[1] 6w CATERPILLERs, pyrin and hereditary immunological disorders

[1] 29w DorsalRel/nuclear factor-κB transcription factor family member found in Drosophila melanogaster. Dorsal-related immunity factor(DIF). A Drosophila melanogaster transcription factor that is a Rel-homologue, and is important for dorsalventral axis determination.

[1] 101w (SNP). Common DNA sequence polymorphisms among individuals. acidic, pyrin, CARD, baculoviral inhibitory repeat (BIR)-like 17,18 and unclassified (FIG. 1; TABLE 1). The largest pyrin-containing subgroup, or members of this group, are also known by various names including PYPAF, PAN, or NALP [19][20][21][22] (TABLE 1). In addition to their evolutionary preservation and implicated roles in immunity, CATERPILLER proteins share certain features of TLRs. Both TLRs and CATERPILLERs contain LRR sequences, have a role in the sensing of microbial products, and regulate inflammatory and adaptive immune responses. Therefore, there is much excitement concerning the roles of members of the novel CATERPILLER gene family.

[2] 51w Although the term 'master regulator' is frequently used under relaxed criteria, CIITA fits the description of a master regulator from several perspectives 65 . First, the lack of CIITA in both humans and mice results in a severe reduction or elimination of Box 1 | Disease resistance (R) proteins of plants.

[3] 183w Immune defence in plants is primarily mediated by plant-disease-resistance (R) proteins, which are polymorphic yet structurally conserved molecules that mediate immune recognition of pathogenic products from bacteria, fungi, viruses, nematodes, insects and even insecticides. The largest subgroup of R proteins has either a Toll interleukin-1 receptor (TIR) or coiled-coil domain, followed by a mid-section nucleotide-binding domain (NBD), and an amino (N)-terminal leucine-rich repeat (LRR) (FIG. 1). The pathogen-derived avr (avirulence) molecules that are recognized by R proteins are encoded by avr genes. This name reflects the observation that recognition of an avr product by R protein results in the mounting of a cellular response to eliminate the pathogen, rendering the pathogen avirulent. A range of avr molecules have been defined, however there is little evidence to show that a direct interaction between these molecules and R proteins takes place. R protein can be considered as a sensor that requires additional cellular proteins for the direct recognition of an avr product. A consequence of this sensor function is a hypersensitive response which leads to an array of responses including the death of host cells.

[4] 75w MHC class II expression and subsequent CD4 + T cells. Second, the lack of CIITA results in the reduction of both constitutive and cytokine-induced MHC class II expression. Third, the introduction of CIITA into almost all of the cells tested results in MHC class II expression. And last, the repertoire of genes that are induced by CIITA is very restricted and the most dramatically induced genes are all important in MHC class II antigen presentation.

[5] 143w CIITA regulates the transcription of all MHC class II genes and several other genes that encode proteins important in antigen presentation. In humans, these include genes encoding the classical surface MHC class II molecules, HLA-DR, HLA-DP and HLA-DQ, as well as genes encoding accessory molecules that are important in peptide loading, such as the nonclassical MHC class II molecules, HLA-DM and HLA-DO, and the invariant chain protein. The promoters of all these genes contain three conserved regulatory elements, W/S, X and Y (FIG. 2). The X and Y elements are recognized by the DNA-binding transcription factors, regulatory factor X (RFX) (which consists of RFX5, RFXANK and RFXAP), and nuclear transcription factor-Y (NFY) (which consists of NFYA, NFYB and NFYC), respectively. Defects in RFXANK, RFX5 and RFXAP are responsible for the symptoms observed in the BLS complementation groups B, C and D, discussed earlier.

[6] 28w High frequency of a particular allele in a population because the population is derived from a small number of founders, one or more of whom carried that allele.

[7] 125w CIITA is not a DNA-binding protein, but it either directly or indirectly interacts with the DNA-binding transcription factors NFY, cAMP-responsive-elementbinding protein (CREB) and RFX 66,67 (FIG. 2). These proteins interact with different regions of CIITA, but the interaction takes place outside of the carboxy (C)-terminal LRR domain. Instead, there is evidence that the LRR domain mediates self-association, which indicates that the C-terminus is an oligomerization domain 68,69 . In addition, the LRR domain is crucial for a number of functions, including nuclear import and export, trans criptosome formation and MHC class II transcription, and deletion of this region results in dominant-negative mutants [70][71][72][73] . CIITA also interacts with the basal trans cription factors, as well as histone-modifying enzymes of both the acetylase and methylase families.

[8] 149w The prevailing model is that CIITA coordinates the assembly of acetylases, methylases and basal transcription factors on MHC class II promoters 65 . Indeed, the lack of CIITA results in a 'bare' promoter that lacks associated DNA-binding proteins, whereas the addition of CIITA can induce MHC class II, HLA-DM and invariant-chainprotein-promoter loading 77,78 . Furthermore, the lack of CIITA also reduces histone acetylation or methylation and diminishes the association of histone acetylases with the promoter and the acetylation of His4-Lys4 (REF. 79). Finally, CIITA itself has histone acetylase activity and can substitute for other histone acetylases during gene activation. One intriguing finding is that CIITA also facilitates the association of histone deacetylase 1 (HDAC1) and HDAC4 with the promoter. As HDACs are typically viewed as modifying enzymes that decrease gene activation, it seems that CIITA might also have a role in the silencing of activated MHC class II genes.

