RESUMEN
Deciphering signaling pathways that regulate the complex interplay between inflammation and cell death is a key challenge in understanding innate immune responses. Over recent years, receptor interacting protein (RIP) kinases have been described to regulate the interplay between inflammation and cell death. Whereas RIP1 and 3, the most well described members of the RIP kinase family, play important roles in necroptosis, RIP2's involvement in regulating inflammation, cell death processes and cancer is less well described and controversially discussed. Here, we demonstrate that RIP2 exerts immune regulatory functions by regulating mitochondrial damage and mitochondrial superoxide production in response to SV40 LT-induced genotoxic stress by the induction of ULK1-phosphorylation, therefore regulating the expression of interferon stimulated genes (ISGs) and NLRP3-inflammasome dependent IL-1ß release. Because RIP2 is upregulated and/or activated in autoimmune/inflammatory disease and cancer, observations from this study promise implications of RIP kinases in human disease.
Asunto(s)
Inflamación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Daño del ADN , Homeostasis , Humanos , Especies Reactivas de Oxígeno/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismoRESUMEN
Bronchopulmonary dysplasia (BPD) is a chronic lung disease of preterm infants. Mouse models of hyperoxia-induced lung injury are often used to study pathogenesis and potential therapeutic approaches of BPD. Beside histological studies, gene expression analysis of lung tissue is typically used as experimental readout. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is the standard method for gene expression analysis; however, the accuracy of the quantitative data depends on the appropriate selection of reference genes. No data on validated reference genes for hyperoxia-induced neonatal lung injury in mice are available. In this study, 12 potential reference genes were systematically analyzed for their expression stability in lung tissue of neonatal mice exposed to room air or hyperoxia and healthy adult controls using published software algorithms. Analysis of gene expression data identified Hprt, Tbp, and Hmbs as the most stable reference genes and proposed combinations of Hprt/Sdha or Hprt/Rpl13a as potential normalization factors. These reference genes and normalization factors were validated by comparing Il6 gene and protein expression and may facilitate accurate gene expression analysis in lung tissues of similar designed studies.
Asunto(s)
Displasia Broncopulmonar/genética , Complejo II de Transporte de Electrones/genética , Hipoxantina Fosforribosiltransferasa/genética , Lesión Pulmonar/patología , Proteínas Ribosómicas/genética , Proteína de Unión a TATA-Box/genética , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/patología , Citocinas/análisis , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Hiperoxia/patología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Oxígeno/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genéticaRESUMEN
A novel, strictly anaerobic, hydrogenotrophic methanogen, strain E09F.3T, was isolated from a commercial biogas plant in Germany. Cells of E09F.3T were Gram-stain-negative, non-motile, slightly curved rods, long chains of which formed large aggregates consisting of intertwined bundles of chains. Cells utilized H2+CO2 and, to a lesser extent, formate as substrates for growth and methanogenesis. The optimal growth temperature was around 40 °C; maximum growth rate was obtained at pH around 7.0 with approximately 6.8 mM NaCl. The DNA G+C content of strain E09F.3T was 39.1 mol%. Phylogenetic analyses based on 16S rRNA and mcrA gene sequences placed strain E09F.3T within the genus Methanobacterium. On the basis of 16S rRNA gene sequence similarity, strain E09F.3T was closely related to Methanobacterium congolense CT but morphological, physiological and genomic characteristics indicated that strain E09F.3T represents a novel species. The name Methanobacterium aggregans sp. nov. is proposed for this novel species, with strain E09F.3T ( = DSM 29428T = JCM 30569T) as the type strain.
Asunto(s)
Reactores Biológicos/microbiología , Methanobacterium/clasificación , Filogenia , Composición de Base , ADN de Archaea/genética , Genes Arqueales , Alemania , Methanobacterium/genética , Methanobacterium/aislamiento & purificación , Datos de Secuencia Molecular , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , TemperaturaRESUMEN
Pro-inflammatory caspase-1 is a key player in innate immunity. Caspase-1 processes interleukin (IL)-1ß and IL-18 to their mature forms and triggers pyroptosis. These caspase-1 functions are linked to its enzymatic activity. However, loss-of-function missense mutations in CASP1 do not prevent autoinflammation in patients, despite decreased IL-1ß production. In vitro data suggest that enzymatically inactive caspase-1 drives inflammation via enhanced nuclear factor κB (NF-κB) activation, independent of IL-1ß processing. Here, we report two mouse models of enzymatically inactive caspase-1-C284A, demonstrating the relevance of this pathway in vivo. In contrast to Casp1-/- mice, caspase-1-C284A mice show pronounced hypothermia and increased levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 when challenged with lipopolysaccharide (LPS). Caspase-1-C284A signaling is RIP2 dependent and mediated by TNF-α but independent of the NLRP3 inflammasome. LPS-stimulated whole blood from patients carrying loss-of-function missense mutations in CASP1 secretes higher amounts of TNF-α. Taken together, these results reveal non-canonical caspase-1 signaling in vivo.