Interleukin-22 drives a metabolic adaptive reprogramming to maintain mitochondrial fitness and treat liver injury - PubMed (original) (raw)

. 2020 Apr 27;10(13):5879-5894.

doi: 10.7150/thno.43894. eCollection 2020.

Wenjing Zai 1 3, Jiajun Fan 1, Xuyao Zhang 1, Xian Zeng 1, Jingyun Luan 1, Yichen Wang 1, Yilan Shen 4, Ziyu Wang 5, Shixuan Dai 1, Si Fang 1 6, Zhen Zhao 1, Dianwen Ju 1

Affiliations

Interleukin-22 drives a metabolic adaptive reprogramming to maintain mitochondrial fitness and treat liver injury

Wei Chen et al. Theranostics. 2020.

Abstract

Rationale: Interleukin 22 (IL-22) is an epithelial survival cytokine that is at present being explored as therapeutic agents for acute and chronic liver injury. However, its molecular basis of protective activities remains poorly understood. Methods: Here we demonstrate that IL-22 inhibits the deteriorating metabolic states induced by stimuli in hepatocytes. Utilizing cell biological, molecular, and biochemical approaches, we provide evidence that IL-22 promotes oxidative phosphorylation (OXPHOS) and glycolysis and regulates the metabolic reprogramming related transcriptional responses. Results: IL-22 controls metabolic regulators and enzymes activity through the induction of AMP-activated protein kinase (AMPK), AKT and mammalian target of rapamycin (mTOR), thereby ameliorating mitochondrial dysfunction. The upstream effector lncRNA H19 also participates in the controlling of these metabolic processes in hepatocytes. Importantly, amelioration of liver injury by IL-22 through activation of metabolism relevant signaling and regulation of mitochondrial function are further demonstrated in cisplatin-induced liver injury and steatohepatitis. Conclusions: Collectively, our results reveal a novel mechanism underscoring the regulation of metabolic profiles of hepatocytes by IL-22 during liver injury, which might provide useful insights from the bench to the clinic in treating and preventing liver diseases.

Keywords: glycolysis; lncRNA H19; mitochondria; oxidative phosphorylation; reactive oxygen species.

© The author(s).

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1

Figure 1

IL-22 regulates mitochondrial function and glycolysis in hepatocytes. (A) Using Seahorse XF96 Extracellular Flux Analyzer to assess the changes in the oxygen consumption rate and extracellular acid rate of hepatocytes. (B and C) OCR and ECAR in hepatocytes treated with 200 mM ethanol, or 5 μg/mL cisplatin, or 0.25 mM palmitic acid, or 10 mM CCl4 in the absence or presence of IL-22 for 24 h (n = 3). (D and E) Representative curves in the OCR and ECAR of hepatocytes after incubated with oligomycin, glucose, FCCP, rotenone, and 2-DG (n = 3). (F) MRC of hepatocytes evaluated by real time changes in OCR (n = 3). (G) Relative glucose consumption in hepatocytes upon IL-22 treatment (n = 3). (H) Glut1 protein expression upon IL-22 treatment under injury stress. Densitometric values were quantified and normalized to control (PBS) group. (I) Localization and expression of Glut1 (green), nuclear (blue), and plasma membrane (red) in hepatocytes treated as in (B) for 24 h. Scale bars, 20 μm; Student's t test (unpaired); *P < 0.05, **P < 0.01, ***P < 0.001. All data are means ± SD of at least three independent experiments.

Figure 2

Figure 2

IL-22 regulates metabolic reprogramming-related transcriptional responses. (A) Heat map of the remarkably altered genes in hepatocytes with injury factors-challenged groups versus IL-22 plus injury factors-challenged groups. (B) Kyoto Encyclopedia of Genes and Genomes analysis (KEGG) of IL-22 targets in hepatocytes with IL-22-protected and -nonprotected groups. (C and D) KEGG of glycolysis and AMPK signaling pathway in IL-22-protected and -nonprotected hepatocytes. (E) Heat map of top altered genes from hepatocytes with IL-22 treatment. (F) Relative mRNA expression level of HK-2, HIF-1α, c-Myc and mtDNA in the absence or presence of IL-22 treatment (n = 3); Student's t test (unpaired); *P < 0.05, **P < 0.01. All data are means ± SD of at least three independent experiments.

Figure 3

Figure 3

IL-22 prevents generation of dysfunctional mitochondria and mitochondrial ROS via AMPK-associated signal mechanism. Hepatocytes were treated with PBS, 200 mM ethanol, or 5 μg/mL cisplatin, or 0.25 mM palmitic acid, or 10 mM CCl4 in the absence or presence of IL-22 for 24 h (n = 3) (A) Mitochondrial mass was stained with MitoTracker Green and assessed by flow cytometry. Mitochondrial ROS and membrane potential were assessed in hepatocytes stained with MitoSOX (C), MitoTracker Green and MitoTracker Red (B), or MitoTracker Green and MitoSOX (D), respectively. (E) Confocal images indicated mitochondrial ROS production in hepatocytes stained with MitoSOX. (F) Western blot analysis suggested that IL-22 induced AMPK/AKT activation in hepatocytes, which could be inhibited by Dorsomorphin. (G) OCR in hepatocytes was measured in the absence or presence of indicated inhibitors and IL-22 for 24 h. Student's t test (unpaired); *P < 0.05, **P < 0.01, ***P < 0.001. All data are means ± SD of at least three independent experiments.

