Decoys and Regulatory "Receptors" of the IL-1/Toll-Like Receptor Superfamily.

TIR8/SIGIRR gene is localized on human chromosome 11 and on murine chromosome 7 ( 97 ). The 410 amino acid-long protein is constituted by a single Ig extracellular domain, a transmembrane domain, an intracellular conserved TIR domain, and a 95 amino acid-long tail at the C-terminal, reminiscent of the intracellular tails of few ILR/TLR family members, in particular IL-1AcPb and TIGIRR (see below) (Figure 2 ). Both in human and mouse, TIR8/SIGIRR has several N- and O-glycosylation sites in the extracellular domain ( 97 , 98 ). The sequence and pattern of expression of TIR8/SIGIRR is conserved among vertebrates, from chicken to humans ( 99 ). In particular, human and mouse protein sequences share 82% homology. TIR8/SIGIRR is expressed in several tissues, particularly in kidney, digestive tract, liver, lung, and lymphoid organs ( 97 , 100 ). TIR8/SIGIRR, a negative regulatory receptor of ILR and TLR signaling . TIR8/SIGIRR is composed by a single extracellular Ig domain, a transmembrane domain, an intracellular conserved TIR domain and a long intracellular tail. Two replaced amino acid in the TIR domain (Cys 222, Leu 305) are potentially involved in non-conventional activation. TIR8/SIGIRR is an orphan receptor, but IL-36Ra has been proposed as a TIR8/SIGIRR ligand in glial cells. TIR8/SIGIRR inhibits ILR and TLR signaling by competing with MyD88 and IRAK recruitment at the TIR domain thus dampening the signaling pathway leading to NF-kB activation. In T cells and epithelial cells, TIR8/SIGIRR inhibits IL-1-dependent activation of the Akt- mTOR pathway and of JNK, thus controlling cell proliferation and survival. In vivo studies in gene-targeted mice demonstrate that Tir8/Sigirr acts as a non-redundant negative regulator in different inflammatory conditions dependent on ILRs or TLRs. TIR8/SIGIRR proximal promoter has a binding site for SP1, which enhances its transcription in basal conditions ( 101 ). LPS stimulation reduces SP1 binding to TIR8/SIGIRR promoter, possibly explaining the TIR8/SIGIRR down-regulation in inflammatory conditions (LPS administration, ulcerative colitis, lung and urinary infections,infestations) ( 100 – 106 ). Recent studies demonstrated lower expression of TIR8/SIGIRR in fetal human enterocytes providing a reasonable explanation to the excessive inflammatory response in the immature intestine ( 107 ). In contrast with these studies, TIR8/SIGIRR up-regulation was shown in human monocytes during sepsis and sterile systemic inflammation ( 108 ). Th2-lymphocytes expressed higher levels of TIR8/SIGIRR compared to Th1 polarized or non-differentiated lymphocytes ( 109 ). Pseudomonas aeruginosa infected mice showed up-regulation of Tir8/Sigirr in the cornea, macrophages, and Langerhans cells through the activity of vasoactive intestinal peptide ( 110 ). Lactobacillus jensenii , a probiotic microorganism, induced up-regulation of TIR8/SIGIRR in porcine Payer’s patch antigen presenting cells through activation of TLR2 ( 111 ). Similarly, LPS-induced Tir8/Sigirr in murine Payer’s patch DCs, but not in spleen DCs ( 112 ). These data suggest that Payer’s patch DCs use Tir8/Sigirr to tune TLRs signaling. The function of TIR8/SIGIRR consists in the specific inhibition of NF-kB and JNK activation following stimulation of ILR or TLR family members ( 102 , 113 ). TIR8 can modulate the signal transduction activated by IL-1RI, IL-18R, T1/ST2, TLR1/2, TLR3, TLR4, TLR7, and TLR9 ( 98 , 102 , 109 , 113 – 115 ) (Figure 2 ). The extracellular Ig-like domain of TIR8/SIGIRR has been show to interfere with the dimerization of IL-1RI and IL-1RAcP. The cytoplasmic TIR domain binds TIR-containing adaptor molecules, which are no more available for