RESUMEN
Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α-NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activation.
Asunto(s)
Enfermedades Autoinmunes , Sistema Nervioso Central , Células Dendríticas , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ácido Láctico , Humanos , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/prevención & control , Autoinmunidad , Sistema Nervioso Central/citología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ácido Láctico/metabolismo , Probióticos/uso terapéutico , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/inmunología , Retroalimentación Fisiológica , Lactasa/genética , Lactasa/metabolismo , Análisis de la Célula IndividualRESUMEN
Dendritic cells (DCs) control the generation of self-reactive pathogenic T cells. Thus, DCs are considered attractive therapeutic targets for autoimmune diseases. Using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies we identified a negative feedback regulatory pathway that operates in DCs to limit immunopathology. Specifically, we found that lactate, produced by activated DCs and other immune cells, boosts NDUFA4L2 expression through a mechanism mediated by HIF-1α. NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs involved in the control of pathogenic autoimmune T cells. Moreover, we engineered a probiotic that produces lactate and suppresses T-cell autoimmunity in the central nervous system via the activation of HIF-1α/NDUFA4L2 signaling in DCs. In summary, we identified an immunometabolic pathway that regulates DC function, and developed a synthetic probiotic for its therapeutic activation.
RESUMEN
Synthetic biology has the potential to transform cell- and gene-based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease-related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium "Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics," which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell-based therapies.
Asunto(s)
Congresos como Asunto/tendencias , Ingeniería Genética/tendencias , Terapia Genética/tendencias , Informe de Investigación , Biología Sintética/tendencias , Animales , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/tendencias , Marcación de Gen/métodos , Marcación de Gen/tendencias , Ingeniería Genética/métodos , Terapia Genética/métodos , Humanos , Células Asesinas Naturales/inmunología , Aprendizaje Automático/tendencias , Biología Sintética/métodos , Linfocitos T/inmunologíaRESUMEN
The steadfast advance of the synthetic biology field has enabled scientists to use genetically engineered cells, instead of small molecules or biologics, as the basis for the development of novel therapeutics. Cells endowed with synthetic gene circuits can control the localization, timing and dosage of therapeutic activities in response to specific disease biomarkers and thus represent a powerful new weapon in the fight against disease. Here, we conceptualize how synthetic biology approaches can be applied to programme living cells with therapeutic functions and discuss the advantages that they offer over conventional therapies in terms of flexibility, specificity and predictability, as well as challenges for their development. We present notable advances in the creation of engineered cells that harbour synthetic gene circuits capable of biological sensing and computation of signals derived from intracellular or extracellular biomarkers. We categorize and describe these developments based on the cell scaffold (human or microbial) and the site at which the engineered cell exerts its therapeutic function within its human host. The design of cell-based therapeutics with synthetic biology is a rapidly growing strategy in medicine that holds great promise for the development of effective treatments for a wide variety of human diseases.
Asunto(s)
Ingeniería Celular/métodos , Ingeniería Genética/mortalidad , Biología Sintética , Tratamiento Basado en Trasplante de Células y Tejidos/tendencias , Redes Reguladoras de Genes , Ingeniería Genética/métodos , Humanos , Biología Sintética/métodos , Biología Sintética/tendenciasRESUMEN
The availability of L-arginine in tumours is a key determinant of an efficient anti-tumour T cell response1-4. Consequently, increases of typically low L-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies5. However, currently no means are available to locally increase intratumoural L-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours6, to L-arginine. Colonization of tumours with these bacteria increased intratumoural L-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by L-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies.
Asunto(s)
Inmunoterapia/métodos , Ingeniería Metabólica , Microorganismos Modificados Genéticamente , Neoplasias Experimentales/terapia , Traslado Adoptivo , Animales , Arginina/metabolismo , Antígeno B7-H1/antagonistas & inhibidores , Línea Celular Tumoral , Escherichia coli , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/microbiología , Probióticos , Proteoma , Biología Sintética , Linfocitos T/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine.
