RESUMO
Chronic overproduction of IL-15 contributes to the pathogenesis of numerous inflammatory and autoimmune disorders. Experimental methods used to reduce the cytokine activity show promise as potential therapeutic approaches to modify IL-15 signaling and alleviate the development and progression of IL-15-related diseases. We previously demonstrated that an efficient reduction of IL-15 activity can be obtained by selective blocking of the specific, high affinity subunit alpha of the IL-15 receptor (IL-15Rα) with small-molecule inhibitors. In this study, we determined the structure-activity relationship of currently known IL-15Rα inhibitors in order to define the critical structural features required for their activity. To validate our predictions, we designed, analyzed in silico, and assessed in vitro function of 16 new potential IL-15Rα inhibitors. All newly synthesized molecules were benzoic acid derivatives with favorable ADME properties and they efficiently reduced IL-15 dependent peripheral blood mononuclear cells (PBMCs) proliferation, as well as TNF-α and IL-17 secretion. The rational design of IL-15 inhibitors may propel the identification of potential lead molecules for the development of safe and effective therapeutic agents.
Assuntos
Interleucina-15 , Leucócitos Mononucleares , Interleucina-15/antagonistas & inibidores , Leucócitos Mononucleares/metabolismo , Peso Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Interleukin (IL)-15 is a pleiotropic cytokine structurally close to IL-2 and sharing with the IL-2Rß and γc receptor (R) subunits. IL-15 plays important roles in innate and adaptative immunity, supporting the activation and proliferation of NK, NK-T, and CD8+ T cells. Over-expression of IL-15 has been shown to participate to the development of inflammatory and autoimmune diseases and diverse T cell malignancies. This study is in continuity of our previous work through which a family of small-molecule inhibitors impeding IL-15/IL-2Rß interaction with sub-micromolar activity has been identified using pharmacophore-based virtual screening and hit optimization methods. With the aim to improve the efficacy and selectivity of our lead inhibitor, specific modifications have been introduced on the basis of optimized SAR and modelisation. The new series of compounds generated have been evaluated for their capacity to inhibit the proliferation as well as the down-stream signaling of IL-15-dependent cells and to bind to IL-15.
Assuntos
Interleucina-15/antagonistas & inibidores , Ftalazinas/química , Ftalazinas/farmacologia , Linhagem Celular , Humanos , Ftalazinas/síntese química , Análise Espectral/métodos , Relação Estrutura-AtividadeRESUMO
Interleukin (IL)-15 plays an important role in several inflammatory diseases. We have previously identified an IL-15 antagonist called P8 peptide, which binds specifically to IL-15 receptor alpha subunit. However, the P8 peptide rapidly degraded by proteases, limiting its therapeutic application. Thus, we replaced each P8 peptide l-amino acid by its corresponding d-isomers. First, we determined the biological activity of the resulting peptides in a proliferation assay by using CTLL-2 cells. The substitution of l-Ala by d-Ala ([A4a]P8 peptide) increased the inhibitory effect of the P8 peptide in CTLL-2 cells in five-fold. In addition to that, the [A4a]P8 peptide dimer showed the most inhibitory effect. To protect the [A4a]P8 peptide and its dimer against exopeptidase activity, we acetylated the N-terminal of these peptides. At least a three-fold reduction in antagonist activity of acetylated peptides was exhibited. However, the substitution of the N-terminal l-Lys residue of [A4a]P8 peptide and its dimer by d-Lys ([K1k;A4a]P8 peptide) did not affect the antagonist effect of the aforementioned peptides. The [K1k;A4a]P8 peptide dimer was stable to the degradation of trypsin, chymotrypsin, and pepsin up until 48 min. Also, the safety and immunogenicity studies in healthy BALB/c mice demonstrated that the administration of this peptide did not affect the clinical parameters of the animals nor generated antipeptide antibodies. Our findings reveal that two distinct d-amino acid substitutions and dimerization increase the biological activity and stability of P8 peptide. The resulting peptide constitutes a novel IL-15 antagonist with potential applicability in inflammatory diseases.
