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Typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis.

ABSTRACT: Macrophages, with diverse functions and variable phenotypes, are considered as an important executor of inflammatory diseases. And it has been proved that autophagy is deeply connected with the development of inflammation, while the exact regulatory mechanism still remains unclear, and the application of autophagy regulators in anti-inflammation needs to be further confirmed. Here, we firstly verified that neochromine S5 (hereinafter referred to as S5) significantly inhibited M1-like macrophage polarization with decrease of the proinflammatory cytokines and downregulation of NF-?B and STAT1 signals. Then, in vivo experiments demonstrated S5 improved cecal ligation and puncture (CLP)-induced sepsis specially based on the regulation of M1-like macrophages. Mechanistic studies indicated that S5 treatment dramatically upregulated cellular autophagy in M1-like macrophage. Furthermore, by multiple methods, S5 was revealed to directly bind with ubiquitin-specific proteases 14 (USP14) at Ser404, Phe405, and Cys414 by hydrogen bond to inhibit its deubiquitinating activity, and block USP14-TRAF6 (TNF receptor associated factor 6) interaction, subsequently promoting ubiquitination of Beclin1, interrupting Beclin1-Bcl2 interaction, and accumulating the autophagosome in macrophages, which finally resulted in the blockade of M1-like macrophage polarization. Animal experiments also confirmed the protection of S5 in CLP mice was dependent on activation of macrophage autophagy. What's more, as a novel USP14 inhibitor, S5 exhibited higher efficiency and safety than IU1, the known USP14 inhibitor. Therefore, this study has demonstrated that typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis. Moreover, we provide a new candidate compound, S5, for sensitizing autophagy to interfere with the macrophage inflammation.

SUBMITTER: Xu F

PROVIDER: S-EPMC7441392 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis.

Xu Fang F Ma Yuxiang Y Huang Wei W Gao Jian J Guo Mengmeng M Li Jianxin J Kong Lingdong L Liang Guang G Du Ronghui R Xu Qiang Q Wu Xudong X

Cell death & disease 20200820 8

Macrophages, with diverse functions and variable phenotypes, are considered as an important executor of inflammatory diseases. And it has been proved that autophagy is deeply connected with the development of inflammation, while the exact regulatory mechanism still remains unclear, and the application of autophagy regulators in anti-inflammation needs to be further confirmed. Here, we firstly verified that neochromine S5 (hereinafter referred to as S5) significantly inhibited M1-like macrophage ...[more]

PMID: 32820146

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Project description:Recent studies have shown that autophagy upregulation can attenuate sepsis-induced acute kidney injury (SAKI). The tumor suppressor p53 has emerged as an autophagy regulator in various forms of acute kidney injury (AKI). Our previous studies showed that p53 acetylation exacerbated hemorrhagic shock-induced AKI and lipopolysaccharide (LPS)-induced endothelial barrier dysfunction. However, the role of p53-regulated autophagy in SAKI has not been examined and requires clarification. In this study, we observed the dynamic changes of autophagy in renal tubular epithelial cells (RTECs) and verified the protective effects of autophagy activation on SAKI. We also examined the changes in the protein expression, intracellular distribution (nuclear and cytoplasmic), and acetylation/deacetylation levels of p53 during SAKI following cecal ligation and puncture (CLP) or LPS treatment in mice and in a LPS-challenged human RTEC cell line (HK-2 cells). After sepsis stimulation, the autophagy levels of RTECs increased temporarily, followed by a sharp decrease. Autophagy inhibition was accompanied by an increased renal tubular injury score. By contrast, autophagy agonists could reduce renal tubular damage following sepsis. Surprisingly, the expression of p53 protein in both the renal cortex and HK-2 cells did not significantly change following sepsis stimulation. However, the translocation of p53 from the nucleus to the cytoplasm increased, and the acetylation of p53 was enhanced. In the mechanistic study, we found that the induction of p53 deacetylation, due to either the resveratrol/quercetin -induced activation of the deacetylase Sirtuin 1 (Sirt1) or the mutation of the acetylated lysine site in p53, promoted RTEC autophagy and alleviated SAKI. In addition, we found that acetylated p53 was easier to bind with Beclin1 and accelerated its ubiquitination-mediated degradation. Our study underscores the importance of deacetylated p53-mediated RTEC autophagy in future SAKI treatments.

Project description:BackgroundThe infiltration and activation of M1-macrophages can promote renal tubular interstitial damage. The study aimed to investigate the effect of V-set and immunoglobulin domain containing 4 (VSIG4) on M1-macrophages activation and acute kidney injury (AKI) mice.MethodsThe M1-macrophage markers cluster of differentiation 86 (CD86) and inducible nitric oxide synthase (iNOS) were detected via flow cytometry. Cell viability and expression of inflammatory factors were analyzed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), as well as quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) assays. Moreover, HK-2 cells stimulated with lipopolysaccharide (LPS) and RAW264.7 cells overexpressing VSIG4 were co-cultured to analyze the effect of VSIG4 suppressing M1-macrophage activation on HK-2 cells via detecting cell proliferation and apoptosis levels. Furthermore, the pathological changes and iNOS expression of kidney tissue in VSIG4 knockout mice with renal ischemia-reperfusion injury (IRI) were detected by hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining.ResultsOverexpression of VSIG4 partially reversed the phenomenon of M1-macrophageactivation caused by LPS-upregulated CD86 and iNOS expression, reduced cell viability, and induced the expression levels of interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) in RAW264.7. In addition, RAW264.7 cells overexpressing VSIG4 could also alleviate the low proliferation and high apoptotic level of HK-2 cells stimulated with LPS. After VSIG4 knockout, the kidney tissue of AKI mice showed obvious lesions and iNOS expression, indicating that VSIG4 knockout promoted the infiltration of M1-macrophages in the damaged kidney tissue and accelerated kidney tissue lesions.ConclusionsOverexpression of VSIG4 might alleviate the lesions of kidney tissue in AKI mice via inhibition of the secretion of inflammatory factors in M1-macrophages.

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Typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis.

Publications

Typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis.

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