Interleukin-22 Ameliorates Neutrophil-Driven Nonalcoholic Steatohepatitis Through Multiple Targets - PubMed (original) (raw)
. 2020 Aug;72(2):412-429.
doi: 10.1002/hep.31031. Epub 2020 Mar 16.
Yong He 1, Xiaogang Xiang 1, Wonhyo Seo 1, Seung-Jin Kim 1, Jing Ma 1, Tianyi Ren 1, Seol Hee Park 1, Zhou Zhou 1, Dechun Feng 1, George Kunos 2, Bin Gao 1
Affiliations
- PMID: 31705800
- PMCID: PMC7210045
- DOI: 10.1002/hep.31031
Interleukin-22 Ameliorates Neutrophil-Driven Nonalcoholic Steatohepatitis Through Multiple Targets
Seonghwan Hwang et al. Hepatology. 2020 Aug.
Abstract
Background and aims: Nonalcoholic fatty liver disease encompasses a spectrum of diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. At present, how simple steatosis progresses to NASH remains obscure and effective pharmacological therapies are lacking. Hepatic expression of C-X-C motif chemokine ligand 1 (CXCL1), a key chemokine for neutrophil infiltration (a hallmark of NASH), is highly elevated in NASH patients but not in fatty livers in obese individuals or in high-fat diet (HFD)-fed mice. The aim of this study was to test whether overexpression of CXCL1 itself in the liver can induce NASH in HFD-fed mice and to test the therapeutic potential of IL-22 in this new NASH model.
Approach and results: Overexpression of Cxcl1 in the liver alone promotes steatosis-to-NASH progression in HFD-fed mice by inducing neutrophil infiltration, oxidative stress, and stress kinase (such as apoptosis signal-regulating kinase 1 and p38 mitogen-activated protein kinase) activation. Myeloid cell-specific deletion of the neutrophil cytosolic factor 1 (Ncf1)/p47phox gene, which encodes a component of the NADPH oxidase 2 complex that mediates neutrophil oxidative burst, markedly reduced CXCL1-induced NASH and stress kinase activation in HFD-fed mice. Treatment with interleukin (IL)-22, a cytokine with multiple targets, ameliorated CXCL1/HFD-induced NASH or methionine-choline deficient diet-induced NASH in mice. Mechanistically, IL-22 blocked hepatic oxidative stress and its associated stress kinases via the induction of metallothionein, one of the most potent antioxidant proteins. Moreover, although it does not target immune cells, IL-22 treatment attenuated the inflammatory functions of hepatocyte-derived, mitochondrial DNA-enriched extracellular vesicles, thereby suppressing liver inflammation in NASH.
Conclusions: Hepatic overexpression of CXCL1 is sufficient to drive steatosis-to-NASH progression in HFD-fed mice through neutrophil-derived reactive oxygen species and activation of stress kinases, which can be reversed by IL-22 treatment via the induction of metallothionein.
Published 2019. This article is a U.S. Government work and is in the public domain in the USA.
Figures
Fig. 1.. Hepatic overexpression of CXCL1 promotes steatosis-to-NASH progression in HFD-fed mice.
(A) Immunohistochemical analysis of CXCL1 in the liver of healthy individuals and NASH patients. Representative images from 5 healthy individuals and 5 NASH patients are shown. Information for the characteristics of each patient is included in Supporting Table 1. (B-E) HFD-fed C57BL/6 mice were infected with Ad-Gfp or Ad-Cxcl1 for 2 weeks (in panels B-C, E) or up to 8 weeks (in panel D). (B) Serum ALT levels (n=6/group). (C) Representative images of various stainings in mouse liver sections (left) and the number of MPO-positive cells per field (100×) and the area positive for F4/80 staining and α-SMA staining (100×) in liver sections (right). Red arrows indicate MPO+ cells (in MPO staining image) or hepatic crown-like structures (in F4/80 staining image). (D) Representative Sirius Red staining images in liver sections of HFD-fed mice infected with Ad-Gfp or Ad-Cxcl1 for up to 8 weeks (top) and the quantification of the images (bottom). Scale bars indicate 200 μm. (E) Heat map illustration of the expression profiles of the genes involved in inflammation and fibrosis in human (left, obtained from microarray in Lake et al., Drug Metab Dispos. 2011;39:1954-1960) and 2-week Ad-_Gfp_- or Ad-_Cxcl1_-infected HFD-fed mice (right, obtained from RT-qPCR). Values represent mean ± SEM. Statistical evaluation was performed by Student’s t-test (**p<0.01). N.A., not available.