signaling, whereas the cytoplasmic tail is not involved in the inhibitory activity ( 102 , 114 ). A computational approach suggests a three-dimensional model for the interaction among the TIR domains of TLR4, TLR7, MyD88, and TIR8/SIGIRR. In this model, TIR8/SIGIRR binds TLR4 and TLR7 through its BB-loop region preventing their dimerization and MyD88 recruitment ( 116 ). TIR8/SIGIRR can also regulate mTOR kinase activity in Th17 lymphocytes ( 117 ) and in intestinal epithelial cells ( 118 ) (Figure 2 ). These results are in agreement with the role of TIR8/SIGIRR in autoimmune diseases and in tumor suppression (see below). Tir8/Sigirr -deficient mice are more susceptible than wild type mice in several infections, such as tuberculosis, candidiasis, aspergillosis, P. aeruginosa infection, in terms of mortality and tissue damage due to an exaggerated inflammatory response ( 103 , 106 , 119 , 120 ) (Figure 2 ). Results obtained with IL-1-blocking antibodies and IL-1RI-deficient mice indicated that in some of these infectious conditions (tuberculosis and P. aeruginosa lung infection), TIR8/SIGIRR played a major role in dampening inflammation induced by IL-1R activation. Similarly, in a colitis mouse model, Tir8/Sigirr -deficient mice developed a more severe gut inflammation compared to wild type mice ( 113 , 121 ). Commensal microflora activates enterocyte TLRs and consequently induces survival of epithelial cells and maintains gut homeostasis ( 122 , 123 ). Lack of Tir8/Sigirr in colon epithelial cells was shown to be associated to constitutive NF-kB and JNK activation and up-regulated expression of Cyclin D1 and Bcl-xL in homeostatic conditions, which returned to the control level after depletion of commensal bacteria ( 121 ) (Figure 2 ). Excessive systemic inflammation was observed in Tir8/Sigirr -deficient mice upon LPS challenge, and reduced inflammation and mortality were described in Tir8/Sigirr overexpressing mice in LPS-dependent acute lung injury model ( 102 , 124 ). However, these phenotypes possibly depend on the genetic background since excessive systemic or local inflammatory reactions to LPS were not confirmed in other studies ( 102 , 113 ). In contrast with these data, in a urinary tract infection model, Tir8/Sigirr inhibited an effective host response against uropathogenic E. coli , as indicated by lower renal bacterial load and dysfunction in TIR8-deficient mice, associated to increased circulating and intrarenal neutrophils at the early phase of infection ( 125 ). Recent data suggest that TIR8/SIGIRR plays a direct role in inhibiting different IL-1-dependent signaling pathways, including IL-1R-mTOR, in Th17 lymphocytes, thus tuning initial Th17 differentiation and preventing Th17 cell-mediated pathogenic effects ( 117 ) (Figure 2 ). This effect was particularly evident in the control of CNS autoimmune inflammation in a model of experimental autoimmune encephalomyelitis ( 117 ). Tir8/Sigirr deficiency was also associated to increased susceptibility to develop autoimmunity in a model of systemic lupus erythematosus (B6lpr/lpr), as well as in a model of lupus nephritis induced by hydrocarbon oil (pristane) ( 115 , 126 ). In the lpr/lpr model, Tir8/Sigirr deficiency was responsible for massive lymphoproliferation, peribronchial inflammation, and mesangio-proliferative glomerulonephritis, due to B and dendritic cell hyper-activation in TLR7- and TLR9-dependent response to autoantigens and nucleosomes ( 115 ). Tir8/Sigirr-deficient mice were also more susceptible than wild type mice to both zymosan-induced and collagen antibody-induced arthritis models, because of excessive inflammation at least in part dependent on IL-1 ( 127 ) (Figure 2 ). In agreement with the results obtained in autoimmunity mouse models, TIR8/SIGIRR was