Hepatic mitochondrial DNA/Toll-like receptor 9/MicroRNA-223 forms a negative feedback loop to limit neutrophil overactivation and acetaminophen hepatotoxicity in mice

Abstract

Acetaminophen (APAP) overdose is a leading cause of acute liver failure worldwide, in which mitochondrial DNA (mtDNA) released by damaged hepatocytes activates neutrophils through binding of Toll-like receptor 9 (TLR9), further aggravating liver injury. Here, we demonstrated that mtDNA/TLR9 also activates a negative feedback pathway through induction of microRNA-223 (miR-223) to limit neutrophil overactivation and liver injury. After injection of APAP in mice, levels of miR-223, the most abundant miRNAs in neutrophils, were highly elevated in neutrophils. Disruption of the miR-223 gene exacerbated APAP-induced hepatic neutrophil infiltration, oxidative stress, and injury and enhanced TLR9 ligand-mediated activation of proinflammatory mediators in neutrophils. An additional deletion of the intercellular adhesion molecule 1 (ICAM-1) gene ameliorated APAP-induced neutrophil infiltration and liver injury in miR-223 knockout mice. In vitro experiments revealed that miR-223-deficient neutrophils were more susceptible to TLR9 agonist-mediated induction of proinflammatory mediators and nuclear factor kappa B (NF-κB) signaling, whereas overexpression of miR-223 attenuated these effects in neutrophils. Moreover, inhibition of TLR9 signaling by either treatment with a TLR9 inhibitor or by disruption of TLR9 gene partially, but significantly, suppressed miR-223 expression in neutrophils post-APAP injection. In contrast, activation of TLR9 up-regulated miR-223 expression in neutrophils in vivo and in vitro. Mechanistically, activation of TLR9 up-regulated miR-223 by enhancing NF-κB binding on miR-223 promoter, whereas miR-223 attenuated TLR9/NF-κB-mediated inflammation by targeting IκB kinase α expression. Collectively, up-regulation of miR-223 plays a key role in terminating the acute neutrophilic response and is a therapeutic target for treatment of APAP-induced liver failure. (Hepatology 2017;66:220-234).

Fig. 1. Upregulation of miR-223 and its role in APAP-induced liver injury

Overnight-fasted WT mice and/or miR-223 KO mice were intraperitoneally injected with 350 mg/kg APAP dissolved in warm PBS. (A) Liver tissues, serum samples, and liver and peripheral neutrophils from WT mice were collected 6h or 24 h after APAP treatment, and subjected to the measurement of miR-223. (B–D) Liver tissues and serum samples were collected 6h or 24h after APAP treatment. Serum ALT and AST levels were measured (panel B). Representative images of liver sections for CYP2E1 protein staining (original magnification X40) are shown in panel C. Hepatic CYP2E1 activity was measured (panel D). Values represent means ± SEM (n=6 in panel A and n=9 in panels B–D). ^*P< 0.05, ^**P< 0.01, ^***P< 0.001.

Fig. 2. MiR-223KO mice are more susceptible to APAP-induced liver injury and inflammation

Non-fasted WT and miR-223KO mice were injected with 350 mg/kg APAP dissolved in warm PBS. Liver tissues and serum samples were collected 0, 6 or 24h after APAP treatment. (A) Serum ALT and AST levels were measured. (B) Necrotic area, MPO⁺ or F4/80⁺ cells per field were quantified. (C) RT-qPCR analyses of Ly6G and various inflammatory genes. (D) Non-fasted miR-223KO and miR-223/ICAM-1dKO mice were injected with 350 mg/kg APAP dissolved in warm PBS. Liver tissues and serum samples were collected 6h and 24h after APAP treatment. MPO⁺ neutrophils in the liver were determined, and serum ALT levels were measured. Values represent means ± SEM (n=6 in panels A–C; n=7 in panel D). ^*P< 0.05, ^**P< 0.01, ^***P< 0.001.

Fig. 3. MiR-223KO mice are more susceptible to APAP-induced ROS in the liver

Non-fasted WT and miR-223KO mice were injected with 350 mg/kg APAP. (A) Liver GSH levels were measured. (B) 4-HNE adducts, N-nitrotyrisine adducts, total JNK (T-JNK) and p-JNK expression were detected by western blot. (C) The mRNA expressions of iNOS, SOD-1. SOD-2, and catalase were measured by RT-qPCR. Values represent means ± SEM (n=6). ^*P< 0.05, ^**P< 0.01.

