosylation, along with formation of sulfonic acids and destruction of iron-sulfur clusters in proteins. One example of how these oxidative species may be toxic to pathogens is the oxidation by ROS of cysteine sulfhydryls that are present in the active sites of many enzymes, inactivating the enzymes. ROS and RNS also can be released from activated neutrophils and macrophages and kill extracellular pathogens.
- 17. An important exampleis expression of the potent antiviral Type 1 interferons, IFNs-alpha and beta, induced by pathways downstream of the TLRs that bind viral components. Activation of the interferon regulatory factors (IRFs) is essential for inducing transcription of the genes encoding IFN-alpha and beta. Combinations of transcription factors contribute to inducing the expression of many of these genes; examples include combinations of NF-kB, IRFs, and/or transcription factors downstream of MAP kinase (MAPK) pathways , such as AP-1, that can be activated by signaling intermediates downstream of certain TLRs. The particular signal transduction pathway(s) activated by a TLR dimer following PAMP binding are largely determined by the TLR and by the initial protein adaptor that binds to the TLR’s cytoplasmic domain. TIR domain (from Toll/IL-1 receptor), referring to the similarity noted earlier between the cytoplasmic domains of TLRs and IL-1 receptors .TIR domains of all TLR dimers serve as binding sites for the TIR domains of adaptors that activate the downstream signaling pathways.
- 18. Signaling from IL-1Receptors Productive ligand binding to the extracellular portion of the IL-1 receptor leads to a conformational alteration in its cytoplasmic domain. This structural alteration in the receptor leads to a series of downstream signaling events. First, binding of the adapter protein MyD88 to the occupied receptor allows recruitment to the receptor complex of one or more members of the IL-1 Receptor Activated Kinase (IRAK) protein family. IRAK-4, is activated by autophosphorylation and phosphorylates its fellow IRAKs, resulting in the generation of binding sites for TNF Receptor Associated Factor 6 (TRAF6), which is associated with a ubiquitin-ligase complex capable of generating polyubiquitin chains. The IRAK-TRAF6 complex now dissociates from the receptor and interacts with a preformed cytosolic complex made up of the kinase TGF beta Associated Kinase 1 (TAK1) and two TAK1-Binding proteins, TAB1 and TAB2. Binding of polyubiquitin chains to the TAB proteins in the TAK1 complex activates it.
- 19. The TAK1 complexnow performs two functions. It phosphorylates and activates the IKK complex, leading to the destruction of IB and the resultant activation of the transcription factor NF-B (see Figure 3-17). In addition, TRAF6 also plays a role in IKK activation by providing ubiquitination sites to which the NEMO component of IKK can bind, resulting in its further activation. TAK1 also activates downstream members of the MAP kinase cascade, which in turn activate the AP-1 transcription factor (see Figure 3-16). Binding of IL-1 family cytokines to their receptors thereby leads to a global alteration in the transcription patterns of the affected cells, which in turn results in the up-regulation of proinflammatory cytokines and adhesion molecules.
- 22. The two keyadaptors are MyD88 (Myeloid differentiation factor 88) and TRIF (TIR-domain-containing adaptor-inducing IFN- factor). Most TLRs, whether found on the cell surface or in endosomes/lysosomes, bind the adaptor protein MyD88 (activating MyD88-dependent signaling pathways). In contrast, TLR3 binds the alternative adaptor protein TRIF (activating TRIF dependent signaling pathways). TLR4 is unique in binding both MyD88 (when it is in the plasma membrane, signaling its endocytosis) and TRIF (when it is in endosomes, after internalization). The presence of two additional adaptors associated with most TLRs: TIRAP (TIR-domain containing adaptor protein) and TRAM (TRIF-related adaptor molecule). They are TIR domain-containing adaptors that serve as sorting receptors: TIRAP helps to recruit MyD88 to TLRs 2/1, 2/6, and 4, and TRAM helps to recruit TRIF to both TLR3 and endosomal/lysosomal TLR4.
- 23. MyD88-Dependent Signaling Pathways Afterassociating with a TLR dimer following ligand binding, MyD88 initiates a signaling pathway that activates the NF-B and MAPK pathways by essentially the same pathway as that activated by IL-1. As shown for plasma membrane TLRs 2/1, 4, and 5, MyD88 recruits and activates several IRAK protein kinases, which then bind and activate TRAF6. TRAF6 ubiquitinates NEMO and TAB proteins, leading to the activation of TAK1, which then phosphorylates the IB kinase (IKK) complex. Activated IKK then phosphorylates the inhibitory IB subunit of NF-B, releasing NF-B to enter the nucleus and activate gene expression. TAK1 does double duty in this TLR signaling cascade. After separating from the IKK complex, it activates MAPK signaling pathways that result in the activation of transcription factors including Fos and Jun, which make up the AP-1 dimer.
- 25. In addition toactivating NF-B and MAPK pathways via the MyD88-dependent pathway, the endosomal TLRs 7, 8, and 9, which bind microbial nucleic acids (especially viral RNA and bacterial DNA), also trigger pathways that activate IRFs. As shown in Figure 5-13, when triggered by these TLRs, the MyD88/IRAK4/TRAF6 complex activates a complex containing TRAF3, IRAK1, and IKK, leading to the phosphorylation, dimerization, activation, and nuclear localization of IRF7. IRF7 induces the transcription of genes for both Type 1 interferons, IFN alpha and beta, which have potent antiviral activity. Thus different TLRs may differentially activate distinct transcription factors (NF-B, certain IRFs, and/or those activated by MAPK pathways), leading to variation in which genes are turned on to help protect us against the invading pathogens.
- 26. TRIF-Dependent Signaling Pathways Forthe two endosomal TLRs that recruit the TRIF adaptor instead of MyD88—TLR3 and endosomal TLR4— the downstream signaling pathways differ somewhat from those activated by MyD88. TRIF recruits the RIP1 protein kinase that in turn recruits and activates TRAF6, which then initiates the same steps as in the MyD88-dependent pathway.
- 27. TLR3 also activatesPI3K, which enhances MAPK pathway activation. In addition, TRIF activates TRAF3 and a complex of TBK-1 and IKK, which phosphorylates and activates IRF7 and a different IRF—IRF3. IRF3 and IRF7 dimerize and move into the nucleus and (together with NF-B and AP-1) induce the transcription of the IFN genes. Thus, all of the intracellular TLRs that bind viral PAMPs in an infected cell induce the synthesis and secretion of Type I interferons, which feed back to potently inhibit the replication of the virus in that infected cell.
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