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. 2011 Oct 15;184(8):928-38.
doi: 10.1164/rccm.201102-0271OC. Epub 2011 Jul 28.

Enhancing Nrf2 pathway by disruption of Keap1 in myeloid leukocytes protects against sepsis

Xiaoni Kong  1 , Rajesh Thimmulappa, Florin Craciun, Christopher Harvey, Anju Singh, Ponvijay Kombairaju, Sekhar P Reddy, Daniel Remick, Shyam Biswal
Affiliations

Enhancing Nrf2 pathway by disruption of Keap1 in myeloid leukocytes protects against sepsis

Xiaoni Kong et al. Am J Respir Crit Care Med. .

Abstract

Rationale: Sepsis syndrome is characterized by inappropriate amplified systemic inflammatory response and bacteremia that promote multiorgan failure and mortality. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates a pleiotropic cytoprotective defense program including antioxidants and protects against several inflammatory disorders by inhibiting oxidative tissue injuries. However, the role of enhanced Nrf2 activity in modulating innate immune responses to microbial infection and pathogenesis of sepsis is unclear.

Objectives: To determine whether Nrf2 in myeloid leukocytes alters inflammatory response and protects against sepsis.

Methods: Mice with deletion of Nrf2 or kelch-like ECH-associated protein (Keap1) in myeloid leukocyte cells and respective floxed controls were subjected to cecal ligation and puncture-induced sepsis and were assessed for survival, organ injury, systemic inflammation, and bacteremia. Using LPS-stimulated peritoneal macrophages, Toll-like receptor (TLR) 4 surface trafficking and downstream signaling events were analyzed.

Measurements and main results: Mortality, organ injury, circulating levels of inflammatory mediators, and bacteremia were markedly reduced in LysM-Keap1(-/-) compared with respective floxed controls (Keap1(f/f) or Nrf2(f/f)) and significantly elevated in LysM-Nrf2(-/-) mice after cecal ligation and puncture. Peritoneal macrophages from septic LysM-Keap1(-/-) mice showed a greater bacterial phagocytic activity compared with LysM-Nrf2(-/-) and floxed controls. LPS stimulation resulted in greater reactive oxygen species-induced cell surface transport of TLR4 from trans-Golgi network and subsequent TLR4 downstream signaling (recruitment of MYD88 and TRIF, phosphorylation of IkB and IRF3, and cytokine expression) in macrophages of LysM-Nrf2(-/-) compared with LysM-Keap1(-/-) mice and floxed controls.

Conclusions: Our study shows that Nrf2 acts as a critical immunomodulator in leukocytes, controls host inflammatory response to bacterial infection, and protects against sepsis.