[9] 140w The structure-function analyses of CIITA demonstrate the existence of three domains: an amino (N)-terminal acidic domain, a mid-region NBD, and a C-terminal LRR region (FIG. 1). The NBD is associated with GTPbinding activity but no GTPase activity has been detected 83 . Similar to the APAF1 protein, CIITA also undergoes self-association and this is dependent on an intact NBD region and on post-transcriptional phosphorylation taking place in the so-called PST (prolineserine-threonine) region that lies N-terminal to the NBD region 68,69,84,85 . A heterotypic association of the GTP-binding domain with the LRR or acidic domain has been found. These data are consistent with a model where, similar to APAF1, CIITA forms a large selfassociative complex in a NTP-dependent fashion, although the detailed mechanism remains to be elucidated. Functionally, intact nucleotide-binding is required for both efficient nuclear import and export 83,86 .

[10] 134w Disease-based gene discovery. CIAS1 (also known as PYPAF1 and NALP3) -the gene that encodes cryopyrin -was identified in 2001 by positional cloning in the search for the genetic basis of two autosomal-dominant autoinflammatory diseases: FCAS and MWS 30 . The availability of large multi-generational families in North America and Europe allowed several groups to identify the linkage of these diseases to a defined region on chromosome 1q44 with no previously recognized genes. Subsequent screening of expressed sequence tags (ESTs) in the region allowed for the discovery of heterozygous missense mutations in CIAS1 in patients with these two similar disorders that were previously considered to be distinct [30][31][32] . Phenotypic similarities between MWS and NOMID (another rare, and more severe, inflammatory disease), prompted the discovery of CIAS1 mutations in these patients as well 87,88 .

[11] 351w CIAS1-associated diseases. These disorders have been identified in patients around the world. FCAS is widespread in North America, owing to a founder effect 89 , and MWS seems to be more prevalent in Europe. As is common with many autosomal-dominant diseases, there are a number of de novo mutations, particularly in patients with NOMID 87 . The CIAS1-associated diseases are all classified as autoinflammatory disorders, which is a new classification of diseases that are characterized by recurrent episodes of systemic inflammation in the absence of infection, and are distinguished from traditional autoimmune diseases by the lack of high-titre autoantibodies or antigenspecific T cells. The three CIAS1-associated diseases share some clinical characteristics, but also have distinguishing features (TABLE 3). Patients with FCAS have recurrent episodes following minimal generalized cold exposures 90 , whereas patients with MWS often have unprecipitated attacks. Approximately 60% of patients with MWS develop progressive neurosensory hearing loss, and up to 25% develop systemic amyloidosis leading to end-stage renal disease 91 . Patients with NOMID have chronic neurological sequelae, including aseptic The DNA-binding proteins with which CIITA associates include nuclear transcription factor-Y (NFY), regulatory factor X (RFX) and cAMP-responsive element binding protein (CREB). In addition, CIITA can associate with basal transcription factors such as polymerase II (POLII), the 32 kDa subunit of TATA-box-binding protein-associated factor (TAF II 32), and the transcription elongation factor b (pTEFb). Finally, CIITA is known to associate with chromatin-modifying enzymes such as histone acetylases (HATs) and methylases. In the absence of functional CIITA, as is the case in type II group A bare lymphocyte syndrome (BLS), the association of all these other factors is greatly reduced as shown by in vivo footprinting and by chromatin immunoprecipitation assays. meningitis, seizures, developmental delay and visual as well as hearing impairment. In addition, patients with NOMID develop progressive deforming overgrowth of the distal femur 92 . These three diseases are now considered to be a continuum of one disease that is referred to as cryopyrin-associated periodic syndrome (CAPS), with increasing severity from FCAS to MWS to NOMID, and this is supported by reports of patients with similar symptoms.

[12] 147w Approximately 40 mutations have been identified in CIAS1 (TABLE 2) and there is some genotype-phenotype correlation 93 . Many specific mutations are associated with a single phenotype (FCAS, MWS or NOMID). However, the same mutations have been observed in both FCAS and MWS and others have been observed in patients with both MWS and NOMID, lending further support to the idea of a disease continuum 93 . There are also patients with classic CAPS phenotypes who do not have mutations in CIAS1, which indicates the involvement of additional genes. Furthermore, there are some genetic variations (such as a substitution of valine with methionine at position 198 (V198M)) in CIAS1 that have been identified in patients with no inflammatory disease and patients with undefined or other inflammatory diseases. So, CIAS1 variations might have a role in more common diseases such as rheumatoid arthritis, systemic lupus erythematosus or gout.

[13] 87w Patients with CAPS have systemic inflammation, as shown by increased levels of C-reactive protein and serum amyloid A, and this inflammation has been attributed to dysregulation of inflammatory cytokines 87,94 . There have been several reports of increased serum cytokines, such as IL-6 and tumour-necrosis factor (TNF), as well as increased peripheral-blood-leukocyte mRNA expression of IL-1β, IL-3 and IL-5 in patients with CAPS 87 . However, the most consistent finding is an increase in the release of IL-1β from peripheral blood leukocytes and/or adherent monocytes 87,94,95 .

[14] 6w A protein variant with reduced function.

[15] 65w Mutational screens of the encoded transcripts identified four different disease-associated conservative missense mutations in the C-terminal B30.2 domain (also known as RFP and SPRY) of the deduced 781-aminoacid full-length pyrin protein. These mutations were not present in chromosomes from ethnically matched non-carrier individuals. The intragenic convergence of haplotypes strongly indicated that there is an independent ancestral origin for modern-day carriers of each of the mutations.

