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, 66 (4), 705-715

MicroRNA-223 Ameliorates Alcoholic Liver Injury by Inhibiting the IL-6-p47 phox-oxidative Stress Pathway in Neutrophils

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MicroRNA-223 Ameliorates Alcoholic Liver Injury by Inhibiting the IL-6-p47 phox-oxidative Stress Pathway in Neutrophils

Man Li et al. Gut.

Abstract

Objectives: Chronic-plus-binge ethanol feeding activates neutrophils and exacerbates liver injury in mice. This study investigates how recent excessive drinking affects peripheral neutrophils and liver injury in alcoholics, and how miR-223, one of the most abundant microRNAs (miRNAs) in neutrophils, modulates neutrophil function and liver injury in ethanol-fed mice.

Designs: Three hundred alcoholics with (n=140) or without (n=160) recent excessive drinking and 45 healthy controls were enrolled. Mice were fed an ethanol diet for 10 days followed by a single binge of ethanol.

Results: Compared with healthy controls or alcoholics without recent drinking, alcoholics with recent excessive drinking had higher levels of circulating neutrophils, which correlated with serum levels of alanine transaminase (ALT) and aspartate transaminase (AST). miRNA array analysis revealed that alcoholics had elevated serum miR-223 levels compared with healthy controls. In chronic-plus-binge ethanol feeding mouse model, the levels of miR-223 were increased in both serum and neutrophils. Genetic deletion of the miR-223 gene exacerbated ethanol-induced hepatic injury, neutrophil infiltration, reactive oxygen species (ROS) and upregulated hepatic expression of interleukin (IL)-6 and phagocytic oxidase (phox) p47phox. Mechanistic studies revealed that miR-223 directly inhibited IL-6 expression and subsequently inhibited p47phox expression in neutrophils. Deletion of the p47phox gene ameliorated ethanol-induced liver injury and ROS production by neutrophils. Finally, miR-223 expression was downregulated, while IL-6 and p47phox expression were upregulated in peripheral blood neutrophils from alcoholics compared with healthy controls.

Conclusions: miR-223 is an important regulator to block neutrophil infiltration in alcoholic liver disease and could be a novel therapeutic target for the treatment of this malady.

Keywords: CYTOKINES; ETHANOL; FATTY LIVER; INFLAMMATION; LEUKOCYTES.

Conflict of interest statement

Competing interests: None declared.

