Nrf2-dependent protection from LPS induced inflammatory response and mortality by CDDO-Imidazolide

Abstract

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.

Fig.1

Elevated levels of ROS and proinflammatory cytokines and chemokines in nrf2-deficient neutrophils in response to LPS stimulus. A) ROS levels in nrf2 +/+ and nrf2 −/− neutrophils stimulated either with vehicle or LPS (100 ng/ml) for 1 h. Each bar is the mean ± SD (n=3) of values presenting the integration of the area under curve expressed in counts. The luminol-dependent chemiluminescence of untreated cells is at the level of the instrument background. B) mRNA expression by QPCR of cytokines (Tnf-α and Il-6) and chemokines (Mcp-1 and Mip2) in nrf2 +/+ and nrf2 −/− neutrophils 3 h after LPS stimulation (100 ng/ml). For inhibiting NADPH oxidase, cells were pretreated with DPI (10 μM) for 20 min, before stimulating with LPS. Data represented are mean fold change ± SD, n=3. * Differs from vehicle control of the same genotype; †differs from LPS treated of the same genotype.

Fig 2

CDDO-Im pretreatment attenuates LPS induced ROS levels as well as expression of cytokines (Tnf-α, Il-6) and chemokines (Mcp1, Mip2) exclusively in nrf2 +/+neutrophils. A) LPS induced ROS levels in nrf2 +/+ and nrf2 −/− peritoneal neutrophils pretreated with CDDO-Im. Peritoneal neutrophils were pretreated with CDDO-Im for 12 h and then stimulated either with vehicle or LPS (100 ng/ml). Each bar is the mean ± SD (n=3) of values presenting the integration of the area under curve for 60 min expressed in counts. B) LPS induced cytokines and chemokines expression in nrf2 +/+ and nrf2 −/− peritoneal neutrophils pretreated with CDDO-Im and or NAC. Peritoneal neutrophils were pretreated either with CDDO-Im or NAC for 12 h then stimulated either with vehicle and or LPS (100 ng/ml) for 3 h. Data presented are mean fold change ± SD (n=3). * Differs from vehicle control of the same genotype; †, differs from LPS treated of the same genotype (P<0.05).

Fig. 3

CDDO-Im pretreatment attenuates LPS shock induced expression of cytokines and chemokines by up-regulating antioxidative genes specifically in the lungs of nrf2 +/+, but not nrf2 −/− mice A) mRNA levels of Gclc, Gclm, and Gpx2 in the lungs of nrf2 +/+ and nrf2 −/− mice after treatment with CDDO-Im. Mice were treated with 3 doses of CDDO-Im (3 μmol/kg bodyweight) 24 h apart by intraperitoneal injection. Lungs were isolated 6 h after the last dose of CDDO-Im. The fold change was calculated using the formula described in methods. Data presented is mean fold change ± SD (n=3). B) LPS induced mRNA expression of cytokines (Tnf-α and Il-6) and chemokines (Mcp1 and Mip2) in the lung of nrf2 +/+ and nrf2 −/− mice pretreated with 3 doses of CDDO-Im. Mice were treated with 3 doses of CDDO-Im (3 μmol/kg bodyweight) 24 h apart by intraperitoneal injection. A lethal dose of LPS (50 mg/kg bodyweight) was administered peritoneally 6 h after the last dose of CDDO-Im. Lungs were isolated for measuring cytokine expression 1 h after LPS treatment. Data represented are mean fold change ± SD (n=3). * Differs from vehicle control of the same genotype; †, differs from LPS treated of the same genotype (P<0.05).

Fig. 4

CDDO-Im pretreatment protects nrf2 +/+ mice mortality from LPS induced septic shock mortality. A) Serum TNF-α levels in CDDO-Im pretreated nrf2 +/+ and nrf2 −/− mice after 1 h of LPS challenge. Data represented are mean ± SD, n=5. B) Mortality after LPS administration in nrf2 +/+ and nrf2 −/− mice. Age-matched male nrf2 +/+ (n=10) and nrf2 −/− mice (n=10) were either pretreated with 3 doses of vehicle or CDDO-Im (3 μmol/kg bodyweight) 24 h apart followed by single intraperitoneal injection of LPS at a dose of 50 mg/kg bodyweight. C) Protection of nrf2 +/+ mice pretreated with CDDO-Im after a higher dose of LPS administration. Nrf2 +/+ (n=10) were either pretreated with 3 doses of vehicle or CDDO-Im (3 μmol/kg bodyweight) 24 h apart followed by single intraperitoneal injection of LPS at a dose of 60 mg/kg bodyweight. Mortality was monitored every 12 h for 5 days.*, Nrf2 +/+ mice had improved survival compared to nrf2 −/− mice †, Nrf2 +/+ mice pretreated with CDDO-Im had improved survival compared to vehicle pretreated mice (P<0.05).