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CircAGFG1modulates autophagy and apoptosis of macrophages infected by Mycobacterium tuberculosis via the Notch signaling pathway.

ABSTRACT: Background:Recent studies have revealed the involvement of circular RNAs (circRNAs) in the control and elimination of invading Mycobacterium tuberculosis (Mtb) by macrophages. However, the regulatory mechanism of circAGFG1 in macrophages infected by Mtb has not been fully explored. In this study, we sought to investigate the role of circAGFG1 on autophagy and apoptosis of Mtb-infected macrophages and reveal its the molecular mechanism. Methods:The expression of circAGFG1 in macrophages from patients with active tuberculosis and in Mtb-treated macrophages in vitro was explored. Then, the effect of circAGFG1 on autophagy and apoptosis of Mtb-infected macrophages was evaluated by flow cytometry, electron microscope, immunofluorescence, and Western blotting. Bioinformatics analysis was used to identify and validate the downstream regulatory pathway of circAGFG1, miRNA-1257/Notch. For further analysis, the role of miRNA-1257 on autophagy and apoptosis was assessed. Results:In vitro, ectopic expression of circAGFG1 upregulated autophagy and reduced apoptosis significantly in Mtb-infected cells. Notch levels were discovered to be increased by the silencing effect of circAGFG1 on miRNA-1257 expression. miRNA-1257 was found to noticeably reduce autophagy and promote macrophage apoptosis. Increased circAGFG1 expression, decreased monocyte apoptosis, and enhanced autophagy were found in macrophages from patients with active tuberculosis. Conclusions:In active tuberculosis, circAGFG1 enhances autophagy and reduces apoptosis via the miRNA-1257/Notch axis; this provides new therapeutic targets for tuberculosis patients.

SUBMITTER: Shi Q

PROVIDER: S-EPMC7290638 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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CircAGFG1modulates autophagy and apoptosis of macrophages infected by Mycobacterium tuberculosis via the Notch signaling pathway.

Shi Qinghong Q Wang Jingying J Yang Zhe Z Liu Yang Y

Annals of translational medicine 20200501 10

<h4>Background</h4>Recent studies have revealed the involvement of circular RNAs (circRNAs) in the control and elimination of invading Mycobacterium tuberculosis (Mtb) by macrophages. However, the regulatory mechanism of circAGFG1 in macrophages infected by Mtb has not been fully explored. In this study, we sought to investigate the role of circAGFG1 on autophagy and apoptosis of Mtb-infected macrophages and reveal its the molecular mechanism.<h4>Methods</h4>The expression of circAGFG1 in macrop ...[more]

PMID: 32566582

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Project description:BAG2 (BCL2 associated athanogene 2) is associated with cell fate determination in response to various pathological conditions. However, the effects of BAG2 on M. tuberculosis-induced endoplasmic reticulum (ER) stress remain elusive. Herein, we report that M. tuberculosis infection of macrophages triggered ER stress and downregulated BAG2 expression. Overexpression of BAG2 enhanced autophagic flux and activated macroautophagy/autophagy targeted to the ER (reticulophagy). In addition, through increasingly localizing SQSTM1 to the ER in BAG2-overexpressing macrophages, we found that the autophagy receptor protein SQSTM1/p62 (sequestosome 1) is associated with the BAG2-induced reticulophagy. Our data also confirmed that BAG2 could render cells resistant to M. tuberculosis-induced cellular damage, and the anti-apoptotic effects of BAG2 in M. tuberculosis-treated macrophages were partially abolished by the autophagic flux inhibitor bafilomycin A1. Furthermore, the dissociation of BECN1 and BCL2 mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) was responsible for BAG2-activated autophagy. In addition, XBP1 downstream of the ERN1/IRE1 signaling pathway was bound to the Bag2 promoter region and transcriptionally inhibited BAG2 expression. Collectively, these results indicated that BAG2 has anti-apoptotic effects on M. tuberculosis-induced ER stress, which is dependent on the promotion of autophagic flux and the induction of selective autophagy. We revealed a potential host defense mechanism that links BAG2 to ER stress and autophagy during M. tuberculosis infection.AbbreviationsATF6: activating transcription factor 6; BECN1: beclin 1; Baf A1: bafilomycin A1; CASP3: caspase 3; DDIT3/CHOP/GADD153: DNA damage inducible transcript 3; DAPI: 4',6-diamidino-2-phenylindole; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; HSPA5/GRP78/BiP: heat shock protein 5; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK/ERK: mitogen-activated protein kinase; SQSTM1/p62: sequestosome 1; UPR: unfolded protein response; XBP1: x-box binding protein 1.

