RESUMO
Interleukin (IL)-1R3 is the co-receptor in three signaling pathways that involve six cytokines of the IL-1 family (IL-1α, IL-1ß, IL-33, IL-36α, IL-36ß and IL-36γ). In many diseases, multiple cytokines contribute to disease pathogenesis. For example, in asthma, both IL-33 and IL-1 are of major importance, as are IL-36 and IL-1 in psoriasis. We developed a blocking monoclonal antibody (mAb) to human IL-1R3 (MAB-hR3) and demonstrate here that this antibody specifically inhibits signaling via IL-1, IL-33 and IL-36 in vitro. Also, in three distinct in vivo models of disease (crystal-induced peritonitis, allergic airway inflammation and psoriasis), we found that targeting IL-1R3 with a single mAb to mouse IL-1R3 (MAB-mR3) significantly attenuated heterogeneous cytokine-driven inflammation and disease severity. We conclude that in diseases driven by multiple cytokines, a single antagonistic agent such as a mAb to IL-1R3 is a therapeutic option with considerable translational benefit.
Assuntos
Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Proteína Acessória do Receptor de Interleucina-1/antagonistas & inibidores , Peritonite/imunologia , Pneumonia/imunologia , Psoríase/imunologia , Células A549 , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células HEK293 , Humanos , Imiquimode/toxicidade , Inflamação/patologia , Interleucina-1/imunologia , Proteína Acessória do Receptor de Interleucina-1/imunologia , Interleucina-1beta/imunologia , Interleucina-33/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/toxicidade , Peritonite/tratamento farmacológico , Peritonite/patologia , Pneumonia/tratamento farmacológico , Pneumonia/patologia , Psoríase/tratamento farmacológico , Psoríase/patologia , Transdução de Sinais/imunologia , Ácido Úrico/toxicidadeRESUMO
Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.
Assuntos
Subunidade alfa de Receptor de Interleucina-18/imunologia , Interleucina-1/imunologia , Leucócitos Mononucleares/imunologia , Receptores de Interleucina-1/imunologia , Transdução de Sinais/imunologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-1/genética , Subunidade alfa de Receptor de Interleucina-18/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-18/genética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , NF-kappa B/imunologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/imunologia , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , c-Mer Tirosina QuinaseRESUMO
The IL-1 Family member IL-38 has been characterized primarily as an antiinflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL-38 in the murine small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL-38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL-38-deficient mice develop more slowly. This reduction in size is likely due to the downregulation of intestinal stemness markers (i.e., Fzd5, Ephb2, and Olfm4) expression compared with wild-type organoids. The IL-38 binding to IL-1R6 and IL-1R9 is still a matter of debate. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL-1R9-deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL-38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL-38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild-type mice, IL-38 stimulation induced low concentrations of IL-1ß which contribute to organoid growth. However, high concentrations of IL-1ß have detrimental effects on the cultures that were prevented by treatment with recombinant IL-38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an antiinflammatory molecule in the SI, maintaining homeostasis.
Assuntos
Mucosa Intestinal , Via de Sinalização Wnt , Animais , Camundongos , Homeostase , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interleucinas/metabolismo , Mucosa Intestinal/metabolismo , Organoides/metabolismo , Células-Tronco/metabolismoRESUMO
Interleukin-1ß (IL-1ß)-mediated inflammation suppresses antitumor immunity, leading to the generation of a tumor-permissive environment, tumor growth, and progression. Here, we demonstrate that nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation in melanoma is linked to IL-1ß production, inflammation, and immunosuppression. Analysis of cancer genome datasets (TCGA and GTEx) revealed greater NLRP3 and IL-1ß expression in cutaneous melanoma samples (n = 469) compared to normal skin (n = 324), with a highly significant correlation between NLRP3 and IL-1ß (P < 0.0001). We show the formation of the NLRP3 inflammasome in biopsies of metastatic melanoma using fluorescent resonance energy transfer analysis for NLRP3 and apoptosis-associated speck-like protein containing a CARD. In vivo, tumor-associated NLRP3/IL-1 signaling induced expansion of myeloid-derived suppressor cells (MDSCs), leading to reduced natural killer and CD8+ T cell activity concomitant with an increased presence of regulatory T (Treg) cells in the primary tumors. Either genetic or pharmacological inhibition of tumor-derived NLRP3 by dapansutrile (OLT1177) was sufficient to reduce MDSCs expansion and to enhance antitumor immunity, resulting in reduced tumor growth. Additionally, we observed that the combination of NLRP3 inhibition and anti-PD-1 treatment significantly increased the antitumor efficacy of the monotherapy by limiting MDSC-mediated T cell suppression and tumor progression. These data show that NLRP3 activation in melanoma cells is a protumor mechanism, which induces MDSCs expansion and immune evasion. We conclude that inhibition of NLRP3 can augment the efficacy of anti-PD-1 therapy.