[16] 260w Mediterranean fever-associated diseases. FMF is common among Jews, Armenians, Arabs, Turks, Italians, and to a lesser extent, other Mediterranean populations. Attacks of FMF consist of apparently unprovoked 1-3 day episodes of fever with severe abdominal or chest pain, monoarticular arthritis, or an erysipeloid rash (TABLE 3). Between these attacks, patients feel well. Histologically, there is a massive influx of polymorphonuclear leukocytes into the affected region(s), neutrophilia and a rapid acute-phase response, but autoantibodies and antigenspecific T cells are generally not found. In a subset of patients, systemic amyloidosis can occur -caused by the deposition of a misfolded fragment of serum amyloid A, an acute-phase protein. This sometimes leads to kidney failure and death. Patients with FMF might also be at increased risk of developing vasculitis 109 , and MEFV mutations might increase the risk of autoinflammatory diseases with a more complex inheritance pattern, such as Behçet's disease 110 , inflammatory bowel disease 111,112 and juvenile idiopathic arthritis 113 . Nearly all of the known FMF-associated mutations in MEFV are missense changes (TABLE 2), as shown by the Infevers database (a database of mutations and polymorphisms found in the genes associated with several autoinflammatory diseases, including FMF). There is only one known frameshift mutation near the 5-prime end of the coding sequence, and there are no null mutations associated with FMF. Missense substitutions seem to be a general feature of a number of genetic disorders with a 'periodic' phenotype (for example, periodic paralysis and sickle-cell anaemia) 114,115 , which allow some level of residual function that may decompensate with environmental triggers.

[17] 201w In the case of FMF, heterozygotes seem to have a selective advantage: carrier frequencies of one in five or more have been documented in several ethnic groups [116][117][118][119] . The concept of selection is further supported by the elucidation of a biochemical inflammatory phenotype that is associated with heterozygous mutations 120,121 . Nevertheless, the identity of the putative infectious agent(s) that selected for MEFV mutations remains to be established. About half of the documented mutations in MEFV are located in exon 10, which encodes the B30.2 domain. This bias is even stronger when the high frequency of individual exon 10 mutations among patients is taken into account. Intriguingly, the mutant sequence in humans is often the wild-type in non-human primates, and examination of d N /d S ratios is consistent with episodic positive selection 122 . Furthermore, rodent pyrins lack most of the B30.2 domain 123 . Taken together, these data indicate that variants of the B30.2 domain have been highly selected, both in primate evolution and in human history, although not in rodents. As noted below, this might be explained by the interaction of the B30.2 domain with a product of a microbial pathogen that is yet to be identified.

[1] 159w The association of the CATERPILLERs and pyrin with various immunological disorders suggests that these proteins are crucial regulators of adaptive and/or innate immunity. Although CIITA and cryopyrin have significant structural similarities that are characteristic of the CATERPILLERs, their functions are very different. By contrast, cryopyrin and pyrin have structural similarity that is limited to their pyrin domains, but they seem to participate in similar inflammatory pathways that regulate cytokines. Although the unequivocal role of CIITA in MHC class II gene transcription is well accepted and the underlying cause of MHC2TAassociated immunodeficiency is well explained, the effects of mutations in other CATERPILLER genes that are associated with immunological diseases and the normal role of CATERPILLER proteins and pyrin are not well understood. Although the CATERPILLERs and pyrin might have unique signalling and microbial sensing functions, the properties of CIITA such as selfassociation, nucleotide binding and large proteome formation might serve as a template to understand the mechanisms of other CATERPILLERs.

[2] 123w Elucidating the mechanism involved in the rare MHC2TA-associated immunodeficiency has led to a better understanding of this protein as a master regulator. Uncovering the mechanism that is responsible for the rare cryopyrin or pyrin-associated immunodeficiency is extremely important for clarifying the role of these proteins in maintaining immune balance. It is also valuable, because it is likely that more common immunological disorders will be found to be associated with variations in these genes, as has been observed in multiple sclerosis, rheumatoid arthritis, and myocardial infarction with SNPs in CIITA. In a striking parallel to the findings for CIITA, several recent papers using cryopyrin-deficient mice also implicate a broader role of cryopyrin in the control of a host of inflammatory responses and possible diseases.

[3] 73w The model of diseased-based gene discovery discussed in this Review has proven to be extremely useful for unravelling novel immunological pathways. Translational research such as this, spanning from disease state to protein structure, has led to improved diagnosis and treatment of patients. The analysis of the CATERPILLER and pyrin proteins has provided additional information on the molecular and cellular mechanisms regulating the inflammatory processes that might have significant implications for all inflammatory diseases.

[1] 116w The discovery of evolutionarily conserved large gene families has been pivotal in the discovery of new innate immune pathways. This is exemplified by the Toll protein in Drosophila melanogaster and its mammalian para- logues, the Toll-like receptors (TLRs) [1][2][3] . The transmembrane Toll molecule was initially identified as important for the specification of dorsal-ventral development in flies 4 . Subsequent analyses showed decreased survival of genetic mutants of Toll after infection with the fungus Aspergillus fumigatus or with Gram-positive bacteria 5 . Toll recruits a complex of adaptors (including myeloid differentiation primary-response gene 88, MyD88, and TUBE) and a serine-threonine kinase (denoted Pelle) leading to the activation of the dorsal and dorsal-related immunity factor (DIF) pathways.

[2] 178w In 1997, the first mammalian TLR was discovered 6 . It was predicted to contain a transmembrane region with an extracellular leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to that of the human interleukin-1 (IL-1) receptor, which is now known as the Toll IL-1R (TIR) domain. Similar to D. melanogaster Toll, mammalian TLRs signal through adaptors (including MyD88; TIRAP (TIR domain-containing adaptor protein, also known as MAL); TRIF (TIRdomain-containing adaptor inducing interferon-β also known as TICAM ) and TRAM (TRIF-related adaptor molecule), and serine-threonine kinases, leading to nuclear factor-κB (NF-κB) activation, cytokine production and changes in membrane-receptor expression. A seminal genetic approach that relies on endotoxin resistance in C3H/HeJ mice showed that the resistance allele corresponds to a missense mutation in the gene encoding TLR4 (REF. 7). This demonstrates that TLR4 is required for mice to mount an efficient response to the endotoxin lipopolysaccharide (LPS), therefore opening the floodgate of discoveries that have shown that TLRs are sensors of microbial products. So far, there are 13 mammalian TLRs that recognize a wide range of microbial products.

