Glycan-mediated functional assembly of IL-1RI: structural insights into completion of the current description for immune response.

Interleukin 1 Receptor type I (IL-1RI) is a multi-domain transmembrane receptor that triggers the inflammatory response. Understanding its detailed mechanism of action is crucial for treating immune disorders. IL-1RI is activated upon formation of its functional assembly that occurs by binding of the IL-1 cytokine and the accessory protein (Il-1RAcP) to it. X-ray crystallography, small-Angle X-ray Scattering and molecular dynamics simulation studies showed that IL-1RI adopts two types of 'compact' and 'extended' conformational states in its dynamical pattern. Furthermore, glycosylation has shown to play a critical role in its activation process. Here, classical and accelerated atomistic molecular dynamics were carried out to examine the role of full glycosylation of IL-1RI and IL-1RAcP in arrangement of the functional assembly. Simulations showed that the 'compact' and 'extended' IL-1RI form two types of 'cytokine-inaccessible-non-signaling' and 'cytokine-accessible-signaling' assemblies with the IL-1RacP, respectively that are both abiding in the presence of glycans. Suggesting that the cytokine binding to IL-1RI is not required for the formation of IL-1RI-IL-1RAcP complex and the 'compact' complex could act as a down-regulatory mechanism. The 'extended' complex is maintained by formation of several persistent hydrogen bonds between the IL-1RI-IL-1RAcP inter-connected glycans. Taken together, it was shown that full glycosylation regulates formation of the IL-1RI functional assembly and play critical role in cytokine biding and triggering the IL-1RI involved downstream pathways in the cell.Communicated by Ramaswamy H. Sarma.

IL-1ß promotes immunoregulatory responses in human blood basophils.

Human basophils are essential effector cells of chronic allergic inflammation. IL-1 family cytokines such as interleukin (IL)-33 and IL-1ß are elevated in serum and bronchoalveolar lavage fluid of allergic asthmatics. IL-33 is known to be a critical regulator of basophil's T2 immune responses. However, the effect of IL-1ß on the function of basophils has not been well investigated. Here, we elucidate whether IL-1ß regulates the function of human basophils and compared the effects of IL-1ß and IL-33 on basophils of healthy and allergic subjects. We found that IL-1ß activates the p38 MAPK signaling pathway and promotes IL-8 release in basophils of healthy donors, while FcεRI-mediated LCT4 and histamine secretion is not affected. Strikingly, in the presence of IL-3, IL-1ß shows more potency than IL-33, as evidenced by the enhanced p38 phosphorylation and NF-κB activation, as well as the release of both IL-13 and IL-8. We found that the enhanced basophil responsiveness is achieved through IL-3-induced IL-1RI surface expression. Importantly, basophils of allergic donors release significantly higher amounts of IL-8 compared to those from healthy donors upon IL-33 and IL-1ß stimulation. Consistently, we detected increased IL-1RI and decreased IL-3 receptor alpha-chain (CD123) and CCR3 expression on basophils of allergic subjects compared to healthy controls, suggesting an in vivo IL-3 priming in allergic donors. In summary, our results suggest enhanced sensitivity of basophils toward IL-33 and IL-1ß in allergic subjects compared to those from healthy controls.

Structure-based Development of Human Interleukin-1ß-Specific Antibody That Simultaneously Inhibits Binding to Both IL-1RI and IL-1RAcP.

Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.

GAG positioning on IL-1RI; A mechanism regulated by dual effect of glycosylation.

IL-1RI is the signaling receptor for the IL-1 family of cytokines that are involved in establishment of the innate and acquired immune systems. Glycosylated extracellular (EC) domain of the IL-1RI binds to agonist such as IL-1ß or antagonist ligands and the accessory protein to form the functional signaling complex. Dynamics and ligand binding of the IL-1RI is influenced by presence of the glycosaminoglycans (GAGs) of the EC matrix. Here a combination of molecular dockings and molecular dynamics simulations of the unglycosylated, partially N-glycosylated and fully N-glycosylated IL-1RI EC domain in the apo, GAG-bound and IL-1ß-bound states were carried out to explain the co-occurring dynamical effect of receptor's glycosylation and GAGs. It was shown that the IL-1RI adopts two types of "extended" and "locked" conformations in its dynamical pattern, and glycosylation maintains the receptor in the latter form. Maintaining the receptor in the locked conformation disfavors IL-1ß binding by burying its two binding site on the IL-1RI EC domain. Glycosylation disfavors GAG binding to the extended IL-1RI EC domain by sterically limiting the GAGs degrees of freedom in targeting its binding site, while it favors GAG binding to the locked IL-1RI by favorable packing interactions.