down modulated together with other anti-inflammatory genes in psoriatic patients ( 128 ). Studies on allergic inflammatory responses showed that Tir8/Sigirr plays an important role also in controlling the axis IL-33 – ST2 which is involved in Th2 cell polarization and Th2 cytokine production ( 109 ) (Figure 2 ). DAMPs generated during renal ischemia/reperfusion are responsible of the activation of intrarenal DCs, macrophages, and neutrophils via TLRs and IL-1R, which are potentially involved in post ischemic renal failure. In models of renal ischemia/reperfusion or kidney transplantation, Tir8/Sigirr-deficient mice showed increased renal injury or severe graft rejection, respectively, associated to excessive cytokine and chemokine production and consequently, leukocyte recruitment and amplified adaptive immune response against donor antigens ( 129 , 130 ) (Figure 2 ). Finally, in agreement with the expression in neurons, microglia, and astrocytes ( 131 ), TIR8/SIGIRR was shown to be a modulator of microglia activation by LPS, and of neuroinflammation ( 132 ). Furthermore, Tir8/Sigirr-deficient mice showed impaired cognitive and synaptic functions associated to up-regulated IL-1R1 and TLR4 signaling in hippocampal tissue in response to IL-1α and high mobility group box 1 ( 133 ). Studies on the anti-inflammatory activity of IL-36Ra in the brain demonstrated at least a partial involvement of TIR8/SIGIRR in down modulating glial cell inflammatory responses through the production of IL-4 ( 18 ). Chronic inflammation is associated with promotion of malignancy and tumor progression and several studies in animals have shown the protumoral role of IL-1 in this context ( 134 , 135 ). Along the same line, in different murine models, TIR8/SIGIRR has been demonstrated to play a key protective role in the pathogenesis of cancer-related inflammation. In a model of colitis-associated cancer (CAC), a colorectal disease that arises in patients suffering from chronic IBD, Tir8/Sigirr-deficient mice were highly susceptible to both inflammation and carcinogenesis in terms of number, size, and severity of lesions ( 121 , 136 ) (Figure 2 ). The mechanism proposed suggests that TIR8/SIGIRR plays a protective role probably by modulating the signaling activated by commensal bacteria through TLRs in the epithelial cells and consequently, downstream events, including production of inflammatory mediators and factors involved in cell survival and proliferation, leukocyte recruitment, and angiogenesis ( 137 ). Moreover, Tir8/Sigirr deficiency in the Apc min/+ mouse model was associated to increased intestinal lesion development due to higher Akt-mTOR activity, a crucial tumorigenic pathway ( 118 , 138 ). The data suggest that Tir8/Sigirr exerts a tumor suppressor activity by controlling IL-1- and TLR-induced mTOR-mediated cell cycle progression and consequent genetic instability ( 118 ). In Chronic Lymphocytic Leukemia (CLL), human malignant B cells express lower levels of TIR8/SIGIRR mRNA than normal B cells ( 139 , 140 ). Similar results were found in the mouse where CD19+ cells express lower levels of Tir8 messenger compared to CD19+ cells isolated from a transgenic mouse model of CLL (TCL1 mice) ( 141 ). In CLL, both genetic (e.g., MyD88 mutations) and micro environmental factors concur to the development, expansion, and progression of the disease ( 139 , 142 ). In a murine CLL model, the absence of Tir8/Sigirr led to a more severe and earlier appearance of monoclonal B-cell expansions and to shortened life span. The disease mimicked the aggressive variant of human CLL, characterized by the appearance of prolymphocytes ( 141 ) (Figure 2 ), suggesting that TIR8/SIGIRR acts as an inhibitor of CLL progression through a still unclear molecular mechanism.

