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regulating inflammasome activation and immunity. Authors' addresses. Magdalena Matusiak1,2, Nina Van Opdenbosch1,2, Mohamed. Lamkanfi1,2.
Magdalena Matusiak Nina Van Opdenbosch Mohamed Lamkanfi

CARD- and pyrin-only proteins regulating inflammasome activation and immunity

Authors’ addresses Magdalena Matusiak1,2, Nina Van Opdenbosch1,2, Mohamed Lamkanfi1,2 1 Department of Medical Protein Research, VIB, Ghent, Belgium. 2 Department of Biochemistry, Ghent University, Ghent, Belgium.

Summary: Membrane-bound and intracellular immune receptors respond to microbial pathogens by initiating signaling cascades that result in production of inflammatory cytokines and antimicrobial factors. These host responses need to be tightly regulated to prevent tissue damage and other harmful consequences of excessive inflammation. CARD-only proteins (COPs) and Pyrin-only proteins (POPs) are human- and primate-specific dominant negative inhibitors that modulate inflammatory and innate immune responses. In addition, several poxviruses encode POPs that interfere with inflammatory and host defense responses. COPs and POPs modulate inflammatory signaling at several checkpoints by sequestering key components of the inflammasome and NF-jB signaling cascades, thus hampering downstream signal transduction. Here, we review and discuss current understanding of the evolutionary history and molecular mechanisms by which roles of host- and virus-encoded COPs and POPs may regulate inflammatory and immune responses. In addition, we address their (patho)physiological roles and highlight topics for further research.

Correspondence to: Mohamed Lamkanfi Department of Medical Protein Research, VIB Albert Baertsoenkaai 3 B-9000 Ghent, Belgium Tel.: +32 9 264 9341 e-mail: [email protected] Acknowledgements Work in ML’s laboratory is supported by grants from VIB, Ghent University (BOF 01N02313, BOF 01J11113, BOF14/GOA/013), the Fund for Scientific ResearchFlanders (grant G030212N), and the European Research Council (Grant 281600). The authors have no conflicts of interest to declare.

Keywords: CARD-only proteins, Pyrin-only proteins, caspase, NF-jB, virus, infection, inflammasome, inflammation

Mechanisms modulating inflammation and innate immunity This article is part of a series of reviews covering Inflammasomes appearing in Volume 265 of Immunological Reviews.

Immunological Reviews 2015 Vol. 265: 217–230

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Immunological Reviews 0105-2896

The ubiquitous presence of pathogens and potentially harmful agents in the environment triggered multicellular organisms to evolve a set of response mechanisms to cope with these threats. The physical barriers of mucosal interfaces and active host defense mechanisms also are critical for mammals to maintain a symbiotic relationship with the microbial communities that inhabit their digestive tract, skin, and other tissues that are exposed to the environment (1). It is now evident that membrane-bound and intracellular immune receptors are involved in proper recognition of invading pathogens to distinguish them from harmless commensal microflora. Several families of such pattern recognition receptors (PRRs) are essential for successfully clearing infections, with members of the Toll-like receptors (TLRs), Nod-like receptors (NLRs), C-type lectin receptors,

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AIM2-like receptors (ALRs), and RIG-I-like receptors amongst the best characterized (2). These immune sensors respond to evolutionarily conserved microbe-associated molecular patterns and host-derived damage-associated molecular patterns. The latter are host-derived factors that may be produced or released upon tissue damage and (sterile) trauma irrespective of the causal agent (3). Activation of PRRs triggers production of inflammatory cytokines and other effector molecules that halt replication and dissemination of infectious agents and coordinately instruct the innate and adaptive immune arms to resolve the threat and return to homeostasis (4, 5). A subset of PRRs may also instruct macrophages, lymphocytes, and other cell types to undergo programmed cell death. As a result of these actions, chronic inflammation may itself become hazardous to the host. Powerful local and systemic inflammatory reactions may result in significant tissue damage and organ dysfunction if left unchecked. This can lead to serious discomfort to patients and may precipitate in potentially life-threatening inflammatory pathologies, including common autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and type II diabetes, or rare but highly debilitating autoinflammatory syndromes (6). Therefore, carefully balancing the beneficial and harmful outcomes of inflammatory responses is critical to the host, and breakdown of these regulatory brakes frequently is associated with inflammatory disease. Various mechanisms for fine-tuning inflammation have been uncovered during the past decade. Transcriptional regulation of pro- and anti-inflammatory gene expression constitutes a first level of immune modulation. Exposure of epithelial cells, fibroblasts, and myeloid cells to TLR agonists, such as the TLR4 ligand lipopolysaccharide (LPS), triggers broad transcriptional re-programming of the cell that involves upregulation and downregulation of hundreds of mRNAs and miRNAs (7). In addition to inducing production of pro-inflammatory cytokines such as TNF, IL-6, and proIL-1b, LPS stimulates expression of procaspase-11 and NLRP3, two inflammasome effectors that contribute importantly to secretion of mature IL-1b and IL-18 (8–11). Simultaneously, LPS triggers upregulation of ubiquitin-editing enzymes such as A20/TNFAIP3 and CYLD that actively repress the NF-jB signaling cascade to prevent the harmful consequences of excessive inflammasome responses and inflammation in general (12–15). LPS also stimulates expression of IjBNS and B-cell lymphoma 3 (BCL-3), two nucleus-localized IjB family members that selectively inhibit NF-jB signaling by exchanging transcriptionally