[3] 189w The TLR field provides examples of immune genes that are similar in higher vertebrates and invertebrates. In early 2000, we and other groups independently described the existence of a novel gene family -found in both plants and mammals -that might also regulate immune responses 8,9 . Later, we described the entire family in detail and named it the CATERPILLER (CARD (caspase-recruitment domain) transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats) gene family 10,11 . This family was identified by the scanning of genes with a similar structure to the MHC class II transactivator (MHC2TA) gene 10,11 . MHC2TA encodes MHC class II transactivator (CIITA), which is a protein with a nucleotide-binding domain (NBD), followed by LRRs 12 . Therefore, by definition, all CATERPILLER proteins contain these two domains. The Inohara-Nunez group described a similar family that they called the nucleotide-binding oligomerization domain (NOD)-leuine-rich repeat (LRR) family (the NOD-LRR family) 13,14 . A more recent review suggested the name NACHT (domain present in NAIP, CIITA, HET-E and TP1)-leucine-rich repeat (LRR) family (the NACHT-LRR family) 15,16 . CATERPILLERs include several subgroups that can be distinguished by their amino (N)-terminal domains:

[4] 12w The frequency with which a genotype manifests itself in a given phenotype.

[5] 210w Several structural and genetic features of CATERPILLER proteins have garnered much attention (FIG. 1). As discussed earlier, these proteins are similar in structure to plant-disease-resistance (denoted R) proteins with the TIR-NBD-LRR or coiled-coil-NBD-LRR structure 11 . R proteins recognize specific products of microbial pathogens and elicit a plant-host response 23 (BOX 1). They are also similar in structure to the APAF1 protein, which has a CARD-NBD-WD40 (also known as Trp-Asp40) structure. During apoptosis, the WD40 domain of APAF1 binds to cytosolic cytochrome C in the presence of ATP or dATP, leading to self-oligomerization followed by association with, and activation of, caspase-9 (REFS 24,25). APAF1 is a useful model, on which CATERPILLER protein activation is based, because the structure of a WD40-deficient APAF1 protein bound to dADP has been resolved 26 . Finally, a remarkable number of genes within this family are associated with immunological disorders that have a high degree of penetrance. For example, mutations in MHC2TA lead to MHC class II deficiency or the type II, group A bare lymphocyte syndrome (BLS) 27,28 . More recently, single nucleotide polymorphisms (SNPs) in the promoter of MHC2TA were found to be genetically associated with multiple diseases with an immunological component, such as rheumatoid arthritis, multiple sclerosis and myocardial infarction 29 .

[6] 104w The gene that encodes cold-induced autoinflammatory syndrome (CIAS1) is associated with three autoinflammatory syndromes: familial-cold autoinflamma tory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal-onset multisystem inflammatory disease (NOMID) [30][31][32] , whereas NOD2 is associated with two autoinflammatory disorders (Crohn's disease and Blau syndrome) [33][34][35] . The gene encoding Mediterranean fever (MEFV), although not part of the CATERPILLER family, encodes the pyrin (also known as marenostrin) protein, which shares the pyrin domain found in the largest subgroup of CATERPILLER proteins. It is mutated in patients with familial Mediterranean fever (FMF), an inherited autoinflammatory disease that occurs mainly in Mediterranean and Middle Eastern populations 36,37 .

[7] 81w This Review focuses on two of the CATERPILLER proteins that are associated with immunological diseases: CIITA and cryopyrin. The pyrin protein is also included here because in addition to the sharing of a pyrin domain, both CIAS1 and pyrin share a similar expression pattern, are involved in similar pathways (caspase-1 pathway, IL-1 regulation, NF-κB pathway, apoptosis), and mutations in these genes result in similar clinical presentations with relatively short episodes of systemic inflammation and the potential development of amyloidosis 38 .

[8] 328w Although the physiological function of the CATERPILLER proteins has remained elusive, two lines of evidence provide additional clues as to their function. First, NOD1, NOD2 and CIAS1 are found to be important for cellular responses to pathogen-derived molecules, which provides evidence for the intriguing hypothesis that some of the CATERPILLER genes are intracellular microbial sensors, as many seem to reside in the cytosol 39,40 . Although it is attractive to separate TLRs (as membrane receptors) from CATERPILLERs (as intracellular sensors), the reality is more complicated and it is noteworthy that several TLRs are also located intracellularly. The cellular location of most CATERPILLER proteins that are produced from transfected constructs is the cytoplasm, which is in agreement with the hypothesis that these are intracellular sensors. However, it is important to note that the localization of most endogenous CATERPILLER proteins remains poorly characterized. Nonetheless, these findings might place certain CATERPILLERs in the important role of microbial sensors in the cytoplasm. Second, several CATERPILLER proteins are found to inhibit the inflamma tory and adaptive immune responses. These include the CLR16.2 (also known as NOD3), PYPAF2 (also known as NALP2 and PAN1), PYPAF3 and Monarch1 (also known as PYPAF7) proteins, which have been shown to reduce a range of responses including those of NF-κB, activator protein 1 (AP1), nuclear factor of activated T cells (NFAT) activity, IL-1 receptor-associated kinase (IRAK) activation, and IL-1 production [41][42][43][44] . In the case of PYPAF2 and Monarch1, siRNA against the protein of interest was used, lending credence to the hypothesis that these are indeed antagonists of inflammatory responses 43,44 . However, the inhibitory role of these proteins is under debate, as several in vitro studies using overexpressed proteins suggest that proteins such as cryopyrin, PYPAF5 and Monarch1 might activate NF-κB when ASC is cotransfected 19,45,46 . However, this pattern of activation in the presence of ASC is not observed by others 47 . Therefore, caution needs to be exercised in resolving the biological function of CATERPILLERs.

