Hepatocyte-derived extracellular vesicles carrying damaged mitochondria drive neutrophil extracellular traps formation and exacerbate acetaminophen-induced liver injury.

OBJECTIVE: Acetaminophen (APAP) overdose causes severe hepatotoxicity, yet how mitochondrial injury in hepatocytes amplifies innate immune activation remains unclear. This study investigated whether extracellular vesicles released from APAP-injured hepatocytes (APAP-EVs) deliver damaged mitochondrial components to neutrophils, promoting NETs formation and worsening liver injury.

METHODS: APAP-induced liver injury was established in C57BL/6 mice. Hepatocyte-derived EVs were isolated and intravenously administered. Liver injury was assessed by ALT, AST, necrosis, apoptosis, and NETs markers. GW4869, GSK484, RU.521, H-151 and ODN 2088 were used to block EVs release, NETs formation, cGAS-STING or TLR9 signaling.

RESULTS: APAP-EVs treatment markedly exacerbated hepatotoxicity, increasing serum ALT (8775 ± 563.7 U/L vs. 6037 ± 436.5 U/L), AST (8952 ± 670.4 U/L vs. 5539 ± 525.8 U/L), and hepatic necrosis (56.10 ± 1.60 % U/L vs. 30.10 ± 1.52 % U/L) compared with APAP group. Blocking EVs release with GW4869 or inhibiting NETs formation with GSK484 significantly attenuated liver injury. Mechanistically, APAP-EVs activated cGAS-STING signaling in neutrophils to induce NETs formation, an effect abolished by the cGAS inhibitor RU.521 and the STING inhibitor H-151, whereas TLR9 inhibition (ODN 2088) had no effect.

CONCLUSION: APAP-injured hepatocytes release EVs enriched with damaged mitochondrial cargo that activate cGAS-STING-dependent NETs formation, thereby amplifying liver injury. Targeting EVs biogenesis or NETs formation may provide effective therapeutic strategies for APAP-induced hepatotoxicity.