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active NF-jB dimers at gene promoters for their inactive counterparts. Consequently, genetic deletion of IjBNS and BCL-3 resulted in more pronounced inflammatory gene expression and increased susceptibility to endotoxic shock (16, 17). A second set of mechanisms that allow balanced inflammatory responses influence the half-life of inflammatory gene transcripts (18). AU-rich element (AREs) motifs in the untranslated mRNA regions of TNF and several other cytokines promote rapid mRNA degradation to ensure transient expression of TNF and other pro- and anti-inflammatory factors including IL-6, IL-1b, transforming growth factor-b (TGFb), and IL-10 (19). Deletion of the ARE-motifs in the 30 UTR of TNF increases the half-life of TNF transcripts and causes a Crohn’s disease-like ileitis in mice (20). miRNAs recently emerged as an alternative mechanism for regulating the mRNA half-life and translation of inflammatory signaling molecules (21, 22). The myeloid-specific miR-223 that targets Nlrp3 is only one out of several miRNAs that control key inflammatory signaling pathways (23, 24). A third strategy that is employed by the immune system for modulating inflammatory signaling is the spatio-temporal regulation of inflammatory gene expression. Human intestinal epithelial cells and murine intestinal organoids express very low TLR2 levels and are broadly unresponsive to TLR ligands (25, 26). Similarly, TLR4 was shown to be expressed at low levels in vaginal, ectocervical, and endocervical epithelial cells of the female reproductive tract (27). This likely serves to prevent chronic inflammatory cytokine secretion in the context of the continuous exposure of these cell types to the rich microflora communities at the mucosal interfaces of the intestinal tract and female reproductive system. The phenomenon of endotoxin tolerance highlights the complex temporal regulation of inflammatory responses. Chronic exposure to LPS renders myeloid cells unresponsive to a renewed challenge with LPS (28). Downregulation of TLR4 cell surface expression and downstream signaling molecules may represent one of the mechanisms underlying endotoxin tolerance (29). This immunosuppressed state is believed to protect against excessive inflammation during Gram-negative infections but also is thought to contribute to mortality in sepsis patients (30). Decoy receptors represent a fourth frequently encountered mechanism to modulate inflammation. Because they lack the domains necessary to transduce the inflammatory signal, decoys dampen inflammation by acting as molecular traps that scavenge the inflammatory factor away from its signaling receptor. The IL-1 type 2 receptor (IL-1R2) is encoded © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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on human chromosome 2q12 and is expressed as either a soluble or membrane-bound isoform by myeloid cells and lymphocytes. Both isoforms potently bind IL-1a and IL-1b to prevent engagement of the signaling receptor IL-1R1 (31–33). IL-1R2 also acts by sequestering the IL-1 receptor accessory protein (IL-1RAcP) that is necessary for signal transduction by IL-1R1 (34). Similarly, secreted IL-18 binding protein (IL-18BP) is produced from a gene on human chromosome 11q13 and it effectively neutralizes IL-18 activity in circulation by preventing its binding to IL-18 receptor (IL-18R) (35). Decoy receptors that arise by alternative splicing of signaling receptor pre-mRNAs also exist. Good examples are the soluble TLR2 and -4 isoforms that lack the transmembrane and intracellular domains normally found in the full-length proteins (36, 37). Soluble TLR2 was recently shown to prevent the damaging consequences of TLR2 overactivation without compromising in vivo clearance of the Gram-positive bacterial pathogen Staphylococcus epidermidis (38). Dominant negative inhibitors represent another large class of molecules that modulate innate immune signal transduction. These proteins generally interfere with inflammatory signal transduction cascades by physically interacting with the immune receptor or crucial downstream adapter molecules, thereby preventing the inflammatory signal to be relaying. For instance, studies in Toll/IL-1R8 (TIR8)/SI-

GIRR-deficient mice established its critical role in regulating TLR- and IL-1R1-mediated inflammatory responses by interfering with the recruitment of TIR domain-containing adapter molecules such as IRAK1 and TRAF6 to the receptive receptor complexes (39–41). The short splice form of the TLR/IL-1R adapter MyD88 also acts as a dominant negative inhibitor of these pathways. MyD88s lacks the intermediate domain of the full-length protein and prevents NF-jB activation at the level of MyD88 and IRAK4 recruitment (42). An endoplasmic reticulum-targeted isoform of the TLR4 adapter TIR-domain-containing adapter protein inducing IFNb (TRIF)-related adapter molecule (TRAM) competes with TRIF for binding to TRAM, thereby inhibiting LPSinduced IFNb production (43). Proteins that are composed solely of a pyrin or related caspase recruitment domain (CARD) motif have emerged as a potentially new class of primate-specific dominant negative immune regulators. Their genes originated from inflammatory gene duplications during evolution of the primate lineage. The first three identified card-only proteins (COPs), COP/Pseudo-ICE (CARD16) (44, 45), INCA (CARD17) (46), and ICEBERG (CARD18) (47), all arose from recent caspase-1 gene duplications (48) (Fig. 1). Functional studies highlighted their potential roles in modulating inflammasome activation and NF-jB signaling. Additional COPs and pyrin-only proteins (POPs) that regulate these inflammatory