IL-18 binding protein (IL-18BP) is a secreted high affinity IL-18 binding molecule, which acts as a potent inhibitor of IL-18 and a modulator of Th1 response. It is constituted by only one Ig-like domain and it is structurally and functionally similar to IL-1RII ( 30 ). Indeed, phylogenetic analysis suggests that IL-18BP and IL-1RII had a common ancestral gene and diverged at the level of fish ( 31 ). Recombinant IL-1F7 also binds to the IL-18BP, further increasing the ability of IL-18BP to neutralize IL-18 activity ( 143 ). Proteins homolog to IL-18BP have been found in poxviruses ( Ectromelia ), which are responsible of neutralization of human IL-18 during the viral infection and of dampening the inflammatory response associated to the infection ( 144 ). Further information about this molecule is available in the review by Dinarello et al. in this issue. The IL-1RAcP is the receptor subunit of the IL-1RI complex, and it is also used by IL-36α/IL-1F6, IL-36β/IL-1F8 and IL-36γ/IL-1F9, and IL-33 receptors. It has been shown that an alternative form of AcP, called AcPb, can be generated by alternative splicing, in which the prototypical AcP C-terminal exon 12 is skipped and an alternative exon 12b is used ( 145 , 146 ). Smith et al. ( 146 ) characterized this molecule and showed its regulatory properties in the brain. The C-terminus encoded by these two alternative exons has 35% amino acid identity, which includes conserved motifs of the TIR domain. Moreover, the exon 12b encodes a sequence of approximately 140 additional amino acids in the C-terminal of the TIR domain that has no homology to other protein sequences and is of unknown function. The general structure of AcPb is similar to that of AcP and suggests that the AcPb cytoplasmic domain is similar to AcP TIR domain. However, there is one area of substantial difference because of changed configuration in the DD loop and aD helix regions of the AcPb TIR domain, which remembers modification of TIR8/SIGIRR TIR domain, and altered charge distribution pattern on its surface. It has been proposed that these modifications could affect interaction with adapter and signaling molecules. Indeed, AcPb is capable of forming a ligand-dependent complex with IL-1R, but it does not lead to the recruitment of the adaptor molecules MyD88 and IRAK4 after stimulation with IL-1, and is unable to mediate specific IL-1 responses. In both human and mouse, the expression of the AcPb is restricted to the CNS (whole brain, fetal brain, cerebellum, and spinal cord). AcP and AcPb are coexpressed in the same cells, but AcPb is the more abundant isoform. It has been proposed that AcPb could also be recruited to other AcP-utilizing receptors, such as ST2 and IL-1Rrp2/IL-36R, which are expressed in the CNS, once they have bound their ligands (IL-33 and IL-36α,β,γ, respectively). In a model of LPS challenge in the CNS, AcPb-deficiency was associated to neuronal loss suggesting that AcPb may dampen the neurotoxic effects of IL-1 by modulating the intracellular signaling and gene expression response to LPS-induced IL-1, or possibly to other cytokines acting through AcP ( 146 ). The inhibitory effect of AcPb could depend on the failure to recruit MyD88 and IRAK4, on the competition with AcP in a IL-1 receptor complex containing multiple IL-1R and AcP molecules, or on unknown functions mediated by the C-terminal tail. TIGIRR-1 and IL-1RAPL (also named TIGIRR-2) are localized on the X chromosome and share between 22 and 48% overall identity to other ILR family members. IL-1RAPL and TIGIRR-1 exons are spread out over a very large segment of genomic DNA (more than 1500 kb for IL-1RAPL and 380 kb for TIGIRR-1). Both TIGIRR-1 and IL-1RAPL contain a signal peptide, three predicted extracellular Ig domains, a single transmembrane domain, and a highly conserved cytoplasmic region containing a C-terminal cytoplasmic extension reminiscent of the Drosophila Toll family, TIR8/SIGIRR, and AcPb cytoplasmic domains. A negative regulatory role has not yet been reported for these two receptors. However, in in vitro studies performed with chimeric molecules, the cytoplasmic domains of TIGIRR-1 and IL-1RAPL fused to the extracellular and transmembrane domains of IL-1RI or AcP could not induce NF-κB, similarly to TIR8/SIGIRR, and in contrast with the cytoplasmic domains of other members of the ILR family ( 10 ). Other functional studies showed that IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas TIGIRR-1 cannot activate JNK. Deletion mutagenesis studies showed