[9] 75w Although gene deletion in mice represents a relevant physiological system, the separate analyses of three distinct mice with Nod2 gene deletion or diseasegene-replacement mutants support a role for NOD2 in either increasing antimicrobial responses, or reducing proinflammatory responses [48][49][50] . It is possible that the CATERPILLER proteins have both activating and inhibitory effects, depending on the expression levels of the protein or interacting proteins, or on expression of other intracellular and extracellular variables that have

[10] 393w Table 1 | CATERPILLER gene family members organized by their at amino-terminal domains CLR nomenclature* (human chromosome location) Other nomenclature Domain structure GenBank accession ‡ References Acidic transactivation (16p13.13) MHC2TA, C2ta AD-NACHT-LRR NM_000246 12 Baculoviral inhibitory repeat (5q13.2) BirC1, NAIP BIR-BIR-BIR-NACHT-LRR NM_004536 17,18 Caspase recruitment domain (7p14) CARD4, NOD1 CARD-NACHT-LRR NM_006092 1,11, 13,14 (16q12) CARD15, NOD2, BLAU, CD, PSORAS1, IBD1 CARD-CARD-NACHT-LRR NM_022162 1,11, 13,14 CLR2.1 (2p22) CARD12, IPAF, CLAN CARD-NACHT-LRR NM_021209 1,11, 13,14 Pyrin CLR19.8 (19q13.43) MATER, NALP5, PYPAF8, NOD14, PAN11 PYD-NACHT-LRR NM_153447 1,11,13,14,19-22 CLR17.1 (17p13.1) CARD7, DEFCAP, NALP1, NAC PYD-NACHT-LRR NM_033004 1,11,13,14,19-22 CLR19.9 (19q13.42) NALP2, NBS1, PYPAF2, PAN1, NBS1 PYD-NACHT-LRR NM_017852 1,11,13,14,19-22 CLR1.1 (1q44) CIAS1, PYPAF1, NALP3 PYD-NACHT-LRR NM_004895 1,11,13,14,19-22 CLR19.5 (19q13.42) PYPAF4, NALP4, PAN2, RNH2 PYD-NACHT-LRR NM_134444 1,11,13,14,19-22 CLR11.4 (11p15.5) PYPAF5, NALP6, PAN3 PYD-NACHT-LRR NM_138329 1,11,13,14,19-22 CLR19.4 (19q13.42) PYPAF3, NALP7, NOD12 PYD-NACHT-LRR NM_206828 1,11,13,14,19-22 CLR19.6 (19q13.42) PYPAF6, NALP11, NOD17 PYD-NACHT-LRR NM_145007 1,11,13,14,19-22 CLR19.3 (19q13.42) PYPAF7, Monarch1, NALP12, RNO2, PAN6 PYD-NACHT-LRR NM_144687 1,11,13,14,19-22 CLR19.2 (19q13.43) NALP8, NOD16, PAN4 PYD-NACHT-LRR NM_176811 1,11,13,14,19-22 CLR19.1 (11p15.4) NALP9, NOD6 PYD-NACHT-LRR NM_176820 1,11,13,14,19-22 CLR11.1 (19q13.42) NALP10, NOD8, PYNOD, PAN5 PYD-NACHT NM_176821 1,11,13,14,19-22 CLR19.7 (19q13.42) NALP13, NOD14 PYD-NACHT-LRR NM_176810 1,11,13,14,19-22 CLR11.2 (11p15.4) NALP14, NOD5, NALP1, GC-LRR PYD-NACHT-LRR NM_176822 1,11,13,14,19-22 Undefined CLR11.3 (11q23) NOD9 X-NACHT-LRR NM_024618 1,11,13,14,19-22 CLR16.1 (16q13) § NOD27, PAN14 X-NACHT-LRR NM_032206 1,11,13,14,19-22 CLR16.2 (16p13.3) || NOD3 X-NACHT-LRR NM_178844 1,11,13,14,19-22 *The CATERPILLER gene family members (abbreviated as CLR) were named according to their human chromosomal localization, followed by the order in which they were discovered. Hence, CLR19.3 is located on human chromosome 19, and was the third CATERPILLER discovered in our laboratory. ‡ See Entrez Gene wesite. § CLR16.2 has been grouped as either a CARD-or a pyrin-containing member. || CLR16.2 is considered by some to be a CARD-containing member. AD, acidic domain; BirC1, baculoviral inhibitor of apoptosis protein (IAP)-repeat-containing protein 1; BLAU, Blau syndrome (susceptibility to); C2ta, MHC class II transactivator; CARD, caspase-recruitment domain; CD, Crohn's disease (susceptibility to); CIASI, gene encoding cold-induced autoinflammatory syndrome; CLR, CATERPILLER (CARD, transcription enhancer, R (purine)-binding, pyrin, lots of leucine repeats); GC-LRR, germ-cell-specific leucine-rich repeat; IBD1, inflammatory bowel disease 1 (susceptibility to); IPAF, ICE-protease activating factor; LRR, leucine-rich repeat; MHC2TA, MHC class II transactivator; NACHT, NAIP CIITA HET-E, and TP1containing; NALP5, NACHT, leucine-rich repeat and PYD containing; NAIP, neuronal apoptosis inhibitor protein; NOD, nucleotide-binding oligomerization domain; PSORAS, psoriatic arthritis (susceptibility to); PYD, pyrin domain; TA, transactivator; X, regulatory element X.

[11] 18w Two genetic mutations that, when present in the same cell or organism, cause the appearance of wild-type traits.

