NOD LRR CARD domains pro-caspase 1 NLRP3 ASC
Fig. 3.20 The inflammasome is composed of several filamentous protein polymers created by
aggregated CARD and pyrin domains.
Top panel: an electron micrograph of structures formed by full-length ASC, the pyrin domain of AIM2, and the CARD domain of caspase 1. The central dark region represents anti-ASC staining with a gold-labeled (15 nm) antibody. The long outward filaments represent the polymer composed of the caspase 1 CARD domain. Bottom panel: Schematic interpretation of NLRP3 inflammasome assembly. In this model, CARD regions of ASC and caspase 1 aggregate into a filamentous structure. The adaptor ASC translates aggregation of NLRP3 into aggregation of pro-caspase 1. Electron micrograph courtesy of Hao Wu.
its CARD-dependent polymerization into discrete caspase 1 filaments. This aggregation seems to trigger the autocleavage of pro-caspase 1, which releases the active caspase 1 fragment from its autoinhibitory domains. Active caspase 1 then carries out the ATP-dependent proteolytic processing of pro- inflammatory cytokines, particularly IL-1β and IL-18, into their active forms (see Fig. 3.19). Caspase 1 activation also induces a form of cell death called pyroptosis (‘fiery death’) through an unknown mechanism that is associated with inflammation because of the release of these pro-inflammatory cytokines upon cell rupture.
For inflammasome activation to produce inflammatory cytokines, a prim- ing step must first occur in which cells induce and translate the mRNAs that encode the pro-forms of IL-1β, IL-18, or other cytokines. This priming step can result from TLR signaling, which may help ensure that inflammasome acti- vation proceeds primarily during infections. For example, the TLR-3 agonist poly I:C (see Section 3-5) can be used experimentally to prime cells for subse- quent triggering of the inflammasome.
Several other NLR family members form inflammasomes with ASC and caspase 1 that activate these pro-inflammatory cytokines. NLRP1 is highly expressed in monocytes and dendritic cells and is activated directly by MDP, similar to NOD2, but can also be activated by other factors. For example, Bacillus anthracis expresses an endopeptidase, called anthrax lethal factor, which allows the pathogen to evade the immune system by killing macrophages. Lethal factor does this by cleaving NLRP1, activating an NLRP1 inflammasome and inducing pyroptosis in the infected macrophages. NLRC4 acts as an adaptor with two other NLR proteins, NAIP2 and NAIP5, that serve to detect various bacterial proteins that enter cells through specialized secretion systems used by pathogens to transport materials into or access nutrients from host cells. One such protein, PrgJ, from the pathogen Salmonella typhimurium, is a component of the type III secretion system (T3SS), a needle-like macromolecular complex. Upon infection of host cells by Salmonella, PrgJ enters the cytoplasm and is recognized by NLRC4 functioning together with NAIP2. Extracellular bacterial flagellin is recognized by TLR5, but flagellin may also enter host cells with PrgJ via the T3SS, and in this case can be recognized by NLRC4 in conjunction with NAIP5. Some NLR proteins may negatively regulate innate immunity, such as NLRP6, since mice lacking this protein exhibit increased resistance to certain pathogens. However, NLRP6 is highly expressed in intestinal epithelium, where it appears to play a positive role in promoting normal mucosal barrier function and is required for the normal secretion of mucus granules into the intestine by goblet cells. NLRP7, which is present in humans but not mice, recognizes microbial acyl ated lipopeptides and forms an inflammasome with ASC and caspase 1 to produce IL-1β and IL-18. Less is known about NLRP12, but like NLRP6, it initially was proposed to have an inhibitory function. Subsequent studies of mice lacking NLRP12 suggest it has a possible role in the detection of and response to certain bacterial species, including Yersinia pestis, the bacterium that causes bubonic plague, although the basis of this recognition is still unclear.