Assuntos
Interleucina-15/antagonistas & inibidores , Peptídeos/farmacologia , Substituição de Aminoácidos/efeitos dos fármacos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dimerização , Feminino , Interleucina-15/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/síntese química , Peptídeos/químicaRESUMO
IL-15 has emerged as a potentially relevant target in the IL-17 response in psoriasis. However, its mechanism is poorly characterized in humans. IL-15 and IL-23 are constitutively expressed in the psoriatic lesion. Also, IL-15 is considered a susceptibility-associated gene in psoriasis, as are IL-23R, and HLACW6. Here, we studied the effect of IL-15 and IL-23 stimulation on the cytokine response of CLA+/CLA- T cells from 9 psoriasis patients and 3 healthy control subjects. To this end, CLA + and CLA- T cells from blood samples were cultured with epidermal cells from skin biopsies and treated with IL-15 and IL-23. After five days of culture, cytokines in supernatant were measured by ELISA or fluorescent bead-based immunoassay. There was a statistically significant increase in IL-17F and IL-17A production (P < .001) in cocultures of psoriasis skin-homing CLA + T cells with epidermal cells when stimulated with IL-15 and IL-23, but this effect was not observed in the cells of healthy controls. Interestingly, this response was reduced by around 50 to 80% by blocking HLA class I and II molecules. Our results point to the synergic action of IL-15 and IL-23 selectively for CLA + cells in psoriasis, leading to the induction of Th17 cell-related cytokines.
Assuntos
Interleucina-15/farmacologia , Interleucina-23/farmacologia , Células T de Memória/metabolismo , Psoríase/imunologia , Psoríase/metabolismo , Anticorpos Neutralizantes/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Técnicas de Cocultura , Células Epidérmicas , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-15/antagonistas & inibidores , Interleucina-15/metabolismo , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Subunidade p19 da Interleucina-23/antagonistas & inibidores , Subunidade p19 da Interleucina-23/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Oligossacarídeos/metabolismo , Cultura Primária de Células , Proteínas Recombinantes/farmacologia , Antígeno Sialil Lewis X/análogos & derivados , Antígeno Sialil Lewis X/metabolismoAssuntos
Transplante de Células-Tronco Hematopoéticas , Interleucina-15 , Vírus JC , Leucoencefalopatia Multifocal Progressiva , Adulto , Humanos , Terapia de Imunossupressão , Interleucina-15/antagonistas & inibidores , Leucoencefalopatia Multifocal Progressiva/etiologia , Leucoencefalopatia Multifocal Progressiva/terapia , Masculino , Estados UnidosRESUMO
Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential "shock and kill" therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.
Assuntos
Antirretrovirais/administração & dosagem , Linfócitos B/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Interleucina-15/antagonistas & inibidores , Células Matadoras Naturais/efeitos dos fármacos , Proteínas/administração & dosagem , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/fisiopatologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Interleucina-15/genética , Interleucina-15/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Macaca mulatta , Proteínas Recombinantes de Fusão , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/fisiologia , Latência Viral/efeitos dos fármacosRESUMO
Pathologic roles of interleukin (IL)-2, IL-9, and IL-15, have been implicated in multiple T-cell malignancies and autoimmune diseases. BNZ-1 is a selective and simultaneous inhibitor of IL-2, IL-9, and IL-15, which targets the common gamma chain signaling receptor subunit. In this first-in-human study, 18 healthy adults (n = 3/cohort) received an intravenous dose of 0.2, 0.4, 0.8, 1.6, 3.2, or 6.4 mg/kg infused over ≤5 minutes on day 1 and were followed for 30 days for safety and pharmacokinetic/pharmacodynamic sample collection. No dose-limiting toxicities, infusion reactions, or serious or severe treatment-emergent adverse events were observed. Headache was the only treatment-emergent adverse event in >1 subject (n = 3). Peak and total BNZ-1 exposure was generally dose proportional, with a terminal elimination half-life of â¼5 days. Pharmacodynamic effects of BNZ-1 on regulatory T cells (Tregs, IL-2), natural killer (NK) cells (IL-15) and CD8 central memory T cells (Tcm, IL-15) were measured by flow cytometry and used to demonstrate target engagement. For Tregs, 0.2 mg/kg was an inactive dose, while a maximum â¼50% to 60% decrease from baseline was observed on day 4 after doses of 0.4 to 1.6 mg/kg, and higher doses produced an 80% to 93% decrease from baseline on day 15. Similar pharmacodynamic trends were observed for natural killer cells and CD8 Tcm, although decreases in CD8 Tcm were more prolonged. These subpopulations returned to/toward baseline by day 31. T cells (total, CD4, and CD8), B cells, and monocytes were unchanged throughout. These preliminary results suggest that BNZ-1 safely and selectively inhibits IL-2 and IL-15, which results in robust, reversible immunomodulation.
