Hepatic Mitochondrial DNA/Toll-Like Receptor 9/MicroRNA-223 Forms a Negative Feedback Loop to Limit Neutrophil Overactivation and Acetaminophen Hepatotoxicity in Mice

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-kappa B) 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-kappa B binding on miR-223 promoter, whereas miR-223 attenuated TLR9/NF-kappa B-mediated inflammation by targeting I kappa B kinase alpha 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.