Assuntos
Melanoma Experimental/imunologia , Células Supressoras Mieloides/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas de Neoplasias/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Neoplasias/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Unchecked inflammation can result in severe diseases with high mortality, such as macrophage activation syndrome (MAS). MAS and associated cytokine storms have been observed in COVID-19 patients exhibiting systemic hyperinflammation. Interleukin-18 (IL-18), a proinflammatory cytokine belonging to the IL-1 family, is elevated in both MAS and COVID-19 patients, and its level is known to correlate with the severity of COVID-19 symptoms. IL-18 binds its specific receptor IL-1 receptor 5 (IL-1R5, also known as IL-18 receptor alpha chain), leading to the recruitment of the coreceptor, IL-1 receptor 7 (IL-1R7, also known as IL-18 receptor beta chain). This heterotrimeric complex then initiates downstream signaling, resulting in systemic and local inflammation. Here, we developed a novel humanized monoclonal anti-IL-1R7 antibody to specifically block the activity of IL-18 and its inflammatory signaling. We characterized the function of this antibody in human cell lines, in freshly obtained peripheral blood mononuclear cells (PBMCs) and in human whole blood cultures. We found that the anti-IL-1R7 antibody significantly suppressed IL-18-mediated NFκB activation, reduced IL-18-stimulated IFNγ and IL-6 production in human cell lines, and reduced IL-18-induced IFNγ, IL-6, and TNFα production in PBMCs. Moreover, the anti-IL-1R7 antibody significantly inhibited LPS- and Candida albicans-induced IFNγ production in PBMCs, as well as LPS-induced IFNγ production in whole blood cultures. Our data suggest that blocking IL-1R7 could represent a potential therapeutic strategy to specifically modulate IL-18 signaling and may warrant further investigation into its clinical potential for treating IL-18-mediated diseases, including MAS and COVID-19.
Assuntos
Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Fatores Imunológicos/farmacologia , Interleucina-18/genética , Receptores de Interleucina-18/genética , Anti-Inflamatórios/metabolismo , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fatores Imunológicos/biossíntese , Inflamação , Interferon gama/genética , Interferon gama/imunologia , Interleucina-18/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Síndrome de Ativação Macrofágica/tratamento farmacológico , NF-kappa B/genética , NF-kappa B/imunologia , Cultura Primária de Células , Receptores de Interleucina-18/antagonistas & inibidores , Receptores de Interleucina-18/imunologia , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Tratamento Farmacológico da COVID-19RESUMO
Interleukin-37 (IL-37), a member of the IL-1 family of cytokines, is a fundamental suppressor of innate and acquired immunities. Here, we used an integrative approach that combines biophysical, biochemical, and biological studies to elucidate the unique characteristics of IL-37. Our studies reveal that single amino acid mutations at the IL-37 dimer interface that result in the stable formation of IL-37 monomers also remain monomeric at high micromolar concentrations and that these monomeric IL-37 forms comprise higher antiinflammatory activities than native IL-37 on multiple cell types. We find that, because native IL-37 forms dimers with nanomolar affinity, higher IL-37 only weakly suppresses downstream markers of inflammation whereas lower concentrations are more effective. We further show that IL-37 is a heparin binding protein that modulates this self-association and that the IL-37 dimers must block the activity of the IL-37 monomer. Specifically, native IL-37 at 2.5 nM reduces lipopolysaccharide (LPS)-induced vascular cell adhesion molecule (VCAM) protein levels by â¼50%, whereas the monomeric D73K mutant reduced VCAM by 90% at the same concentration. Compared with other members of the IL-1 family, both the N and the C termini of IL-37 are extended, and we show they are disordered in the context of the free protein. Furthermore, the presence of, at least, one of these extended termini is required for IL-37 suppressive activity. Based on these structural and biological studies, we present a model of IL-37 interactions that accounts for its mechanism in suppressing innate inflammation.
