Hepatic Mitochondrial DNA-TLR9-microRNA223 Forms A Negative Feedback Loop To Control Acetaminophen-induced Acute Liver Injury And Inflammation

Acetaminophen (APAP) overdose is one of leading causes of acute liver failure worldwide, which is accompanied with significant neutrophil infiltration in the liver; however, the mechanisms underlying hepatic neutrophil infiltration and neutrophilic inflammation remain obscure. Recently, microRNAs (miRNAs) have been reported to regulate cell proliferation, differentiation, and apoptosis and so on. Myeloid-specific miR-223 was previously reported to act as a fine-tuner of the generation and function of neutrophils, and the role of miR-223 in APAP-induced liver injury and neutrophils infiltration remains largely unknown. In this study, we generated miR-223 knockout (miR-223KO) mice, and explore the molecular mechanisms of miR-223 in APAP-induced liver injury and the function of neutrophils.In this study, APAP-induced acute liver injury was generated by injection of wild-type (WT) and miR-223KO mice with APAP (350mg/kg, intraperitoneal injection) after overnight-fasting or without overnight fasting. Serum alanine transaminase (ALT) and aspartate transaminase (AST) levels and miR-223 level were detected 6h or 24h after APAP injection. Liver tissues were performed by histological and immunohistochemical staining. Neutrophils from liver, bone marrow and peripheral blood were isolated by MACS, and real-time qPCR and western blot were performed to detect mRNA and protein expression.In this study, we found that miR-223 levels in liver and serum were significantly elevated after APAP injection. Furthermore, compared with WT mice, miR-223KO mice were more susceptible to APAP-induced liver injury, as indicated by higher levels of serum ALT, AST, and liver necrosis after overnight-fasting or without fasting. As expected, miR-223KO mice showed greater degree of neutrophils and macrophages cells infiltration in the liver after APAP injection than WT mice. Accordingly, inflammatory response and oxidative damage manifested by 4-Hydroxynonenal (4-HNE) and N-nitrotyrosine expression were more pronounced in the APAP-treated miR-223KO mice compared with that of WT mice. Intriguingly, necrotic cells induced a strong influx of neutrophils into the peritoneal cavity, and this response was significantly increased in miR-223KO mice. Furthermore, toll-like receptor 9 (TLR9) ligand or free DNA released from necrotic hepatocytes increased miR-223 expression in neutrophils in vitro and in vivo through a TLR9/NF-κB dependent mechanism, TLR9 antagonist suppressed miR-223 upregulation in neutrophils after APAP injection. In addition, neutrophils lacking of miR-223 showed more production of pro inflammatory cytokines and chemokines after TLR9 ligand stimulation in vitro. Mechanistically, miR-223 was found to form a negative feedback loop to control TLR9/NF-κB-mediated inflammatory response partly by regulating IKKa expression. Taken together, deletion of miR-223 increases neutrophilic inflammatory response and oxidative stress in the liver, and subsequently exacerbates APAP-induced hepatotoxicity. These findings suggest that miR-223 is a key mediator controlling the acute neutrophilic response and function, and may be used a therapeutic target for the treatment of APAP-induced liver failure.