Fig. 1. Classification and chromosomal location of human COPs and POPs. Several lines of evidence suggest that the genes encoding human COPs and POPs originated from the duplication or transposition of fragments of pre-existing genes. The genes encoding the COPs COP/PseudoICE, INCA and ICEBERG cluster together with CASP1, CASP5, CASP4 and CASP12 on human chromosome 11q22. COP, INCA and ICEBERG share a great degree of similarity to the CARD domain of CASP1. PYDC1 encoding POP1 likely originates from the PYD-domain-encoding region of the PYCARD gene, and PYDC2 arose from the PYD-domain-encoding region of NLRP2. POP3 is located in between the IFI16 and PYHIN1 genes in close proximity to AIM2 on chromosome 1q23. PYDC3 that encodes POP3 has genomic organization and sequence that is homologous to AIM2, suggesting that it may have been formed consequent to duplication of the PYD-domain-encoding region of AIM2. The NLRP2P gene that encodes POP4 likely originated from retrotransposition of an NLRP2/NLRP7-homologous mRNA. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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signaling axes have been discovered, and several viruses were recently shown to encode POPs that interfere with inflammasome and NF-jB activation (49). In the following paragraphs, we review and discuss the potential mechanisms by which COPs and POPs may regulate inflammatory and immune responses. CARD-only proteins The CARD motif together with the structurally related pyrin domain (PYD), death domain (DD), and death effector domain (DED) comprise the death domain superfamily. All members of this superfamily are characterized by a similar secondary protein structure that consists of six or seven antiparallel a-helices (50). They engage in homotypic proteinprotein interactions with the other members of the same subfamily. Each protein containing a death fold motif thus may interact with another protein that carries a similar domain, and so a PYD interacts with a PYD, a CARD with a CARD, a DED with DED, and a DD with DD. Importantly, these death fold motifs allow immune and death receptors to recruit intracellular adapters and effector proteins to assemble large multi-protein complexes such as the apoptosome (51), death-inducing signaling complex (DISC) (52), and inflammasomes (53) that promote caspase activation and/or relay signals that culminate in NF-jB activation. Caspase-1 COPs COP/Pseudo-ICE (CARD16) (44, 45), INCA (CARD17) (46), and ICEBERG (CARD18) (47) represent a first subgroup of COPs. They appear to be a relatively new mechanism for modulating inflammatory and immune signaling, as the genes encoding these COPs are absent in the genetic codes of rodents (48). They are relatively short proteins of approximately 100 amino acids that essentially comprise a CARD domain. They share a common origin as all three arose from the caspase-1 gene by a series of recent gene duplication events that occurred after the bifurcation of rodents and primates (48). Orthologs of CARD16 (98% amino acid identity) and CARD17 (95% amino acid identity) are encoded in the chimpanzee genome, indicating that the duplications that gave rise to these caspase-1 COPs occurred before the split of the primate and hominid lineages. Notably, CARD18 is absent from the chimpanzee genome (54) but present in that of rhesus monkeys (100% amino acid identity), which diverged earlier from the human–chimpanzee lineage. This suggests that ICEBERG was deleted in chimpanzees after bifurcating from humans.

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The genes encoding COP/Pseudo-ICE, INCA, and ICEBERG are mapped to human chromosome 11q22.3 adjacent to caspase-1 (Fig. 1). Given the high sequence homology shared with the caspase-1 CARD motif, COP/Pseudo-ICE, INCA, and ICEBERG are thought to act as dominant negative inhibitors of caspase-1 activation. This inflammatory protease undergoes proximity-induced auto-activation in inflammasomes, cytosolic scaffolds that are assembled in a pathogen- or stimulus-dependent manner (53). Several inflammasomes are distinguished with those assembled by NLRP1, NLRP3, NLRC4, and AIM2 among the best characterized. NLRP3 triggers inflammasome formation in response to a diverse set of cellular stress signals, including bacterial, viral, and fungal pathogens and medically relevant crystals and particles (53). The Nlrp1b inflammasome responds to anthrax lethal toxin (LeTex), a virulence factor of Bacillus anthracis, and caspase-1 activation in this complex does not require its autoproteolysis (55). The NLRC4 inflammasome responds to intracellular bacterial pathogens such as Salmonella enterica serovar Typhimurium, Shigella flexneri, Pseudomonas aeruginosa, Burkholderia thailandensis, and Legionella pneumophila that express flagellin and/or a type III secretion system (53). The AIM2 inflammasome senses Francisella tularensis, vaccinia virus, and a number of additional DNA viruses by means of its HIN200 domain that detects bacterial and viral DNA in the cytosol of infected cells. Finally, the Pyrin inflammasome activates caspase-1 in response to infection with Burkholderia cenocepacia, an important cause of respiratory inflammation in cystic fibrosis patients (56). This inflammasome specifically responds to Rho-inactivating toxins of Clostridium difficile, Vibrio parahaemolyticus, Histophilus somni, Clostridium botulinum, and Burkholderia cenocepacia (57). Once activated, caspase-1 proteolytically matures pro-IL-1b and pro-IL-18 into their secreted forms, and it commits the cell to inducing a pro-inflammatory and lytic cell death mode termed pyroptosis (3). ICEBERG shares 52% amino acid sequence identity with caspase-1 CARD, and both proteins were shown to be highly expressed in the heart, placenta, and other tissues. Moreover, ICEBERG expression is highly upregulated by LPS and other pro-inflammatory stimuli, and its expression in THP-1 cells inhibited LPS-induced caspase-1 activation and IL-1b secretion (45, 47). This is thought to result from the efficient interaction of ICEBERG with caspase-1 CARD (Fig. 2), although it differs from COP/Pseudo-ICE in that it does not bind the NF-jB-stimulating kinase receptor-interacting serine/threonine-protein kinase 2 (RIPK2) (45, 47). Taken together, these observations demonstrate that ICE© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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Fig. 2. Modulation of inflammasome signaling by COPs and POPs. Inflammasomes are cytosolic multi-protein complexes that are assembled in response to conserved pathogen-and damage-associated molecular patterns. Interactions between inflammasome components rely on homotypic CARD-CARD and PYD-PYD interactions. The COPs COP/Pseudo-ICE, INCA, ICEBERG, CARD8 and caspase-12 were all shown to interfere with inflammasome-mediated caspase-1 maturation and IL-1b secretion. Also human POP2 and the poxvirus POPs M013L and gp013L inhibit NLRPmediated recruitment of ASC. POP3 binds specifically to the PYD domain of the ALR AIM2, thereby inhibiting inflammasome assembly by preventing ASC recruitment.