[12] 18w A region of DNA associated with a specific trait that varies in a quantitative fashion, rather than dichotomously

[13] 95w not yet been identified. Clearly, much work needs to be done in additional physiological systems, but the findings that genetic mutations within these proteins result in autoinflammatory diseases in humans is consistent with the contention that CATERPILLER proteins have important roles in the regulation of inflammation and immunity. Against this backdrop, this Review focuses on immunological disorders that are associated with two CATERPILLER genes (MHC2TA and CIAS1) as well as the related MEFV gene, and the molecular function of their encoded proteins. This follows recent discussion on the NOD2 gene and Crohn's disease 51 .

[14] 162w Disease-based gene discovery. The founding member of the CATERPILLER gene family is the MHC2TA gene that is found on human chromosome 16p13, which encodes a master transcription regulator of MHC class II genes, known as CIITA 10 . MHC2TA is defective in the rare form of autosomal recessive hereditary immuno deficiency, BLS 12 . BLS is a collection of MHC class II deficiencies that are defined by four complementation groups and changes in the MHC2TA gene represents the defect in group A. In all four groups of patients with BLS, the structural genes for MHC class II are intact, and the defect lies in the transcriptional activation of MHC class II genes. MHC2TA was discovered by complementation cloning using an Epstein-Barr virus (EBV) vector-based library and an in vitro-generated, MHC-deficient cell line as a recipient. The genetic defects for the other three complementation groups (groups B-D) have been defined, and all affect transcription factors important for MHC class II expression 28,52,53 .

[15] 404w MHC2TA-associated diseases. BLS is mainly found in families from northern Africa, although it is also found in families from Spain and Turkey 28,52,53 . Clinical manifestations among the four complementation groups are indistinguishable and occur within the first year of life 28,52,53 . These include frequent bacterial, viral, protozoan and fungal infections, accompanied by infection of the gastrointestinal tract and respiratory system. With rare exceptions, patients do not survive beyond the age of ten. Antigen presentation is defective 54 and as expected, laboratory findings show reduced or no expression of MHC class II molecules accompanied by a specific reduction of CD4 + T cells, although residual CD4 + T cells have been found 55 . As predicted by the immunological function of MHC class II molecules, both T cell and antibody responses are defective, which accounts for the frequent infections. Bone-marrow transplantation has resulted in modest success 56 . MHC class I expression is also reduced in the majority of patients with BLS. Interestingly, BLS was initially defined in patients with reduced MHC class I expression, and CIITA controls human MHC class I promoters through a site in the promoter called the α-motif 57,58 . This provides a plausible molecular mechanism to explain the lack of both MHC class I and II in some patients with BLS. Among the group A patients with BLS, the defects within the MHC2TA structural gene can be divided into three groups with additional defects located in the promoter region (TABLE 2). In the first group, an individual exon within the LRR is skipped 28 , and in one case this results in a dramatic decrease in CIITA nuclear trans location 59 . Other patient-derived cells carry a nonsense mutation that results in truncated proteins 60 . A final group of patients have missense mutations, and they exhibit varying degrees of MHC class II loss and symptoms that range from severe to mild immuno deficiency 11,61 . In one case, siblings with a missense mutation from leucine to proline at position 469 (L469P) showed mild symptoms or remained asymptomatic 61 . The implication of this study is that more variations within MHC2TA are probably present in the population, allowing varying degrees of MHC class II transcription that do not result in the total loss of MHC class II expression, but could tip the balance of MHC class II gene expression, and therefore tip the balance of autorecognition versus autoimmunity.

[16] 158w In addition to mutations within the structural gene, recent studies have found SNPs in the promoter III region of MHC2TA (MHC2TA has four promoters 62 ) that are associated with various disorders with an immunological component 29,63 . The promoter III region is both a constitutive promoter in human B and T cells, and an interferon-γ (IFNγ)-inducible promoter in several human cell lines 64 . Zamvil and colleagues found a SNP in the nucleotide at position 155 of this promoter. A consortium used previously defined rat quantitative trait locus for MHC class II expression and defined a SNP within the rat MHC2TA promoter III that corresponds to a polymorphism between alanine and glycine at position 168. This SNP is associated with increased susceptibility to rheumatoid arthritis, multiple sclerosis and myocardial infarction, all of which are disorders with an inflammatory or immunological component. These studies point to MHC2TA as an important genetic linkage to immunological disorders other than BLS.

[17] 14w Enzymes such as acetylases and methylases that cause the covalent modification of histone tails.

[18] 16w The process of identifying a gene based on its chromosomal position, including genetic and physical mapping.

[19] 21w A short partial cDNA sequence that represents a part of an expressed gene and can be mapped to a chromosomal position.

[20] 144w Cryopyrin is expressed primarily in neutrophils and monocytes 19,30 , which are the main effector cells of the innate immune system. Although it is not well understood, cryopyrin might have roles in cell signalling by regulating cytokine responses at the transcriptional (NF-κB) and post-translational (caspase-1) levels, and in sensing bacterial ligands and endogenous signals that activate cytokine responses in these cells. This could explain many of the systemic and tissue inflammatory symptoms that are seen in patients. However, cryopyrin is also expressed in human chondrocytes, and in the skin and eyes in mice 88,96 . These are the same tissues in which patients with CAPS experience symptoms such as cartilage overgrowth, inflammatory rash and ocular inflammation. However, the function of cryopyrin in these cells and tissues, including a possible role in the regulation of apoptosis, has not been elucidated and further study is required.