Analysis of Surface Levels of IL-1 Receptors and Macrophage Scavenger Receptor I in Peripheral Immune Cells of Patients With Alzheimer Disease.

Increased concentrations of interleukin 1 (IL-1) in the cerebrospinal fluid and serum of patients with Alzheimer disease (AD) reduced phagocytic capacity point to an inflammatory activation of mononuclear phagocytes in AD. Interleukin 1 receptors (IL-1R) and the macrophage scavenger receptor I (MSRI) are important players in IL-1 signaling and phagocytosis. In 20 patients with AD and 17 controls, IL-1RI, IL-1RII, and MSRI were assessed on peripheral blood mononuclear cells by flow cytometry. IL-1ß, soluble IL-1 receptors, and IL-1R antagonist (IL-1Ra) were measured by enzyme-linked immunosorbent assay. The fraction of IL-1RI+ monocytes was increased by 10% and the expression of MSRI was reduced by 12% in AD. A 3.6% increased fraction of IL-1RI+ lymphocytes was accompanied by a 6.1% reduced expression of IL-1RII. The IL-1RI on monocytes and lymphocytes discriminated patients with AD with an accuracy of 0.79 and 0.75, respectively. The IL-1Ra was elevated in AD. Changes in the expression of IL-1 receptors and MSRI on peripheral blood cells fit to the concept of a proinflammatory state of the peripheral immune system. However, the observed differences are not strong enough to suggest their application as biomarkers for AD.

Molecular cloning and expression analysis of interleukin-1ß and interleukin-1 receptor type I genes in yellow catfish (Pelteobagrus fulvidraco): Responses to challenge of Edwardsiella ictaluri.

Interleukin-1ß (IL-1ß) is one of the pivotal early pro-inflammatory cytokines, which play important roles in regulating immune response and inducing a series of inflammatory reactions to infections. Interleukin-1 type I receptor (IL-1RI) is a receptor of the IL-1ß that can mediate IL-1-dependent activation. In this study, partial cDNA sequences of the Pf_IL-1ß and Pf_IL-1RI genes were cloned from yellow catfish (Pelteobagrus fulvidraco). The open reading frames (ORF) of Pf_IL-1ß and Pf_IL-1RI genes encode putative peptides of 280 and 543 amino acids, respectively. The deduced amino acid sequences of these two genes shared highly conserved structures with those from other teleosts. Quantitative real-time PCR results showed that the Pf_IL-1ß mRNA had relatively high expression levels in trunk kidney and blood, and the Pf_IL-1RI mRNA was highly expressed in blood and had relatively high expression level in liver. Ontogenetic expression analyses indicate that the Pf_IL-1ß and Pf_IL-1RI genes may play important roles during the embryonic developmental stages. The mRNA expression levels of Pf_IL-1ß and Pf_IL-1RI genes were up-regulated in the trunk kidney, head kidney, blood, spleen, heart and liver after Edwardsiella ictaluri challenge. Western blot analyses showed that Pf_IL-1ß protein was highly expressed in the spleen and head kidney, but not in the fin of adult individuals. These results suggest that the Pf_IL-1ß and Pf_IL-1RI genes may play significant roles in the immune regulation and defense against E. ictaluri in the yellow catfish.

The IL-1RI Co-Receptor TILRR (FREM1 Isoform 2) Controls Aberrant Inflammatory Responses and Development of Vascular Disease.

Expression of the interleukin-1 receptor type I (IL-1RI) co-receptor Toll-like and interleukin-1 receptor regulator (TILRR) is significantly increased in blood monocytes following myocardial infarction and in the atherosclerotic plaque, whereas levels in healthy tissue are low. TILRR association with IL-1RI at these sites causes aberrant activation of inflammatory genes, which underlie progression of cardiovascular disease. The authors show that genetic deletion of TILRR or antibody blocking of TILRR function reduces development of atherosclerotic plaques. Lesions exhibit decreased levels of monocytes, with increases in collagen and smooth muscle cells, characteristic features of stable plaques. The results suggest that TILRR may constitute a rational target for site- and signal-specific inhibition of vascular disease.

Characterization of IL-1ß and two types of IL-1 receptors in miiuy croaker and evolution analysis of IL-1 family.

