RESUMEN
Phagocytes such as dendritic cells (DC) and macrophages employ phagocytosis to take up pathogenic bacteria into phagosomes, digest the bacteria and present the bacteria-derived peptide antigens to the adaptive immunity. Hence, efficient antigen presentation depends greatly on a well-regulated phagocytosis process. Lipids, particularly phosphoinositides, are critical components of the phagosomes. Phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3 ] is formed at the phagocytic cup, and as the phagosome seals off from the plasma membrane, rapid disappearance of PI(3,4,5)P3 is accompanied by high levels of phosphatidylinositol-3-phosphate (PI3P) formation. The sorting nexin (SNX) family consists of a diverse group of Phox-homology (PX) domain-containing cytoplasmic and membrane-associated proteins that are potential effectors of phosphoinositides. We hypothesized that SNX3, a small sorting nexin that contains a single PI3P lipid-binding PX domain as its only protein domain, localizes to phagosomes and regulates phagocytosis in DC. Our results show that SNX3 recruits to nascent phagosomes and silencing of SNX3 enhances phagocytic uptake of bacteria by DC. Furthermore, SNX3 competes with PI3P lipid-binding protein, early endosome antigen-1 (EEA1) recruiting to membranes. Our results indicate that SNX3 negatively regulates phagocytosis in DC possibly by modulating recruitment of essential PI3P lipid-binding proteins of the phagocytic pathways, such as EEA1, to phagosomal membranes.
Asunto(s)
Células Dendríticas/inmunología , Fagocitosis/fisiología , Fagosomas/inmunología , Nexinas de Clasificación/inmunología , Animales , Bacterias/genética , Bacterias/inmunología , Línea Celular Transformada , Línea Celular Tumoral , Células Dendríticas/citología , Humanos , Membranas Intracelulares/inmunología , Ratones , Fagosomas/genética , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/inmunología , Estructura Terciaria de Proteína , Nexinas de Clasificación/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/inmunologíaRESUMEN
Since being introduced globally as aspirin in 1899, acetylsalicylic acid has been widely used as an analgesic, anti-inflammation, anti-pyretic, and anti-thrombotic drug for years. Aspirin had been reported to down-regulate surface expression of CD40, CD80, CD86, and MHCII in myeloid dendritic cells (DC), which played essential roles in regulating the immune system. We hypothesized that the down-regulation of these surface membrane proteins is partly due to the ability of aspirin in regulating trafficking/sorting of endocytosed surface membrane proteins. By using an established epidermoid carcinoma cell line (A-431), which overexpresses the epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR), we show that aspirin (1) reduces cell surface expression of EGFR and (2) accumulates endocytosed-EGFR and -TfnR in the early/sorting endosome (ESE). Further elucidation of the mechanism suggests that aspirin enhances recruitment of SNX3 and SNX5 to membranes and consistently, both SNX3 and SNX5 play essential roles in the aspirin-mediated accumulation of endocytosed-TfnR at the ESE. This study sheds light on how aspirin may down-regulate surface expression of EGFR by inhibiting/delaying the exit of endocytosed-EGFR from the ESE and recycling of endocytosed-EGFR back to the cell surface.
Asunto(s)
Aspirina/farmacología , Carcinoma de Células Escamosas/metabolismo , Receptores ErbB/metabolismo , Nexinas de Clasificación/metabolismo , Secuencia de Bases , Transporte Biológico Activo/efectos de los fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/ultraestructura , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cartilla de ADN/genética , Endocitosis/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Microscopía Inmunoelectrónica , Modelos Biológicos , Receptores de Transferrina/metabolismo , Transducción de Señal/efectos de los fármacos , Nexinas de Clasificación/antagonistas & inhibidores , Nexinas de Clasificación/genéticaRESUMEN
MHC class II expression is controlled mainly at transcriptional level by class II transactivator (CIITA), which is a non-DNA binding coactivator and serves as a master control factor for MHC class II genes expression. Here, we describe the function of a novel splice-isoform of CIITA, DC-expressed caspase inhibitory isoform of CIITA (or DC-CASPIC), and we show that the expression of DCCASPIC in DC is upregulated upon lipopolysaccharides (LPS) induction. DC-CASPIC localizes to mitochondria, and protein-protein interaction study demonstrates that DC-CASPIC interacts with caspases and inhibits its activity in DC. Consistently, DC-CASPIC suppresses caspases-induced degradation of nitric oxide synthase-2 (NOS2) and subsequently promotes the synthesis of nitric oxide (NO). NO is an essential regulatory molecule that modulates the capability of DC in stimulating T cell proliferation/activation in vitro; hence, overexpression of DC-CASPIC in DC enhances this stimulation. Collectively, our findings reveal that DC-CASPIC is a key molecule that regulates caspases activity and NO synthesis in DC.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Células Dendríticas/metabolismo , Óxido Nítrico/biosíntesis , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Adaptadoras de Señalización CARD/genética , Línea Celular , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Humanos , Técnicas In Vitro , Lipopolisacáridos/farmacología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transactivadores/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Dendritic cells (DC) are professional antigen-presenting cells that possess specific and efficient mechanisms to initiate immune responses. Upon encounter with pathogens, immature DC will go through a maturation process that converts them to highly immunogenic mature DC. Despite the fact that nitric oxide (NO) was produced in large amounts in maturing DC, it is still unclear whether NO is the key molecule that initiates and enhances DC maturation and T cell proliferation, respectively. Here, we report that NO donor and overexpression of either nitric-oxide synthase 2 (NOS2) or nitric-oxide synthase 3 (NOS3) alone can induce surface expression of major histocompatibility complex class II (MHC II) and both the essential co-stimulatory molecules CD80 and CD86 in immature DC. Consistently, NO donor-treated immature DC were capable of enhancing T cell proliferation in vitro in the absence of lipolysaccharide. Interestingly, NOS2 interacts with CD74 (the MHC II-associated invariant chain), and the degradation of CD74 by caspases in immature DC was inhibited upon treatment with NO donor. Because the trafficking of MHC II is CD74-dependent, the increase in cell surface localization of MHC II in maturing DC is in part due to the increase in CD74 protein expression in the presence of NOS2 and NO.