RESUMO
PURPOSE: Target-dependent TCB activity can result in the strong and systemic release of cytokines that may develop into cytokine release syndrome (CRS), highlighting the need to understand and prevent this complex clinical syndrome. EXPERIMENTAL DESIGN: We explored the cellular and molecular players involved in TCB-mediated cytokine release by single-cell RNA-sequencing of whole blood treated with CD20-TCB together with bulk RNA-sequencing of endothelial cells exposed to TCB-induced cytokine release. We used the in vitro whole blood assay and an in vivo DLBCL model in immunocompetent humanized mice to assess the effects of dexamethasone, anti-TNFα, anti-IL6R, anti-IL1R, and inflammasome inhibition, on TCB-mediated cytokine release and antitumor activity. RESULTS: Activated T cells release TNFα, IFNγ, IL2, IL8, and MIP-1ß, which rapidly activate monocytes, neutrophils, DCs, and NKs along with surrounding T cells to amplify the cascade further, leading to TNFα, IL8, IL6, IL1ß, MCP-1, MIP-1α, MIP-1ß, and IP-10 release. Endothelial cells contribute to IL6 and IL1ß release and at the same time release several chemokines (MCP-1, IP-10, MIP-1α, and MIP-1ß). Dexamethasone and TNFα blockade efficiently reduced CD20-TCB-mediated cytokine release whereas IL6R blockade, inflammasome inhibition, and IL1R blockade induced a less pronounced effect. Dexamethasone, IL6R blockade, IL1R blockade, and the inflammasome inhibitor did not interfere with CD20-TCB activity, in contrast to TNFα blockade, which partially inhibited antitumor activity. CONCLUSIONS: Our work sheds new light on the cellular and molecular players involved in cytokine release driven by TCBs and provides a rationale for the prevention of CRS in patients treated with TCBs. See related commentary by Luri-Rey et al., p. 4320.
Assuntos
Anticorpos Biespecíficos , Fator de Necrose Tumoral alfa , Humanos , Camundongos , Animais , Quimiocina CCL3 , Quimiocina CCL4 , Anticorpos Biespecíficos/farmacologia , Interleucina-8 , Quimiocina CXCL10 , Interleucina-6 , Síndrome da Liberação de Citocina , Células Endoteliais , Inflamassomos , Citocinas , Linfócitos T , Dexametasona/farmacologia , RNARESUMO
Knowledge of a protein's spatial dynamics at the subcellular level is key to understanding its function(s), interactions, and associated intracellular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that controls immune responses via tryptophan metabolism, mainly through its enzymic activity. When phosphorylated, however, IDO1 acts as a signaling molecule in plasmacytoid dendritic cells (pDCs), thus activating genomic effects, ultimately leading to long-lasting immunosuppression. Whether the two activities-namely, the catalytic and signaling functions-are spatially segregated has been unclear. We found that, under conditions favoring signaling rather than catabolic events, IDO1 shifts from the cytosol to early endosomes. The event requires interaction with class IA phosphoinositide 3-kinases (PI3Ks), which become activated, resulting in full expression of the immunoregulatory phenotype in vivo in pDCs as resulting from IDO1-dependent signaling events. Thus, IDO1's spatial dynamics meet the needs for short-acting as well as durable mechanisms of immune suppression, both under acute and chronic inflammatory conditions. These data expand the theoretical basis for an IDO1-centered therapy in inflammation and autoimmunity.
Assuntos
Indolamina-Pirrol 2,3,-Dioxigenase , Fosfatidilinositol 3-Quinases , Células Dendríticas/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação , Fosfatidilinositol 3-Quinases/genética , Transdução de SinaisRESUMO
Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell-mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos Imunológicos/farmacologia , Neoplasias Colorretais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunoterapia , Neovascularização Patológica , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/imunologia , Angiopoietina-2/genética , Angiopoietina-2/imunologia , Animais , Linhagem Celular , Neoplasias Colorretais/irrigação sanguínea , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/imunologia , Camundongos , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/terapia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N-acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases.