[21] 257w So far, functional analyses of cryopyrin and other CATERPILLER proteins show that many of these proteins intersect with the NF-κB pathway, but their true functions remain to be determined. Whereas some genes that have N-terminal pyrin domains seem to have an inhibitory effect on NF-κB activation 41,43 , other CATERPILLER genes with N-terminal CARD domains, such as NOD2, have been shown to have a role in the activation of NF-κB, using in vitro overexpression reporter assays 97 and genetically modified mice [48][49][50] . In the case of NOD2, peripheral blood monocytes from individuals with disease-associated NOD2 mutations are defective in their response to muramyl dipeptide 98 . In in vitro studies, cryopyrin has been shown to activate NF-κB in the presence of another pyrin-domain-containing protein called apoptosis-associated speck-like protein containing a CARD (ASC) 19,41,99 . However, like other pyrin-domaincontaining CATERPILLER proteins, cryopyrin has been shown to have an inhibitory effect on TNF and TNF-receptor-associated factor 6 (TRAF6)-induced NF-κB activation in the absence of ASC 47,100,101 . Cryopyrin also has an inhibitory effect on TRAF6-induced NF-κB activation in cells that are stably transfected and express physiological levels of ASC 47 . Therefore, it is possible that cryopyrin and other CATERPILLER proteins can be inhibitory or activating for NF-κB depending on other factors such as the levels of ASC. Recent studies in cryopyrin-null mice do not suggest a direct role for cryopyrin in inhibitor of NF-kB (IκB) phosphorylation in response to TLR4 or TLR7 agonists, but instead indicate that it has a crucial role in caspase-1 activation [102][103][104] .

[22] 195w The pyrin and NBD domains of cryopyrin are also involved in protein-protein interactions in a complex that has been referred to as the inflammasome (FIG. 3), which regulates post-translational cytokine processing 105 . Immunoprecipitation experiments have demonstrated that cryopyrin interacts with ASC and CARDINAL (CARD-inhibitor of NF-κB-activating ligand), two adaptor proteins that are involved in the activation of caspase-1, leading to the release of IL-1β 94 . Cryopyrin colocalizes with ASC in cytoplasmic specks that are visible by cytochemistry 19,47 . Recent in vivo studies with cryopyrin-and ASC-null mice and ex vivo studies using macrophages from these mice demonstrate that cryopyrin and ASC are both necessary components for the activation of inflammasome-mediated inflammation [102][103][104] . In vitro transfection experiments and ex vivo studies CARD CARD LRR Inflammasome activation • Fever • Inflammation Reduction in: • Fever • Inflammation Stimuli: • Bacterial RNA • Imidazoquinoline compounds • Toxins from Grampositive bacteria: nigericin and maitotoxins • ATP • Uric acid CARDINAL Procaspase-1 Caspase I complex NBD FIIND CARDINAL PYD PYD CARD CARD Pro IL-1β IL-1β Pro IL-1β IL-1β Caspase-1 CARD CARD LRR Inflammasome activation inhibited FIIND PYD PYD CARD CARD Cryopyrin a b ASC ASC NBD Cryopyrin

[23] 301w with monocytes from patients with CAPS indicate that CIAS1 mutations lead to an increase in the activation of caspase-1 and therefore an increase in IL-1β release and resultant systemic inflammation that is consistent with the inflammatory nature of the cryopyrin-associated diseases 94,106 . The LRR domain seems to interfere with the interaction of the NBD domain and CARDINAL, which results in the inhibition of inflammasome activation, and mutations in CIAS1 might reverse this inhibition 94 . It is not known if other proteins participate in these pathways either as activators or inhibitors, or how cold temperatures trigger inflammation in patients with FCAS. The elucidation of the upstream and downstream pathways of cryopyrin will hopefully answer these questions. Some CATERPILLER proteins have been shown to sense, although not necessarily bind, microbialassociated molecular patterns, similar to the TLRs. This is consistent with a role in innate immunity. Specifically, NOD2 detects muramyl dipeptide, which is an active component of bacterial peptidoglycan and a common contaminant of LPS. Cryopyrin, like NOD2, might be a sensor of muramyl dipeptide through its C-terminal LRR domain 107 , although experiments with cryopyrinnull mice do not support this function 102,103 . Instead, cryopyrin-associated inflamma somes have been shown to be necessary for activation by a number of exo genous or endogenous ligands including bacterial RNA, imidazoquinoline compounds 102 , the Gram-positive bacterial toxins nigericin and maitotoxin, ATP 103 and uric-acid crystals 104 . This indicates that cryopyrin is a specific sensor of pathogens or a general sensor of stress or danger signals 104 from cells that are dying or under attack. It is not clear if cryopyrin directly senses these ligands or if additional proteins help mediate this sensing. Future studies of null as well as mutant cryopyrin mice will further elucidate the protein's many roles in inflammatory pathways.

[24] 135w Disease-based gene discovery. MEFV is the locus on human chromosome 16 that is mutated in familial Mediterranean fever (FMF), a recessively inherited systemic autoinflammatory disease that is common in the Mediterranean basin and the Middle East 108 . The gene was discovered by two positional-cloning consortia in 1997, and encodes what was then a novel protein denoted as pyrin or marenostrin 36,37 . Using polymorphic microsatellite markers and SNPs spanning the relevant region of chromosome 16p, the two teams deduced the presence of FMF-carrier haplotypes among several affected populations. Remarkably, subgroups of these haplotypes had sets of shared markers that all map to the same genomic region, which indicates that the modern-day carrier chromosomes were derived from a limited number of common ancestors, and localize the FMF-causing locus to the small genomic region of overlap.

[25] 29w A 170 amino-acid globular domain found at the C-terminus of several proteins, first identified as the product of an isolated exon of the same name encoded in the MHC.

[26] 28w The identification of a shared set of DNA markers within a gene among unrelated individuals with a specific disease, suggesting a common ancestral origin of the diseaseassociated chromosomes.

[27] 26w A disorder common in the Middle and Far East, characterized by oral and genital ulcerations, ocular inflammation, skin lesions, and a number of other inflammatory manifestations.

[28] 20w The ratio of nonsynonymous to synonymous substitutions, which varies directly with the probability of evolutionary selection at a given locus.

[29] 15w A family of proteins involved in the regulation of intracellular signaling, cell-cycle control, and apoptosis.

