Influence of C57BL/6 Sub‐Strain on the Innate Immune Response and Liver Recovery Following Acetaminophen‐Induced Liver Injury

Acetaminophen (APAP) overdose is a leading cause of acute liver failure, and the inadequacy of the current antidote N-acetylcysteine motivates the search for newer therapeutic interventions. This is facilitated by the replication of human pathophysiology by the mouse model, with the C57BL/6 strain being most used. While variations in susceptibility to liver injury between the C57BL/6(J) and (N) substrains are well recognized, substrain influence on the innate immune response and regenerative outcomes which influence development of acute liver failure are unknown. We compared these aspects temporally between 6J and 6N mice following a 300 mg/kg APAP overdose. Consistent with prior findings, 6N mice exhibited higher liver injury compared to 6J mice. Neutrophil and macrophage infiltration followed similar patterns, but with different temporal dynamics, with 6J mice showing a rapid but transient response, while 6N mice exhibited delayed and prolonged immune activity. Importantly, these immune dynamics were accompanied by delayed regeneration and sustained p21 expression, indicating impaired regeneration in 6N mice. Taken together, both substrains displayed broadly similar immune behavior in terms of cytokine expression and cell recruitment patterns, with the delayed immune resolution and regeneration observed in 6N mice being reflective of the extent of injury rather than intrinsic differences in immune function. Thus, our study emphasizes the need to evaluate multiple time points to fully capture the evolving nature of liver injury and recovery during APAP hepatotoxicity. It also underscores the importance of clearly considering and reporting mouse substrain and vendor, as these factors can significantly shape experimental outcomes and may explain discrepancies between studies using this clinically relevant model.