Objectives: The pathophysiology of sepsis is complex, and unfortunately poorly understood. Sepsis induced apoptosis is known to contribute to end organ injury and ultimately death. Recent evidence suggests that autophagy is also activated in sepsis and is believed to primarily play a protective role in the progression of the disease. In addition, apoptosis and autophagy share a number of common signaling pathways and mediators, and therefore may be linked to one another. However, how the balance between these two cellular processes affects sepsis induced outcomes has not been described. In this study, we show that the transcription factor interferon regulatory factor-1 (IRF-1) is pivotal in regulating the balance between apoptosis and autophagy in a model of endotoxemia.

Methods: Male IRF1-KO and wildtype C57BL/6 mice were injected with a lethal dose of intraperitoneal LPS (35mg/kg). Control mice received injections of sterilized PBS. 96h survival rates were assessed. Splenic,lung and liver tissues were analyzed by western blot, flow cytometry, immunofluorescent staining, TUNEL staining and transmission electron microscopy. In vitro, murine RAW 264.7 cells were transfected with pEGFP-LC3 and analyzed by immunofluorescent microscopy, western-blot and RT-PCR.

Results: In vivo, IRF-1 knockout mice were significantly protected from endotoxin induced mortality(p<0.01). This protection was associated with less end organ apoptosis as seen with fewer TUNEL postivie cells and decreased cleaved Caspase-3 activation in splenic, lung and liver tissues in IRF-1 knockout mice compared to their wildtype countertypes. Interestingly, IRF-1 knockout mice exhibited evidence of increased autophagic flux with an increase of the active form of LC3, LC3-II, in the same tissues. In vitro, LPS stimulation increase autophagy in both harvested splenocytes and peritoneal macrophages in IRF-1 knockout compared to wildtype cells as measured by LC3 activation and autophagic vesicle formation. Conversely, in response to LPS stimulation, IRF-1 knockout macrophages experienced decreased apoptosis as seen by lower caspase-3 cleavage levels and chromatin condensation. To determine the signaling pathway for LPS induced IRF-1 activation leading to autophagy, we examined the role of the Janus associated kinase (JAK) pathway. IRF-1 activation was induced in a JAK-dependent manner as RAW264.7 stimulated with LPS in the presence of a JAK inhibitor demonstrated decreased induction of IRF-1. JAK inhibition during LPS stimulation in RAW264.7 cells also recapitulated the effects seen in IRF-1 knockout cells with diminished apoptosis and increased autophagy. Furthermore, the mechanism of IRF-1 regulation of autophagy may involve mammalian target of rapamycin (mTOR), a negative regulator of autophagy since JAK inhibition significantly decreased mTOR activation.

Conclusion: Our study defines a novel role for the transcription factor IRF-1 in regulating the balance between apoptosis and autophagy in the setting of endotoxemia. IRF-1 appears to shift this balance in the direction of apoptosis, while IRF-1 knockdown and/or inhibition results in the opposite effect (i.e. promoting autophagy while inhibiting apoptosis). Understanding the regulation of IRF-1 activity may lead to potential targets to improve sepsis related outcomes.