Assuntos
Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-15/antagonistas & inibidores , Interleucina-2/antagonistas & inibidores , Interleucina-9/antagonistas & inibidores , Peptídeos/efeitos adversos , Peptídeos/farmacocinética , Adulto , Linfócitos B/efeitos dos fármacos , Feminino , Voluntários Saudáveis , Humanos , Infusões Intravenosas , Células Matadoras Naturais/efeitos dos fármacos , Masculino , Peptídeos/administração & dosagem , Linfócitos T/efeitos dos fármacosAssuntos
Astrócitos/imunologia , Isquemia Encefálica , Linfócitos T CD8-Positivos/imunologia , Interleucina-15 , Células Matadoras Naturais/imunologia , Acidente Vascular Cerebral , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/terapia , Revascularização Cerebral/métodos , Proteína Glial Fibrilar Ácida/metabolismo , Interleucina-15/antagonistas & inibidores , Interleucina-15/imunologia , Ativação Linfocitária/imunologia , Camundongos , Modelos Animais , Traumatismo por Reperfusão/imunologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapiaRESUMO
Interleukin-15 (IL-15) and IL-2 drive T-cell malignancies including T-cell large granular lymphocyte leukemia (T-LGLL) and HTLV-1 driven adult T-cell leukemia (ATL). Both cytokines share common γ-chain receptors and downstream signaling pathways. T-LGLL is characterized by clonal expansion of cytotoxic T cells and is associated with abnormal JAK/STAT signaling. ATL is an aggressive CD4+ T-cell neoplasm associated with HTLV-1. T-LGLL and ATL share dependence on IL-2 and IL-15 for survival and both diseases lack effective therapies. BNZ-1 is a pegylated peptide designed to specifically bind the γc receptor to selectively block IL-2, IL-15, and IL-9 signaling. We hypothesized that treatment with BNZ-1 would reduce cytokine-mediated proliferation and viability. Our results demonstrated that in vitro treatment of a T-LGLL cell line and ex vivo treatment of T-LGLL patient cells with BNZ-1 inhibited cytokine-mediated viability. Furthermore, BNZ-1 blocked downstream signaling and increased apoptosis. These results were mirrored in an ATL cell line and in ex vivo ATL patient cells. Lastly, BNZ-1 drastically reduced leukemic burden in an IL-15-driven human ATL mouse xenograft model. Thus, BNZ-1 shows great promise as a novel therapy for T-LGLL, ATL, and other IL-2 or IL-15 driven hematopoietic malignancies.
Assuntos
Benzodiazepinas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Interleucina-15/antagonistas & inibidores , Interleucina-2/antagonistas & inibidores , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Janus Quinases/metabolismo , Leucemia de Células T/metabolismo , Camundongos , Fosforilação , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Advances in immunotherapy utilizing immune checkpoint inhibitors (ICIs) have transformed the treatment landscapes of several malignancies in recent years. Oncologists are now tasked with extending these benefits to a greater number of patients and tumor types. Metastatic castration-resistant prostate cancer (mCRPC) infrequently responds to ICIs, while the cellular vaccine approved for mCRPC, sipuleucel-T, provides a 4-month survival benefit but does not produce clinical responses as monotherapy. However, many novel and generally well-tolerated immune oncology agents with potential for immune synergy and/or additive effects are undergoing clinical development. This availability presents opportunities to develop adaptive-design combination clinical trials aimed to generate, expand, and facilitate antitumor immune responses. Here we describe a currently accruing phase I/II trial (NCT03493945) testing a brachyury-targeted antitumor vaccine, TGF-ß TRAP/anti-PD-L1 antibody, an IL-15 agonist, and an IDO1 inhibitor in mCRPC. TRIAL REGISTRATION: This trial ( NCT03493945 ) was registered in National Clinical Trials on April 11th 2018.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Proteínas Fetais/uso terapêutico , Oximas/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Sulfonamidas/uso terapêutico , Proteínas com Domínio T/uso terapêutico , Extratos de Tecidos/uso terapêutico , Vacinas Virais/uso terapêutico , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Humanos , Imunoterapia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Interleucina-15/antagonistas & inibidores , Masculino , Proteínas/uso terapêutico , Proteínas Recombinantes de Fusão , Fator de Crescimento Transformador beta/antagonistas & inibidores , Resultado do Tratamento , Vacinas de DNARESUMO
