Sepsis induced lethality is characterized by amplified host innate immune response. Nrf2, a bZIP transcription factor, regulates a battery of cellular antioxidative genes and maintains cellular redox homeostasis. This study demonstrates that increasing Nrf2 activity by a potent small molecule activator, CDDO-Im (1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole), protects from deregulation of lipopolysaccharide (LPS) induced innate immune response. In response to LPS stimuli, nrf2-deficient (nrf2 -/-) peritoneal neutrophils showed increased NADPH oxidase-dependent ROS generation, proinflammatory cytokines (Tnf-alpha and Il-6) and chemokines (Mip2 and Mcp-1) relative to wild-type (nrf2 +/+) cells. Pretreatment of peritoneal neutrophils with CDDO-Im induced antioxidative genes (Ho-1, Gclc, Gclm, and Nqo1) and attenuated LPS induced ROS generation as well as expression of proinflammatory cytokines exclusively in nrf2 +/+ neutrophils but not in nrf2 -/- cells. In corroboration with in vitro studies, pretreatment with CDDO-Im induced Nrf2-dependent antioxidative genes, attenuated LPS induced proinflammatory cytokine expression, and decreased mortality specifically in the nrf2 +/+ mice. In conclusion, the results suggest that Nrf2 is associated with oxidative regulation of LPS induced innate immune response in neutrophils. Activation of Nrf2-dependent compensatory antioxidative pathways by CDDO-Im protects from LPS induced inflammatory response and mortality.

MitoSOX-based assays are widely used to detect mitochondrial reactive oxygen species (ROS), especially superoxide. To this end, 5 μM MitoSOX is commonly used. In this ROS Protocols article, we described the flow cytometric protocol involving the use of various concentrations of MitoSOX (1, 2.5, 5 μM) for detecting mitochondrial ROS in control and mitochondrial DNA-deficient (MD) melanoma B16-F10 cells. We also compared the MitoSOX-based flow cytometry with lucigenin-derived chemiluminometry for their ability to reliably detect the relative differences in mitochondrial ROS formation in the control and MD cells. Our results suggested that 1 μM, rather than the commonly used 5 μM, appeared to be the optimal concentration of MitoSOX for detecting mitochondrial ROS via flow cytometry.

Sepsis is characterized by an inappropriate host immune-inflammatory response and sustained oxidative damage. Nrf2, a bZIP oxidant-responsive transcription factor, regulates a battery of cytoprotective genes including antioxidants and maintains cellular redox homeostasis. Mouse studies have demonstrated a critical role of Nrf2 in improving survival during sepsis. This preclinical ex vivo study using neutrophils and peripheral blood mononuclear cells (PBMCs) as a surrogate cells evaluates the efficacy of CDDO-Im and CDDO-Me [imidazole and methyl ester derivative of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO)] to activate the Nrf2 pathway and protect from lipopolysaccharide (LPS)-induced inflammatory response in humans. CDDO-Im treatment significantly induced Nrf2-dependent antioxidative genes (HO-1, GCLC, GCLM, and NQO1) in PBMCs isolated from six normal subjects. CDDO-Im increased nuclear accumulation of Nrf2 protein. Pretreatment of PBMC by CDDO-Im significantly attenuated LPS-induced cytokine expression. Similar increases in levels of antioxidant genes and suppression of LPS-induced cytokine expression was observed after CDDO-Me pretreatment. CDDO-Im also greatly inhibited LPS, fMLP, TNF-α, and TPA-induced ROS generation in neutrophils. In conclusion, these results demonstrate that activation of the Nrf2-dependent antioxidative pathway by CDDO-Im or CDDO-Me protects against the LPS-induced inflammatory response and suggest that they can be potential therapeutic candidates for intervening sepsis syndrome.