BERG acts as a dominant negative regulator of caspase-1 activation to help balance the levels of secreted IL-1b. Interestingly, ICEBERG expression levels were suggested to inversely correlate with the risk for cardiovascular disease because patients with increased risk for left ventricular assist device (LVAD) implantation showed significantly lower levels of cardiac ICEBERG mRNA and increased levels of caspase-1 and proIL-1b mRNA when compared to stable heart failure patients that did not require LVAD implantation (58). COP/Pseudo-ICE is closely related to caspase-1 CARD with a protein sequence identity of 92% (45). Caspase-1 and COP/Pseudo-ICE show a nearly identical expression pat© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

tern, suggesting that the transcriptional regulation of the two genes may be very similar (45). Similar to ICEBERG (45, 47), COP/Pseudo-ICE also self-associated. However, in contrast to ICEBERG, ectopically expressed COP/Pseudo-ICE co-immunoprecipitated both caspase-1 and RIPK2 (45). By engaging RIPK2 and the IKK complex, COP/Pseudo-ICE stimulated NF-jB activation (45, 59) (Fig. 3). Moreover, secretion of IL-1b was hampered when COP/Pseudo-ICE was co-expressed with caspase-1 and pro-IL-1b (44, 45) (Fig. 2). These two functional outcomes may compete because human procaspase-1 variants with decreased enzymatic activity were shown to more effectively promote

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Fig. 3. COPs regulating NOD1 and NOD2 signaling. Peptidoglycan recognition by the NOD-like receptors (NLRs) NOD1 and NOD2 triggers recruitment and ubiquitination of receptor interacting protein kinase 2 (RIPK2), which subsequently leads to the activation of transforming growth factor b (TGFb)-activated kinase 1 (TAK1). The COP CARD8 binds to NOD1 and NOD2 to inhibit downstream signaling. NOD2-S and caspase-12 also hamper NOD1- and NOD2mediated NF-jB activation. Conversely, NOD2-C2 and COP/PseudoICE were shown to enhance NOD1- and NOD2-mediated NF-jB activation.

inflammation through RIPK2 and NF-jB than wildtype procaspase-1 (60). Notably, COP/Pseudo-ICE reduced RIPK2 binding to caspase-1, consequently leading to inhibition of RIPK2-mediated caspase-1 activation and NF-jB activation (44, 45). This observation initially was somewhat puzzling given that the CARDs of COP/Pseudo-ICE, RIPK2 and caspase-1 were thought to be complementary (61). However, a detailed structure-function analysis clarified these observations by demonstrating that the two oppositely

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charged surface patches of caspase-1 CARD contain two distinct interaction sites that differentially interacted with RIPK2 and the inflammasome adapter ASC (62). More work is needed to characterize the potential roles of COP/PseudoICE in modulating inflammatory responses in inflammatory and infectious diseases. INCA (Inhibitory CARD) is the third member of the human caspase-1 COP subfamily. It shares 81% amino acid sequence identity with caspase-1 CARD (46). The mRNA expression profile of INCA differs significantly from that of caspase-1 in that INCA expression was detected both in tissues with high and low caspase-1 expression levels (46). However, the transcriptional mechanisms regulating expression of the two genes appear common, at least to some extent, because both caspase-1 and INCA mRNAs were markedly upregulated by IFNc in the human monocytic cell lines THP-1 and U937, whereas LPS and TNF treatment had little effect (46). Coimmunoprecipitation studies showed that INCA is able to selfassociate, that it interacted with COP/Pseudo-ICE and ICEBERG, and that it could bind to procaspase-1 (46). Consequently, INCA efficiently inhibited LPS-induced secretion of IL-1b from THP-1 monocytes (46), most likely by preventing recruitment and activation of procaspase-1 in inflammasomes (Fig. 2). However, INCA failed to interact with RIPK2, and it was unable to modulate RIPK2-induced NF-jB signaling, a feature shared with ICEBERG but not COP/Pseudo-ICE (45– 47). This differential regulation of NF-jB signaling by, respectively, caspase-1 CARD and COP/Pseudo-ICE, but not INCA and ICEBERG, formed the basis for identification of two critical positions, aspartic acid at position 27 (Asp27) and arginine at position 45 (Arg45), in caspase-1 CARD for stimulating NF-jB activation (62). Mutating these residues to, respectively, glycine (Asp27Gly) or aspartic acid (Arg45Asp) abrogated the ability of caspases-1 CARD to stimulate NF-jB activation (62). Arg45 is located in a basic surface patch and proved critical for interacting with RIPK2. Moreover, the mutation precluded auto-oligomerization of procaspase-1 (62). Conversely, Asp27, which is located in an acidic surface patch, was not important for RIPK2 recruitment and oligomerization of procaspase-1 (62). Thus, although Asp27 and Arg45 both were required for stimulating NF-jB signaling, Arg45 sufficed to trigger caspase-1 CARD oligomerization and RIPK2 recruitment. The role of Asp27 was clarified by its critical role in recruiting ASC. Mutation of Asp27 precluded caspase-1 from interacting with ASC and inducing IL-1b maturation (62). Moreover, ASC recruitment proved to exert a strong synergistic effect on RIPK2 binding to caspase-1 and consequently its ability to drive NF-jB activation (62). Inter© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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estingly, COP/Pseudo-ICE contains both Asp27 and Arg45, whereas INCA has an Asp27Gly mutation, implying that the latter may be unable to potently trigger NF-jB because of its failure to recruit ASC. On the other hand, ICEBERG may be impaired in RIPK2 recruitment because it holds an Arg45Asp mutation. However, co-immunoprecipitation studies addressing the potential interactions of ASC with ICEBERG, COP/ Pseudo-ICE and INCA are needed to validate this hypothesis and shed more light on the mechanisms by which caspases-1 COPs modulate inflammasome and NF-jB signaling. Human caspase-12 Human caspase-12 resides on chromosome 11q22, adjacent to caspase-1 and the genes encoding the caspase-1 COPs COP/ Pseudo-ICE (CARD16) (44, 45), INCA (CARD17) (46) and ICEBERG (CARD18) (47) (Fig. 1). Whereas mouse caspase12 is a genuine protease (63), human caspase-12 acquired mutations that are thought to impede its proteolytic activity (63, 64). The caspase-12 gene of Caucasians and most people around the globe has acquired a C>T single nucleotide polymorphism (SNP) (rs497116) that results in a premature stop at amino acid position 125 (64). Intriguingly, a fraction of people of North and Sub-Saharan African, Middle Eastern, and South-East Asian descent encodes a readthrough mutation at this position that allows expression of full-length caspase-12 with frequencies ranging from 4% up to 60% in some Sub-Saharan ethnic groups (63, 65–67). However, this long form likely lacks proteolytic activity as well because the evolutionarily conserved catalytic SHG box normally found in caspases is mutated to SHS in human caspase-12 (64), and mutation of the SHG box in human caspase-1 abolished its ability to process proIL-1b (68). Interestingly, the caspase-12 genes of chimpanzees, Rhesus monkeys, and gibbons encode intact SHG boxes and do not contain the premature stop-causing rs497116 SNP, implying that these mutations in humans are evolutionarily recent and occurred after the split of the hominid and primate lineages. In agreement, bio-informatics analyses suggested that the short isoform of human Casp12 was under strong selective pressure from the environment and this led to its nearly complete fixation in modern humans that migrated out of Africa some 60–100 thousand years ago (67). However, the nature of the environmental factor(s) that selected for the short isoform of human caspases-12 is controversial. For instance, increasing pressure of sepsis as a result of a growing population size and the colonization of new continents by modern humans that migrated out of Africa was pro© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