Acute ischemic stroke is followed by a complex interplay between the brain and the immune system in which ischemia-reperfusion leads to a detrimental inflammatory response that causes brain injury. In the brain, IL-15 is expressed by astrocytes, neurons and microglia. Previous study showed that ischemia-reperfusion induces expression of IL-15 by astrocytes. Transgenic over-expression of IL-15 in astrocytes aggravates ischemia-reperfusion brain damage by increasing the levels and promoting the effector functions of CD8+ T and NK cells. Treatment of neonatal rats with IL-15 neutralizing antibody before hypoxia-ischemia induction reduces the infarct volume. However, as stroke-induced inflammatory responses differ between neonate and adult brain, the effects of IL-15 blockade on the injury and immune response arising from stroke in adult animals has remained unclear. In this study, we examined the effect of post-ischemia/reperfusion IL-15 blockade on the pathophysiology of cerebral ischemia-reperfusion in adult mice. Using a cerebral ischemia-reperfusion model, we compared infarct size and the infiltrating immune cells in the brain of wild type (WT) mice and Il15-/- mice lacking NK and memory CD8+ T cells. We also evaluated the effects of IL-15 neutralizing antibody treatment on brain infarct volume, motor function, and the status of brain-infiltrating immune cells in WT mice. Il15-/- mice show a smaller infarct volume and lower numbers of activated brain-infiltrating NK, CD8+ T, and CD4+ T cells compared to WT mice after cerebral ischemia-reperfusion. Post-ischemia/reperfusion IL-15 blockade reduces infarct size and improves motor and locomotor activity. Furthermore, IL-15 blockade reduces the effector function of NK, CD8+ T, and CD4+ T cells in the ischemia-reperfusion brain of WT mice. Ablation of IL-15 responses after cerebral ischemia-reperfusion ameliorates brain injury in adult mice. Therefore, targeting IL-15 is a potential effective therapy for ischemic stroke.
Assuntos
Interleucina-15/antagonistas & inibidores , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Lesões Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Interleucina-15/metabolismo , Células Matadoras Naturais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Ratos , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologiaRESUMO
Somatostatin and its analogues are known to have modulatory effects on immune response and their anti-proliferative, anti-angiogenic, and analgesic properties make them attractive candidates for a therapeutic use in immune-mediated diseases, such as rheumatoid arthritis. Here, we demonstrate the ability of the somatostatin analogue octreotide to inhibit interleukin-15 and to increase interleukin-10 production by rheumatoid arthritis fibroblast-like synovial cells maintained in a chronic inflammatory state. We also prove that the inhibitory effect of octreotide on interleukin-15 and tumor necrosis factor-α production depended on the increase in interleukin-10, since neutralizing anti-interleukin-10 antibody was able to partially reverse this inhibition. In addition, our observations suggest an octreotide control on purinergic signaling, with an inhibitory effect on purinergic P2X and P2Y receptors activation. This would have great implications, considering the roles of P2 receptors in the onset of inflammation. Data here reported extend knowledge on the biological action of octreotide and underline its multiple effects on immune response, which could make octreotide an attractive and valid support for the therapy of diseases where several inflammatory mediators are involved, such as rheumatoid arthritis, and in which the simultaneous action on different aspects can be a successful strategy.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Inflamação/tratamento farmacológico , Octreotida/farmacologia , Somatostatina/análogos & derivados , Sinoviócitos/patologia , Artrite Reumatoide/patologia , Humanos , Interleucina-10/agonistas , Interleucina-15/antagonistas & inibidores , Octreotida/uso terapêutico , Purinérgicos , Sinoviócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Interleukin (IL)-15 is an inflammatory cytokine that constitutes a validated therapeutic target in some immunopathologies, including rheumatoid arthritis (RA). Previously, we identified an IL-15 antagonist peptide named [K6T]P8, with potential therapeutic application in RA. In the current work, the metabolic stability of this peptide in synovial fluids from RA patients was studied. Moreover, [K6T]P8 peptide was labeled with 99m Tc to investigate its stability in human plasma and its biodistribution pattern in healthy rats. The biological activity of [K6T]P8 peptide and its dimer was evaluated in CTLL-2 cells, using 3 different additives to improve the solubility of these peptides. The half-life of [K6T]P8 in human synovial fluid was 5.88 ± 1.73 minutes, and the major chemical modifications included peptide dimerization, cysteinylation, and methionine oxidation. Radiolabeling of [K6T]P8 with 99m Tc showed a yield of approximately 99.8%. The 99m Tc-labeled peptide was stable in a 30-fold molar excess of cysteine and in human plasma, displaying a low affinity to plasma proteins. Preliminary biodistribution studies in healthy Wistar rats suggested a slow elimination of the peptide through the renal and hepatic pathways. Although citric acid, sucrose, and Tween 80 enhanced the solubility of [K6T]P8 peptide and its dimer, only the sucrose did not interfere with the in vitro proliferation assay used to assess their biological activity. The results here presented, reinforce nonclinical characterization of the [K6T]P8 peptide, a potential agent for the treatment of RA and other diseases associated with IL-15 overexpression.