Asunto(s)
Escherichia coli/inmunología , Inmunoterapia/métodos , Proteínas de la Membrana/inmunología , Neoplasias/terapia , Transducción de Señal/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Línea Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética/métodos , Humanos , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/genética , Neoplasias/inmunología , Fagocitos/inmunología , Fagocitos/metabolismo , Transducción de Señal/genética , Biología Sintética/métodos , Biología Sintética/tendenciasRESUMEN
Atopic asthma is a prevalent respiratory disease that is characterized by inflammation, mucus hypersecretion, and airway hyperresponsiveness. The complexity of this heterogeneous disorder has commanded the need to better define asthma phenotypes based on underlying molecular mechanisms of disease. Although classically viewed as a type 2-regulated disease, type 17 helper T (Th17) cells are known to be influential in asthma pathogenesis, predominantly in asthmatics with neutrophilia and severe refractory disease. Bromodomain and extra-terminal domain (BET) chromatin adaptors serve as immunomodulators by directly regulating Th17 responses and Th17-mediated pathology in murine models of autoimmunity and infection. Based on this, we hypothesized that BET proteins may also play an essential role in neutrophil-dominant allergic airway disease. Using a murine model of neutrophil-dominant allergic airway disease, we demonstrate that BET inhibition limits pulmonary inflammation and alters the Th17-related inflammatory milieu in the lungs. In addition, inhibition of BET proteins improved lung function (specifically quasi-static lung compliance and tissue elastance) and reduced mucus production in airways. Overall, these studies show that BET proteins may have a critical role in asthma pathogenesis by altering type 17 inflammation, and thus interfering with BET-dependent chromatin signaling may provide clinical benefits to patients suffering from asthma.
Asunto(s)
Asma/patología , Neutrófilos/inmunología , Factores de Transcripción/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Inflamación/patología , Pulmón/patología , Ratones , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Células Th17/inmunología , Factores de Transcripción/metabolismoRESUMEN
Epigenetic control of gene expression is enforced in part through histone modifications. Bromodomain and extra terminal domain (BET) proteins function as crucial chromatin readers, responsible for interpretation of the chromatin code in diverse cellular contexts, ultimately impacting gene transcription. BET proteins can play a major role in inflammation by profoundly affecting the biology of the Thelper 17 (TH17) lineage. We summarize recent studies focusing on BET inhibition as a viable therapeutic alternative for the control of autoimmune diseases driven by aberrant activation of TH17 cells.
Asunto(s)
Proteínas Nucleares/antagonistas & inhibidores , Dominios Proteicos , Células Th17/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Animales , Diferenciación Celular , Citocinas/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Células Th17/citología , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Significant morbidity in cystic fibrosis (CF) results from chronic lung inflammation, most commonly due to Pseudomonas aeruginosa infection. Recent data suggest that IL-17 contributes to pathological inflammation in the setting of abnormal mucosal immunity, and type 17 immunity-driven inflammatory responses may represent a target to block aberrant inflammation in CF. Indeed, transcriptomic analysis of the airway epithelium from CF patients undergoing clinical bronchoscopy revealed upregulation of IL-17 downstream signature genes, implicating a substantial contribution of IL-17-mediated immunity in CF lungs. Bromodomain and extraterminal domain (BET) chromatin modulators can regulate T cell responses, specifically Th17-mediated inflammation, by mechanisms that include bromodomain-dependent inhibition of acetylated histones at the IL17 locus. Here, we show that, in vitro, BET inhibition potently suppressed Th17 cell responses in explanted CF tissue and inhibited IL-17-driven chemokine production in human bronchial epithelial cells. In an acute P. aeruginosa lung infection murine model, BET inhibition decreased inflammation, without exacerbating infection, suggesting that BET inhibition may be a potential therapeutic target in patients with CF.
RESUMEN
Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.
Asunto(s)
Proteína de Unión a CREB/antagonistas & inhibidores , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Acetilación/efectos de los fármacos , Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/metabolismo , Factores de Transcripción Forkhead/metabolismo , Histonas/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo[b]isoxazolo[4,5-d]azepines and benzotriazolo[4,3-d][1,4]diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice.