Fig. 2.. Neutrophil infiltration mediates oxidative stress and liver injury in Ad-_Cxcl1_-infected, HFD-fed mice via an NCF1/p47phox-dependent manner.
(A-C) HFD- or chow-fed C57BL/6 mice were infected with Ad-Gfp or Ad-Cxcl1 for 2 weeks. Quantification of the area positive for MDA staining or 4-HNE staining in liver sections is shown in panel A. The representative images are included in Supporting Fig. S5A. Expression of NADPH oxidase 2 complex in the liver was determined by RT-qPCR (n=6/group) in panel B. Hepatic expression of stress kinases was examined by immunoblot analysis (top) and the blots were quantified by densitometry (bottom) in panel C. (D) A schematic diagram depicting the coculture of neutrophils with AML12 (top) and the expression of stress kinases in AML12 cells (bottom). Neutrophils (1×106 cells) were placed onto the insert (3 μm pore size), and a recombinant mouse CXCL1 protein (100 ng/mL) was added to the bottom chamber (6 hr). DPI (5 μM) was added to the bottom chamber 30 min prior to CXCL1 treatment. AML12 cells were then collected for immunoblot analysis. (E-F) HFD-fed Ncf1 Lyz−/− and WT mice were infected with Ad-Gfp or Ad-Cxcl1 for 2 weeks. Deletion of the Ncf1 gene in bone marrow-derived neutrophils of Ncf1 Lyz−/− mice was confirmed by immunoblot analysis (top, panel E). Serum ALT levels (bottom left, panel E) and quantification of α-SMA+ area (bottom right, panel E) from HFD-fed, Ad-_Cxcl1_-infected WT and Ncf1 Lyz−/− mice. Representative α-SMA staining images are included in Supporting Fig. S5E. Hepatic expression of stress kinases was determined by immunoblot analysis in panel F. Values represent mean ± SEM. Statistical evaluation was performed by Student’s t-test or one-way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05; **p<0.01).
Fig. 3.. IL-22Fc therapy ameliorates CXCL1-induced NASH in HFD-fed mice.
HFD-fed C57BL/6J mice were subjected to a 2-week infection with Ad-Gfp or Ad-Cxcl1 and/or an IL-22Fc treatment. Detailed experimental design is illustrated in Supporting Fig. S7A. (A) Serum ALT levels (n=4-6/group). (B) Quantification of the area positive for MDA and 4-HNE staining. The representative images are included in Supporting Fig. S8A. (C) Hepatic expression of stress kinase, CASP3, and STAT3 was determined by immunoblot analysis (left) and the blots were quantified by densitometry (right). (D) Histological analysis of collagen (Sirius Red) and the expression of F4/80 and α-SMA. Red arrows indicate hepatic crown-like structures. Scale bars indicate 200 μm. (E) Quantification of the histological analysis of F4/80, Sirius Red, and α-SMA included in panel (D). (F) RT-qPCR analysis of hepatic expression of inflammatory (left) and fibrogenic (right) genes (n=4-6/group). Values represent mean ± SEM. Statistical evaluation was performed by one-way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05; **p<0.01). N.S., not significant; C1. CASP3, cleaved form of CASP3.
Fig. 4.. ASK1 inhibition fails to ameliorate CXCL1-induced NASH and IL-22Fc improves NASH in an ASK1-independent manner.
(A-C) HFD-fed C57BL/6J mice (B6J), Ask1 −/− mice and their WT littermates were infected with Ad-Cxcl1 for two weeks. B6J group was also given vehicle or GS-4997 treatment (n=7–8/group). (A) Serum ALT levels. (B) Quantification of the histological analysis of Sirius Red and α-SMA in liver sections. The representative images are included in Supporting Fig. S11. (C) Quantification of the histological analysis of liver fibrosis in Ask1 −/− mice and WT littermates. The representative images are included in Supporting Fig. S12. (D) Ask1 −/− mice and WT littermates were given HFD + Ad-Cxcl1, followed by a treatment with IgG2 or IL-22Fc (n=8/group). Serum ALT levels were measured. Values represent mean ± SEM. Statistical evaluation was performed by Student’s t-test or one-way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05; **p<0.01).
Fig. 5.. MT contributes to the ability of IL-22Fc to attenuate ROS levels, stress kinase activation, and apoptotic signaling in hepatocytes.