Assuntos
Tolerância Imunológica , Imunidade Inata , Interleucina-1/metabolismo , Linhagem Celular , Cristalografia por Raios X , Humanos , Tolerância Imunológica/imunologia , Tolerância Imunológica/fisiologia , Interleucina-1/genética , Interleucina-1/fisiologia , Espectroscopia de Ressonância Magnética , Multimerização ProteicaRESUMO
The IL-1 family member IL-37 broadly suppresses innate inflammation and acquired immunity. Similar to IL-1α and IL-33, IL-37 is a dual-function cytokine in that IL-37 translocates to the nucleus but also transmits a signal via surface membrane receptors. The role of nuclear IL-37 remains unknown on the ability of this cytokine to inhibit innate inflammation. Here, we compared suppression of innate inflammation in transgenic mice expressing native human IL-37 (IL-37Tg) with those of transgenic mice carrying the mutation of aspartic acid (D) to alanine (A) at amino acid 20 (IL-37D20ATg). The mutation D20A prevents cleavage of caspase-1, a step required for IL-37 nuclear translocation. In vitro, peritoneal macrophages from IL-37Tg mice reduced LPS-induced IL-1ß, IL-6, TNFα and IFNγ by 40-50% whereas in macrophages from IL-37D20ATg mice this suppression was not observed, consistent with loss of nuclear function. Compared with macrophages from IL-37Tg mice, significantly less or no suppression of LPS-induced MAP kinase and NFκB activation was also observed in macrophages from IL-37D20ATg mice. In vivo, levels of IL-1ß, IL-6, and TNFα in the lungs and liver were markedly reduced during endotoxemia in IL-37Tg mice but not observed in IL-37D20ATg mice. However, suppression of innate inflammation remains intact in the IL-37D20A mice once the cytokine is released from the cell and binds to its receptor. These studies reveal a nuclear function for suppression of innate inflammation and are consistent with the dual function of IL-37 and a role for caspase-1 in limiting inflammation.
Assuntos
Imunidade Inata/genética , Interleucina-1/fisiologia , Animais , Núcleo Celular/metabolismo , Citocinas/metabolismo , Feminino , Interleucina-1/genética , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , NF-kappa B/metabolismo , Transporte ProteicoRESUMO
Activation of the NLRP3 inflammasome induces maturation of IL-1ß and IL-18, both validated targets for treating acute and chronic inflammatory diseases. Here, we demonstrate that OLT1177, an orally active ß-sulfonyl nitrile molecule, inhibits activation of the NLRP3 inflammasome. In vitro, nanomolar concentrations of OLT1177 reduced IL-1ß and IL-18 release following canonical and noncanonical NLRP3 inflammasome activation. The molecule showed no effect on the NLRC4 and AIM2 inflammasomes, suggesting specificity for NLRP3. In LPS-stimulated human blood-derived macrophages, OLT1177 decreased IL-1ß levels by 60% and IL-18 by 70% at concentrations 100-fold lower in vitro than plasma concentrations safely reached in humans. OLT1177 also reduced IL-1ß release and caspase-1 activity in freshly obtained human blood neutrophils. In monocytes isolated from patients with cryopyrin-associated periodic syndrome (CAPS), OLT1177 inhibited LPS-induced IL-1ß release by 84% and 36%. Immunoprecipitation and FRET analysis demonstrated that OLT1177 prevented NLRP3-ASC, as well as NLRP3-caspase-1 interaction, thus inhibiting NLRP3 inflammasome oligomerization. In a cell-free assay, OLT1177 reduced ATPase activity of recombinant NLRP3, suggesting direct targeting of NLRP3. Mechanistically, OLT1177 did not affect potassium efflux, gene expression, or synthesis of the IL-1ß precursor. Steady-state levels of phosphorylated NF-κB and IkB kinase were significantly lowered in spleen cells from OLT1177-treated mice. We observed reduced IL-1ß content in tissue homogenates, limited oxidative stress, and increased muscle oxidative metabolism in OLT1177-treated mice challenged with LPS. Healthy humans receiving 1,000 mg of OLT1177 daily for 8 d exhibited neither adverse effects nor biochemical or hematological changes.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamassomos/antagonistas & inibidores , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nitrilas/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Caspase 1/metabolismo , Células Cultivadas , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nitrilas/química , Nitrilas/uso terapêuticoRESUMO