Fig. 4. APAP-induced liver injury and inflammatory responses in WT or miR-223KO mice are dependent on TLR9

TLR9 antagonist (ODN2088) or PBS were injected into WT or miR-223KO mice immediately after and at 6h after APAP injection, serum and liver samples were collected at 24h after APAP treatment. Serum ALT was measured (panel A). MPO⁺ cells per field in the liver were determined (panel B). RT-qPCR analysis of Ly6G and various inflammatory genes in the liver (panel C and D). Values represent means ± SEM (n=8). ^*P< 0.05.

Fig. 5. MiR-223 negatively regulates TLR9-mediated NF-κB signaling by targeting IKKα in neutrophils

(A, B) Bone marrow neutrophils from WT and miR-223KO mice were treated with TLR9 ligand (ODN1585) for 0, 3, and 6h in vitro. RT-qPCR analysis of various inflammatory genes (panel A) and TLR9 and its downstream genes (panel B). (C) The bioinformatics approach analysis of the target prediction of miR-223. (D) Dual luciferase reporter assay was performed to verify binding between miR-223 and IKKα mRNA. The 293T cells were transfected with luciferase vector containing either WT or mutated 3′UTR of IKKα, and also transfected with miR-223 mimics or NS-miRNA. Results of relative luciferase activity are shown. (E) IKKα and NF-κB p65 protein expressions in the liver and bone marrow neutrophils from WT and miR-223KO were detected by western blot. Values represent means ± SEM (n=4 in panels A–B and n=7 in panel D). ^*P< 0.05, ^**P< 0.01.

Fig. 6. MiR-223 inhibitsTLR9-mediated NF-κB signaling in human neutrophil-like cells (dHL-60 cells)

dHL-60 cells were transfected with nonspecific miRNA mimics (NS-miR) or miR-223 mimics (panels A, B), nonspecific miR-inhibitor or miR-223 inhibitor (panel C) by Lipofectamine RNAiMAX Reagent according to the manufacturer’s instructions at a final concentration of 20 nM for 48 h. After 48 h, the cells were stimulated with human TLR9 agonist (ODN2216) for the indicated time. (A) RT-qPCR analysis of inflammatory genes. (B, C) Western blot analyses. (D) Transfected dHL-60 cells were stimulated with ODN2216 for 30 min, and NF-κB p65 activity was measured by using an NF-κB p65 transcription factor assay kit. Values represent means ± SEM (n=4–8). ^*P< 0.05.

Fig. 7. Upregulation of miR-223 expression in neutrophils during APAP-induced liver injury is mediated via a TLR9-dependent pathway

(A) TLR9 antagonist (ODN2088) or PBS were injected into WT mice immediately after and at 6h after APAP injection. Liver neutrophils and peripheral neutrophils were isolated 24h after APAP injection and subjected to miR-223 detection. (B) WT and TLR9KO mice were intraperitoneally injected with 350 mg/kg APAP dissolved in warm PBS. Liver neutrophils and peripheral neutrophils were isolated 6h after APAP injection and subjected to miR-223 detection. (C) WT mice were injected with TLR9 agonist (ODN1585) or TLR9 control (ODN 1585 control) for 6h. MiR-223 expression in liver neutrophils and peripheral neutrophils was measured. Values represent means ± SEM (n=8 for panel A and n=7 for panels B–C). ^*P< 0.05, ^***P< 0.001.

Fig. 8. Upregulation of miR-223 in neutrophils after APAP-induced liver injury depends on mtDNA/TLR9 and NF-κB activation

(A) Microparticles and exosomes were isolated from serum after APAP injection, and then mtDNA levels were measured by RT-qPCR. (B) Primary hepatocytes were treated with or without APAP (5 mM) for 24h, and then microparticles were isolated. These microparticles were used to incubate with bone marrow neutrophils for 6h. MiR-223 expression was measured. (C) Bone marrow neutrophils were treated with TLR9 agonist (ODN1585) or control (ODN1585 control) in vitro. MiR-223 expression was measured. (D) The NF-κB binding site is identified in the miR-223 promoter region. Bone marrow neutrophils were treated with a TLR9 agonist (ODN1585) for 1h. A representative ChIP assay blot is shown. Values represent means ± SEM (n=6 in panel A and n=4 in panels B–C). ^*P< 0.05, ^**P< 0.01. (E) A model depicting the dichotomy role of TLR9 activation in inducing and terminating inflammation and liver injury after APAP injection. During APAP hepatotoxicity, DNA released from damaged hepatocytes activates NF-κB via a TLR9-dependent pathway and subsequently upregulates expression of many inflammatory genes, thereby inducing liver inflammation and injury. Meanwhile, mtDNA/TLR9/NF-κB signaling also elevates miR-223 expression in neutrophils, which acts a negative feedback loop to control APAP-induced acute liver injury and inflammation by downregulating IKKα expression.