Assuntos
Artrite Reumatoide/sangue , Interleucina-15/antagonistas & inibidores , Peptídeos/síntese química , Tecnécio/química , Animais , Linhagem Celular , Humanos , Técnicas In Vitro , Camundongos , Peptídeos/química , Peptídeos/farmacocinética , Estabilidade Proteica , Ratos , Ratos Wistar , Líquido Sinovial/química , Distribuição TecidualRESUMO
Obesity is a global epidemic affecting chronic inflammatory diseases. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease that can occur as an extraintestinal manifestation of inflammatory bowel disease (IBD). Previously we reported that patients with PSC who are obese have a higher risk of advanced liver disease. Currently it is unknown how obesity accelerates or worsens PSC. We evaluated the progression of PSC in an antigen-driven cholangitis mouse model of diet-induced obesity. Obesity was induced in our murine model of immune-mediated cholangitis (OVAbil). OVAbil mice were fed standard chow or high-fat/sucrose diet for twelve weeks followed by induction of biliary inflammation by OVA-specific T cell transfer. Histopathological damage in portal tracts was scored and serum collected. Neutralizing antibodies against IL-15 were administered daily until study termination. Obese mice developed exacerbated liver inflammation and damage. Immune cell phenotyping in liver revealed greater numbers of neutrophils and CD8+ T cells in obese mice. Higher levels of cytokines and chemokines were found in obese mice with cholangitis. Immuno-neutralizing antibodies against IL-15 greatly attenuated cholangitis in obese mice. Obesity exacerbated experimental PSC in part by overproduction of IL-15. Timely targeting of IL-15 may slow the progression of PSC.
Assuntos
Anticorpos Neutralizantes/imunologia , Colangite Esclerosante/imunologia , Colangite Esclerosante/patologia , Interleucina-15/imunologia , Animais , Anticorpos Neutralizantes/farmacologia , Sistema Biliar/metabolismo , Colangite/imunologia , Colangite/patologia , Colangite/prevenção & controle , Colangite Esclerosante/prevenção & controle , Colestase/patologia , Citocinas/imunologia , Citocinas/fisiologia , Dieta , Dieta Hiperlipídica , Modelos Animais de Doenças , Feminino , Doenças Inflamatórias Intestinais/patologia , Interleucina-15/antagonistas & inibidores , Interleucina-15/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/complicações , Obesidade/imunologia , Obesidade/patologia , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Metastases are responsible for the vast majority of cancer-related deaths. Although tumor cells can become invasive early during cancer progression, metastases formation typically occurs as a late event. How the immune response to primary tumors may dictate this outcome remains poorly understood, which hampers our capacity to manipulate it therapeutically. Here, we used a two-step experimental model, based on the highly aggressive B16F10 melanoma, that temporally segregates the establishment of primary tumors (subcutaneously) and the formation of lung metastases (from intravenous injection). This allowed us to identify a protective innate immune response induced by primary tumors that inhibits experimental metastasis. We found that in the presence of primary tumors, increased numbers of natural killer (NK) cells with enhanced IFNγ, granzyme B, and perforin production were recruited to the lung upon metastasis induction. These changes were mirrored by a local accumulation of patrolling monocytes and macrophages with high expression of MHC class II and NOS2. Critically, the protective effect on metastasis was lost upon patrolling monocyte or NK cell depletion, IL15 neutralization, or IFNγ ablation. The combined analysis of these approaches allowed us to establish a hierarchy in which patrolling monocytes, making IL15 in response to primary tumors, activate NK cells and IFNγ production that then inhibit lung metastasis formation. This work identifies an innate cell network and the molecular determinants responsible for "metastasis immunosurveillance," providing support for using the key molecular mediator, IL15, to improve immunotherapeutic outcomes. Cancer Immunol Res; 5(9); 812-20. ©2017 AACR.