Inflammasome activation can also involve proteins of the PYHIN family, which contain an N-terminal pyrin domain but lack the LRR domains present in the NLR family. In place of an LRR domain, PYHIN proteins have a HIN (H inversion) domain, so named for the HIN DNA recombinase of Salmonella that mediates DNA inversion between flagellar H antigens. There are four PYHIN proteins in humans, and 13 in mice. In one of these, AIM2 (absent in melanoma 2), the HIN domain recognizes double-stranded DNA genomes and triggers caspase 1 activation through pyrin domain interactions with ASC. AIM2 is located in the cytoplasm and is important for responses in vitro to vaccinia virus, and its in vivo role has been demonstrated by the increased susceptibility of AIM2-deficient mice to infection by Francisella tularensis,
the causative agent of tularemia. The related protein IFI16 (interferon induc- ible protein 16) contains two HIN domains; it is primarily located in the cell nucleus and recognizes viral double-stranded DNA, and will be described below in Section 3-11.
A ‘non-canonical’ inflammasome (caspase 1-independent) pathway uses the protease caspase 11 to detect intracellular LPS. The discovery of this pathway was initially confused as being dependent on caspase 1 because of a specific genetic difference between experimental mouse strains. Caspase 11 is encoded by the murine Casp4 gene and is homologous to human caspases 4 and 5. The mice in which the caspase 1 gene (Casp1) was initially disrupted and studied were originally found to be resistant to lethal shock (see Section 3-20) induced by administration of LPS. This led researchers to conclude that caspase 1 acted in the inflammatory response to LPS. But researchers later discovered that this mouse strain also carried a natural mutation that inactivated the related Casp4 gene. Because the Casp1 and Casp4 genes reside within 2 kilobases of each other on mouse chromosome 9, they failed to segregate independently during subsequent experimental genetic backcrosses to other mouse strains. Thus, mice initially thought to lack only caspase 1 protein in fact lacked both caspase 1 and caspase 11. Later, mice lacking only caspase 1 were generated by expressing functional Casp4 as a transgene; these mice became susceptible to LPS-induced shock. Mice were also generated that lacked only caspase 11, and these were found to be resistant to LPS-induced shock. These results indi- cated that caspase 11, and not caspase 1 as originally thought, is responsible for LPS-induced shock. Caspase 11 is responsible for inducing pyroptosis, but not for processing of IL-1β or IL-18. It was suspected that TLR-4 was not the sensor for LPS that activated the non-canonical imflammasome, since mice lacking TLR-4 remain susceptible to LPS-induced shock. Recent evidence has suggested that caspase 11 itself is the intracellular LPS sensor, making it an example of a protein that is both a sensor and an effector molecule.
Inappropriate inflammasome activation has been associated with various diseases. Gout has been known for many years to cause inflammation in the cartilaginous tissues by the deposition of monosodium urate crystals, but how urate crystals caused inflammation was a mystery. Although the precise mechanism is still unclear, urate crystals are known to activate the NLRP3 inflammasome, which induces the inflammatory cytokines associated with the symptoms of gout. Mutations in the NOD domain of NLRP2 and NLRP3 can activate inflammasomes inappropriately, and they are the cause of some inherited autoinflammatory diseases, in which inflammation occurs in the absence of infection. Mutations in NLRP3 in humans are associated with hereditary periodic fever syndromes, such as familial cold inflammatory syn- drome and Muckle–Wells syndrome (discussed in more detail in Chapter 13). Macrophages from patients with these conditions show spontaneous produc- tion of inflammatory cytokines such as IL-1β. We will also discuss how patho- gens can interfere with formation of the inflammasome in Chapter 13.
3-10 The RIG-I-like receptors detect cytoplasmic viral RNAs and activate MAVS to induce type I interferon production and pro-inflammatory cytokines.
