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Roquin-1 Regulates Macrophage Immune Response and Participates in Hepatic Ischemia-Reperfusion Injury.

ABSTRACT: With the development of liver surgery, ischemia-reperfusion (IR) injury has received increasing attention. Roquin-1 has been shown to play an important role in innate immune and immune balance. We demonstrate that Roquin-1 expression increased at 1 h after IR and then decreased in C57B/L mice. The immunofluorescence double-label showed that Roquin-1 was mainly expressed in macrophages (mø). Furthermore, we used clodronate liposomes to remove mø, and injected the bone marrow-derived mø (BMDM) through the tail vein in 1 h before IR. We found that liver IR injury was aggravated by Roquin-1 interference. The results of PCR and ELISA suggested that after interference with Roquin-1, mø increased toward M1 and decreased toward M2. Then, interference with Roquin-1 promoted the polarization of mø to M1 and inhibited the polarization of M2. By Western blot technology and AMPK? and mTOR inhibitors, we found that Roquin-1 promotes the phosphorylation of mTOR and STAT3 by inhibiting the phosphorylation of AMPK?. We used AICAR to activate AMPK? in mø and found that the level of ubiquitination of AMPK? was decreased after activation of AMPK?. Furthermore, by bioinformatics methods, we identified potential ubiquitination sites on AMPK?. By the point mutation experiments in vitro, we confirmed that the ubiquitination of these sites is regulated by Roquin-1. Meanwhile, Roquin-1 interference inhibited the activation and function of AMPK?. This topic describes the protection of liver IR injury by Roquin-1 and discusses its main mechanism for regulating AMPK? activity through ubiquitination and affecting the polarization of mø.

SUBMITTER: Zheng L

PROVIDER: S-EPMC7026771 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Roquin-1 Regulates Macrophage Immune Response and Participates in Hepatic Ischemia-Reperfusion Injury.

Zheng Lei L Ling Wei W Zhu Deming D Li Zhi Z Kong Lianbao L

Journal of immunology (Baltimore, Md. : 1950) 20200129 5

With the development of liver surgery, ischemia-reperfusion (IR) injury has received increasing attention. Roquin-1 has been shown to play an important role in innate immune and immune balance. We demonstrate that Roquin-1 expression increased at 1 h after IR and then decreased in C57B/L mice. The immunofluorescence double-label showed that Roquin-1 was mainly expressed in macrophages (mø). Furthermore, we used clodronate liposomes to remove mø, and injected the bone marrow-derived mø (BMDM) thr ...[more]

PMID: 31996460

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Project description:BackgroundHepatic ischemia-reperfusion injury (IRI) is considered as an effecting factor for hepatocellular carcinoma (HCC) recurrence. Th17/Treg cells are a pair of essential components in adaptive immune response in liver IRI, and forkhead box O1 (FOXO1) has the properties of maintaining the function and phenotype of immune cells. Herein, we illuminated the correlation and function between Th17/Treg cell balance and FOXO1 in IRI-induced HCC recurrence.MethodsRNA sequencing was performed on naive CD4+ T cells from normal and IRI model mice to identify relevant transcription factors. Western blotting, qRT-PCR, immunohistochemical staining, and flow cytometry were performed in IRI models to indicate the effect of FOXO1 on the polarization of Th17/Treg cells. Then, transwell assay of HCC cell migration and invasion, clone formation, wound healing assay, and Th17 cells adoptively transfer was utilized to assess the function of Th17 cells in IRI-induced HCC recurrence in vitro and in vivo.ResultsOwning to the application of RNA sequencing, FOXO1 was screened and assumed to perform a significant function in hepatic IRI. The IRI model demonstrated that up-regulation of FOXO1 alleviated IR stress by attenuating inflammatory stress, maintaining microenvironment homeostasis, and reducing the polarization of Th17 cells. Mechanistically, Th17 cells accelerated IRI-induced HCC recurrence by shaping the hepatic pre-metastasis microenvironment, activating the EMT program, promoting cancer stemness and angiogenesis, while the upregulation of FOXO1 can stabilize the liver microenvironment homeostasis and alleviate the negative effects of Th17 cells. Moreover, the adoptive transfer of Th17 cells in vivo revealed its inducing function in IRI-induced HCC recurrence.ConclusionsThese results indicated that FOXO1-Th17/Treg axis exerts a crucial role in IRI-mediated immunologic derangement and HCC recurrence, which could be a promising target for reducing the HCC recurrence after hepatectomy. Liver IRI affects the balance of Th17/Treg cells by inhibiting the expression of FOXO1, and the increase of Th17 cells has the ability to induce HCC recurrence through EMT program, cancer stemness pathway, the formation of premetastatic microenvironment and angiogenesis.

Project description:Oxidative stress can induce inflammation, promoting macrophage polarization and liver fibrosis following hepatic ischemia-reperfusion (I/R). Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has anti-oxidant and anti-inflammatory activity. However, how PGC-1α regulates macrophage polarization following hepatic I/R remains largely unknown. Male C57BL/6 wild-type mice were pre-treated with vehicle or trichostatin A (TSA) for 2 days and subjected to surgical induction of I/R. Liver injury and fibrosis in individual mice were examined longitudinally and the expression levels of IL-6, STAT3, M2-type macrophage markers, Collagen I and α-SMA in the liver of mice were analyzed by immunohistochemistry, RT-qPCR and Western blot. The potential interaction of PGC-1α with phosphorylated NF-kBp65 was determined by immunoprecipitation. The impacts of PGC-1α deficiency in hepatocytes on their IL-6 production and macrophage polarization were tested in a Transwell co-culture system. Moreover, the M2-type macrophage polarization and liver fibrosis were examined in hepatocyte-specific PGC-1α knockout mice and AAV8-mediated PGC-1α over-expressing mice following liver I/R. The down-regulated PGC-1α expression by I/R was negatively correlated with IL-6 levels in the liver of I/R mice and PGC-1α deficiency enhanced IL-6 expression, STAT3 activation and M2-type macrophage polarization in the I/R mice, which were abrogated by TSA treatment. In addition, PGC-1α directly interacted with phosphorylated NF-kBp65 in I/R livers. Hepatocyte-specific PGC-1α deficiency increased IL-6 production and promoted macrophage polarization toward M2 type when co-culture. More importantly, administration with AAV8-PGC-1α rescued the I/R-induced liver fibrosis by inhibiting the IL-6/JAK2/STAT3 signaling and M2-type macrophage polarization in the liver. These results suggest that PGC-1α may alleviate the I/R-induced liver fibrosis by attenuating the IL-6/JAK2/STAT3 signaling to limit M2-type macrophage polarization. PGC-1α may be a therapeutic target for the treatment of liver fibrosis.

Dataset Information

Roquin-1 Regulates Macrophage Immune Response and Participates in Hepatic Ischemia-Reperfusion Injury.

Publications

Roquin-1 Regulates Macrophage Immune Response and Participates in Hepatic Ischemia-Reperfusion Injury.

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