Obesity and the metabolic syndrome are characterized by chronic, low-grade inflammation mainly originating from expanding adipose tissue and resulting in inhibition of insulin signaling and disruption of glycemic control. Transgenic mice expressing human interleukin 37 (IL-37), an anti-inflammatory cytokine of the IL-1 family, are protected against metabolic syndrome when fed a high-fat diet (HFD) containing 45% fat. Here, we examined whether treatment with recombinant IL-37 ameliorates established insulin resistance and obesity-induced inflammation. WT mice were fed a HFD for 22 weeks and then treated daily with IL-37 (1 µg/mouse) during the last 2 weeks. Compared with vehicle only-treated mice, IL-37-treated mice exhibited reduced insulin in the plasma and had significant improvements in glucose tolerance and in insulin content of the islets. The IL-37 treatment also increased the levels of circulating IL-1 receptor antagonist. Cultured adipose tissues revealed that IL-37 treatment significantly decreases spontaneous secretions of IL-1ß, tumor necrosis factor α (TNFα), and CXC motif chemokine ligand 1 (CXCL-1). We also fed mice a 60% fat diet with concomitant daily IL-37 for 2 weeks and observed decreased secretion of IL-1ß, TNFα, and IL-6 and reduced intracellular levels of IL-1α in the liver and adipose tissue, along with improved plasma glucose clearance. Compared with vehicle treatment, these IL-37-treated mice had no apparent weight gain. In human adipose tissue cultures, the presence of 50 pm IL-37 reduced spontaneous release of TNFα and 50% of lipopolysaccharide-induced TNFα. These findings indicate that IL-37's anti-inflammatory effects can ameliorate established metabolic disturbances during obesity.
Assuntos
Tecido Adiposo/metabolismo , Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Resistência à Insulina , Interleucina-1/uso terapêutico , Síndrome Metabólica/tratamento farmacológico , Obesidade/fisiopatologia , Animais , Biomarcadores/sangue , Dieta Hiperlipídica , Teste de Tolerância a Glucose , Humanos , Interleucina-1/genética , Síndrome Metabólica/metabolismo , Síndrome Metabólica/fisiopatologia , Camundongos , Camundongos Transgênicos , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêuticoRESUMO
Similar to IL-1α and IL-33, IL-1 family member IL-37b translocates to the nucleus and is associated with suppression of innate and adaptive immunity. Here we demonstrate an extracellular function of the IL-37 precursor and a processed form. Recombinant IL-37 precursor reduced LPS-induced IL-6 by 50% (P < 0.001) in highly inflammatory human blood-derived M1 differentiated macrophages derived from selective subjects but not M2 macrophages. In contrast, a neutralizing monoclonal anti-IL-37 increased LPS-induced IL-6, TNFα and IL-1ß (P < 0.01). The suppression by IL-37 was consistently observed at low picomolar but not nanomolar concentrations. Whereas LPS induced a 12-fold increase in TNFα mRNA, IL-37 pretreatment decreased the expression to only 3-fold over background (P < 0.01). Mechanistically, LPS-induced p38 and pERK were reduced by IL-37. Recombinant IL-37 bound to the immobilized ligand binding α-chain of the IL-18 receptor as well as to the decoy receptor IL-1R8. In M1 macrophages, LPS increased the surface expression of IL-1R8. Compared with human blood monocytes, resting M1 cells express more surface IL-1R8 as well as total IL-1R8; there was a 16-fold increase in IL-1R8 mRNA levels when pretreated with IL-37. IL-37 reduced LPS-induced TNFα and IL-6 by 50-55% in mouse bone marrow-derived dendritic cells, but not in dendritic cells derived from IL-1R8-deficient mice. In mice subjected to systemic LPS-induced inflammation, pretreatment with IL-37 reduced circulating and organ cytokine levels. Thus, in addition to a nuclear function, IL-37 acts as an extracellular cytokine by binding to the IL-18 receptor but using the IL-1R8 for its anti-inflammatory properties.
