Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis
Rajesh K. Thimmulappa, Hannah Lee, Tirumalai Rangasamy, Sekhar P. Reddy, Masayuki Yamamoto, Thomas W. Kensler, Shyam Biswal
Rajesh K. Thimmulappa, Hannah Lee, Tirumalai Rangasamy, Sekhar P. Reddy, Masayuki Yamamoto, Thomas W. Kensler, Shyam Biswal
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Research Article

Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis

Abstract

Host genetic factors that regulate innate immunity determine susceptibility to sepsis. Disruption of nuclear factor-erythroid 2–related factor 2 (Nrf2), a basic leucine zipper transcription factor that regulates redox balance and stress response, dramatically increased the mortality of mice in response to endotoxin- and cecal ligation and puncture–induced septic shock. LPS as well as TNF-α stimulus resulted in greater lung inflammation in Nrf2-deficient mice. Temporal analysis of pulmonary global gene expression after LPS challenge revealed augmented expression of large numbers of proinflammatory genes associated with the innate immune response at as early as 30 minutes in lungs of Nrf2-deficient mice, indicating severe immune dysregulation. The expression profile indicated that Nrf2 has a global influence on both MyD88-dependent and -independent signaling. Nrf2-deficient mouse embryonic fibroblasts showed greater activation of NF-κB and interferon regulatory factor 3 in response to LPS and polyinosinic-polycytidylic acid [poly(I:C)] stimulus, corroborating the effect of Nrf2 on MyD88-dependent and -independent signaling. Nrf2’s regulation of cellular glutathione and other antioxidants is critical for optimal NF-κB activation in response to LPS and TNF-α. Our study reveals Nrf2 as a novel modifier gene of sepsis that determines survival by mounting an appropriate innate immune response.

Authors

Rajesh K. Thimmulappa, Hannah Lee, Tirumalai Rangasamy, Sekhar P. Reddy, Masayuki Yamamoto, Thomas W. Kensler, Shyam Biswal

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Figure 8

LPS and/or TNF-α stimulus induces greater NF-κB activation in Nrf2-deficient MEFs.

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LPS and/or TNF-α stimulus induces greater NF-κB activation in Nrf2-defic...
(A) Nuclear extracts from Nrf2+/+ and Nrf2–/– MEFs were assayed for NF-κB DNA-binding activity by EMSA 30 minutes after LPS (0.5 μg/ml) and or TNF-α (10 ng/ml). The major NF-κB bands contained p65 and p55 subunits, as determined by the supershift analysis using p65 and p55 antibody. (B) Quantification of NF-κB DNA-binding was performed by densitometric analysis. All values are mean α SEM (n = 3) and are represented relative to respective vehicle control. (C) NF-κB–mediated reporter activity in MEFs of both genotypes challenged with LPS (0.5 μg/ml) and TNF-α (10 ng/ml). At 24 hours after transfection with p–NF-κB–Luc vector, cells were treated with LPS and/or TNF-α for 3 hours, and then luciferase activity was measured. Data are mean α SEM from 3 independent experiments (n = 3) and are represented relative to respective vehicle control. (D) Immunoblot of IκB-α and p–IκB-α protein in Nrf2+/+ and Nrf2–/– MEFs after LPS (0.5 μg/ml) or TNF-α (10 ng/ml) stimulus. (E and F) Quantification of IκB-α (E) and p–IκB-α (F) protein in Nrf2+/+ and Nrf2–/– MEFs by densitometric analysis. Data are mean α SEM (n = 3) and are shown as relative to respective vehicle control. (G) IKK activity in Nrf2+/+ and Nrf2–/– MEFs after LPS (0.5 μg/ml) or TNF-α (10 ng/ml) stimulus. (H) Quantification of IKK activity in Nrf2+/+ and Nrf2–/– MEFs by densitometric analysis. Densitometric units are normalized to IKKα. Data are mean α SEM (n = 3) and are relative to respective controls. *Differs from vehicle control of the same genotype. †Differs frowild-type treatment group. P < 0.05.