Project description:BackgroundTuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis (Mtb) remains a significant health problem worldwide with a third of the world population infected and nearly nine million new cases claiming 1.1 million deaths every year. The outcome following infection by Mtb is determined by a complex and dynamic host-pathogen interaction in which the phenotype of the pathogen and the immune status of the host play a role. However, the molecular mechanism by which Mtb strains induce different responses during intracellular infection of the host macrophage is not fully understood. To explore the early molecular events triggered upon Mtb infection of macrophages, we studied the transcriptional responses of murine bone marrow-derived macrophages (BMM) to infection with two clinical Mtb strains, CDC1551 and HN878. These strains have previously been shown to differ in their virulence/immunogenicity in the mouse and rabbit models of pulmonary TB.ResultsIn spite of similar intracellular growth rates, we observed that compared to HN878, infection by CDC1551 of BMM was associated with an increased global transcriptome, up-regulation of a specific early (6 hours) immune response network and significantly elevated nitric oxide production. In contrast, at 24 hours post-infection of BMM by HN878, more host genes involved in lipid metabolism, including cholesterol metabolism and prostaglandin synthesis were up-regulated, compared to infection with CDC1551.In association with the differences in the macrophage responses to infection with the 2 Mtb strains, intracellular CDC1551 expressed higher levels of stress response genes than did HN878.ConclusionsIn association with the early and more robust macrophage activation, intracellular CDC1551 cells were exposed to a higher level of stress leading to increased up-regulation of the bacterial stress response genes. In contrast, sub-optimal activation of macrophages and induction of a dysregulated host cell lipid metabolism favored a less stressful intracellular environment for HN878. Our findings suggest that the ability of CDC1551 and HN878 to differentially activate macrophages during infection probably determines their ability to either resist host cell immunity and progress to active disease or to succumb to the host protective responses and be driven into a non-replicating latent state in rabbit lungs.

Project description:Tuberculosis is one of the most important infectious diseases worldwide and macrophage apoptosis is the major host defense mechanism against TB. We attempted to characterize the role of miRNA (miR)-125b-5p on mycobacterium tuberculosis (Mtb) infection and macrophages behaviors in vitro. According to fluorescence-activated cell separation (FACS), primary monocytes (CD14+) in TB patients were accumulated, and apoptotic monocytes were decreased. Peripheral blood mononuclear cells (PBMCs)-derived macrophages (MDMs) and monocytic cells THP-1-derived macrophage-like cells (TDMs) in vitro were used to be infected with H37Rv. After infection, colony-forming units assay revealed the increase of bacterial activity, FACS demonstrated the decrease of apoptosis rate of MDMs and TDMs, as well as promoted levels of IL-6, TNF-α, Bax, and Bim and suppressed levels of IL-10 and Bcl-2, examined by enzyme-linked immunosorbent assay (ELISA) and western blot assay. Expression of miR-125b-5p and DNA damage-regulated autophagy modulator 2 (DRAM2) was examined, and real-time PCR and western blot assay showed that miR-125b-5p was upregulated, whereas DRAM2 was downregulated in primary monocytes and H37Rv-infected macrophages (MDMs and TDMs). Moreover, blocking miR-125b-5p could attenuated H37Rv-induced bacterial activity and inflammatory response of MDMs and TDMs, accompanied with apoptosis inhibition. Whereas these effects of miR-125b-5p knockdown were abolished by downregulating DRAM2. In mechanism, DRAM2 was a downstream target of miR-125b-5p, as evidenced by dual-luciferase reporter assay. Collectively, silencing miR-125b-5p could protect human macrophages against Mtb infection through promoting apoptosis and inhibiting inflammatory response via targeting DRAM2, suggesting a novel target for Mtb eliminating. Abbreviations: TB: tuberculosis; PBMCs: peripheral blood mononuclear cells; Mtb: mycobacterium tuberculosis; AFB: acid fast bacilli; FITC: fluorescein isothiocyanate; MDMs: monocytes-derived macrophages; TDMs: THP-1-derived macrophage-like cells; ERFP: Mtb-enhanced red fluorescent protein; CFU: colony-forming units; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell separation; PI: propidium iodide; DRAM2: DNA damage-regulated autophagy modulator 2; Real-time PCR: real-time polymerase chain reaction; in-miR-125b-5p: miR-125b-5p inhibitor; si-DRAM2: siRNA against DRAM2.

Dataset Information

CircAGFG1modulates autophagy and apoptosis of macrophages infected by Mycobacterium tuberculosis via the Notch signaling pathway.

Publications

CircAGFG1modulates autophagy and apoptosis of macrophages infected by Mycobacterium tuberculosis via the Notch signaling pathway.

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