Assuntos
Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/química , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , Biocatálise , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos Knockout , Esclerose Múltipla/enzimologia , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Serotonina/análogos & derivados , Serotonina/química , Serotonina/metabolismo , Triptofano/metabolismoRESUMO
Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first step in the kynurenine pathway of tryptophan (Trp) degradation that produces several biologically active Trp metabolites. L-kynurenine (Kyn), the first byproduct by IDO1, promotes immunoregulatory effects via activation of the Aryl hydrocarbon Receptor (AhR) in dendritic cells (DCs) and T lymphocytes. We here identified the nuclear coactivator 7 (NCOA7) as a molecular target of 3-hydroxyanthranilic acid (3-HAA), a Trp metabolite produced downstream of Kyn along the kynurenine pathway. In cells overexpressing NCOA7 and AhR, the presence of 3-HAA increased the association of the two molecules and enhanced Kyn-driven, AhR-dependent gene transcription. Physiologically, conventional (cDCs) but not plasmacytoid DCs or other immune cells expressed high levels of NCOA7. In cocultures of CD4+ T cells with cDCs, the co-addition of Kyn and 3-HAA significantly increased the induction of Foxp3+ regulatory T cells and the production of immunosuppressive transforming growth factor ß in an NCOA7-dependent fashion. Thus, the co-presence of NCOA7 and the Trp metabolite 3-HAA can selectively enhance the activation of ubiquitary AhR in cDCs and consequent immunoregulatory effects. Because NCOA7 is often overexpressed and/or mutated in tumor microenvironments, our current data may provide evidence for a new immune check-point mechanism based on Trp metabolism and AhR.
Assuntos
Ácido 3-Hidroxiantranílico/metabolismo , Células Dendríticas/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Células Dendríticas/imunologia , Feminino , Humanos , Cinurenina/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Coativadores de Receptor Nuclear/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Introduction: Fingolimod, the first oral disease-modifying treatment (DMT) in multiple sclerosis (MS), is a sphingosine 1-phosphate receptor (S1PR) ligand. Approved in 2010, fingolimod has been extensively studied and has been credited with several mechanisms of actions that contribute to its efficacy in MS, among which is the regulation of lymphocyte circulation between the central nervous system and the periphery. Concerns about toxicity, off-target effects, and real-life performance have been raised over time in post-marketing studies of such that next-generation sphingosine-1 phosphate receptor ligands are now being developed. Areas covered: Herein, the authors expand upon previous systematic reviews obtained via PubMed and through their expert opinion on fingolimod use in clinical practice. Long-term data including long-term efficacy, safety, tolerability, and management especially within growing DMT options and pre-treatment constellation in MS patients are discussed, together with the results of an increased understanding of the chemistry underlying the structure-activity relationship. Expert opinion: Despite the limitations illustrated in this article, fingolimod still constitutes a paradigm shift in MS treatment. However, although immunomodulation via S1PRs on lymphocytes has represented a major breakthrough in the clinical management of MS, modifying the evolution of progressive MS will likely require the development of approaches other than merely targeting S1PRs.
Assuntos
Cloridrato de Fingolimode/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Moduladores do Receptor de Esfingosina 1 Fosfato/uso terapêutico , Administração Oral , Animais , Desenvolvimento de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Cloridrato de Fingolimode/farmacologia , Humanos , Imunossupressores/efeitos adversos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Esclerose Múltipla/fisiopatologia , Moduladores do Receptor de Esfingosina 1 Fosfato/efeitos adversos , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , Relação Estrutura-AtividadeRESUMO
The cytokine interleukin IL-35 is known to exert strong immunosuppressive functions. Indoleamine 2,3-dioxygenase 1 (IDO1) and Arginase 1 (Arg1) are metabolic enzymes that, expressed by dendritic cells (DCs), contribute to immunoregulation. Here, we explored any possible link between IL-35 and the activity of those enzymes. We transfected a single chain IL-35Ig gene construct in murine splenic DCs (DC35 ) and assessed any IDO1 and Arg1 activities as resulting from ectopic IL-35Ig expression, both in vitro and in vivo. Unlike Ido1, Arg1 expression was induced in vitro in DC35 , and it conferred an immunosuppressive phenotype on those cells, as revealed by a delayed-type hypersensitivity assay. Moreover, the in vivo onset of a tolerogenic phenotype in DC35 was associated with the detection of CD25+ CD39+ , rather than Foxp3+ , regulatory T cells. Therefore, Arg1, but not Ido1, expression in DC35 appears to be an early event in IL-35Ig-mediated immunosuppression.