Interleukin (IL)-1ß is a prototypical proinflammatory cytokine that belongs to the IL-1 family. This cytokine possesses two receptor types, namely, IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII). IL-1RI, is an IL-1 receptor that plays a crucial role in immune responses and IL-1RII is a decoy receptor for IL-1ß signaling inhibitors in mammals. IL-1ß, together with its two types of receptors, has been characterized in mammals and implicated in immunity. However, IL-1ß and IL-1 receptors in teleost species have been rarely investigated. In this study three genes, namely, IL-1ß, IL-1RI, and IL-1RII, were identified and characterized from miiuy croaker. Structural and comparative analysis revealed that miiuy croaker IL-1ß, IL-1RI and IL-1RII, particularly their functional domains, were conservative in most of the species. Simultaneously, synteny phylogenetic analysis indicated that IL-1ß and IL-18 were widely distributed in vertebrates and hence might be the ancestors of the IL-1 family. Challenge experiment demonstrated that IL-1ß, IL-1RI and IL-1RII expression in miiuy croaker was induced by LPS and poly (I:C). IL-1RI expression was also induced by the overexpressed miiuy croaker IL-1ß protein which in cell supernatant, whereas IL-1RII was not induced.

Proinflammatory signaling regulates voluntary alcohol intake and stress-induced consumption after exposure to social defeat stress in mice.

Proinflammatory activity has been postulated to play a role in addictive processes and stress responses, but the underlying mechanisms remain largely unknown. Here, we examined the role of interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α) in regulation of voluntary alcohol consumption, alcohol reward and stress-induced drinking. Mice with a deletion of the IL-1 receptor I gene (IL-1RI KO) exhibited modestly decreased alcohol consumption. However, IL-1RI deletion affected neither the rewarding properties of alcohol, measured by conditioned place preference (CPP), nor stress-induced drinking induced by social defeat stress. TNF-α signaling can compensate for phenotypic consequences of IL1-RI deletion. We therefore hypothesized that double deletion of both IL-1RI and TNF-1 receptors (TNF-1R) may reveal the role of these pathways in regulation of alcohol intake. Double KOs consumed significantly less alcohol than control mice over a range of alcohol concentrations. The combined deletion of TNF-1R and IL-1RI did not influence alcohol reward, but did prevent increased alcohol consumption resulting from exposure to repeated bouts of social defeat stress. Taken together, these data indicate that IL-1RI and TNF-1R contribute to regulation of stress-induced, negatively reinforced drinking perhaps through overlapping signaling events downstream of these receptors, while leaving rewarding properties of alcohol largely unaffected.

Effect of ozone combined with arthroscopy on expression of IL1R I,CXCL13 and IL24 gene in synovium of osteoarthritis of knee joint / 实用医学杂志

Objective To observe the effect of medical ozone on the expression of IL1R,CXCL-13 and IL24 gene in synovial membrane of osteoarthritis of knee joint. Methods Sixty-five cases of knee osteoarthritis including 11 lost-cases were selected ,and randomly divided into combination group and arthroscopy group with 27 cases in each group. After arthroscopic surgery ,combination group performed intra-articular injection of 40μg/mL concentration of medical ozone 40 mL/week for 2 weeks but arthroscopic surgery group had no ozone injection. Differences of the expression of IL1R I,CXCL13 and IL24 gene and protein in synovium were compared before and after the treatment in two groups by RT-PCR and Western blot. Results Expression of IL1R I gene and protein in synovium of combination group was significantly lower than that of arthroscopy group and it showed statistical significance(P<0.01). Expression of CXCL13 gene and IL24 gene and protein in synovium of combination group was higher than those of arthroscopy group and it had statistical significance (P < 0.05). Conclusions Medical ozone can reduce the symptoms of arthritis and slow synovitis progress through influencing the expression of IL1R, CXCL-13 and IL24 gene. The effect of ozone combined with arthroscopic is better than that of simple arthroscopic debridement,but cannot stop and reverse the progression of the disease completely.

Effect of ozone combined with arthroscopy on expression of IL1R I,CXCL13 and IL24 gene in synovium of osteoarthritis of knee joint / 实用医学杂志