Assuntos
Imunidade Inata , Inflamação/imunologia , Interleucina-1/química , Interleucina-1/metabolismo , Receptores de Interleucina-1/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Endotoxemia/metabolismo , Endotoxemia/patologia , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/química , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Imobilizadas/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Testes de Neutralização , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-1/química , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Both IL-1α and IL-1ß are highly inflammatory cytokines mediating a wide spectrum of diseases. A recombinant form of the naturally occurring IL-1R antagonist (IL-1Ra), which blocks IL-1R1, is broadly used to treat autoimmune and autoinflammatory diseases; however, blocking IL-1 increases the risk of infection. In this study, we describe the development of a novel form of recombinant IL-1Ra, termed chimeric IL-1Ra. This molecule is a fusion of the N-terminal peptide of IL-1ß and IL-1Ra, resulting in inactive IL-1Ra. Because the IL-1ß N-terminal peptide contains several protease sites clustered around the caspase-1 site, local proteases at sites of inflammation can cleave chimeric IL-1Ra and turn IL-1Ra active. We demonstrate that chimeric IL-1Ra reduces IL-1-mediated inflammation in vitro and in vivo. This unique approach limits IL-1 receptor blockade to sites of inflammation, while sparing a multitude of desired IL-1-related activities, including host defense against infections and IL-1-mediated repair.
Assuntos
Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Receptores de Interleucina-1/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Linhagem Celular , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Interleucina-1beta/química , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Elastase Pancreática/metabolismo , Fragmentos de Peptídeos , Domínios e Motivos de Interação entre Proteínas/genética , Proteólise , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
ITF2357 (generic givinostat) is an orally active, hydroxamic-containing histone deacetylase (HDAC) inhibitor with broad anti-inflammatory properties, which has been used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits both Class I and II HDACs and reduces caspase-1 activity in human peripheral blood mononuclear cells and the secretion of IL-1ß and other cytokines at 25-100 nm; at concentrations >200 nm, ITF2357 is toxic in vitro. ITF3056, an analog of ITF2357, inhibits only HDAC8 (IC50 of 285 nm). Here we compared the production of IL-1ß, IL-1α, TNFα, and IL-6 by ITF2357 with that of ITF3056 in peripheral blood mononuclear cells stimulated with lipopolysaccharide (LPS), heat-killed Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokines from 100 to 1000 nm; at 1000 nm, the secretion of IL-1ß was reduced by 76%, secretion of TNFα was reduced by 88%, and secretion of IL-6 was reduced by 61%. The intracellular levels of IL-1α were 30% lower. There was no evidence of cell toxicity at ITF3056 concentrations of 100-1000 nm. Gene expression of TNFα was markedly reduced (80%), whereas IL-6 gene expression was 40% lower. Although anti-CD3/28 and Candida stimulation of IL-1ß and TNFα was modestly reduced, IFNγ production was 75% lower. Mechanistically, ITF3056 reduced the secretion of processed IL-1ß independent of inhibition of caspase-1 activity; however, synthesis of the IL-1ß precursor was reduced by 40% without significant decrease in IL-1ß mRNA levels. In mice, ITF3056 reduced LPS-induced serum TNFα by 85% and reduced IL-1ß by 88%. These data suggest that specific inhibition of HDAC8 results in reduced inflammation without cell toxicity.
Assuntos
Citocinas/metabolismo , Regulação Enzimológica da Expressão Gênica , Inibidores de Histona Desacetilases/química , Proteínas Repressoras/antagonistas & inibidores , Animais , Apoptose , Candida/metabolismo , Caspase 1/metabolismo , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Histona Desacetilases/metabolismo , Humanos , Inflamação , Concentração Inibidora 50 , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVES: In the present study, we generated a new protein, recombinant human alpha-1-anti-trypsin (AAT)-IgG1 Fc fusion protein (AAT-Fc), and evaluated its properties to suppress inflammation and interleukin (IL)-1ß in a mouse model of gouty