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.