RESUMO
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.
Assuntos
Células Dendríticas/imunologia , Fagocitose/fisiologia , Fagossomos/imunologia , Nexinas de Classificação/imunologia , Animais , Bactérias/genética , Bactérias/imunologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Células Dendríticas/citologia , Humanos , Membranas Intracelulares/imunologia , Camundongos , Fagossomos/genética , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/imunologia , Estrutura Terciária de Proteína , Nexinas de Classificação/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/imunologiaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Células Dendríticas/metabolismo , Óxido Nítrico/biossíntese , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas Adaptadoras de Sinalização CARD/genética , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Técnicas In Vitro , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transativadores/genética , Regulação para Cima/efeitos dos fármacosRESUMO
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.