Assuntos
Arginase/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica/imunologia , Interleucinas/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Apirase/imunologia , Apirase/metabolismo , Arginase/genética , Arginase/metabolismo , Células Dendríticas/metabolismo , Feminino , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Tolerância Imunológica/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-4/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Purpose: Despite promising clinical activity, T-cell-engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug.Experimental Design: We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically. We also applied a single administration of obinutuzumab (Gazyva pretreatment, Gpt; Genentech/Roche) prior to the first infusion of CD20-TCB as a way to safely administer such a potent drug.Results: CD20-TCB is associated with a long half-life and high potency enabled by high-avidity bivalent binding to CD20 and head-to-tail orientation of B- and T-cell-binding domains in a 2:1 molecular format. CD20-TCB displays considerably higher potency than other CD20-TCB antibodies in clinical development and is efficacious on tumor cells expressing low levels of CD20. CD20-TCB also displays potent activity in primary tumor samples with low effector:target ratios. In vivo, CD20-TCB regresses established tumors of aggressive lymphoma models. Gpt enables profound B-cell depletion in peripheral blood and secondary lymphoid organs and reduces T-cell activation and cytokine release in the peripheral blood, thus increasing the safety of CD20-TCB administration. Gpt is more efficacious and safer than SUD.Conclusions: CD20-TCB and Gpt represent a potent and safer approach for treatment of lymphoma patients and are currently being evaluated in phase I, multicenter study in patients with relapsed/refractory non-Hodgkin lymphoma (NCT03075696). Clin Cancer Res; 24(19); 4785-97. ©2018 AACR See related commentary by Prakash and Diefenbach, p. 4631.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Neoplasias Hematológicas/tratamento farmacológico , Rituximab/administração & dosagem , Animais , Antígenos CD20/genética , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Humanos , Macaca fascicularis , Camundongos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
A defect in indoleamine 2,3-dioxygenase 1 (IDO1), which is responsible for immunoregulatory tryptophan catabolism, impairs development of immune tolerance to autoantigens in NOD mice, a model for human autoimmune type 1 diabetes (T1D). Whether IDO1 function is also defective in T1D is still unknown. We investigated IDO1 function in sera and peripheral blood mononuclear cells (PBMCs) from children with T1D and matched controls. These children were further included in a discovery study to identify SNPs in IDO1 that might modify the risk of T1D. T1D in children was characterized by a remarkable defect in IDO1 function. A common haplotype, associated with dysfunctional IDO1, increased the risk of developing T1D in the discovery and also confirmation studies. In T1D patients sharing such a common IDO1 haplotype, incubation of PBMCs in vitro with tocilizumab (TCZ) - an IL-6 receptor blocker - would, however, rescue IDO1 activity. In an experimental setting with diabetic NOD mice, TCZ was found to restore normoglycemia via IDO1-dependent mechanisms. Thus, functional SNPs of IDO1 are associated with defective tryptophan catabolism in human T1D, and maneuvers aimed at restoring IDO1 function would be therapeutically effective in at least a subgroup of T1D pediatric patients.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Triptofano/metabolismo , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Criança , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Feminino , Regulação Enzimológica da Expressão Gênica , Estudos de Associação Genética , Humanos , Tolerância Imunológica , Imunoterapia , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Análise Multivariada , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-6/efeitos dos fármacosRESUMO