TLR-3, TLR-7, and TLR-9 detect extracellular viral RNAs and DNAs that enter the cell from the endocytic pathway. By contrast, viral RNAs produced within a cell are sensed by a separate family of proteins called the RIG-I-like receptors (RLRs). These proteins serve as viral sensors by binding to viral RNAs using an RNA helicase-like domain in their carboxy terminal. The RLR helicase-like domain has a ‘DExH’ tetrapeptide amino acid motif and is a subgroup of DEAD-box family proteins. The RLR proteins also contain two amino- terminal CARD domains that interact with adaptor proteins and activate
Hereditary Periodic Fever Syndromes
signaling to produce type I interferons when viral RNAs are bound. The first of these sensors to be discovered was RIG-I (retinoic acid-inducible gene I). RIG-I is widely expressed across tissues and cell types and serves as an intra- cellular sensor for several kinds of infections. Mice deficient in RIG-I are highly susceptible to infection by several kinds of single-stranded RNA viruses, including paramyxoviruses, rhabdoviruses, orthomyxoviruses, and flavi- viruses, but not picornaviruses.
RIG-I discriminates between host and viral RNA by sensing differences at the 5ʹ end of single-stranded RNA transcripts. Eukaryotic RNA is transcribed in the nucleus and contains a 5ʹ-triphosphate group on its initial nucleotide that undergoes subsequent enzymatic modification called capping by the addition of a 7-methylguanosine to the 5ʹ-triphosphate. Most RNA viruses, however, do not replicate in the nucleus, where capping normally occurs, and their RNA genomes do not undergo this modification. Biochemical studies have deter- mined that RIG-I senses the unmodified 5ʹ-triphosphate end of the ssRNA viral genome. Flavivirus RNA transcripts have the unmodified 5ʹ-triphosphate, as do the transcripts of many other ssRNA viruses, and they are detected by RIG-I. In contrast, the picornaviruses, which include poliovirus and hepati- tis A, replicate by a mechanism that involves the covalent attachment of a viral protein to the 5ʹ end of the viral RNA, so that the 5ʹ-triphosphate is absent, which explains why RIG-I is not involved in sensing them.
MDA-5 (melanoma differentiation-associated 5), also called helicard, is similar in structure to RIG-I, but it senses dsRNA. In contrast to RIG-I-deficient mice, mice deficient in MDA-5 are susceptible to picornaviruses, indicating that these two sensors of viral RNAs have crucial but distinct roles in host defense. Inactivating mutations in alleles of human RIG-I or MDA-5 have been reported, but these mutations were not associated with immunodeficiency. The RLR family member LGP2 (encoded by DHX58) retains a helicase domain but lacks CARD domains. LGP2 appears to cooperate with RIG-I and MDA-5 in the recognition of viral RNA, since mice lacking LGP2 have impaired anti- viral responses normally mediated by RIG-I or MDA-5. This cooperative viral recognition by LGP2 appears to depend on its helicase domain, since in mice, mutations that disrupt its ATPase activity result in impaired IFN-β production in response to various RNA viruses.
Sensing of viral RNAs activates signaling by RIG-I and MDA-5 that leads to type I interferon production appropriate for defense against viral infection (Fig. 3.21). Before infection by viruses, RIG-I and MDA-5 are in the cytoplasm
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virus triphosphateRNA
RIG-I MDA-5 helicase domain CTD CARD Cytoplasmic replication of virus produces uncapped RNA
with a 5'-triphosphate
Viral RNA alters conformation of RIG-I, and induces binding and aggregation with MAVS in a manner
requiring K63-linked polyubiquitin and TRIM25
Aggregated MAVS recruits TRAFs and induces the generation of free K63-linked polyubiquitin chains that
activate the IRFs and NFκB pathways
TRIM25 helicase K63- polyubiquitin MAVS MAVS mitochondrion CTD NFκB IκB IRF-3 TBK1 TRAFs IKKα IKKβ IKKγ (NEMO) MAVS CARD
Fig. 3.21 RIG-I and other RLRs are cytoplasmic sensors of viral RNA.
