25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

Background: Interferons and viral infections stimulate the production of 25-hydroxycholesterol. Results: 25-Hydroxycholesterol significantly alters cholesterol ester and sphingolipid levels and activates the integrated stress response. Conclusion: 25-Hydroxycholesterol activates the GCN2/eIF2/ATF4 integrated stress response likely by causing cysteine depletion and/or by generating oxidative stress. Significance: Altering important membrane lipids and activating the integrated stress response may contribute to the antiviral activity of 25-hydroxycholesterol.25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.