Muscles of sarcopenic people show hypotrophic myofibers and infiltration with adipose and, at later stages, fibrotic tissue. The origin of infiltrating adipocytes resides in fibro-adipogenic precursors and nonmyogenic mesenchymal progenitor cells, and in satellite cells, the adult stem cells of skeletal muscles. Myoblasts and brown adipocytes share a common Myf5+ progenitor cell: the cell fate depends on levels of bone morphogenetic protein 7 (BMP-7), a TGF-ß family member. S100B, a Ca2+-binding protein of the EF-hand type, is expressed at relatively high levels in myoblasts from sarcopenic humans and exerts anti-myogenic effects via NF-κB-dependent inhibition of MyoD, a myogenic transcription factor acting upstream of the essential myogenic factor, myogenin. Adipogenesis requires high levels of ROS, and myoblasts of sarcopenic subjects show elevated ROS levels. Here we show that: (1) ROS overproduction in myoblasts results in upregulation of S100B levels via NF-κB activation; and (2) ROS/NF-κB-induced accumulation of S100B causes myoblast transition into brown adipocytes. S100B activates an NF-κB/Ying Yang 1 axis that negatively regulates the promyogenic and anti-adipogenic miR-133 with resultant accumulation of the brown adipogenic transcription regulator, PRDM-16. S100B also upregulates BMP-7 via NF-κB/Ying Yang 1 with resultant BMP-7 autocrine activity. Interestingly, myoblasts from sarcopenic humans show features of brown adipocytes. We also show that S100B levels and NF-κB activity are elevated in brown adipocytes obtained by culturing myoblasts in adipocyte differentiation medium and that S100B knockdown or NF-κB inhibition in myoblast-derived brown adipocytes reconverts them into fusion-competent myoblasts. At last, interstitial cells and, unexpectedly, a subpopulation of myofibers in muscles of geriatric but not young mice co-express S100B and the brown adipocyte marker, uncoupling protein-1. These results suggest that S100B is an important intracellular molecular signal regulating Myf5+ progenitor cell differentiation into fusion-competent myoblasts or brown adipocytes depending on its levels.
Assuntos
Adipócitos Marrons/metabolismo , MicroRNAs/metabolismo , Mioblastos/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/fisiologia , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Adipócitos Marrons/citologia , Animais , Proteína Morfogenética Óssea 7/genética , Proteína Morfogenética Óssea 7/metabolismo , Humanos , Masculino , Camundongos , MicroRNAs/genética , Mioblastos/citologia , Espécies Reativas de Oxigênio/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Transfecção , Fator de Transcrição YY1/metabolismoRESUMO
In vertebrates, during an early wave of hematopoiesis in the yolk sac between embryonic day E7.0 and E9.0, cells of mesodermal leaflet addressed to macrophage lineage enter in developing central nervous system (CNS) and originate the developing native microglial cells. Depending on the species, microglial cells represent 5-20% of glial cells resident in adult brain. Here, we briefly discuss some canonical functions of the microglia, i.e., cytokine secretion and functional transition from M1 to M2 phenotype. In addition, we review studies on the non-canonical functions of microglia such as regulation of phagocytosis, synaptic pruning, and sculpting postnatal neural circuits. In this latter context the contribution of microglia to some neurodevelopmental disorders is now well established. Nasu-Hakola (NHD) disease is considered a primary microgliopathy with alterations of the DNAX activation protein 12 (DAP12)-Triggering receptor expressed on myeloid cells 2 (TREM-2) signaling and removal of macromolecules and apoptotic cells followed by secondary microglia activation. In Rett syndrome Mecp2-/- microglia shows a substantial impairment of phagocytic ability, although the role of microglia is not yet clear. In a mouse model of Tourette syndrome (TS), microglia abnormalities have also been described, and deficient microglia-mediated neuroprotection is obvious. Here we review the role of microglial cells in neurodevelopmental disorders without inflammation and on the complex role of microglia in developing CNS.