Objective To observe the effect of medical ozone on the expression of IL1R,CXCL-13 and IL24 gene in synovial membrane of osteoarthritis of knee joint. Methods Sixty-five cases of knee osteoarthritis including 11 lost-cases were selected ,and randomly divided into combination group and arthroscopy group with 27 cases in each group. After arthroscopic surgery ,combination group performed intra-articular injection of 40μg/mL concentration of medical ozone 40 mL/week for 2 weeks but arthroscopic surgery group had no ozone injection. Differences of the expression of IL1R I,CXCL13 and IL24 gene and protein in synovium were compared before and after the treatment in two groups by RT-PCR and Western blot. Results Expression of IL1R I gene and protein in synovium of combination group was significantly lower than that of arthroscopy group and it showed statistical significance(P<0.01). Expression of CXCL13 gene and IL24 gene and protein in synovium of combination group was higher than those of arthroscopy group and it had statistical significance (P < 0.05). Conclusions Medical ozone can reduce the symptoms of arthritis and slow synovitis progress through influencing the expression of IL1R, CXCL-13 and IL24 gene. The effect of ozone combined with arthroscopic is better than that of simple arthroscopic debridement,but cannot stop and reverse the progression of the disease completely.

miR-192 targeting IL-1RI regulates the immune response in miiuy croaker after pathogen infection in vitro and in vivo.

Activation of innate and acquired immune responses is regulated by detailed mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling the diverse of biophysical and biochemical processes at the post-transcriptional level. However, the physiological roles of miRNAs in aquatic organisms are largely unclear. In this study, we explored the potential roles of mmi-miR-192 in regulating interleukin 1 receptor type I (IL-1RI) involved in immune and inflammatory response in miiuy croakers. This was further evaluated by negative expression profiles in both LPS exposure macrophages and Vibrio anguillarum challenged miiuy croaker. By means of promoter analysis, mmi-miR-192 was found to be an AP-1 dependent gene. Importantly, the dual luciferase reporter assay presented the regulation between mmi-miR-192 and IL-1RI. The result of miiuy croaker miR-192 reduced the wild-type IL-1RI but not the mutant one luciferase levels suggested that mmi-miR-192 modulated IL-1RI expression by directly targeting the 3'UTR of IL-1RI mRNA. Overall, our study revealed the mechanism that the miR-192-IL1RI pathway regulated bacteria infection in miiuy croakers.

Identification of critical regions within the TIR domain of IL-1 receptor type I.

Interleukin-1 receptor type I (IL-1RI) belongs to a superfamily of proteins characterized by an intracellular Toll/IL-1 receptor (TIR) domain. This domain harbors three conserved regions called boxes 1-3 that play crucial roles in mediating IL-1 responses. Boxes 1 and 2 are considered to be involved in binding of adapter molecules. Amino acids possibly crucial for IL-1RI signaling were predicted via homology models of the IL-1RI TIR domain based on the crystal structure of IL-1RAPL. The role of ten of these residues was investigated by site-directed mutagenesis and a functional luciferase assay reflecting NF-κB activity in transiently transfected Jurkat cells. In particular, the mutants E437K/D438K, E472A/E473A and S465A/S470A/S471A/E472A/E473A showed decreased and the mutant E437A/D438A increased IL-1 responsiveness compared to the mouse IL-1RI wild type. In conclusion, the αC' helix (Q469-E473 in mouse IL-1RI) is probably involved in heterotypic interactions of IL-1RI with IL-1RAcP or MyD88.

IL-1ß increases necrotic neuronal cell death in the developing rat hippocampus after status epilepticus by activating type I IL-1 receptor (IL-1RI).

Interleukin-1ß (IL-1ß) is associated with seizure-induced neuronal cell death in the adult brain. The contribution of IL-1ß to neuronal injury induced by status epilepticus (SE) in the immature brain remains unclear. In the present study, we investigated the effects of IL-1ß administration on hippocampal neuronal cell death associated with SE in the immature brain, and the role of the type I receptor of IL-1ß (IL-1RI). SE was induced with lithium-pilocarpine in 14-days-old (P14) rat pups. Six hours after SE onset, pups were i.c.v. injected in the right ventricle with IL-1ß (0, 0.3, 3, 30, or 300 ng), 30 ng of IL-1RI antagonist (IL-1Ra) alone, or 30 ng of IL-1Ra plus 3ng of IL-1ß. As control groups, pups without seizures were injected with 3 ng of IL-1ß or vehicle. Twenty-four hours after SE onset, neuronal cell death in the CA1 field of dorsal hippocampus was assessed by hematoxylin-eosin, Fluoro-Jade B and in vivo propidium iodide (PI) staining; expression of active caspase-3 (aCas-3) was also determined, using immunohistochemistry. The concentration-response curve of IL-1ß showed a bell-shape. Only pups injected with 3 ng of IL-1ß after SE showed a significant increase in the number of cells with eosinophilic cytoplasm and pyknotic nuclei, as well as F-JB positive cells with respect to the vehicle group. This effect was prevented when IL-1ß was injected with IL-1Ra. Injection of 3 ng of IL-1ß increased the number of PI-positive cells in CA1 area after SE. Injection of 3 ng of IL-1ß did not produce hippocampal cell death in rats without seizures. Active caspase-3 expression was not observed after treatments in hippocampus. The activation of the IL-1ß/IL-1RI system increases necrotic neuronal cell death caused by SE in rat pups.