that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor ( 147 ). TIGIRR-1 is highly conserved in human and mouse (94.5% identical at the amino acid level) and it is expressed in skin, liver, placenta, and fetal brain. IL-1RAPL, whose crystal structure has been determined ( 147 ), is expressed in heart, brain, ovary, skin, and to a lesser extent in tonsil, fetal liver, prostate, testis, small intestine, placenta, and colon. IL-1RAPL was identified as the gene responsible for hereditary non-syndromic mental retardation and autism linked to chromosome region Xp22.1–21.3 ( 148 , 149 ). It is expressed in brain structures involved in the hippocampal memory system, and it has a role in brain development and function ( 150 ). No information are available about a potential role of IL-1RAPL in inflammation and defense, however, its C-terminal extension is reported to interact with neuronal calcium sensor-1 and regulate neurite outgrowth ( 150 – 152 ). Recently, a new member called DIGIRR was added to the ILR family ( 153 ). DIGIRR was discovered in teleost fish and showed high homology with TIR8/SIGIRR. DIGIRR is characterized by an extracellular portion comprising two Ig-like domains, a transmembrane domain and TIR domain carrying two amino acid substitutions (Arg419-Tyr420), which are responsible for the loss of signaling. The DIGIRR mRNA was found expressed in several tissues and in leukocytes and was upregulated by LPS, oppositely to TIR8/SIGIRR, suggesting a different mechanism of response to inflammatory stimuli between the two molecules. At the subcellular level, DIGIRR showed a peculiar distribution within the Golgi apparatus. Different lines of evidence suggest that DIGIRR acts as negative regulator of LPS- and IL-1β-induced inflammation. Indeed, siRNA knock down of DIGIRR lead to increased production of IL-β-induced pro-inflammatory cytokines in liver, kidney, and leukocytes. Moreover, in vitro administration of DIGIRR to zebrafish embryos significantly inhibited LPS- and IL-1β-induced activation of NF-kB. The discovery of DIGIRR could help to understand the evolution of ILR family members. Indeed, the authors suggest the hypothesis that DIGIRR and TIR8/SIGIRR derive from a common ancestral molecule that lost respectively one or two Ig-like extracellular domains, and Ser or Arg-Tyr- amino acids in the TIR domain. DIGIRR might represent an evolutionary intermediate molecule between IL-1R and TIR8/SIGIRR, demonstrating a shift from a potent receptor to a negative regulator.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Studies conducted in the early 1990s suggesting that the non-signaling IL-1RII acts as a molecular trap for the agonist and the AcP, led to the formulation of the decoy paradigm, which has then been extended to other cytokine families and chemokines. Decoy receptors are now recognized as a general strategy to tune the actions of primary inflammatory cytokines and chemokines. The list of ILR/TLR family receptors acting as negative regulators now includes TIR8/SIGIRR, which acts by modulating ILRs- or TLRs-dependent signaling. In addition to these molecules, soluble forms of signaling receptors or AcP act as decoys or negative regulators by trapping the ligands. For instance, T1/ST2 exists also as a soluble isoform obtained by differential mRNA processing, which acts as an antagonistic decoy receptor for IL-33 ( 154 ), and has been proposed in the therapy of arthritis ( 155 ). Similarly, soluble IL-1AcP, generated by alternative splicing, forms a complex with IL-1β and IL-1RII playing a protective role in arthritis ( 156 ) and has been pharmacologically exploited. For several of these molecules further studies have to be performed to unequivocally define their role in disease and their potential as therapeutic targets. For instance, unfortunately there are no genetic evidence on the consequence of IL-1RII-gene deficiency or data supporting the relevance of TIR8/SIGIRR in human disease. In addition, the clinical development of IL-RII pharmacological targeting has not been reported. Finally, pharmacological approaches targeting TIR8/SIGIRR functions have not been developed yet and they will be necessary to assess whether TIR8/SIGIRR might be a therapeutic target in inflammatory conditions. However, the existence of IL-1RII, together with IL-1Ra, TIR8/SIGIRR, brain AcPb, and other soluble receptors acting as molecular traps emphasizes the need for tight control of the IL-1 system, which mediates potentially devastating local and systemic inflammatory reactions.