RESUMEN
Interleukin (IL) 17-producing T helper (T(H)17) cells have been selected through evolution for their ability to control fungal and bacterial infections. It is also firmly established that their aberrant generation and activation results in autoimmune conditions. Using a characterized potent and selective small molecule inhibitor, we show that the bromodomain and extra-terminal domain (BET) family of chromatin adaptors plays fundamental and selective roles in human and murine T(H)17 differentiation from naive CD4(+) T cells, as well as in the activation of previously differentiated T(H)17 cells. We provide evidence that BET controls T(H)17 differentiation in a bromodomain-dependent manner through a mechanism that includes the direct regulation of multiple effector T(H)17-associated cytokines, including IL17, IL21, and GMCSF. We also demonstrate that BET family members Brd2 and Brd4 associate with the Il17 locus in T(H)17 cells, and that this association requires bromodomains. We recapitulate the critical role of BET bromodomains in T(H)17 differentiation in vivo and show that therapeutic dosing of the BET inhibitor is efficacious in mouse models of autoimmunity. Our results identify the BET family of proteins as a fundamental link between chromatin signaling and T(H)17 biology, and support the notion of BET inhibition as a point of therapeutic intervention in autoimmune conditions.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Células Th17/inmunología , Células Th17/patología , Animales , Comunicación Autocrina/genética , Autoinmunidad/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Citocinas/genética , Citocinas/metabolismo , Sitios Genéticos/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Unión Proteica/genética , Transcripción GenéticaRESUMEN
Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by 'mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.
Asunto(s)
Antiinflamatorios/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Inflamación , Macrófagos/efectos de los fármacos , Acetilación/efectos de los fármacos , Animales , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Benzodiazepinas , Células Cultivadas , Epigenómica , Estudio de Asociación del Genoma Completo , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Inflamación/tratamiento farmacológico , Inflamación/prevención & control , Estimación de Kaplan-Meier , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Infecciones por Salmonella/tratamiento farmacológico , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/fisiopatología , Infecciones por Salmonella/prevención & control , Salmonella typhimurium , Sepsis/tratamiento farmacológico , Sepsis/prevención & control , Choque Séptico/tratamiento farmacológico , Choque Séptico/prevención & controlRESUMEN
Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.
Asunto(s)
Benzodioxoles/farmacología , Fenilpropionatos/farmacología , Neumonía/tratamiento farmacológico , Primates , Receptores de Leucotrieno B4/antagonistas & inhibidores , Animales , Benzodioxoles/uso terapéutico , Benzodioxoles/toxicidad , Perros , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Femenino , Cobayas , Células HL-60 , Humanos , Hipersensibilidad/complicaciones , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Masculino , Ratones , Ozono/farmacología , Fenilpropionatos/uso terapéutico , Fenilpropionatos/toxicidad , Neumonía/inducido químicamente , Neumonía/complicaciones , Neumonía/metabolismo , Ratas , Receptores de Leucotrieno B4/metabolismo , Fumar/efectos adversos , Pruebas de ToxicidadRESUMEN
Many nervous system pathologies are associated with increased levels of apolipoprotein D (ApoD), a lipocalin also expressed during normal development and aging. An ApoD homologous gene in Drosophila, Glial Lazarillo, regulates resistance to stress, and neurodegeneration in the aging brain. Here we study for the first time the protective potential of ApoD in a vertebrate model organism. Loss of mouse ApoD function increases the sensitivity to oxidative stress and the levels of brain lipid peroxidation, and impairs locomotor and learning abilities. Human ApoD overexpression in the mouse brain produces opposite effects, increasing survival and preventing the raise of brain lipid peroxides after oxidant treatment. These observations, together with its transcriptional up-regulation in the brain upon oxidative insult, identify ApoD as an acute response protein with a protective and therefore beneficial function mediated by the control of peroxidated lipids.