Chondrocalcin is internalized by chondrocytes and triggers cartilage destruction via an interleukin-1ß-dependent pathway.

Chondrocalcin is among the most highly synthesized polypeptides in cartilage. This protein is released from its parent molecule, type II pro-collagen, after secretion by chondrocytes. A participation of extracellular, isolated chondrocalcin in mineralization was proposed more than 25 years ago, but never demonstrated. Here, exogenous chondrocalcin was found to trigger MMP13 secretion and cartilage destruction ex vivo in human cartilage explants and did so by modulating the expression of interleukin-1ß in primary chondrocyte cultures in vitro. Chondrocalcin was found internalized by chondrocytes. Uptake was found mediated by a single 18-mer peptide of chondrocalcin, which does not exhibit homology to any known cell-penetrating peptide. The isolated peptide, when artificially linked as a tetramer, inhibited gene expression regulation by chondrocalcin, suggesting a functional link between uptake and gene expression regulation. At the same time, the tetrameric peptide potentiated chondrocalcin uptake by chondrocytes, suggesting a cooperative mechanism of entry. The corresponding peptide from type I pro-collagen supported identical cell-penetration, suggesting that this property may be conserved among C-propeptides of fibrillar pro-collagens. Structural modeling localized this peptide to the tips of procollagen C-propeptide trimers. Our findings shed light on unexpected function and mechanism of action of these highly expressed proteins from vertebrates.

IL-1 and T Helper Immune Responses.

CD4 T cells play a critical role in mediating adaptive immunity to a variety of pathogens as well as in tumor immunity. If not adequately regulated, CD4 T cells can be also involved in autoimmunity, asthma, and allergic responses. During TCR activation in a particular cytokine milieu, naïve CD4 T cells may differentiate into one of several lineages of T helper (Th) cells, including Th1, Th2, and Th17, as defined by their pattern of cytokine production and function. IL-1, the prototypic proinflammatory cytokine, has been shown to influence growth and differentiation of immunocompetent lymphocytes. The differential expression of IL-1RI on human CD4 T cell subsets confers distinct capacities to acquire specific effector functions. In this review, we summarize the role of IL-1 on CD4 T cells, in terms of differentiation, activation, and maintenance or survival.

Tollip-induced down-regulation of MARCH1.

In addition to their classical antigen presenting functions, MHC class II molecules potentiate the TLR-triggered production of pro-inflammatory cytokines. Here, we have addressed the effect of Tollip and MARCH1 on the regulation of MHC II trafficking and TLR signaling. Our results show that MARCH1-deficient mice splenocytes are impaired in their capacity to produce pro-inflammatory cytokines in response to poly(I:C) and that TLR3 and MHC II molecules interact in the endocytic pathway. Knocking down Tollip expression in human CIITA(+) HeLa cells increased expression of HLA-DR but reduced the proportion of MHC II molecules associated with the CLIP peptide. Truncation of the HLA-DR cytoplasmic tails abrogated the effect of Tollip on MHC class II expression. While overexpression of Tollip did not affect HLA-DR levels, it antagonized the function of co-transfected MARCH1. We found that Tollip strongly reduced MARCH1 protein levels and that the two molecules appear to compete for binding to MHC II molecules. Altogether, our results demonstrate that Tollip regulates MHC class II trafficking and that MARCH1 may represent a new Tollip target.

EXPRESSIONS OF IL-1? AND IL-1RI mRNA IN THE RAT CAROTID BODY / 解剖学报

Objective To study the expressions of IL-1 receptor type Ⅰ(IL-1RI) mRNA and IL-1? protein in the rat carotid body. Methods In situ hybridization,immunofluorescence double staining and Western blotting methods were used. Results The result of in situ hybridization showed that the positive signal of IL-1? mRNA was mainly located in the glomus cells of the carotid body.The result of immunofluorescence double staining showed that IL-1? protein also expressed in the glomus cells of the organ.The Western blotting proved that the IL-1? immunoreactive band appeared at 18kD,consistent with the molecular weight of the cytokine.Conclusion The glomus cells of the rat carotid body not only express IL-1RI mRNA,but also IL-1?.