First panel: before detecting viral RNA, RIG-I and MDA-5 are cytoplasmic and in inactive, auto-inhibited conformations. The adaptor protein MAVS is attached to the mitochondrial outer membrane. Second panel: detection of uncapped 5ʹ-triphosphate RNA by RIG-I, or viral dsRNA by MDA-5, changes the conformation of their CARD domains to become free to interact with the amino-terminal CARD domain of MAVS. This interaction involves the generation of K63-linked polyubiquitin from the E3 ligases TRIM25 or Riplet, although structural details are still unclear. Third panel: the aggregation induces a proline-rich region of MAVS to interact with TRAFs (see text) and leads to the generation of additional K63-linked polyubiquitin scaffold. As in TLR signaling, this scaffold recruits TBK1 and IKK complexes (see Figs. 3.15 and 3.16) to activate IRF and NFκB, producing type I interferons and pro-inflammatory cytokines, respectively.
in an autoinhibited configuration that is stabilized by interactions between the CARD and helicase domains. These interactions are disrupted upon infection when viral RNA associates with the helicase domains of RIG-I or MDA-5, freeing the two CARD domains for other interactions. The more amino-proximal portion of the two CARD domains can then recruit E3 ligases, including TRIM25 and Riplet (encoded by RNF153), which initiate K63-linked polyubiquitin scaffolds (see Section 3-7), either as free polyubiquitin chains or on linkages within the second CARD domain. Precise details are unclear, but this scaffold appears to help RIG-I and MDA-5 interact with a downstream adaptor protein called MAVS (mitochondrial antiviral signaling protein). MAVS is attached to the outer mitochondrial membrane and contains a CARD domain that may bind RIG-I and MDA-5. This aggregation of CARD domains, as in the inflammasome, may initiate aggregation of MAVS. In this state, MAVS propagates signals by recruiting various TRAF family E3 ubiquitin ligases, including TRAF2, TRAF3, TRAF5, and TRAF6. The relative importance of each E3 ligase may differ between cell types, but their further production of K63- linked polyubiquitin leads to activation of TBK1 and IRF3 and production of type I interferons, as described for TLR-3 signaling (see Fig. 3.16), and also to activation of NFκB. Some viruses have evolved countermeasures to thwart the protection conferred by RLRs. For example, even though the negative-sense RNA genome of influenza virus replicates in the nucleus, some viral RNA transcripts produced during influenza infection are not capped but must be translated in the cytoplasm. The influenza A nonstructural protein 1(NS1) inhibits the activity of TRIM25, and thereby interrupts the antiviral actions that RIG-I might exert against infection.
3-11 Cytosolic DNA sensors signal through STING to induce production of type I interferons.
Innate sensors that recognize cytoplasmic RNA use specific modifications, such as the 5ʹ cap, to discriminate between host and viral origin. Host DNA is generally restricted to the nucleus, but viral, microbial, or protozoan DNA may become located in the cytoplasm during various stages of infection. Several innate sensors of cytoplasmic DNA have been identified that can lead to the production of type I interferon in response to infections. One compo- nent of the DNA-sensing pathway, STING (stimulator of interferon genes), was identified in a functional screen for proteins that can induce expression of type I interferons. STING (encoded by TMEM173) is anchored to the endo- plasmic reticulum membrane by an amino-terminal tetraspan transmem- brane domain; its carboxy-terminal domain extends into the cytoplasm and interacts to form an inactive STING homodimer.