RESUMO
Secondary lymphedema is a common complication after cancer treatment, but the pathomechanisms underlying the disease remain unclear. Using a mouse tail lymphedema model, we found an increase in local and systemic levels of the lymphangiogenic factor vascular endothelial growth factor (VEGF)-C and identified CD68+ macrophages as a cellular source. Surprisingly, overexpression of VEGF-C in a transgenic mouse model led to aggravation of lymphedema with increased immune cell infiltration and vascular leakage compared with wild-type littermates. Conversely, blockage of VEGF-C by overexpression of soluble VEGF receptor-3 reduced edema development, diminishing inflammation and blood vascular leakage. Similar findings were obtained in a hind limb lymph node excision lymphedema model. Flow cytometry analyses and immunofluorescence stainings in lymphedematic tissue showed that VEGF receptor-3 expression was restricted to lymphatic endothelial cells. Our data suggest that endogenous VEGF-C causes blood vascular leakage and fluid influx into the tissue, thus actively contributing to edema formation. These data may provide the basis for future clinical therapeutic approaches.
Assuntos
Linfedema/metabolismo , Linfedema/fisiopatologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Humanos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Distribuição Aleatória , Papel (figurativo)RESUMO
Bortezomib (BTZ) is a first-in-class proteasome inhibitor approved for the therapy of multiple myeloma that also displays unique regulatory activities on immune cells. The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme exerting potent immunoregulatory effects when expressed in dendritic cells (DCs), the most potent antigen-presenting cells capable of promoting either immunity or tolerance. We previously demonstrated that, in inflammatory conditions, IDO1 is subjected to proteasomal degradation in DCs, turning these cells from immunoregulatory to immunostimulatory. In non-obese diabetic (NOD) mice, an experimental model of autoimmune diabetes, we also identified an IDO1 defect such that the DCs do not develop tolerance toward pancreatic islet autoantigens. We found that BTZ rescues IDO1 protein expression in vitro in a particular subset of DCs, i.e., plasmacytoid DCs (pDCs) from NOD mice. When administered in vivo to prediabetic mice, the drug prevented diabetes onset through IDO1- and pDC-dependent mechanisms. Although the drug showed no therapeutic activity when administered alone to overtly diabetic mice, its combination with otherwise suboptimal dosages of autoimmune-preventive anti-CD3 antibody resulted in disease reversal in 70% diabetic mice, a therapeutic effect similar to that afforded by full-dosage anti-CD3. Thus, our data indicate a potential for BTZ in the immunotherapy of autoimmune diabetes and further underline the importance of IDO1-mediated immune regulation in such disease.
RESUMO
Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings.
Assuntos
Arginase/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Transdução de Sinais/imunologia , Animais , Arginase/metabolismo , Arginina/imunologia , Arginina/metabolismo , Western Blotting , Células Dendríticas/metabolismo , Feminino , Perfilação da Expressão Gênica , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma , Triptofano/imunologia , Triptofano/metabolismoRESUMO
Cancer metastases arise from a multi-step process that requires metastasizing tumor cells to adapt to signaling input from varying tissue environments [1]. As an early metastatic event, cancer cell dissemination occurs through different migration programs, including multicellular, collective, and single-cell mesenchymal or amoeboid migration [2-4]. Migration modes can interconvert based on changes in cell adhesion, cytoskeletal mechanotransduction [5], and/or proteolysis [6], most likely under the control of transcriptional programs such as the epithelial-to-mesenchymal transition (EMT) [7, 8]. However, how plasticity of tumor cell migration and EMT is spatiotemporally controlled and connected upon challenge by the tumor microenvironment remains unclear. Using 3D cultures of collectively invading breast and head and neck cancer spheroids, here we identify hypoxia, a hallmark of solid tumors [9], as an inducer of the collective-to-amoeboid transition (CAT), promoting the dissemination of amoeboid-moving single cells from collective invasion strands. Hypoxia-induced amoeboid detachment was driven by hypoxia-inducible factor 1 (HIF-1), followed the downregulation of E-cadherin, and produced heterogeneous cell subsets whose phenotype and migration were dependent (â¼30%) or independent (â¼70%) of Twist-mediated EMT. EMT-like and EMT-independent amoeboid cell subsets showed stable amoeboid movement over hours as well as leukocyte-like traits, including rounded morphology, matrix metalloproteinase (MMP)-independent migration, and nuclear deformation. Cancer cells undergoing pharmacological stabilization of HIFs retained their constitutive ability for early metastatic seeding in an experimental model of lung metastasis, indicating that hypoxia-induced CAT enhances cell release rather than early organ colonization. Induced by metabolic challenge, amoeboid movement may thus constitute a common endpoint of both EMT-dependent and EMT-independent cancer dissemination programs.
