The pro-inflammatory effects of miR-155 promote liver fibrosis and alcohol-induced steatohepatitis

Abstract

Background & aims: Alcoholic liver disease (ALD) ranges from fatty liver to inflammation and cirrhosis. miRNA-155 is an important regulator of inflammation. In this study, we describe the in vivo role of miR-155 in ALD.

Methods: Wild-type (WT) (C57/BL6J) or miR-155 knockout (KO) and TLR4 KO mice received Lieber DeCarli diet for 5weeks. Some mice received corn oil or CCl4 for 2 or 9weeks.

Results: We found that miR-155 KO mice are protected from alcohol-induced steatosis and inflammation. The reduction in alcohol-induced fat accumulation in miR-155 KO mice was associated with increased peroxisome proliferator-activated receptor response element (PPRE) and peroxisome proliferator-activated receptors (PPAR)α (miR-155 target) binding and decreased MCP1 production. Treatment with a miR-155 inhibitor increased PPARγ expression in naïve and alcohol treated RAW macrophages. Alcohol increased lipid metabolism gene expression (FABP4, LXRα, ACC1 and LDLR) in WT mice and this was prevented in KO mice. Alcohol diet caused an increase in the number of CD163(+) CD206(+) infiltrating macrophages and neutrophils in WT mice, which was prevented in miR-155 KO mice. Kupffer cells isolated from miR-155 KO mice exhibited predominance of M2 phenotype when exposed to M1 polarized signals and this was due to increased C/EBPβ. Pro-fibrotic genes were attenuated in miR-155 KO mice after alcohol diet or CCl4 treatment. Compared to WT mice, attenuation in CCl4 induced hydroxyproline and α-SMA was observed in KO mice. Finally, we show TLR4 signaling regulates miR-155 as TLR4 KO mice showed no induction of miR-155 after alcohol diet.

Conclusions: Collectively our results demonstrated the role of miR-155 in alcohol-induced steatohepatitis and fibrosis in vivo.

Keywords: Alcohol; Fibrosis; Inflammation; PPARα; PPARγ; microRNA.

Figure 1. miR-155 deficiency attenuates chronic alcohol-induced steatosis and liver injury

C57BL/6 wild type (WT) or miR-155 KO mice (n=8–10) were fed with Lieber DeCarli diet containing either 5% ethanol or isocaloric control diet for 5 weeks. Formalin fixed liver sections were stained with Hematoxylin and eosin (A) Oil-O-red stain (B). Slides were observed under light microscope (200X) and representative slides are shown. Histological scores were recorded by a pathology expert (C). Triglyceride levels were measured from the livers (D). ALT levels were measured from plasma (E). Oxidative stress was measured from whole liver cell lysates by TBARs assay (F). Data is presented as mean ± SEM. * indicates p<0.05 vs. pair fed (PF) mice and # p<0.05 compared to pair fed KO mice.

Figure 2. Alcohol diet results in increased PPRE and PPARα DNA binding in miR-155 KO mice

WT or miR-155 KO mice were fed with Lieber DeCarli diet as described in methods. Total RNA was used to determine PPARα levels by qPCR (A). 5ug of nuclear proteins were used to detect PPRE binding by EMSA and PPARα antibody was used for supershift assay and density (B). Primary hepatocytes were isolated from WT and miR-155 KO mice (chow fed) and treated them with or without MCP1 (100ng/ml) for 36h. 10ug of nuclear proteins was used to detect PPRE binding by EMSA (D). RAW 264.7 macrophages were transfected with either miR-155 inhibitor or control inhibitor using lipofectamine siRNA reagent as described in methods. Cells were treated with or without alcohol (50mM), cells were harvested after 48h of transfection and total RNA was used to quantify PPARγ mRNA levels (E). Expression of lipid metabolism and fatty acid uptake gene expression was evaluated by qPCR. FABP4 (F), LXRα (G), ACC1 (H), and LDLR (I), and HMGCR (J).MCP1 mRNA levels were determined by qPCR (K). 18s was used to normalize Cq values. Whole cell liver lysates (L) and plasma (M) were used to detect MCP1 by ELISA. (D). Data is presented as mean ± SEM. * indicates p<0.05 vs. pair-fed mice, # p<0.05 vs PF mice treated with LPS. $ p<0.05 vs PF KO mice.