Assuntos
Interferon gama/imunologia , Interleucina-15/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon gama/antagonistas & inibidores , Interleucina-15/antagonistas & inibidores , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Monócitos/imunologia , Monócitos/patologia , Metástase NeoplásicaRESUMO
Although allogeneic stem cell transplantation (allo-SCT) can elicit graft-versus-tumor (GVT) immunity, patients often relapse due to residual tumor cells. As essential orchestrators of the immune system, vaccination with dendritic cells (DC) is an appealing strategy to boost the GVT response. Nevertheless, durable clinical responses after DC vaccination are still limited, stressing the need to improve current DC vaccines. Aiming to empower DC potency, we engineered monocyte-derived DCs to deprive them of ligands for the immune checkpoint regulated by programmed death 1 (PD-1). We also equipped them with interleukin (IL)-15 "transpresentation" skills. Transfection with short interfering (si)RNA targeting the PD-1 ligands PD-L1 and PD-L2, in combination with IL15 and IL15Rα mRNA, preserved their mature DC profile and rendered the DCs superior in inducing T-cell proliferation and IFNγ and TNFα production. Translated into an ex vivo hematological disease setting, DCs deprived of PD-1 ligands (PD-L), equipped with IL15/IL15Rα expression, or most effectively, both, induced superior expansion of minor histocompatibility antigen-specific CD8+ T cells from transplanted cancer patients. These data support the combinatorial approach of in situ suppression of the PD-L inhibitory checkpoints with DC-mediated IL15 transpresentation to promote antigen-specific T-cell responses and, ultimately, contribute to GVT immunity. Cancer Immunol Res; 5(8); 710-5. ©2017 AACR.
Assuntos
Vacinas Anticâncer/administração & dosagem , Células Dendríticas/transplante , Interleucina-15/genética , Receptor de Morte Celular Programada 1/genética , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Efeito Enxerto vs Tumor/efeitos dos fármacos , Efeito Enxerto vs Tumor/imunologia , Humanos , Interleucina-15/antagonistas & inibidores , Monócitos/imunologia , Monócitos/transplante , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , RNA Interferente Pequeno/genética , Transplante de Células-Tronco , Transfecção , Transplante Homólogo , VacinaçãoRESUMO
Interleukin (IL)-15 is a pleiotropic cytokine, which is structurally close to IL-2 and shares with it the IL-2 ß and γ receptor (R) subunits. By promoting the activation and proliferation of NK, NK-T, and CD8+ T cells, IL-15 plays important roles in innate and adaptative immunity. Moreover, the association of high levels of IL-15 expression with inflammatory and autoimmune diseases has led to the development of various antagonistic approaches targeting IL-15. This study is an original approach aimed at discovering small-molecule inhibitors impeding IL-15/IL-15R interaction. A pharmacophore and docking-based virtual screening of compound libraries led to the selection of 240 high-scoring compounds, 36 of which were found to bind IL-15, to inhibit the binding of IL-15 to the IL-2Rß chain or the proliferation of IL-15-dependent cells or both. One of them was selected as a hit and optimized by a structure-activity relationship approach, leading to the first small-molecule IL-15 inhibitor with sub-micromolar activity.