Asunto(s)
Apolipoproteínas D/metabolismo , Estrés Oxidativo , Envejecimiento/efectos de los fármacos , Animales , Apolipoproteínas D/genética , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Aprendizaje/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Lipocalinas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Sistema Nervioso/efectos de los fármacos , Sistema Nervioso/patología , Estrés Oxidativo/efectos de los fármacos , Paraquat/farmacología , Análisis de Supervivencia , Transgenes , Regulación hacia Arriba/efectos de los fármacosRESUMEN
IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4Ralpha/IL-13Ralpha1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-kappaB. As a result, we hypothesized that NF-kappaB activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-kappaB activity. Three pharmacologic approaches were used to inhibit NF-kappaB including 1) PS1145, a small molecule inhibitor of IkappaBalpha kinase (IKK2), 2) antennapedia-linked NF-kappaB essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-kappaB component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-kappaB is an attractive target for immunotherapy of IL-13-mediated diseases.
Asunto(s)
Interleucina-13/inmunología , Subunidad p50 de NF-kappa B/inmunología , Fibrosis Pulmonar/inmunología , Hipersensibilidad Respiratoria/inmunología , Transducción de Señal/inmunología , Adenoviridae , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Apoptosis/inmunología , Caspasas/genética , Caspasas/inmunología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Inflamación/terapia , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/inmunología , Interleucina-13/genética , Ratones , Ratones Mutantes , Ratones Transgénicos , Moco/inmunología , Subunidad p50 de NF-kappa B/antagonistas & inhibidores , Subunidad p50 de NF-kappa B/genética , Péptidos/farmacología , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/patología , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/terapia , Piridinas/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Hipersensibilidad Respiratoria/genética , Hipersensibilidad Respiratoria/patología , Hipersensibilidad Respiratoria/terapia , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Sphingosine kinase (Sphk) phosphorylates sphingosine into sphingosine-1-phosphate (S1P), but its recently identified isoform Sphk2 has been suggested to have distinct subcellular localization and substrate specificity. We demonstrate here that, surprisingly, Sphk2(-/-) CD4(+) T cells exhibit a hyperactivated phenotype with significantly enhanced proliferation and cytokine secretion in response to IL-2 as well as reduced sensitivity to regulatory T cell-mediated suppression in vitro, apparently independent of effects upon S1P. Such findings appear to reflect a requirement for Sphk2 to suppress IL-2 signaling because, in Sphk2(-/-) CD4(+) T cells, IL-2 induced abnormally accentuated STAT5 phosphorylation and small interfering RNA knockdown of STAT5 abrogated their hyperactive phenotype. This pathway physiologically modulates autoinflammatory responses, because Sphk2(-/-) T cells induced more rapid and robust inflammatory bowel disease in scid recipients. Thus, Sphk2 regulates IL-2 pathways in T cells, and the modulation of Sphk2 activity may be of therapeutic utility in inflammatory and/or infectious diseases.
Asunto(s)
Autoinmunidad/inmunología , Interleucina-2/inmunología , Interleucina-2/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/inmunología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal/inmunología , Animales , Enfermedades Inflamatorias del Intestino/enzimología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Lisofosfolípidos/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Linfocitos T/enzimología , Linfocitos T/inmunologíaRESUMEN
CCL1 is the predominant chemokine secreted from IgE-activated human and mouse mast cells in vitro, colocalizes to mast cells in lung biopsies, and is elevated in asthmatic airways. CCR8, the receptor for CCL1, is expressed by approximately 70% of CD4(+) T lymphocytes recruited to the asthmatic airways, and the number of CCR8-expressing cells is increased 3-fold in the airways of asthmatic subjects compared with normal volunteers. In vivo, CCL1 expression in the lung is reduced in mast cell-deficient mice after aeroallergen provocation. Neutralization of CCL1 or CCR8 deficiency results in reduced mucosal lung inflammation, airway hyperresponsiveness, and mucus hypersecretion to a similar degree as detected in mast cell-deficient mice. Adenoviral delivery of CCL1 to the lungs of mast cell-deficient mice restores airway hyperresponsiveness, lung inflammation, and mucus hypersecretion to the degree observed in wild-type mice. The consequences of CCR8 