Figure 3. Chronic alcohol-induced inflammation and macrophage infiltration are attenuated in miR-155 KO mice

WT or miR-155 KO mice were fed with Lieber DeCarli diet as described in methods. Whole cell liver lysates (A and B, left panel) and plasma (A and B, right panel) were used to detect TNFα (A) and IL-1β (B) by ELISA. RNA was used to determine CD68 (C) and MIP2 (D) mRNA levels by qPCR. MNCs isolated from WT and miR-155 KO mice after alcohol feeding and were stained with combination of antibodies for monocytes, macrophages and neutrophils as described in methods and processed for flow cytometery. % of inflammatory monocytes (E), and M2 macrophages (F) is shown. Kupffer cells were isolated from WT and KO mice and treated with or without LPS (100ng/ml), IL-4 (20ng/ml) and IFNγ (20ng/ml) for 18h. Total RNA was evaluated for M1 and M2 markers and their regulators. Arginase1 (G), IL-10 (H), MRC1 (I), IL-1β (J), iNOS (K), C/EBPβ (L), STAT3 (M), C/EBPα (N), HiF1α (O), and miR-155 levels (P). 18s was used to normalize Cq values. Data is presented as mean ± SEM. * indicates p<0.05 vs. pair fed mice, # p<0.05 vs PF mice treated with LPS. $ p<0.05 vs PF mice KO mice. ns: non significant.

Figure 4. Reduction in CCl₄-induced liver fibrosis in miR-155 KO mice

WT or miR-155 KO mice were fed with Lieber DeCarli diet. MNCs were stained for neutrophils (A). Liver RNA was used to determine TGFβ (B) procollagen 1α (C) and vimentin (D) mRNA levels by qPCR. 18s was used to normalize Cq values. Data is presented as mean ± SEM. * indicates p<0.05 vs. pair fed mice, ns: non significant. WT or miR-155 KO mice (n=6) were treated either with corn oil or CCl₄ for 2 weeks as described in methods. Total RNA was used to determine miR-155 levels by qPCR (E). Formalin fixed liver sections were stained with Hematoxylin and eosin (F, left panel) and Sirius red stain (F, right panel). Slides were observed under light microscope (100X) and representative slides are shown. RNA was used to determine TNFα (G), MCP1 (H), α smooth muscle actin (I), TGF β (J), collagen 1α (K), MMP9 (L), MMP12 (M), Timp1 (N), Snail1 (P) mRNA levels by qPCR. Timp1 levels were measured from plasma by ELISA (O). 18s or SnoRNA202 was used to normalize Cq values for mRNA, and mouse miR-155 respectively. Data is presented as mean ± SEM. * indicates p<0.05 vs. corn oil treated mice.

Figure 5. miR-155 promotes liver fibrosis

WT or miR-155 KO mice (n=9) were treated either with corn oil or CCl₄ for 9 weeks. Formalin fixed liver sections were stained with Sirius red stain (A). Slides were observed under light microscope (100X) and representative slides are shown. The amount of hydroxyproline was measured from the liver (B). Western blot analysis for α SMA and density units (C), miR-155 levels were determined from alcoholic cirrhotic livers by qPCR (D). RNU48 was used to normalize Cq values. Data is presented as mean ± SEM. * indicates p<0.05 vs. corn oil treated mice.

Figure 6. Alcohol induces miR-155 in the liver at the transcriptional level via the TLR4 pathway

WT or miR-155 KO mice were fed with Lieber DeCarli diet. Endotoxin levels were measured from plasma (A). WT mice were either administered with saline or LPS (2.5mg/kg, ip) for indicated times and miR-155 levels were measured from livers (B). MNCs or hepatocytes were isolated from perfused WT livers as described in methods. Cells were treated with LPS (100ng/ml) for 6h and miR-155 levels were detected (C). WT or miR-155 KO pair- or alcohol- fed mice were injected with LPS (0.5mg/kg, ip) for 3h and livers were isolated and miR-155 levels were detected (D). C57BL/6 WT or TLR4 KO mice (n=6–8) were fed with Lieber DeCarli diet. (E). Primary miR-155 levels were detected using TaqMan gene expression assay. GAPDH was used to normalize Cq values (F). Data is presented as mean ± SEM. * indicates p<0.05 vs. pair fed mice (A, D and F). * indicates p<0.05 vs. saline treated mice (B) or cells in medium only (C).