Assuntos
Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal , Neoplasias de Cabeça e Pescoço/patologia , Fator 1 Induzível por Hipóxia/metabolismo , Mecanotransdução Celular , Neoplasias da Mama/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Hipóxia Tumoral , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Glioblastoma (GBM) is the most malignant and aggressive among primary brain tumors, characterized by very low life expectancy. In vivo, glioma and glioblastoma in particular contain large numbers of immune cells (myeloid cells) such as microglia and tumour-infiltrating macrophages (or glioma associated macrophages). These glioma-infiltrating myeloid cells comprise up to 30% of total tumor mass and have been suggested to play several roles in glioma progression including proliferation, survival, motility and immunosuppression. Although tumor microglia and macrophages can acquire proinflammatory (M1) phenotype being capable of releasing proinflammatory cytokines, phagocytosing and presenting antigens, their effector immune function in gliomas appears to be suppressed by the acquisition of an anti-inflammatory (M2) phenotype. In the present work we review the microglia-glioma interactions to highlight the close relationship between the two cell types and the factors that can influence their properties (chemokines, cytokines, S100B protein). A future therapeutic possibility might be to simultaneously targeting, for example with nanomedicine, glioma cells and microglia to push the microglia towards an antitumor phenotype (M1) and/or prevent glioma cells from "conditioning" by microglia.
Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Microglia/patologia , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/fisiopatologia , Comunicação Celular , Glioma/genética , Glioma/fisiopatologia , Humanos , Tolerância Imunológica , Microglia/fisiologia , Células Supressoras Mieloides/patologia , Células Supressoras Mieloides/fisiologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/fisiologia , Fenótipo , Microambiente TumoralRESUMO
OBJECTIVE: The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood-filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. APPROACH AND RESULTS: We generated an inducible, lymphatic-specific podoplanin knockout mouse model (PdpnΔLEC) and induced gene deletion postnatally. PdpnΔLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, PdpnΔLEC mice had blood-filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood-filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence-activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in PdpnΔLEC mice. CONCLUSIONS: These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes.
Assuntos
Circulação Sanguínea , Movimento Celular , Células Dendríticas/metabolismo , Células Endoteliais/metabolismo , Endotélio Linfático/metabolismo , Linfonodos/metabolismo , Glicoproteínas de Membrana/deficiência , Ducto Torácico/metabolismo , Animais , Padronização Corporal , Células Dendríticas/patologia , Células Endoteliais/patologia , Endotélio Linfático/patologia , Eritrócitos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Linfonodos/patologia , Linfangiogênese , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais , Ducto Torácico/patologiaRESUMO
The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the initial, rate-limiting step in tryptophan (Trp) degradation, resulting in tryptophan starvation and the production of immunoregulatory kynurenines. IDO1's catalytic function has long been considered as the one mechanism responsible for IDO1-dependent immune suppression by dendritic cells (DCs), which are master regulators of the balance between immunity and tolerance. However, IDO1 also harbours immunoreceptor tyrosine-based inhibitory motifs, (ITIM1 and ITIM2), that, once phosphorylated, bind protein tyrosine phosphatases, (SHP-1 and SHP-2), and thus trigger an immunoregulatory signalling in DCs. This mechanism leads to sustained IDO1 expression, in a feedforward loop, which is particularly important in restraining autoimmunity and chronic inflammation. Yet, under specific conditions requiring that early and protective inflammation be unrelieved, tyrosine-phosphorylated ITIMs will instead bind the suppressor of cytokine signalling 3 (SOCS3), which drives IDO1 proteasomal degradation and shortens the enzyme half-life. To dissect any differential roles of the two IDO1's ITIMs, we generated protein mutants by replacing one or both ITIM-associated tyrosines with phospho-mimicking glutamic acid residues. Although all mutants lost their enzymic activity, the ITIM1 - but not ITIM2 mutant - did bind SHPs and conferred immunosuppressive effects on DCs, making cells capable of restraining an antigen-specific response in vivo. Conversely, the ITIM2 mutant would preferentially bind SOCS3, and IDO1's degradation was accelerated. Thus, it is the selective phosphorylation of either ITIM that controls the duration of IDO1 expression and function, in that it dictates whether enhanced tolerogenic signalling or shutdown of IDO1-dependent events will occur in a local microenvironment.