posed to have selected for the short isoform of human caspases-12 (65), but another study failed to observe significant differences in the cytokine profiles of Africans expressing the short or long isoforms of caspases-12 (69). Several other infectious diseases such as the plague and malaria have been proposed as well, but these were unable to explain the peculiar geographic distribution of human caspases-12 alleles (69, 70). Thus, further research is needed to explain the remarkable global distribution of caspases-12 alleles and to shed light on the environmental factors that may be responsible for the marked selection of the short caspase-12 isoform. The analysis of caspase-12-deficient mice has suggested a number of mechanisms by which this caspase may regulate inflammatory and cell death responses. It was originally implicated in inducing endoplasmic reticulum (ER) stressinduced apoptosis (71), but this was subsequently contested by other studies (72, 73). Caspase-12 was also shown to act as a dominant negative inhibitor of caspase-1, thereby causing impaired inflammasome signaling (73) (Fig. 2). In this regard, caspase-12 knockout mice that were subjected to the colon ascendens stent peritonitis (CASP) model of bacterial sepsis cleared bacteria more effectively and they were more resistant to CASP-induced lethality relative to caspase-12expressing control mice (73). Conversely, caspase-12-deficient mice were hypersensitive to LPS-induced endotoxemia with 90% of the caspase-12 knockout group succumbing to shock in the first 72 h (73). Aside from its role in regulating inflammasome responses, caspase-12 was shown to recruit RIPK2 away from the IjB kinase (IKK) complex, thereby inhibiting MAPK and NF-jB signaling (74) (Fig. 3). Consequently, caspase-12 knockout mice cleared the malaria parasite Plasmodium chabaudi more effectively than wildtype mice because of enhanced NF-jB activation levels (75). Caspase-12 also was suggested to augment type I interferon (IFN) production by the RIG-I pathway, hence leading to increased IFNb production in response to West Nile virus infection (76). In conclusion, mouse caspase-12 has been implicated in several innate immune pathways, hinting to an important role in regulating inflammatory responses. CARD8 CARD8 distinguishes itself from other COPs in that it contains a function-to-find (FIIND) domain upstream of the carboxy-terminal CARD motif. The FIIND motif is uniquely found in CARD8 and the NLR protein NLRP1, which assembles an inflammasome in response to anthrax lethal toxin

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(53). Interestingly, CARD8 is thought to have emerged from a partial duplication of the NLRP1 gene that gave rise to a novel protein containing related FIIND and CARD motifs. Notably, rodents do not encode a CARD8 gene, unlike humans and primates (77). Human CARD8 was demonstrated to inhibit NF-jB activation by sequestering the regulatory IKK component IKKc/NEMO (78). It was also shown to interact with the nucleotide-binding domains (NBDs) of NOD2 and NLRP3, thereby inhibiting muramyl dipeptideinduced NF-jB activation in intestinal epithelial cells (79) (Fig. 3) or promoting IL-1b production by the NLRP3 inflammasome (80), respectively. On the other hand, the CARD of CARD8 shares a high degree of homology to caspase-1 CARD. The two proteins were shown to interact, and this inhibited caspase-1-mediated IL-1b secretion in THP-1 cells (81) (Fig. 2). Taken together, these observations suggest that human CARD8 may function as a dominant negative regulator of NF-jB signaling and that it may positively or negatively regulate caspase-1 activation in inflammasomes. Notably, CARD8 genomic variants have been associated with increased risk for cancer, rheumatoid arthritis, inflammatory bowel disease, and several other inflammatory diseases, warranting further analysis of its functions. NOD2-S and NOD2-C2 The NLR family member NOD2 is a well-characterized cytosolic immune receptor that activates NF-jB in response to bacterial muramyl dipeptide (82). NOD2-S and NOD2-C2 are COPs that originate from the NOD2-encoding CARD15 gene. NOD2-S (short) contains the first CARD and part of the second CARD of full-length NOD2 (83), whereas NOD2-C2 corresponds to the two CARD motifs (84). NOD2-C2 mRNA is ubiquitously expressed with highest expression levels found in placenta and leukocytes (84). NOD2-C2 is able to induce NF-jB activation in the absence of muramyl dipeptide (Fig. 3), in agreement with the notion that the NBD and LRR motifs of full-length NOD2 suppress NF-jB signaling in the absence of the NOD2 ligand (82, 84). NOD2-C2 may also regulate muramyl dipeptideinduced NOD2 signaling by acting as a dominant negative version of the full-length molecule (84). Unlike NOD-C2, the NOD2-S isoform inhibits NF-jB activation when ectopically expressed (Fig. 3), although it is capable of recruiting RIPK2 (83). This is in agreement with previous work showing that NOD2 requires both its CARD motifs to recruit RIPK2 and activate NF-jB (82). NOD2-S transcripts result from exclusion of the third exon, which

