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Exosomal miR-223 derived from natural killer cells inhibits hepatic stellate cell activation by suppressing autophagy.

ABSTRACT: BACKGROUND:Activation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrosis. We previously demonstrated that exosomes derived from natural killer (NK) cells (NK-Exo) attenuated TGF-?1-induced HSC activation. Herein, this study was designed to investigate the mechanism underlying the action of NK-Exo. METHODS:NK-Exo was isolated from NK-92MI cells and then administered into TGF-?1-treated LX-2 (human HSC line) cells. MiR-223 expression in NK-Exo was downregulated by transfecting NK-92MI cells with miR-223 inhibitor followed by exosome isolation. The HSC activation was evaluated by determining cell proliferation using CCK-8 assay and measuring the protein levels of ?-SMA and CoL1A1 using western blot in LX-2 cells. The expression of miR-223 was detected by qRT-PCR. The interaction between miR-223 and ATG7 was analyzed by a dual-luciferase activity assay. The autophagy was evaluated by measuring the autophagy-related proteins using western blot. RESULTS:miR-223 was highly expressed in NK-Exo and inhibition of miR-223 expression in NK-Exo abrogated the inhibitory effect of NK-Exo on TGF-?-induced HSC activation. ATG7 was confirmed as a direct target of miR-223. Furthermore, treatment with the autophagy activator rapamycin and ATG7 overexpression in LX-2 cells abolished the HSC activation-suppressive effect of NK-Exo. CONCLUSION:NK-Exo attenuated TGF-?-induced HSC activation by transferring miR-223 that inhibited autophagy via targeting ATG7.

SUBMITTER: Wang L

PROVIDER: S-EPMC7465359 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Exosomal miR-223 derived from natural killer cells inhibits hepatic stellate cell activation by suppressing autophagy.

Wang Ling L Wang Yinghao Y Quan Jun J

Molecular medicine (Cambridge, Mass.) 20200901 1

<h4>Background</h4>Activation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrosis. We previously demonstrated that exosomes derived from natural killer (NK) cells (NK-Exo) attenuated TGF-β1-induced HSC activation. Herein, this study was designed to investigate the mechanism underlying the action of NK-Exo.<h4>Methods</h4>NK-Exo was isolated from NK-92MI cells and then administered into TGF-β1-treated LX-2 (human HSC line) cells. MiR-223 expression in NK-Exo was downregulated ...[more]

PMID: 32873229

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Project description:BackgroundThis study investigated the mechanisms underlying the preventive effect of dihydromyricetin (DHM) against liver fibrosis involving hepatic stellate cells (HSCs) and hepatic natural killer (NK) cells.MethodsA carbon tetrachloride (CCl4)-induced liver fibrosis model was established in C57BL/6 mice to study the antifibrotic effect of DHM based on serum biochemical parameters, histological and immunofluorescence stainings, and the expression of several fibrosis-related markers. Based on the immunoregulatory role of DHM, the effect of DHM on NK cell activation ex vivo was evaluated by flow cytometry. Then, we investigated whether DHM-induced autophagy was involved in HSCs inactivation using enzyme-linked immunosorbent assays, transmission electron microscopy, and western blot analysis. Thereafter, the role of DHM in NK cell-mediated killing was studied by in vitro coculture of NK cells and HSCs, with subsequent analysis by flow cytometry. Finally, the mechanism by which DHM regulates NK cells was studied by western blot analysis.ResultsDHM ameliorated liver fibrosis in C57BL/6 mice, as characterized by decreased serum alanine transaminase and aspartate transaminase levels, decreased expressions of collagen I alpha 1 (CoL-1α1), collagen I alpha 2 (CoL-1α2), tissue inhibitor of metalloproteinases 1 (TIMP-1), α-smooth muscle actin (α-SMA) and desmin, as well as increased expression of matrix metalloproteinase 1 (MMP1). Interestingly, HSCs activation was significantly inhibited by DHM in vivo and in vitro. As expected, DHM also upregulated autophagy-related indicators in liver from CCl4-treated mice. DHM also prevented TGF-β1-induced activation of HSCs in vitro by initiating autophagic flux. In contrast, the autophagy inhibitor 3-methyladenine markedly abolished the antifibrotic effect of DHM. Surprisingly, the frequency of activated intrahepatic NK cells was significantly elevated by DHM ex vivo. Furthermore, DHM enhanced NK cell-mediated killing of HSCs by increasing IFN-γ expression, which was abolished by an anti-IFN-γ neutralizing antibody. Mechanistically, DHM-induced IFN-γ expression was through AhR-NF-κB/STAT3 pathway in NK cells.ConclusionThese results demonstrated that DHM can ameliorate the progression of liver fibrosis and inhibition of HSCs activation by inducing autophagy and enhancing NK cell-mediated killing through the AhR-NF-κB/STAT3-IFN-γ signaling pathway, providing new insights into the preventive role of DHM in liver fibrosis.

Project description:Background & aimsAutophagy plays a crucial role in hepatic homeostasis and its deregulation has been associated with chronic liver disease. However, the effect of autophagy on the release of fibrogenic extracellular vesicles (EVs) by platelet-derived growth factor (PDGF)-stimulated hepatic stellate cells (HSCs) remains unknown. Herein, we aimed to elucidate the role of autophagy, specifically relating to fibrogenic EV release, in fibrosis.MethodsIn vitro experiments were conducted in primary human and murine HSCs as well as LX2 cells. Small EVs were purified by differential ultracentrifugation. Carbon tetrachloride (CCl4) or bile duct ligation (BDL) were used to induce fibrosis in our mouse model. Liver lysates from patients with cirrhosis or healthy controls were compared by RNA sequencing.ResultsIn vitro, PDGF and its downstream molecule SHP2 (Src homology 2-containing protein tyrosine phosphatase 2) inhibited autophagy and increased HSC-derived EV release. We used this PDGF/SHP2 model to further investigate how autophagy affects fibrogenic EV release. RNA sequencing identified an mTOR (mammalian target of rapamycin) signaling molecule that was regulated by SHP2 and PDGF. Disruption of mTOR signaling abolished PDGF-dependent EV release. Activation of mTOR signaling induced the release of multivesicular body-derived exosomes (by inhibiting autophagy) and microvesicles (by activating ROCK1 signaling). These mTOR-dependent EVs promoted in vitro HSC migration. To assess the importance of this mechanism in vivo, SHP2 was selectively deleted in HSCs, which attenuated CCl4- or BDL-induced liver fibrosis. Furthermore, in the CCl4 model, mice receiving circulating EVs derived from mice with HSC-specific Shp2 deletion had less fibrosis than mice receiving EVs from control mice. Correspondingly, SHP2 was upregulated in patients with liver cirrhosis.ConclusionThese results demonstrate that autophagy in HSCs attenuates liver fibrosis by inhibiting the release of fibrogenic EVs.Lay summaryDuring liver fibrosis and cirrhosis, activated hepatic stellate cells (HSCs) are the key cell type responsible for fibrotic tissue deposition. Recently, we demonstrated that activated HSCs release nano-sized vesicles enriched with fibrogenic proteins. In the current study, we unveil the mechanism by which these fibrogenic vesicles are released, moving a step closer to the long-term goal of therapeutically targeting this process.

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Exosomal miR-223 derived from natural killer cells inhibits hepatic stellate cell activation by suppressing autophagy.

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Exosomal miR-223 derived from natural killer cells inhibits hepatic stellate cell activation by suppressing autophagy.

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