Figure 4

Figure 4

IL-22 maintains mitochondrial fitness through activation of mTOR signaling. Hepatocytes were stimulated with or without (control) 200 mM ethanol, or 5 μg/mL cisplatin, or 0.25 mM palmitic acid, or 10 mM CCl4 in the absence or presence of IL-22, rapamycin, or LY294002 for indicated times (n = 3). (A and B) Comparison of mTOR signaling activation in hepatocytes or STAT3-WT and STAT3-KD hepatocytes was assessed by western blot analysis. (C) Mitochondrial membrane potentials were assessed in hepatocytes stained with MitoTracker Red and MitoTracker Green. (D) Real time changes in the ECAR of hepatocytes after incubating with glucose, oligomycin, and 2-DG (n = 3) were analyzed. (E) Relative glucose consumption was measured for LY294002 or rapamycin treated hepatocytes at 24 hours. (F) Basal respiration capacity (OXPHOS) of hepatocytes was measured. (G) Comparison of glycolytic enzymes expression in hepatocytes versus LY294002 and rapamycin treated hepatocytes. Student's t test (unpaired); *P < 0.05, **P < 0.01, ***P < 0.001. All data are means ± SD of at least three independent experiments.

Figure 5

Figure 5

LncRNA H19 mediates the links between IL-22/IL-22R1 and the AMPK/AKT/mTOR signaling pathways. (A) Bland-Altman plot illustrating relative gene expression in hepatocytes stimulated with or without (control) ethanol, or cisplatin, or palmitic acid, or CCl4 in the absence or presence of IL-22 for the indicated times. (B) Confocal images suggested lncRNA H19 overexpression in hepatocytes after IL-22 treatment, which required STAT3. Control-siRNA (si-Ctr) treated mice as a vehicle control group (n = 5). (C) Real time PCR analysis suggesting that IL-22 induced lncRNA H19 overexpression in hepatocytes, which could be prevented by STAT3 knockdown (n = 3). (D) Comparison of AMPK/AKT/mTOR signaling activation in hepatocytes versus si-H19 treated hepatocytes (lncRNA H19 knockdown). (E and F) Basal OCR and ECAR in hepatocytes at the absence or presence of IL-22, or si-H19 for 24 h (n = 3). (G) Dysfunctional mitochondria were assessed in hepatocytes stained with MitoTracker Green and MitoTracker Red. (H) Confocal images suggested mitochondrial ROS production in hepatocytes stained with MitoSOX. Student's t test (unpaired); *P < 0.05, **P < 0.01, ***P < 0.001. All data are means ± SD of at least three independent experiments.

Figure 6

Figure 6

IL-22 attenuates hepatic oxidative stress, mitochondrial dysfunction and damage in vivo. (A) Schematic diagram of the animal experimental protocols to assess the effects of IL-22 (0.5 mg/kg and 1.5 mg/kg) in cisplatin (20 mg/kg) induced liver injury. _N_-acetyl-L-cysteine (NAC, 150 mg/kg) as a positive control group and PBS as a vehicle control group (n = 5). Representative HE (B), MitoSOX (E), JC-1 (F), and Ki-67 staining (G) images of the liver sections were presented. (C and D) Serum AST levels, serum ALT levels, liver weights and spleen weights were measured (H) Comparison of AMPK/AKT/mTOR activation in liver extracts from IL-22-treated mice versus control subjects was assessed by western blot analysis. (I) Schematic diagram of the animal experimental protocols to assess the effects of IL-22 (2.5 mg/kg) in high-fat-diet fed mice (n = 8). PBS treated mice as a vehicle control group (n = 5). (J) Representative HE, MitoSOX, and JC-1 staining images of the liver sections were presented. (K) Liver extracts were subjected to western blot analysis with various antibodies as indicated. Western blotting suggested that IL-22 induced AMPK/AKT/mTOR activation in liver extracts from IL-22-treated mice. (L) The mRNA expression levels of the indicated genes in the liver (n = 3). Student's t test (unpaired); *P ≤ 0.05 and **P ≤ 0.01 compared with cisplatin-treated and HFD-fed mice. #P ≤ 0.05 and ##P ≤ 0.01 compared with PBS-treated and chow diet fed mice. All data are means ± SD of at least three independent experiments.

Figure 7

Figure 7

IL-22 attenuates hepatic oxidative stress, mitochondrial dysfunction and damage through the induction of lncRNA H19. (A) Schematic diagram of the animal experimental protocols to assess the effects of IL-22 (2.5 mg/kg) and H19 shRNA (1x1010 virus particles per mouse) in high-fat-diet fed mice (n = 8). PBS and Control shRNA (Ctr shRNA) treated mice as a vehicle control group (n = 5). (B) Representative HE, MitoSOX, and JC-1 staining images of the liver sections were presented. (C) Western blotting suggested that IL-22 induced AMPK/AKT/mTOR activation in liver via the induction of lncRNA H19. (D) Densitometric values were quantified and normalized to control group (n = 3; mean ± SD; *P < 0.05, **P < 0.01). The values of control group were set to 1. (E) The mRNA expression levels of the indicated genes in the liver (n = 3). *P ≤ 0.05 compared with H19 shRNA-treated mice. #P ≤ 0.05 compared with IL-22-treated and high-fat-diet fed mice. All data are means ± SD of at least three independent experiments.

Figure 8

Figure 8

Our works demonstrate a critical role of IL-22 in regulating hepatocellular metabolism to treat liver injury via activating STAT3-lncRNA H19-AMPK-AKT-mTOR axis. These findings describe a novel mechanism underscoring the regulation of metabolic states of hepatocytes by IL-22 during liver injury with potentially broad therapeutic insights.

Comment in

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