arthritis. METHODS: A combination of monosodium urate (MSU) crystals and the fatty acid C16.0 (MSU/C16.0) was injected intra-articularly into the knee to induce gouty arthritis. Joint swelling, synovial cytokine production and histopathology were determined after 4â h. AAT-Fc was evaluated for inhibition of MSU/C16.0-induced IL-1ß release from human blood monocytes and for inhibition of extracellular IL-1ß precursor processing. RESULTS: AAT-Fc markedly suppressed MSU/C16.0-induced joint inflammation by 85-91% (p<0.001). Ex vivo production of IL-1ß and IL-6 from cultured synovia were similarly reduced (63% and 65%, respectively). The efficacy of 2.0â mg/kg AAT-Fc in reducing inflammation was comparable to 80â mg/kg of plasma-derived AAT. Injection of AAT-Fc into mice increased circulating levels of endogenous IL-1 receptor antagonist by fourfold. We also observed that joint swelling was reduced by 80%, cellular infiltration by 95% and synovial production of IL-1ß by 60% in transgenic mice expressing low levels of human AAT. In vitro, AAT-Fc reduced MSU/C16.0-induced release of IL-1ß from human blood monocytes and inhibited proteinase-3-mediated extracellular processing of the IL-1ß precursor into active IL-1ß. CONCLUSIONS: A single low dose of AAT-Fc is highly effective in reducing joint inflammation in this model of acute gouty arthritis. Considering the long-term safety of plasma-derived AAT use in humans, subcutaneous AAT-Fc emerges as a promising therapy for gout attacks.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Gotosa/tratamento farmacológico , Supressores da Gota/uso terapêutico , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Proteína Antagonista do Receptor de Interleucina 1/biossíntese , Interleucina-1beta/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , alfa 1-Antitripsina/uso terapêutico , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Gotosa/imunologia , Artrite Gotosa/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Supressores da Gota/administração & dosagem , Supressores da Gota/farmacologia , Humanos , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/farmacologia , Injeções Intra-Articulares , Injeções Intraperitoneais , Interleucina-1beta/metabolismo , Receptores de Lipopolissacarídeos/análise , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , alfa 1-Antitripsina/administração & dosagem , alfa 1-Antitripsina/farmacologiaRESUMO
IL-32 is a multifaceted cytokine with a role in infections, autoimmune diseases, and cancer, and it exerts diverse functions, including aggravation of inflammation and inhibition of virus propagation. We previously identified IL-32 as a critical regulator of endothelial cell (EC) functions, and we now reveal that IL-32 also possesses angiogenic properties. The hyperproliferative ECs of human pulmonary arterial hypertension and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and, significantly, the cytokine colocalized with integrin αVß3. Vascular endothelial growth factor (VEGF) receptor blockade, which resulted in EC hyperproliferation, increased IL-32 three-fold. Small interfering RNA-mediated silencing of IL-32 negated the 58% proliferation of ECs that occurred within 24 h in scrambled-transfected controls. Reduction of IL-32 neither affected apoptosis (insignificant changes in Bak-1, Bcl-2, Bcl-xL, lactate dehydrogenase, annexin V, and propidium iodide) nor VEGF or TGF-ß levels, but siIL-32-transfected adult and neonatal ECs produced up to 61% less NO, IL-8, and matrix metalloproteinase-9, and up to 3-fold more activin A and endostatin. In coculture-based angiogenesis assays, IL-32γ dose-dependently increased tube formation up to 3-fold; an αVß3 inhibitor prevented this activity and reduced IL-32γ-induced IL-8 by 85%. In matrigel plugs loaded with IL-32γ, VEGF, or vehicle and injected into live mice, we observed the anticipated VEGF-induced increase in neocapillarization (8-fold versus vehicle), but unexpectedly, IL-32γ was equally angiogenic. A second signal such as IFN-γ was required to render cells responsive to exogenous IL-32γ; importantly, this was confirmed using a completely synthetic preparation of IL-32γ. In summary, we add angiogenic properties that are mediated by integrin αVß3 but VEGF-independent to the portfolio of IL-32, implicating a role for this versatile cytokine in pulmonary arterial hypertension and neoplastic diseases.