[30] 208w Although the precise molecular mechanisms by which wild-type and mutant pyrins function are not completely understood, the dramatic inflammatory phenotype of FMF and the high carrier frequencies in certain populations suggest an important role for pyrin in the regulation of innate immunity. Consistent with this view, MEFV and pyrin are expressed at high levels in neutrophils, eosinophils, monocytes and dendritic cells, but not in lymphocytes 36,37,124,125 . Pyrin is also expressed in synovial, peritoneal and skin-derived fibroblasts, but not in chondrocytes or endothelial cells 125 . The subcellular localization of pyrin seems complex. Transfected full-length pyrin clearly localizes to the cytoplasm, while a rare isoform that is encoded by an alternative splice variant lacking exon 2 can enter the nucleus [125][126][127][128][129] . Furthermore, native pyrin is cytoplasmic in monocytes, but predominantly nuclear in granulocytes, dendritic cells and synovial fibroblasts 125 . Recently two members of the 14.3.3 protein family were shown to bind pyrin through phosphorylated serine residues that are encoded in exon 2 of pyrin; mutants that are unable to bind 14.3.3 proteins entered the nucleus 130 . These findings might account for the observed differences in pyrin localization in the different cell types, although the pyrin-14.3.3 protein interaction remains to be demonstrated for the endogenous proteins.

[31] 276w Although the N-terminal half of pyrin seemed to be unique when MEFV was initially cloned, the N-terminal 92 amino acids were subsequently recognized as the proto type for the motif that bears its name 10,131 . This domain binds ASC 132 and through this interaction pyrin might regulate IL-1β processing, NF-κB activation, and apoptosis, although both inhibitory and enhancing effects have been observed depending on the experimental system. Studies of mice that harbour a truncated, hypomorphic variant of pyrin, and complementing studies of pyrin-transfected mouse RAW monocytic cells that endogenously express ASC, have indicated an inhibitory role for wild-type pyrin in IL-1β processing 133 . This gave rise to the sequestration hypothesis (FIG. 4) of pyrin action. By contrast, recent studies in which human pyrin was transfected into 293T human embryonic kidney cells have indicated a proinflammatory effect of pyrin on IL-1β processing and inflammasome formation 47 . Furthermore, in different studies, co-expression of pyrin with ASC has been shown to inhibit 99,134 , accentuate 100 , or not affect 47 ASC-dependent NF-κB activation. Stehlik and colleagues 100 have provided evidence that the effect of pyrin (and cryopyrin) on NF-κB activation might depend on the context of other stimuli, such as TNF and IL-1. Finally, although peritoneal macro phages from the aforementioned pyrin-truncation mutant mice have a defect in apoptosis, pyrin seems to be anti-apoptotic in some transfection systems through interaction with ASC 99,132,134 . Taken together, these studies suggest a context-dependent role for pyrin, although studies of pyrin-null and knock-in mice, and the response of patients with FMF to targeted biological therapies, are likely to provide additional insights into the factors that govern context-dependent regulation.

[32] 159w The mechanism by which mutations in the B30.2 domain of pyrin cause FMF is unknown. Some researchers have suggested that this C-terminal domain of pyrin might bind one or more pathogenassociated molecular patterns (PAMPs) 47,122 that are analogous to the LRR of cryopyrin. This hypothesis is attractive, given the large number of disease-associated mutations in this domain, the apparent heterozygote selection in Mediterranean populations, and the evidence for positive selection for variants in this domain in primates. Moreover, the B30.2 domain of the tripartite motif-5α (TRIM5α) protein has been shown to block infection by certain retroviruses 135,136 , and to have similarly undergone positive selection. According to this hypothesis, FMF-associated mutations would lead to increased PAMP-binding, and if microbial species from which the PAMP was derived were relatively ubiquitous, this could lead to apparently unprovoked episodes of inflammation. However, at present the role of the B30.2 domain of pyrin in the binding of PAMPs, however attractive, is purely conjectural.

[33] 328w Evidence for dysregulation of IL-1 in the cryopyrinassociated disorders has prompted many investigators to target this cytokine with available therapeutic agents. IL-1 receptor antagonist (IL-1Ra), which is a daily injectable medication approved for the treatment of rheumatoid arthritis, seems to be extremely effective in patients with CAPS. A lessening of daily symptoms including rash and pain, and a reduction in laboratory markers of chronic inflammation such as C-reactive protein and serum amyloid A have been demonstrated in multiple case reports on patients with MWS and NOMID 137,138 . Similar responses have been seen in patients with FCAS that are treated with maintenance IL-1Ra and with IL-1Ra administered before a cold challenge. This treatment prevents attacks and blocks acute inflammation, including leukocytosis and increased serum IL-6 levels in patients with FCAS 139 . Additional IL-1-targeted therapies are being studied in CAPS patients. It remains to be seen if targeting IL-1 will have similar benefits in FMF. indicate that the pyrin domain (PYD) of pyrin interacts with the PYD of ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)), and can compete with cryopyrin for ASC-binding, thereby reducing ASC availability in the cryopyrin inflammasome. The net effect would then be a reduction of caspase-1 activation and a reduction in interleukin-1β (IL-1β) processing. b | The pyrin inflammasome hypothesis. In a different system, Yu and colleagues 85 have shown that pyrin can potentiate IL-1β processing. These authors suggest that a pathogen-associated molecular pattern (PAMP) might bind the carboxy (C)-terminal B30.2 domain of pyrin in a similar fashion to the binding of muramyl dipeptide to the leucine-rich repeat of cryopyrin. This might lead to a conformational change in pyrin that would allow it to participate in a (still not completely understood) macromolecular complex, leading to caspase-1 activation by induced proximity, similar to the cryopyrin inflammasome. Protein X is an unknown protein that would bind the B30.2 domain of pyrin and the CARD of pro-caspase-1, thereby permitting the aggregation of two caspase-1 molecules.