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subsequently leads to a premature stop codon due to a frameshift in exon 4 (83). NOD2-S mRNA was found to be expressed at high levels in the human colon samples, and its expression was further upregulated by the anti-inflammatory cytokine IL-10 (83). NOD2-S inhibits NOD2-mediated NFjB activation as well as NOD2- and RIPK2-mediated secretion of IL-8 and IL-1b when ectopically expressed in 293T cells (83). Together, these observations suggest alternative splicing of NOD2 as a powerful mechanism to modulate NOD2-mediated signaling and prevent overt NOD2-driven inflammatory tissue damage in the intestinal tract. Pyrin-only proteins A major challenge in investigating the physiological importance of COPs and POPs is that both have evolved rather recently in evolution as evidenced by their apparent absence from the mouse genome. However, POPs are increasingly recognized as a new class of dominant negative proteins that may potentially modulate inflammatory responses through a number of mechanisms. The human genome encodes 4 distinct POP genes, known as PYDC1 (85), PYDC2 (86, 87), PYDC3 (88) and NLRP2P (89), that may modulate inflammasome and NF-jB signaling by interfering with PYD-PYD interactions in these pathways. Because of the high degree of sequence similarity shared with PYD-motifs in other proteins, human POPs are thought to have originated from recent gene duplication or retro-transposition events (Fig. 1). POP1 resembles the PYD domain of ASC, POP2 has homology to NLRP2 PYD, POP3 is reminiscent of the AIM2 and IFI16 PYD motifs, and POP4 is thought to have arisen from an NLRP2/7 ancestral gene. In addition, several virally encoded POPs were recently identified. We discuss each of these POPs in additional detail in the following paragraphs. Human POP1 The POP1 mRNA contains a 270 bp long open reading frame that is transcribed from the PYDC1 gene on chromosome 16p12.1 (85). The gene lies in close proximity of the PYCARD gene that encodes the inflammasome adapter ASC (Fig. 1). Notably, the structural organization of PYDC1 gene is highly similar to the 50 portion of the PYCARD gene that encodes the PYD domain of ASC, suggesting that PYDC1 resulted from a partial gene duplication of PYCARD (85). In agreement, the PYD motif of POP1 shares 64% amino acid sequence identity with ASC PYD. POP1 was predominantly expressed in monocytes, macrophages, and granulocytes, in concordance with © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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an immune-modulating role for the protein (85). Ectopically expressed POP1 co-localized with ASC in specks and it coimmunoprecipitated with the ASC PYD domain (85). The interaction of POP1 with ASC PYD was studied in great detail by NMR. This analysis revealed extended positively and negatively charged surfaces at opposite ends of the POP1 PYD domain (90), a feature shared with the resolved ASC PYD (91) and NLRP1 PYD (92) structures. The strong electrostatic dipole moment suggests that electrostatic interactions are important in homotypic PYD-PYD interactions. Indeed, mutagenesis experiments demonstrated that the negatively charged residues Asp6, Glu13, Asp48, and Asp54 located in the H1 and H4 a-helices of ASC PYD interact with positively

charged residues in the H2 and H3 a-helices of POP1 (93). These observations suggest that POP1 may modulate inflammatory responses as a dominant negative inhibitor of ASC. However, rather than inhibiting inflammasome signaling, POP1 overexpression turned out to enhance ASC-dependent IL-1b secretion (85). In addition, it led to robust inhibition of TNF-, IL-1b-, NOD1-, and Bcl-10-induced NF-jB activation. The latter was explained by its ability to bind to IKKa and IKKb, thus impairing their kinase activity (85) (Fig. 4). Further work addressing the physiological roles of POP1 by gene ablation may highlight the precise roles of POP1 in inflammatory signaling and its potential contributions to inflammatory diseases.