(A, B) HFD-fed C57BL/6 mice were given Ad-Gfp, Ad-Cxcl1, or Ad-Cxcl1 + IL-22Fc (n=4-6/group). RT-qPCR (panel A) and immunoblot (panel B) analysis of hepatic mRNA and protein levels of Mt1 and Mt2, respectively. (C) AML12 cells were pretreated with IL-22Fc for 20 hr, exposed to H2O2 for 2 hr, and subjected to flow cytometric analysis of DCF. Results obtained from cells untreated with DCFDA dye represent the unstained condition. MFI represents the mean fluorescence intensity of triplicates. (D-F) Primary WT and Mt1/2 −/− hepatocytes were pretreated with IL-22Fc for 20 hr, followed by a treatment with H2O2 for 30 min (panel D), 10 min (panel E), and 5 hr (panel F). ROS was measured by flow cytometric analysis of DCF (panel D). Immunoblot analyses were performed (panels E, F). Quantification of the CASP3 cleavage normalized to intact CASP3 from three independent experiments are shown in panel F (right). Values represent mean ± SEM. Statistical evaluation was performed by one-way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05; **p<0.01).
Fig. 6.. IL-22Fc ameliorates CXCL1-induced NASH phenotypes in an MT-dependent manner.
HFD-fed WT and Mt1/2 −/− mice were treated with Ad-Cxcl1 and subsequently with IgG2 or IL-22Fc in accordance with the experimental design illustrated in Supporting Fig. S7A (n=5–6/group). (A) Serum ALT levels. (B) Paraffin-embedded liver sections were subjected to MDA and 4-HNE staining and the area positive for MDA or 4-HNE staining was quantified. The representative images of each staining are included in Supporting Fig. S15C. (C) Hepatic expression of ASK1, p38, STAT3, and CASP3 was determined by immunoblot analysis (left) and the blots were quantified by densitometry (right). (D, E) Hepatic expression of inflammatory (panel D) and fibrogenic (panel E) genes was examined by RT-qPCR analysis. Values represent mean ± SEM. Statistical evaluation was performed by one-way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05; **p<0.01).
Fig. 7.. IL-22Fc reduces the inclusion of mtDNA in EVs and inhibits their inflammatory properties in an MT-dependent manner.
(A) A schematic illustration of the treatment of RAW264.7 cells with hepatocyte-derived EVs (left). Mouse primary hepatocytes were treated with IL-22Fc or IgG2 followed by a treatment with H2O2 or PBS. EVs from culture medium were isolated and incubated with RAW264.7 cells (5 μg/mL, 3 hr), and RT-qPCR analysis of inflammatory genes was performed (n=3; right). (B) The amount of EVs isolated from primary hepatocytes was measured by BCA assay that quantifies EV protein (left) and EXOCET kit that measures the number of EVs based on the activity of EV-enriched acetylcholinesterase (right). (C) DNA isolated from hepatocyte-derived EVs was subjected to qPCR using the primers for Atp6, Cox3, and Nd2. (D) RAW264.7 cells were pretreated with control or ODN2088 (2 μM, 1 hr), treated with EVs (5 μg/mL, 3 hr) from hepatocytes exposed to vehicle or H2O2 for 5 hr, and subjected to RT-qPCR analysis of inflammatory genes (n=4). (E, F) DNA extracted from the serum EVs of mice given Ad-Gfp, Ad-Cxcl1, or Ad-Cxcl1 + IL-22Fc was subjected to qPCR to measure the levels of Atp6, Cox3, and Nd2 (n=5–6/group). Values represent mean ± SEM. Statistical evaluation was performed by one-way ANOVA with Tukey’s post hoc test for multiple comparisons (*p<0.05; **p<0.01).
Fig. 8.. A model depicting the therapeutic intervention of neutrophil-driven NASH by IL-22Fc that acts on multiple targets.
Adenoviral overexpression of CXCL1 in hepatocytes stimulates hepatic neutrophil infiltration and ROS production in a p47phox-dependent manner, which activates ROS-sensitive kinases (ASK1, MEKKs, and MLKs) and their downstream stress kinases (p38 and JNK) and subsequently enhances apoptotic signaling involving CASP3 cleavage. ROS-damaged hepatocytes enhance inflammation by releasing mtDNA-including EVs that activate macrophages. IL-22Fc alleviates CXCL1-induced NASH by attenuating hepatocyte death, inflammation, and fibrosis through MT induction in addition to its MT-independent effects (e.g., liver regeneration and inhibition of lipogenesis and cell death).
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