Assuntos
Imunossupressores/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Tirosina/imunologia , Animais , Citocinas/imunologia , Células Dendríticas/imunologia , Feminino , Meia-Vida , Tolerância Imunológica/imunologia , Cinurenina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/imunologia , Domínios Proteicos/imunologia , Transdução de Sinais/imunologia , Proteínas Supressoras da Sinalização de Citocina/imunologia , Triptofano/imunologiaRESUMO
Vascular endothelial growth factor-C (VEGF-C)-induced lymphangiogenesis and increased tissue drainage have been reported to inhibit acute and chronic inflammation, and an activated lymphatic endothelium might mediate peripheral tolerance. Using transgenic mice overexpressing VEGF-C in the skin, we found that under inflammatory conditions, VEGF-C-mediated expansion of the cutaneous lymphatic network establishes an immune-inhibitory microenvironment characterised by increased regulatory T (Treg) cells, immature CD11c+CD11b+ dendritic cells (DCs) and CD8+ cells exhibiting decreased effector function. Strikingly, lymphatic endothelial cell (LEC)-conditioned media (CM) potently suppress DC maturation with reduced expression of MHCII, CD40, and IL-6, and increased IL-10 and CCL2 expression. We identify an imbalance in prostaglandin synthase expression after LEC activation, favoring anti-inflammatory prostacyclin synthesis. Importantly, blockade of LEC prostaglandin synthesis partially restores DC maturity. LECs also produce TGF-ß1, contributing to the immune-inhibitory microenvironment. This study identifies novel mechanisms by which the lymphatic endothelium modulates cellular immune responses to limit inflammation.
Assuntos
Células Dendríticas/citologia , Células Endoteliais/metabolismo , Linfonodos/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Apresentação de Antígeno , Células da Medula Óssea/citologia , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Movimento Celular , Quimiocina CCL2/metabolismo , Células Dendríticas/metabolismo , Citometria de Fluxo , Humanos , Tolerância Imunológica , Inflamação , Interleucina-10/metabolismo , Linfonodos/patologia , Linfangiogênese/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Fenótipo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
A multischool outbreak of salmonellosis caused by Salmonella enterica serovar Napoli was investigated in the province of Milan from October to November 2014, following an increase in school absenteeism coinciding with two positive cases. Epidemiological studies detected 47 cases in four primary schools: 46 children and 1 adult woman (51.4% males and 48.6% females, median age 8.9). From these, 14 cases (29.8%) were severe and resulted in hospitalization, including 6 children (12.8%) who developed an invasive salmonellosis. The epidemic curve revealed an abnormally long incubation period, peaking 1 week after the first confirmed case. Twenty-five available isolates were typed by pulsed-field gel electrophoresis showing an identical pattern. The isolate belongs to ST474, an ST composed exclusively of Salmonella Napoli human strains isolated in France and Italy. Antibiotic resistance analysis showed resistance to aminoglycosides, correlating with the presence of the aminoglycoside resistance gene aadA25 in its genome. Trace-back investigations strongly suggested contaminated ham as the most likely food vehicle, which was delivered by a common food center on 21 October. Nevertheless, this ingredient could not be retrospectively investigated since it was no longer available at the repository. This represents the largest Salmonella Napoli outbreak ever reported in Italy and provides a unique scenario for studying the outcome of salmonellosis caused by this emerging and potentially invasive nontyphoidal serotype.