Fig. 4. POPs modulating the TLR4 signaling pathway. TLR4 is present on the plasma membrane and in endosomes. LPS recognition induces dimerization of TLR4-MD2 dimers, which triggers recruitment of the downstream adapter proteins myeloid differentiation primary-response protein 88 (MyD88) and MyD88-adapter-like protein (MAL), and TIR-domain containing adapter protein inducing IFNb (TRIF) and TRIF-related adapter molecule (TRAM) that engage MyD88-dependent and -independent signaling, respectively. Signaling proteins of the IL-1R-associated kinase (IRAK) and TNF receptor-associated factors (TRAF) families are also recruited. This results in activation of mitogen-associated protein kinase (MAPK), nuclear factor-jB (NF-jB), and interferon-regulatory factor (IRF) activation. POP1 binds to inhibitor of NF jB (IjB) kinase a and b (IKKa and IKKb), thus impairing NF-jB activation. POP2 and POP4 interfere with p65 transactivation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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Human POP2 POP2 transcripts are produced from the single-exon PYDC2 gene that is located at the telomeric end of chromosome 3 (3q28) (86, 87). POP2 mRNA expression was detected primarily in myeloid and lymphoid cells, testis, and placenta (87). In addition, it was expressed in several human hematopoietic cell lines, including the leukemia cell line K526, the monocytoid macrophage cell lines U937 and THP-1, the Ramos B and Jurkat T lymphoma cells, and the promyelocytic leukemia cell line HL-60, although they appeared virtually absent from 293T cells (86, 87). POP2 transcripts were upregulated by pro-inflammatory stimuli such as LPS, TNF, and PMA (86, 94), suggesting a role for POP2 in modulating inflammatory responses. In agreement, ectopic expression of POP2 inhibited TNF-induced NF-jB activation in 293T, HeLa and COS-7 cells (87, 94) (Fig. 4). Moreover, LPS- and Pam3CSK4-induced cytokine secretion was reduced in mouse J774A.1 cells that stably expressed POP2 (87). POP2 consists of a PYD domain of 97 amino acids that shares highest sequence homology with the NLRP2 PYD motif (67% amino acid sequence identity) (86, 87). This suggested that it also may interfere with inflammasome signaling. Indeed, overexpressed POP2 interacted with the PYD domains of ASC and NLRP2, and inhibited caspase-1 maturation and IL-1b secretion by the NLRs NLRP1, NLRP2, and NLRP3 (86, 87, 94) (Fig. 2). Interestingly, mutagenesis studies showed the first a-helix of POP2 to be sufficient for inhibiting NLRP3 inflammasome activation (94). The acidic residues Glu6, Asp8, and Glu16 in this a-helix proved critical for inflammasome inhibition (94). Consistent with the notion that PYD-PYD interactions involve oppositely charged electrostatic surface patches (91), the requirement for acidic residues in POP2 also suggests that POP2 interacts with the positively charged surface patch of the ASC PYD motif (94). Interestingly, the first a-helix of POP2 also was required for NF-jB inhibition, but the acidic residues that interacted with ASC were dispensable (94). This suggests that POP2 utilizes partially overlapping but distinct surface regions of the first a-helix to interfere with inflammasome and NF-jB signaling, respectively. Although further testing at the endogenous level is needed, these observations suggest that POP2 may represent a general modulator of ASC-dependent inflammasome activation (Fig. 2) and NF-jB signaling (Fig. 4). Human POP3 POP3 is a recently identified human POP of 113 amino acids that is encoded by the PYDC3 gene in the IFN-inducible gene

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cluster on chromosome 1q23 that contains the genes for the ALR proteins IFI16, PYHIN1, and AIM2 (88) (Fig. 1). Similar to these ALRs, POP3 mRNA expression was highly upregulated by IFN-b in primary macrophages and several human monocytic cell lines (88). Moreover, these ALRs share high sequence homology with the POP3 PYD motif, with the AIM2 PYD domain having 61% sequence identity with POP3. It is therefore thought that PYDC3 may have arisen from a partial gene duplication of the AIM2 gene. This structural association with ALRs is also reflected in its functional roles. POP3 was demonstrated to interact with the PYD domains of AIM2 and IFI16, but not with those of ASC and NLRP3 (88). Moreover, POP3 co-localized with endogenous AIM2 and IFI16 in human primary macrophages that had been infected with modified vaccinia virus Ankara (MVA) and Kaposi’s sarcomaassociated herpesvirus, respectively (88). Consistently, POP3 disrupted ASC-AIM2 interactions in MVA-infected THP1 cells and in inflammasome reconstituted 293T cells. In addition, siRNA-based knockdown of POP3 in primary macrophages enhanced AIM2-mediated IL-1b and IL-18 secretion in response to MVA infection and poly(dA:dT) transfection, whereas NLRP1, NLRP3, and NLRC4 inflammasome-dependent cytokine secretion remained unaltered (88). Together, these observations establish POP3 as a specific dominant negative regulator of ALR-induced inflammasome activation that competes with ASC for binding to the PYD domains of AIM2 and IFI16 (Fig. 2). These results were validated in macrophages of transgenic mice that express POP3 from a myeloid/macrophage-specific CD68 promoter. Poly(dA:dT) transfection of thioglycollate-elicited peritoneal macrophages from these mice elicited reduced amounts of secreted IL-1b, and bone marrow-derived macrophages had impaired caspase-1 cleavage, IL-1b and IL-18 secretion in response to AIM2 stimuli, but no other inflammasome triggers. In agreement, POP3transgenic mice had impaired AIM2- and viral DNA-dependent host defense responses in terms of decreased IL-1b and IL-18 serum levels and IFNc production when infected with mouse cytomegalovirus (88). Together, these results establish POP3 as a type I IFN-induced POP that regulates AIM2 inflammasome activation in humans. Human POP4 The NLRP2P gene that encodes POP4 on chromosome Xp11 was initially described as a processed pseudogene in humans and higher primates that originated from retro-transposition of an NLRP2/7-like mRNA (95). However, NLRP2P was recently shown to produce a transcript that is ubiquitously © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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expressed in human tissues and upregulated by LPS stimulation in monocytes (89). NLRP2P transcripts encode POP4, a relatively short protein of 45 amino acids with high sequence homology to POP2. POP4 is likely composed of only two a-helices, the first of which is nearly identical to the first a-helix of POP2 with the exception of the Glu6 and Glu16 residues in POP2 that have been implicated in ASCdependent inflammasome modulation (94). This suggested that POP4 may specifically interfere with NF-jB activation, whereas POP2 inhibited both inflammasome and NF-jB signaling (86, 87, 94). Indeed, ectopic expression of POP4 in 293T cells significantly reduced TNF-induced NF-jB activation, but it failed to modulate inflammasome responses (89). Consistently, macrophage cell lines that stably expressed POP4 responded to TLR2 and TLR4 stimulation with significantly reduced NF-jB-dependent IL-6 and TNF gene transcription, possibly by interfering with RelA/p65 phosphorylation (89) (Fig. 4). Myxoma virus M013L Myxoma virus is a poxvirus that causes myxomatosis in rabbits (96). The virus triggers a generalized disseminated infection that is characterized by the formation of necrotic lesions called myxomas. Infected rabbits die shortly after inoculation, at least in part because of supervening bacterial infections concomitant with suppression of the host’s immune system. The ability of Myxoma virus and other poxviruses to cause immunosuppression results from the concerted action of a range of immunomodulatory proteins encoded by in the viral genome(97). During evolution, these and other viruses have pirated and modified hostderived genes to subvert the host’s innate and adaptive immune responses (98). One of these virulence genes, the rabbit Myxoma virus M013L gene encodes a 126 amino acid protein product of which the first 81 residues compose a PYD domain that resembles that of human POP1 and ASC (99). Deletion of M013L attenuated Myxoma virus virulence in rabbits because the mutant virus caused less secondary lesions, failed to induce lethality and was quickly resolved. This was correlated with the enhanced induction of acute inflammatory responses at the primary site of infection as reflected in the increased production of pro-inflammatory cytokines such as IL-1b, IL-6, and macrophage chemoatraction protein 1 relative to the levels induced by infection with wildtype Myxoma virus (99). Impaired virulence and attenuated dissemination of M013L-deficient Myxoma virus further © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