Assuntos
Interleucina-15/antagonistas & inibidores , Ftalazinas/química , Triazóis/química , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Bases de Dados de Compostos Químicos , Humanos , Interleucina-15/química , Interleucina-15/metabolismo , Subunidade beta de Receptor de Interleucina-2/química , Subunidade beta de Receptor de Interleucina-2/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Ftalazinas/síntese química , Ftalazinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Estereoisomerismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/farmacologiaRESUMO
Interleukin 2 and IL-15 are two closely related cytokines, displaying important functions in the immune system. They share the heterodimeric CD122/CD132 receptor to deliver their signals within target cells. Their specificity of action is conferred by their α receptor chains, IL-2Rα and IL-15Rα. By combining an increased affinity for CD122 and an impaired recruitment of CD132, we have generated an original molecule named IL-2Rß/γ (CD122/CD132) inhibitor (BiG), targeting the CD122/CD132 receptor. BiG efficiently inhibited IL-15- and IL-2-dependent functions of primary cells, including CD8 T and NK cells, in vitro and in vivo. We also report a differential dynamic of action of these cytokines by highlighting a major role played by the IL-2Rα receptor. Interestingly, due to the presence of IL-2Rα, BiG had no impact on IL-2-dependent regulatory T cell proliferation. Thus, by acting as a fine switch in the immune system, BiG emphasizes the differential roles of these two cytokines.
Assuntos
Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-15/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/genética , Interleucina-2/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Proliferação de Células , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/imunologia , Interleucina-15/antagonistas & inibidores , Interleucina-15/metabolismo , Subunidade alfa de Receptor de Interleucina-15/genética , Subunidade alfa de Receptor de Interleucina-15/imunologia , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Interleucina-2/antagonistas & inibidores , Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade beta de Receptor de Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Camundongos , Ligação Proteica , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Upregulation of interleukin 15 (IL-15) contributes directly i.a. to the development of inflammatory and autoimmune diseases. Selective blockade of IL-15 aimed to treat rheumatoid arthritis, psoriasis and other IL-15-related disorders has been recognized as an efficient therapeutic method. The aim of the study was to identify small molecules which would interact with IL-15 or its receptor IL-15Rα and inhibit the cytokine's activity. Based on the crystal structure of IL-15Rα·IL-15, we created pharmacophore models to screen the ZINC database of chemical compounds for potential IL-15 and IL-15Rα inhibitors. Twenty compounds with the highest predicted binding affinities were subjected to in vitro analysis using human peripheral blood mononuclear cells to validate in silico data. Twelve molecules efficiently reduced IL-15-dependent TNF-α and IL-17 synthesis. Among these, cefazolin - a safe first-generation cephalosporin antibiotic - holds the highest promise for IL-15-directed therapeutic applications.
Assuntos
Interleucina-15/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
The prevention and treatment of type-2 diabetes mellitus (T2DM) and diabetic nephropathy (DN), which are disorders with high incidence rates, is of primary importance. In this study, we analyzed the effect of 1,25-(OH)2D3 and lipopolysaccharide (LPS) in combination with interleukin (IL)-15 on the inflammatory immune response and expression of vitamin D receptor (VDR) in mononuclear cells of T2DM and DN uremia (DNU) patients. The human acute monocytic leukemia cell line THP-1 was treated with peripheral blood serum isolated from 30 healthy controls and T2DM and DNU patients each, cultured in the presence or absence of 1,25-(OH)2D3, and subsequently treated with LPS and IL-15. The VDR mRNA and protein expression in THP-1 cells was detected by real-time polymerase chain reaction and western blot (and immunofluorescence assay), respectively, and IL-6 and IL-10 concentrations in the culture supernatant were detected by enzyme-linked immunosorbent assay. LPS treatment induced a significant decrease in VDR mRNA expression in T2DM and DNU serum-treated THP-1 cells compared to the control cells (P < 0.05). The VDR protein expression in DNU serum-treated THP-1 cells was also significantly down-regulated (P < 0.05). LPS treatment induced IL-6 secretion in serum-treated THP-1 cells (P < 0.05), while 1,25-(OH)2D3 treatment inhibited IL-6 secretion to some extent. These findings suggested that LPS down-regulates the expression of VDR in mononuclear cells of T2DM and DNU patients and induces an imbalance in the pro-inflammatory and anti-inflammatory cytokine response, while 1,25-(OH)2D3 partially reversed the effect of LPS and protected patients with T2DM and DNU.