STING is known to serve as a sensor of intracellular infection, based on its recognition of bacterial cyclic dinucleotides (CDNs), including cyclic diguanyl ate monophosphate (c-di-GMP) and cyclic diadenylate monophos- phate (c-di-AMP). These molecules are bacterial second messengers and are produced by enzymes present in most bacterial genomes. CDNs activate STING signaling by changing the conformation of the STING homodimer. This homodimer recruits and activates TBK1, which in turn phosphorylates and activates IRF3, leading to type I interferon production (Fig. 3.22), similar to signaling by TLR-3 and MAVS (see Figs. 3.16 and 3.21). TRIF (downstream of TLR3), MAVS, and STING each contain a similar amino acid sequence motif at their carboxy termini that becomes serine-phosphorylated when these mole- cules are activated. It appears that this motif, when phosphorylated, recruits both TBK1 and IRF3, allowing IRF3 to be efficiently phosphorylated and acti- vated by TBK1.
STING also plays a role in viral infections, since mice lacking STING are sus- ceptible to infection by herpesvirus. But until recently, it was unclear whether
STING recognized viral DNA directly or acted only downstream of an unknown viral DNA sensor. It was found that the introduction of DNA into cells, even without live infection, generated another second messenger molecule that activated STING. This second messenger was identified as cyclic guano- sine monophosphate-adenosine monophosphate (cyclic GMP-AMP), or cGAMP. cGAMP, like bacterial CDNs, binds both subunits of the STING dimer and activates STING signaling. This result also suggested the presence of a DNA sensor acting upstream of STING. Purification of the enzyme that pro- duces cGAMP in response to cytosolic DNA identified a previously unknown enzyme, which was named cGAS, for cyclic GAMP synthase. cGAS contains a protein motif present in the nucleotidyltransferase (NTase) family of enzymes, which includes adenylate cyclase (the enzyme that generates the second mes- senger molecule cyclic AMP) and various DNA polymerases. cGAS can bind directly to cytosolic DNA, and this stimulates its enzymatic activity to produce cGAMP from GTP and ATP in the cytoplasm, activating STING. Mice harbor- ing an inactivated cGAS gene show increased susceptibility to herpesvirus infection, demonstrating its importance in immunity.
There are several other candidate DNA sensors, but less is known about the mechanism of their recognition and signaling, or their in vivo activity. IFI16 (IFN-γ-inducible protein 16) is a PYHIN family member related to AIM2, but appears to function in DNA sensing and acts through STING, TBK1, and IRF3 rather than activating an inflammasome pathway. DDX41 (DEAD box poly- peptide 41) is an RLR related to RIG-I and is a member of the DEAD-box fam- ily, but appears to signal through STING rather than MAVS. MRE11A (meitotic recombination 11 homolog a) can sense cytosolic double-stranded DNA to activate the STING pathway, but its role in innate immunity is currently unknown.
3-12 Activation of innate sensors in macrophages and dendritic cells triggers changes in gene expression that have far-reaching effects on the immune response.
Besides activating effector functions and cytokine production, another outcome of the activation of innate sensing pathways is the induction of co-stimulatory molecules on tissue dendritic cells and macrophages (see Section 1-15). We will describe these in more detail later in the book, but mention them now because they provide an important link between innate Immunobiology | chapter 3 | 03_104
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Double-stranded DNA from viruses activates cGAS to produce cGAMP from ATP and GTP
cGAMP or other bacterial-derived cyclic dinucleotides bind to the STING dimer present on the ER membrane and activate its signaling
STING activates the kinase TBK1 to phosphorylate IRF3, which enters the nucleus and
induces expression of type I interferon genes
virus ATP GTP cGAS cGAMP dsDNA c-di-GMP c-di-AMP nucleus ER STING bacterium IRF3 type I interferon genes TBK1
Fig. 3.22 cGAS is a cytosolic sensor of DNA that signals through STING to activate type I interferon production.
First panel: cGAS resides in the cytoplasm and serves as a sensor of double-stranded DNA (dsDNA) from viruses. When cGAS binds dsDNA, its enzymatic activity is stimulated, leading to production of cyclic- GMP-AMP (cGAMP). Bacteria that infect cells produce second messengers such as cyclic dinucleotides, including cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP). Second panel: cGAMP and