correlated with its decreased ability to infect monocytes and lymphocytes. In the absence of M013L, the poxvirus is sensed by NLRP3 and several TLRs in infected THP-1 cells to induce inflammasome- and NF-jB-mediated protective responses, respectively (100). This suggests that M013L may interfere directly with inflammasome and NF-jB signaling. In agreement, ectopically expressed M013L co-localized and physically interacted with the ASC PYD domain, thus inhibiting caspase-1 activation and secretion of IL-1b and IL-18 by several inflammasome stimuli (99). M013L simultaneously interfered with TLR2, TLR6, TLR7, and TLR9dependent antiviral responses by binding to NF-jB1/p105 to prevent nuclear translocation of p65 (100, 101). Thus, M013L appears to have evolved to simultaneously interfere with NLRP3 inflammasome-mediated (Fig. 2) and TLR-mediated antiviral responses (Fig. 4). Shope Fibroma virus gp013L and other poxvirusencoded vPOPs Myxoma virus is not the only poxvirus known to produce a viral POP protein that interferes with inflammasome and NF-jB activation. Other members of the Poxviridae family that have been shown to produce viral POPs (vPOPs) include Shope Fibroma virus (vPOP gene: S013L, vPOP protein gp013L) (102), Swinepox virus (vPOP: SPV14L) (103), Yaba-like disease virus (vPOP: 18L) (104), and Mule deer poxvirus (vPOP: DPV83gp024) (105), all of which bear significant sequence homology to Myxoma virus M013L. For instance, the Shope Fibroma virus vPOP gp013L is a protein of 107 amino acids that shares 59% sequence identity with Myxoma virus M013L. Like human POP1 and Myxoma virus M013L, Shope Fibroma virus gp013L interacted with ASC in vitro and they co-localized in perinuclear specks in cells. Moreover, gp013L inhibited IL-1b secretion in inflammasome-reconstituted cells (106), demonstrating its ability to act as a dominant negative inhibitor of PYD-mediated signal transduction pathways (Figs 2 and 4). Future perspectives and outstanding questions A decade of research into COPs and POPs has revealed their extensive roles in modulating inflammatory and immune signaling. They originated from recent gene duplications, explaining their apparent absence in rodents. Both COPs and POPs have been implicated in modulating inflammasome and NF-jB activation pathways to safeguard homeostasis. Apart from the caspase-1 COPs COP/Pseudo-ICE, INCA, and ICEBERG, dominant negative regulators that modulate in-

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flammasome and NF-jB signaling have been identified at the level of the corresponding NLRs (CARD8, NOD2-S, NOD2-C2, POP2) as well as ASC (POP1) and caspase-1 (human caspase-12). This suggests a tight network of regulatory factors, and future work may potentially uncover their physiological roles and associations with disease. Given that COPs and POPs are limited to primates and humans, their functions have so far mainly been analyzed by overexpressing or stably-expressing cell lines. Further studies addressing the roles of these regulators at the endogenous level and in vivo are required to decipher their physiological functions. Not only humans and primates but also viruses were shown to encode POPs that are capable of modulating immune signaling in infected host cells. The poxvirus

M013L protein for instance nicely illustrates how a single POP is able to inhibit both inflammasome and NF-jB activation (101). Although myxoma virus causes lethal disease in rabbits, it is nonpathogenic to humans. However, it may be a prime candidate for cancer virotherapy, given that the virus is able to replicate in human cancer cells (100). It also suggests that viral POPs may be employed to therapeutically modulate NF-jB and inflammasome signaling in inflammatory disorders. Past research has revealed the existence of COPs and POPs and suggested their involvement in regulating inflammasome and NF-jB signaling. The future may see an increased understanding of their (patho) physiological roles and potentially novel therapeutic applications for inflammatory and autoimmune diseases may emerge.

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