Assuntos
Interleucinas/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ativinas/metabolismo , Animais , Apoptose/fisiologia , Células Cultivadas , Endostatinas/metabolismo , Hipertensão Pulmonar Primária Familiar , Glioblastoma/embriologia , Glioblastoma/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Integrina alfaVbeta3/metabolismo , Interferon gama/metabolismo , Interleucina-8/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Óxidos de Nitrogênio/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Acute graft-versus-host disease (GvHD) is a major complication that prevents successful outcomes after allogeneic bone marrow transplantation (BMT), an effective therapy for hematological malignancies. Several studies demonstrate that donor T cells and host antigen-presenting cells along with several proinflammatory cytokines are required for the induction of GvHD and contribute to its severity. Increasing evidence demonstrates that human serum-derived αalpha-1- anti-trypsin (AAT) reduces production of proinflammatory cytokines, induces anti-inflammatory cytokines, and interferes with maturation of dendritic cells. Using well-characterized mouse models of BMT, we have studied the effects of AAT on GvHD severity. Administration of AAT early after BMT decreased mortality in three models of GvHD and reduced serum levels of proinflammatory cytokines in the allogeneic recipients compared with vehicle (albumin) treated animals. AAT treatment reduced the expansion of alloreactive T effector cells but enhanced the recovery of T regulatory T cells, (Tregs) thus altering the ratio of donor T effector to T regulatory cells in favor of reducing the pathological process. However, despite altering the ratio in vivo, AAT had no direct effects on either the donor T effector cells or T regulatory cells Tregs in vitro. In contrast, AAT suppressed LPS-induced in vitro secretion of proinflammatory cytokines such as TNF-α and IL-1ß, enhanced the production of the anti-inflammatory cytokine IL-10, and impaired NF-κB translocation in the host dendritic cells. In light of its long history of safety in humans, these findings suggest that administration of AAT represents a novel unique and viable strategy to mitigate clinical GvHD.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Doença Enxerto-Hospedeiro/prevenção & controle , Neoplasias Hematológicas/terapia , alfa 1-Antitripsina/uso terapêutico , Animais , Citocinas/sangue , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sobrevida , Linfócitos T/efeitos dos fármacos , Transplante Homólogo/efeitos adversos , alfa 1-Antitripsina/administração & dosagemRESUMO
The functional role of IL-1 family member 10, recently renamed IL-38, remains unknown. In the present study we aimed to elucidate the biological function of IL-38 and to identify its receptor. Heat-killed Candida albicans was used to stimulate memory T-lymphocyte cytokine production in freshly obtained human peripheral blood mononuclear cells from healthy subjects. The addition of recombinant IL-38 (152 amino acids) inhibited the production of T-cell cytokines IL-22 (37% decrease) and IL-17 (39% decrease). The reduction in IL-22 and IL-17 caused by IL-38 was similar to that caused by the naturally occurring IL-36 receptor antagonist (IL-36Ra) in the same peripheral blood mononuclear cells cultures. IL-8 production induced by IL-36γ was reduced by IL-38 (42% decrease) and also was reduced by IL-36Ra (73% decrease). When human blood monocyte-derived dendritic cells were used, IL-38 as well as IL-36Ra increased LPS-induced IL-6 by twofold. We screened immobilized extracellular domains of each member of the IL-1 receptor family, including the IL-36 receptor (also known as "IL-1 receptor-related protein 2") and observed that IL-38 bound only to the IL-36 receptor, as did IL-36Ra. The dose-response suppression of IL-38 as well as that of IL-36Ra of Candida-induced IL-22 and IL-17 was not that of the classic IL-1 receptor antagonist (anakinra), because low concentrations were optimal for inhibiting IL-22 production, whereas higher concentrations modestly increased IL-22. These data provide evidence that IL-38 binds to the IL-36R, as does IL-36Ra, and that IL-38 and IL-36Ra have similar biological effects on immune cells by engaging the IL-36 receptor.
Assuntos
Interleucinas/metabolismo , Receptores de Interleucina-1/metabolismo , Linfócitos T/imunologia , Antígenos de Fungos/imunologia , Candida albicans/efeitos dos fármacos , Candida albicans/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Proteínas Imobilizadas/metabolismo , Interleucina-1/antagonistas & inibidores , Interleucina-1/metabolismo , Interleucina-17/biossíntese , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Interleucinas/biossíntese , Lipopolissacarídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores de Interleucina , Linfócitos T/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Interleucina 22RESUMO
IL-18 binding protein (IL-18BP) was originally discovered in 1999 while attempting to identify an IL-18 receptor ligand binding chain (also known as IL-18Rα) by subjecting concentrated human urine to an IL-18 ligand affinity column. The IL-18 ligand chromatography purified molecule was analyzed by protein microsequencing. The result revealed a novel 40 amino acid polypeptide. To isolate the complete open reading frame (ORF), various human and mouse cDNA libraries were screened using cDNA probe derived from the novel IL-18 affinity column bound molecule. The identified entire ORF gene was thought to be an IL-18Rα gene. However, IL-18BP has been proven to be a unique soluble antagonist that shares homology with a variety of viral proteins that are distinct from the IL-18Rα and IL-18Rß chains. The IL-18BP cDNA was used to generate recombinant IL-18BP (rIL-18BP), which was indispensable for characterizing the role of IL-18BP in vitro and in vivo. Mammalian cell lines were used to produce rIL-18BP due to its glycosylation-dependent activity of IL-18BP (approximately 20 kDa). Various forms of rIL-18BP, intact, C-terminal his-tag, and Fc fusion proteins were produced for in vitro and in vivo experiments. Data showed potent neutralization of IL-18 activity, which seems promising for clinical application in immune diseases involving IL-18. However, it was a long journey from discovery to clinical use although there have been various clinical trials since IL-18BP was discovered in 1999. This review primarily covers the discovery of IL-18BP along with how basic research influences the clinical development of IL-18BP.