Figures

Figure 1
Figure 1
The level of peripheral blood neutrophils correlates positively with serum ALT and AST levels in alcoholics with recent excessive drinking. Forty-five healthy controls and 300 alcoholics were enrolled, and alcoholics were stratified into two groups: alcoholics with (n=140) and without (n=160) recent excessive drinking (see table 1). The correlation analyses between the levels of circulating neutrophils and serum ALT and AST levels were performed. Significant correlations (p values) are labelled in the figure.
Figure 2
Figure 2
miR-223 levels in serum and neutrophils from alcoholics and ethanol-fed mice. (A) Blood samples were collected from healthy controls and excessive drinkers. miR-223 levels in serum were measured by real-time quantitative PCR. (B) C57BL/6J mice were pair-fed or fed an ethanol-containing diet for 10 days plus one binge (E10d+1B). Mice were euthanized 9 hours postgavage, and serum was collected for miR-223 measurement. (C) Various types of cells were isolated from naïve C57BL6/J livers and subjected to RT-qPCR for miR-223. (D–F) Mice were subjected to control and ethanol feeding as described in (B). miR-223 and pri-miR-223 levels in mouse peripheral and liver neutrophils were measured. Values represent means±SEM (n=6–14). *p<0.05; **p<0.01; ***p<0.001. HSC, hepatic stellate cells.
Figure 3
Figure 3
Deletion of the miR-223 gene exacerbates chronic-plus-binge ethanol-induced liver injury, elevation of circulating and hepatic neutrophils. Wild-type (WT) mice and miR-223−/− mice were subjected to E10d+1B or pair feeding, and mice were euthanised 9 hours postgavage. (A) Serum ALT, AST and hepatic triglyceride (TG) levels were measured. (B) The percentage and number of neutrophils were determined by a haematology analyser (two upper panels). The percentage of Gr1+CD11b+ circulating neutrophils were analysed by flow cytometer (lower panel). (C) Liver tissues were subjected to immunostaining with an anti-MPO antibody. The number of MPO+ cells was counted. (D) Liver tissues were subjected to real-time PCR analyses of Ly6G (a marker for neutrophils). (E) Liver leucocytes were isolated and subjected to flow cytometric analyses. The percentage of neutrophils (Gr1+CD11b+) cells was analysed. Values represent means±SEM (n=4–10). *p<0.05; **p<0.01; ***p<0.001.
Figure 4
Figure 4
Macrophages and hepatic expression of proinflammatory mediators in ethanol-fed and pair-fed wild-type (WT) and miR-223−/− mice. WT mice and miR-223−/− mice were subjected to E10d+1B or pair feeding, and mice were euthanised 9 hours postgavage. (A and B) Liver tissues were subjected to immunostaining with an anti-F4/80 antibody. Representative photographs are shown in panel A (scale bar: 100 μm). The number of F4/80 was counted in panel B. Hepatic F4/80 mRNA was detected by real-time PCR and is shown in panel B. (C and D) Liver tissues were subjected to real-time PCR analyses. Values represent means±SEM (n=5–10). *p<0.05; **p<0.01; ***p<0.001.
Figure 5
Figure 5
miR-223−/− mice are more susceptible to chronic-plus-binge ethanol-induced oxidative stress in the liver and circulating neutrophils. WT mice and miR-223−/− mice were subjected to E10d+1B or pair feeding, and mice were euthanised 9 hours postgavage. (A and B) Liver tissues were subjected to immunostaining with an anti-malonaldehyde (MDA) or anti-4-hydroxynonenal (HNE) antibody. Representative images are shown in panel A. Relative staining is quantified and shown in panel B (scale bar: 200 μm). (C and D) Hepatic and peripheral neutrophils were isolated and stimulated with or without phorbol 12-myristate 13-acetate (PMA). The reactive oxygen species (ROS) levels were measured by flow cytometric analyses. The ROS levels are calculated and shown. Values represent means±SEM (n=4–8). *p<0.05; **p<0.01; ***p<0.001.
Figure 6
Figure 6
MiR-223 regulates reactive oxygen species (ROS)-associated genes in the liver from ethanol-fed mice. WT mice and miR-223−/− mice were subjected to E10d+1B or pair feeding, and mice were euthanised 9 hours postgavage. Liver tissues were subjected to real-time PCR analyses of ROS-associated genes. Values represent means±SEM (n=4–11). *p<0.05; **p<0.01;***p<0.001.
Figure 7
Figure 7
MiR-223 regulates circulating neutrophils by targeting the IL-6–p47phox axis in ethanol-fed mice and alcoholics with recent drinking. (A) WT mice and miR-223−/− mice were subjected to E10d+1B or pair feeding, and mice were euthanised 9 hours postgavage. Circulating neutrophils were isolated and subjected to real-time PCR analyses. (B) Circulating neutrophils were isolated, and IL-6 (50 ng/mL) was added to the culture medium for 6 hours, and expression of p47phox was determined by real-time PCR analyses. (C) WT and p47phox−/− mice were subjected to E10d+1B feeding, and mice were euthanised 9 hours postgavage. Serum ALT and AST were measured (left panel). Liver neutrophils were isolated and treated with or without phorbol 12-myristate 13-acetate (PMA), and reactive oxygen species (ROS) was measured and calculated (right panel). (D) Circulating neutrophils were isolated from healthy controls and alcoholics with recent drinking, followed by real-time PCR analyses. (E) A summarised figure depicting the miR-223 as a modulator to control hepatic neutrophil infiltration and liver injury after chronic-plus-binge ethanol drinking. Values represent means±SEM (n=4–16). *p<0.05; **p<0.01.

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