RESUMEN
OBJECTIVE: Macrophages polarize to proinflammatory M1 or anti-inflammatory M2 states with distinct physiological functions. This transition within the M1-M2 phenotypes decides the nature, duration and severity of an inflammatory response. Although there is a substantial understanding of the fate of these phenotypes, the underlying molecular mechanism of transition within the M1-M2 phenotypes is not well understood. We have investigated the role of neuronal nitric oxide synthase (NOS1)-mediated regulation of activator protein 1 (AP-1) transcription factor in macrophages as a critical effector of macrophage phenotypic change. MATERIALS AND METHODS: Raw 264.7 and THP1 macrophages were stimulated with LPS (250 ng/ml) to activate the inflammatory signaling pathway. We analyzed the effect of pharmacological NOS1 inhibitor: TRIM (1-(2- Trifluoromethylphenyl) imidazole) on LPS-induced inflammatory response in macrophages. RESULTS: We determined that NOS1-derived nitric oxide (NO) facilitate Fos and Jun interaction which induces IL-12 & IL-23 expression. Pharmacological inhibition of NOS1 inhibits ATF2 and Jun dimer. Switching of Fos and Jun dimer to ATF2 and Jun dimerization controls phenotype transition from IL-12high IL-23high IL-10low to IL-12low IL-23lowIL-10high phenotype, respectively. CONCLUSION: These findings highlight a key role of the TLR4-NOS1-AP1 signaling axis in regulating macrophage polarization.
Asunto(s)
Macrófagos/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción Activador 2/metabolismo , Animales , Línea Celular , Citocinas/metabolismo , Dimerización , Humanos , Lipopolisacáridos , Macrófagos/efectos de los fármacos , Ratones , Modelos Moleculares , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-fos/metabolismo , Células RAW 264.7 , Transducción de SeñalRESUMEN
Salivary RNA-based biomarkers are not available for any physiological condition in farm animals. Hence, an objective of this study was to perform salivary transcript analysis in buffaloes. Saliva, after removal of the cells and particulate matter, was directly used for RT-PCR without RNA isolation. Direct saliva transcript analysis (DSTA) showed a suggestively significant higher expression of the Heat shock protein 70 (HSP70) and Toll-like receptor 4 (TLR4) at oestrus than the diestrous period in buffaloes by a non-parametric Mann-Whitney U test. Therefore, DSTA without RNA isolation is an easy method to identify salivary RNA markers for oestrus detection in buffaloes.
Asunto(s)
Búfalos/genética , Estro/genética , Perfilación de la Expresión Génica , Saliva/metabolismo , Transcripción Genética/genética , Animales , Biomarcadores/metabolismo , Femenino , Proteínas HSP70 de Choque Térmico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/genéticaRESUMEN
PROBLEM: In granulosa cells, TLR4-mediated LPS-induced immune response interferes with ovarian granulosa cell function. METHOD OF STUDY: LPS-induced pro-inflammatory gene expression was monitored by real-time PCR and NF-κB was determined by the immuno-blotting and immuno-staining in granulosa cells in vitro. RESULTS: The LPS (1 µg/mL) increased pro-inflammatory gene expression which was reverted back by treatment with TLR4 signaling inhibitor. Cotreatment of CLA (10 µm) with LPS did not show any effect on LPS-induced pro-inflammatory gene expression but granulosa cells pre-treated with CLA for 24 hr, attenuated LPS-induced pro-inflammatory gene expression and nuclear NF-κB. GW9662, a PPARγ-inhibitor, further increased the expression of pro-inflammatory genes. CONCLUSION: The present findings reiterated that pre-treatment with CLA can prevent LPS-induced granulosa cells dysfunction.