RESUMEN
MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.
Asunto(s)
Perfilación de la Expresión Génica/métodos , MicroARNs/genética , Anotación de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Animales , Células Cultivadas , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , MicroARNs/metabolismoRESUMEN
The mycobacterial cord factor trehalose-6,6-dimycolate (TDM) and its synthetic analog trehalose-6,6-dibehenate (TDB) are potent adjuvants for Th1/Th17 vaccination that activate Syk-Card9 signaling in APCs. In this study, we have further investigated the molecular mechanism of innate immune activation by TDM and TDB. The Syk-coupling adapter protein FcRgamma was essential for macrophage activation and Th17 adjuvanticity. The FcRgamma-associated C-type lectin receptor Mincle was expressed in macrophages and upregulated by TDM and TDB. Recombinant Mincle-Fc fusion protein specifically bound to the glycolipids. Genetic ablation of Mincle abolished TDM/TDB-induced macrophage activation and induction of T cell immune responses to a tuberculosis subunit vaccine. Macrophages lacking Mincle or FcRgamma were impaired in the inflammatory response to Mycobacterium bovis bacillus Calmette-Guérin. These results establish that Mincle is a key receptor for the mycobacterial cord factor and controls the Th1/Th17 adjuvanticity of TDM and TDB.
Asunto(s)
Adyuvantes Inmunológicos/metabolismo , Factores Cordón/metabolismo , Glucolípidos/metabolismo , Lectinas Tipo C/fisiología , Proteínas de la Membrana/fisiología , Mycobacterium bovis/metabolismo , Animales , Línea Celular , Factores Cordón/inmunología , Glucolípidos/inmunología , Humanos , Interleucina-17/biosíntesis , Interleucina-17/fisiología , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Mycobacterium bovis/inmunología , Receptores de IgG/deficiencia , Receptores de IgG/genética , Receptores de IgG/metabolismo , Staphylococcus aureus/inmunología , Staphylococcus aureus/metabolismo , Células TH1/inmunología , Células TH1/metabolismoRESUMEN
Candida albicans is a causative agent in mycoses of the skin, oral cavity, and gastrointestinal tract. Identification of receptors, and their respective ligands, that are engaged by immune cells when in contact with C. albicans is crucial for understanding inflammatory responses leading to invasive candidiasis. Mincle is a recently identified macrophage-expressed receptor that is important for host responses to C. albicans. The carbohydrate-recognition domain of human and mouse Mincle were expressed, purified under denaturing conditions, and successfully refolded. In addition to oligomers, there are isolatable monomeric and dimeric forms of the protein that occur under two different buffer solutions. The human and mouse homologues bound yeast extract, and the isolated dimeric and monomeric species also demonstrated the recognition of whole C. albicans yeast cells. The data are indicative of several functional states mediating the interaction of Mincle and yeast at the surface of the macrophage.
Asunto(s)
Candida albicans/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Extractos Celulares/farmacología , Células Cultivadas , Dimerización , Humanos , Lectinas Tipo C/fisiología , Macrófagos/metabolismo , Masculino , Proteínas de la Membrana/fisiología , Ratones , Unión Proteica , SolubilidadRESUMEN
The recognition of carbohydrate moieties by cells of the innate immune system is emerging as an essential element in antifungal immunity, but despite the number and diversity of lectins expressed by innate immune cells, few carbohydrate receptors have been characterized. Mincle, a C-type lectin, is expressed predominantly on macrophages, and is here shown to play a role in macrophage responses to the yeast Candida albicans. After exposure to the yeast in vitro, Mincle localized to the phagocytic cup, but it was not essential for phagocytosis. In the absence of Mincle, production of TNF-alpha by macrophages was reduced, both in vivo and in vitro. In addition, mice lacking Mincle showed a significantly increased susceptibility to systemic candidiasis. Thus, Mincle plays a novel and nonredundant role in the induction of inflammatory signaling in response to C. albicans infection.