IL-1R like receptors (ILRs) belong, together with Toll-like receptors (TLRs), to a superfamily of phylogenetically conserved proteins involved in innate immunity and inflammation ( 1 – 5 ). The common characteristic of the members of this family is the presence of a conserved domain in the cytoplasmic region, called TIR domain, originally defined as the Toll/IL-1-resistance and now generally assumed as an acronym for Toll/IL-1R domain. The TIR domain is involved in the activation of an evolutionarily conserved signaling pathway leading to NF-kB translocation to the nucleus and activation of protein kinases such as p38, c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinases (ERKs), and mitogen-activated protein kinases (mAPKs) ( 6 ). The ILR subfamily includes the receptors and the accessory proteins (AcP) for IL-1α (IL-1F1) and IL-1β (IL-1F2), IL-18/IL-1F4, IL-33/IL-F11, and other IL-1 family members (IL-36α/IL-1F6, IL-36β/IL-1F8, and IL-36γ/IL-1F9), which are involved in the initiation of an amplification cascade of innate resistance and inflammation and contribute to the activation and orientation of adaptive immunity ( 7 – 9 ). Some members of the family remain orphan receptors with still unknown ligands and functions. For instance, in the IL-1R subfamily, TIR8/SIGIRR, TIGIRR-1, and IL-1RAPL have no characterized ligands so far ( 2 , 10 , 11 ). The activation of the ILR-dependent signaling cascade is tightly regulated. Indeed, the deregulated activation of these receptors, which lead to the production of proteins related to inflammation and immunity, potentially mediates damaging local and systemic inflammatory reactions. Several pathological conditions depend, at least in part, on the inflammatory potential of the IL-1 family members mentioned above. For instance, the IL-1 system represents a relevant therapeutic target in arthritis, type 2 diabetes, psoriasis, sepsis, ischemia and reperfusion, atherosclerosis, graft rejection, cancer ( 12 – 15 ). The regulatory mechanisms identified so far in the IL-1 system (ligands, receptors, signaling pathway) act extracellularly or intracellularly ( 16 , 17 ). IL-1R antagonists (IL-1Ra)/IL-1F3 and IL-36Ra/IL-1F5 are polypeptide antagonists competing with IL-1 and IL-36α/IL-1F6, IL-36β/IL-1F8, and IL-36γ/IL-1F9, respectively, for receptor binding ( 3 , 7 , 18 – 20 ). IL-1RII lacks a signaling domain and by binding IL-1 prevents its interaction with a signaling receptor complex and therefore acts as a decoy, dominant-negative molecule, and scavenger. The negative regulator of ILR and TLR signaling, TIR8 (also known as SIGIRR), acts intracellularly. IRAK-M and MyD88s are intracellular negative regulators of ILRs and TLRs signaling ( 21 , 22 ). Finally, ILR or TLR signaling proteins or transcription factors are targets of miRNAs, such as miR-155, miR-21, miR-146a, miR-132, miR-9, and miR-147, whose transcription is induced by inflammatory mediators [lipopolysaccharide (LPS), TNFα, IL-1 β] through NF-kB ( 23 – 25 ). Here, we summarize our current understanding of the structure and function of negative regulatory receptors of the ILR family, in particular IL-1RII, which has served to defining the decoy receptor paradigm, and TIR8/SIGIRR, focusing on their regulatory roles in different pathological disorders dependent on ILRs and TLRs activity, and finally describe other largely uncharacterized members of the family with a negative regulatory potential, TIGIRR-1, IL-1RAPL, IL-1RAPb.