RESUMO
Introduction: Interleukin-18 (IL-18), a pro-inflammatory cytokine belonging to the IL-1 Family, is a key mediator ofautoinflammatory diseases associated with the development of macrophage activation syndrome (MAS).High levels of IL-18 correlate with MAS and COVID-19 severity and mortality, particularly in COVID-19patients with MAS. As an inflammation inducer, IL-18 binds its receptor IL-1 Receptor 5 (IL-1R5), leadingto the recruitment of the co-receptor, IL-1 Receptor 7 (IL-1R7). This heterotrimeric complex subsequentlyinitiates downstream signaling, resulting in local and systemic inflammation. Methods: We reported earlier the development of a novel humanized monoclonal anti-human IL-1R7 antibody whichspecifically blocks the activity of human IL-18 and its inflammatory signaling in human cell and wholeblood cultures. In the current study, we further explored the strategy of blocking IL-1R7 inhyperinflammation in vivo using animal models. Results: We first identified an anti-mouse IL-1R7 antibody that significantly suppressed mouse IL-18 andlipopolysaccharide (LPS)-induced IFNg production in mouse splenocyte and peritoneal cell cultures. Whenapplied in vivo, the antibody reduced Propionibacterium acnes and LPS-induced liver injury and protectedmice from tissue and systemic hyperinflammation. Importantly, anti-IL-1R7 significantly inhibited plasma,liver cell and spleen cell IFNg production. Also, anti-IL-1R7 downregulated plasma TNFa, IL-6, IL-1b,MIP-2 production and the production of the liver enzyme ALT. In parallel, anti-IL-1R7 suppressed LPSinducedinflammatory cell infiltration in lungs and inhibited the subsequent IFNg production andinflammation in mice when assessed using an acute lung injury model. Discussion: Altogether, our data suggest that blocking IL-1R7 represents a potential therapeutic strategy to specificallymodulate IL-18-mediated hyperinflammation, warranting further investigation of its clinical application intreating IL-18-mediated diseases, including MAS and COVID-19.
Assuntos
Inflamação , Lipopolissacarídeos , Animais , Camundongos , Lipopolissacarídeos/imunologia , Inflamação/imunologia , Humanos , Interleucina-18/metabolismo , Interleucina-18/imunologia , Modelos Animais de Doenças , COVID-19/imunologia , Camundongos Endogâmicos C57BL , Síndrome de Ativação Macrofágica/imunologia , SARS-CoV-2/imunologiaRESUMO
Our immune system possesses sophisticated mechanisms to cope with invading microorganisms, while pathogens evolve strategies to deal with threats imposed by host immunity. Human plasma protein α1-antitrypsin (AAT) exhibits pleiotropic immune-modulating properties by both preventing immunopathology and improving antimicrobial host defence. Genetic associations suggested a role for AAT in candidemia, the most frequent fungal blood stream infection in intensive care units, yet little is known about how AAT influences interactions between Candida albicans and the immune system. Here, we show that AAT differentially impacts fungal killing by innate phagocytes. We observed that AAT induces fungal transcriptional reprogramming, associated with cell wall remodelling and downregulation of filamentation repressors. At low concentrations, the cell-wall remodelling induced by AAT increased immunogenic ß-glucan exposure and consequently improved fungal clearance by monocytes. Contrastingly, higher AAT concentrations led to excessive C. albicans filamentation and thus promoted fungal immune escape from monocytes and macrophages. This underscores that fungal adaptations to the host protein AAT can differentially define the outcome of encounters with innate immune cells, either contributing to improved immune recognition or fungal immune escape.