Introduction: Sepsis results in the exuberant production of nitric oxide (NO) secondary to inducible NO synthase (iNOS). This overproduction of NO is thought to contribute to mitochondrial dysfunction, bioenergetic failure and organ dysfunction. Adaptive cell signaling responses, such as autophagy, prevent the accumulation of dysfunctional mitochondria and other organelles, preventing cell death and organ dysfunction. The purpose of these experiments was to test the hypothesis that sepsis-induced autophagy is dependent on iNOS and subsequent reactive oxygen species (ROS) cell signaling.

Methods: Hepatocytes were harvested from C57BL/6 mice and iNOS knockout mice and treated with 100ng of LPS for 12 hours. Cells were then either fixed for immunohistochemistry or cell lysates prepared for Western blotting. LC3 protein levels were measured on Western blot analysis and punctate LC3 was used as a marker of autophagy on immunohistochemistry, both well known methods of measuring autophagy. Hepatocytes were also treated with or without EUK-134, a superoxide dismutase/catalase mimetic that acts as a ROS scavenger, and LC3 protein levels were then measured from whole cell lysates. ROS production was determined by DCF fluorescence in primary hepatocytes as well as the HepG2 cell line or mitochondrial deficient (rho zero) HepG2 cells. For experimental sepsis, C57BL/6 and iNOS knockout mice underwent cecal ligation and perforation (CLP). Sham animals underwent laparotomy and bowel manipulation without CLP. Liver tissue samples were then examined by immunohistochemistry or protein extraction done for western blot analysis. Immunohistochemistry and Western blot analysis were done for LC3.

Results: In vivo, CLP results in increased hepatic autophagic signaling as determined by electron microscopy and immunofluorescence. iNOS knockout mice had a decreased autophagic signaling compared to wild type animals in this model of sepsis. As expected, LPS significantly induced iNOS protein expression in C57BL/6 hepatocytes as determined by Western blot analysis and immunohistochemistry. LPS induces autophagic signaling in hepatocytes as determined by Western blotting and immunohistochemical evaluation for autophagic proteins, including LC3; however this induction was significantly attenuated in iNOS knockout hepatocytes. LPS or NO donor, PAPA-NONOate (10 uM) results in increased hepatocyte ROS production as determined by DCF fluorescence. This ROS production was absent in rho zero cells, indicating that ROS are produced by the mitochondria. Moreover, hepatocytes treated with EUK-134 had no evidence of autophagic induction after LPS stimulation, suggesting that induction of this pathway is dependent on ROS signaling.

Conclusion: These data suggest that sepsis-induced iNOS/NO leads altered mitochondrial function and ROS production. These reactive molecules may induce protective adaptive signaling such as autophagy. The contribution of NO to adaptive signaling and autophagic induction, as well as the elucidation of the role of autophagy in organ dysfunction requires further investigation.