RESUMO
BACKGROUND: Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis. As an important component of host immunity, macrophages are not only the first line of defense against M. tuberculosis but also the parasitic site of M. tuberculosis in the host. Glucocorticoids can cause immunosuppression, which is considered to be one of the major risk factors for active tuberculosis, but the mechanism is unclear. OBJECTIVE: To study the effect of methylprednisolone on the proliferation of mycobacteria in macrophages and try to find key molecules of this phenomenon. METHODS: The macrophage line RAW264.7 infected by M. smegmatis was treated with methylprednisolone, and the intracellular bacterial CFU, Reactive Oxygen Species (ROS), cytokine secretion, autophagy, and apoptosis were measured. After the cells were treated with NF-κB inhibitor BAY 11-7082 and DUSP1 inhibitor BCI, respectively, the intracellular bacterial CFU, ROS, IL-6, and TNF-α secretion were detected. RESULTS: After treatment with methylprednisolone, the CFU of intracellular bacteria increased, the level of ROS decreased, and the secretion of IL-6 and TNF-α decreased in infected macrophages. After BAY 11-7082 treatment, the CFU of M. smegmatis in macrophages increased, and the level of ROS production and the secretion of IL-6 by macrophages decreased. Transcriptome high-throughput sequencing and bioinformatics analysis suggested that DUSP1 was the key molecule in the above phenomenon. Western blot analysis confirmed that the expression level of DUSP1 was increased in the infected macrophages treated with methylprednisolone and BAY 11-7082, respectively. After BCI treatment, the level of ROS produced by infected macrophages increased, and the secretion of IL-6 increased. After the treatment of BCI combined with methylprednisolone or BAY 11-7082, the level of ROS produced and the secretion of IL-6 by macrophages were increased. CONCLUSION: methylprednisolone promotes the proliferation of mycobacteria in macrophages by suppressing cellular ROS production and IL-6 secretion through down-regulating NF-κB and up-regulating DUSP1 expression. BCI, an inhibitor of DUSP1, can reduce the level of DUSP1 in the infected macrophages and inhibit the proliferation of intracellular mycobacteria by promoting cellular ROS production and IL-6 secretion. Therefore, BCI may become a new molecule for host-directed therapy of tuberculosis, as well as a new strategy for the prevention of tuberculosis when treated with glucocorticoids.
RESUMO
Stanford type A aortic dissection (TA-AD) is a life-threatening disease. Most cases of aortic dissection (AD) are sporadic rather than inherited. Unlike that of inherited AD, the pathogenesis of sporadic AD is still unclear. In the current study, we aimed to explore the pathogenesis of sporadic AD through transcriptome sequencing data analyses. We downloaded sporadic TA-AD transcriptome profiles from Gene Expression Omnibus (GEO) and found response to DNA damage stimulus was activated in AD. Furthermore, by conducting mouse AD tissue single cell RNA sequencing and immunostaining, we found that DNA damage mainly occurred in smooth muscle cells (SMCs) and fibroblasts. Next, we examined the repair patterns in response to DNA damage and found the linker molecules RBBP8/NOTCH1 between DNA damage/repair and extracellular matrix (ECM) organization through protein-protein interaction analysis. Thus, we proposed that DNA damage could contribute to AD by regulating ECM changes. To explore the underlying mechanism, we knocked down the DNA repair-related gene RBBP8 in aortic SMCs, which could exacerbate DNA damage, and observed decreased expression level of NOTCH1. Inhibition of NOTCH1 with crenigacestat in vivo accelerated ß-aminopropionitrile-induced formation of AD and increased mortality. Meanwhile, phenotype switching of SMCs was induced by Notch1 knockdown or inhibition; this switching occurred via a pathway involving downregulation of contractile marker gene expression and upregulation of MMP2 expression, which might aggravate ECM degradation. In conclusion, excessive DNA damage is a characteristic pathological change of sporadic aortic dissection, which might contribute to ECM changes and AD development via action on the NOTCH1 pathway.
