*Purpose: Hepatic ischemia-reperfusion injury (IRI) poses a significant risk in liver transplantation and hepatic resection and is further exacerbated by hepatic steatosis. Type 1 Natural Killer T-cells (iNKT cells) play a critical role in hepatic IRI. In this study, we evaluate the role of iNKT in hepatic steatosis complicated IRI, an area that is largely understudied.

*Methods: C57BL/6 mice were placed on a normal diet (ND) or high-fat diet (HFD) composed of 20% and 60% fat, respectively. Mice were subjected to sham operations versus partial hepatic ischemia in the left and median lobes for 45 minutes, which simulated the average warm ischemia time during transplantation. Reperfusion occurred for 24 hours post-procedure. A subset of mice from both groups was allocated to receive oral N-Acetylcysteine (po-NAC) supplementation. NAC was administered as a 1% solution in drinking water ad libitum.

*Results: IRI in HFD mice was exacerbated when compared to ND mice. This was evident in the evaluation of serum alanine aminotransferase (ALT) levels, Suzuki scoring of H&E stained sections, and influx of neutrophils (Gr-1) and macrophages (CD-68) on stained sections. Polychromatic flow cytometry demonstrated a pronounced increase in CD45+CD3+NK1.1+ iNKT cells when compared to ND IRI and HFD sham mice. This was verified by measuring ex vivo cytokine expression in the HFD and ND mice following IRI, proving higher INF-γ expression in the HFD mice. We hypothesized this was secondary to increased activation of iNKT by phospholipid ligands. To test this, we performed a lipidomic analysis of hepatic tissue, which revealed a higher composition of triacylglycerols (TAG) and phospholipids (PL) in the HFD mice. Since NAC can reduce hepatic steatosis, we then examined the reaction to IRI in HFD+po-NAC mice. Interestingly, not only did HFD+po-NAC mice have less hepatic steatosis as evident on H&E sections, but also had a vast reduction in IRI. Type 1 NKT cells were largely reduced in these mice and consistent with ND Sham mice. This was consistent with lipidomic evaluation showing a decrease in TAG and PL, which we postulate resulted in fewer iNKT cell-activating ligands.

*Conclusions: Hepatic Steatosis results in profound IRI via iNKT cell-dependent mechanism secondary to PL activation and is ameliorated by po-NAC. This provides the foundation for further investigation to prevent IRI in liver transplantation.

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