deficiency, including a marked reduction in Th2 cytokine levels, are comparable with those observed by depletion of CD4(+) T lymphocytes. Thus, mast cell-derived CCL1- and CCR8-expressing CD4(+) effector T lymphocytes play an essential role in orchestrating lung mucosal inflammatory responses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Quimiocinas CC/fisiología , Hipersensibilidad/inmunología , Hipersensibilidad/patología , Mastocitos/inmunología , Receptores de Quimiocina/fisiología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Animales , Asma/inmunología , Asma/metabolismo , Asma/patología , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/patología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Quimiocina CCL1 , Quimiocinas CC/biosíntesis , Quimiocinas CC/genética , Citocinas/biosíntesis , Citocinas/genética , Femenino , Humanos , Hipersensibilidad/genética , Inmunoglobulina E/farmacología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Mastocitos/metabolismo , Mastocitos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Endogámicas WKY , Receptores CCR8 , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Mucosa Respiratoria/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Células Th2/inmunología , Células Th2/metabolismo , Células Th2/patología , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
IL-13 is a major effector at sites of Th2 inflammation and tissue remodeling. In these locations, it frequently coexists with the CCR5 chemokine receptor and its ligands MIP-1alpha/CCL3 and MIP-1beta/CCL4. We hypothesized that CCR5 induction and activation play important roles in the pathogenesis of IL-13-induced tissue responses. To test this hypothesis, we evaluated the effects of IL-13 on the expression of CCR5 in the murine lung. We also compared the effects of lung-targeted transgenic IL-13 in mice treated with anti-CCR5 or an Ab control and mice with wild-type or null CCR5 loci. These studies demonstrate that IL-13 is a potent stimulator of epithelial cell CCR5 expression. They also demonstrate that CCR5 neutralization or a deficiency of CCR5 significantly decreases IL-13-induced inflammation, alveolar remodeling, structural and inflammatory cell apoptosis, and respiratory failure and death. Lastly, these studies provide mechanistic insights by demonstrating that CCR5 is required for optimal IL-13 stimulation of select chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-1/CCL-2), matrix metalloproteinase-9 and cell death regulators (Fas, TNF, TNFR1, TNFR2, Bid), optimal IL-13 inhibition of alpha1-antitrypsin, and IL-13-induction of and activation of caspases-3, -8, and-9. Collectively, these studies demonstrate that CCR5 plays a critical role in the pathogenesis of IL-13-induced inflammation and tissue remodeling.
Asunto(s)
Inflamación/etiología , Interleucina-13/fisiología , Receptores CCR5/fisiología , Animales , Secuencia de Bases , ADN/genética , Daño del ADN , Inflamación/inmunología , Inflamación/patología , Interleucina-13/antagonistas & inhibidores , Interleucina-13/genética , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Pruebas de Neutralización , Péptido Hidrolasas/metabolismo , Receptores CCR5/deficiencia , Receptores CCR5/genética , Transducción de SeñalRESUMEN
The Th2 cell-dependent inflammatory response is a central component of asthma, and the ways in which it is regulated is a critical question. The cysteinyl leukotrienes (cys-LTs) are 5-lipoxygenase pathway products implicated in asthma, in particular, by their function as smooth muscle constrictors of airways and microvasculature. To elucidate additional roles for cys-LTs in the pathobiology of pulmonary inflammation, we used an OVA sensitization and challenge protocol with mice lacking leukotriene C(4) synthase (LTC(4)S), the terminal enzyme for cys-LT generation. Ag-induced pulmonary inflammation, characterized by eosinophil infiltration, goblet cell hyperplasia with mucus hypersecretion, and accumulation and activation of intraepithelial mast cells was markedly reduced in LTC(4)S(null) mice. Furthermore, Ag-specific IgE and IgG1 in serum, Th2 cell cytokine mRNA expression in the lung, and airway hyperresponsiveness to methacholine were significantly reduced in LTC(4)S(null) mice compared with wild-type controls. Finally, the number of parabronchial lymph node cells from sensitized LTC(4)S(null) mice and their capacity to generate Th2 cell cytokines ex vivo after restimulation with Ag were also significantly reduced. In contrast, delayed-type cutaneous hypersensitivity, a prototypic Th1 cell-dependent response, was intact in LTC(4)S(null) mice. These findings provide direct evidence of a role for cys-LTs in regulating the initiation and/or amplification of Th2 cell-dependent pulmonary inflammation.