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Figures

Figure 1.
Figure 1.
Generation and characterization of conditional knockout mice. (A) Schematic representation of kelch-like ECH-associated protein (Keap1) gene targeting vector for tissue-specific deletion of Exons 2 and 3. LA = long arm; SA = short arm. (B) Specific recombination of the conditional Keap1 allele in the LysM-Keap1−/− mice lungs, liver, kidney, spleen, bone marrow–derived macrophages, and neutrophils. The 288-bp band represents Exons 2 and 3 deleted Keap1 allele and 2,954-bp band represents the floxed or the wild-type allele. No deletion was detected in the macrophages and neutrophils from Keap1f/f mice. (C) mRNA expression by quantitative polymerase chain reaction (PCR) of Keap1, Gclm, and Nqo1 genes in bone marrow–derived macrophages and peritoneal neutrophils from Keap1−/− and Keap1f/f mice. P < 0.05. (D) Schematic representation of nuclear factor–erythroid 2 p45-related factor 2 (Nrf2) gene targeting vector for tissue-specific deletion of Exon 5. LA = long arm; SA = short arm. (E) Specific recombination of the conditional Nrf2 allele in the LysM-Nrf2−/− lungs, liver, kidney, spleen, bone marrow–derived macrophages, and neutrophils. PCR-based genotyping of the DNA revealed a 2,600-bp band of wild-type or the floxed allele and a 467-bp band representing the Exon 5 deleted allele. No deletion was detected in the macrophages and neutrophils from Nrf2f/f mice. (F) mRNA expression by reverse transcriptase PCR of Nrf2, Gclm, and Nqo1 genes in bone marrow–derived macrophages and peritoneal neutrophils from Nrf2−/− and Nrf2f/f mice. *Significant compared with respective flox control mice. P < 0.05. Data were analyzed by Student t test. BMDM = bone marrow-derived macrophages; RFC = relative fold change.
Figure 2.
Figure 2.
Myeloid cell–specific deletion of Keap1 protects against cecal ligation and puncture (CLP)–induced sepsis pathogenesis. (A) Survival curves of mice (LysM-Nrf2−/−, Keap1f/f, Nrf2f/f, and LysM-Keap1−/−) after CLP (n = 10/gp); details are provided in the Methods section. Data were analyzed using log-rank test. *Significant compared with respective flox control mice; P < 0.01. (B and C) Serum level of blood urea nitrogen (BUN) and aspartate aminotransferase (AST) in LysM-Nrf2−/−, Keap1f/f, Nrf2f/f, and LysM-Keap1−/− mice 24 hours after CLP (n = 5/gp). (D) Histopathologic analysis of lung from Nrf2f/f, LysM-Nrf2−/−, Keap1f/f, and LysM-Keap1−/− mice 24 hours after CLP (n = 5/gp). Images (×40) from lung sections of three different mice are shown. Data were analyzed using Student t test. *Significant compared with respective flox control mice; †Significant compared with LysM-Nrf2−/− P < 0.05.
Figure 3.
Figure 3.
LysM–Keap1−/− mice are associated with markedly low systemic inflammation compared with LysM–Nrf2−/− and control floxed mice after CLP. (A) Serum cytokines (IL-6, tumor necrosis factor [TNF]-α, IL-10, and monocyte chemoattractant protein-1) in mice alive at 24 hours after CLP (22-guage, a less severe CLP model) as assessed by microarray immunoassay. Data were represented as scatter plot, and each point represents an individual mouse. The horizontal line represents mean value. Data were analyzed by U test and Student t test. *Significant compared with flox control group; P < 0.05. †Significant compared with LysM-Keap1−/− group; P < 0.01. (B) Serum high mobility group 1 levels in mice 24 hours after CLP as analyzed by immunoblot. Each band represents the level of HMGB1 in an individual mouse. For this analysis, 5 μl of serum from Keap1f/f and LysM-Keap1−/− mice and 3 μl of serum from Nrf2f/f and LysM-Nrf2−/− was used.
Figure 4.
Figure 4.
Keap1 deletion in myeloid cells augments antibacterial defenses, whereas Nrf2 deletion disposes to greater bacterial burden. (A) Blood CFUs in mice 24 hours after CLP. No blood CFU was detected in sham surgery group (data not shown); (B) Bacterial burden in peritoneal activity 24 hours after CLP; (C) Inflammatory cells in the peritoneal cavity 24 hours after CLP; (D) Bacterial phagocytic activity of peritoneal macrophages isolated 24 hours after CLP. (E and F) Phagocytosis and killing of bacteria by peritoneal macrophages isolated from healthy mice. Data were analyzed by Student t test; *Significant compared with flox control group; P < 0.05, †Significant compared with LysM-Keap1−/− group; P < 0.01.
Figure 5.
Figure 5.
Nrf2–dependent redox regulation of Toll-like receptor (TLR)4 surface expression in macrophages. (A) mRNA expression of IL-6, TNF-α, and IFN-β in LPS-stimulated peritoneal macrophages isolated from Nrf2f/f, LysM-Nrf2−/−, Keap1f/f, and LysM-Keap1−/− mice as assessed by quantitative polymerase chain reaction. (B) Levels of reactive oxygen species (ROS) in macrophages at, 5, 20, and 45 minutes after LPS challenge as assessed by fluorescence-activated cell sorter (FACS) analysis. (C) Surface expression of TLR4 on macrophages 5, 20, and 45 minutes after LPS treatment by FACS analysis. (D and E) LPS-induced surface expression of TLR4 in macrophages pretreated with N-acetylcysteine (NAC) (0.5 mM) (D) or monensin (10 μM) (E) for 30 minutes by FACS analysis. Surface TLR4 expression was assessed 45 minutes after LPS challenge. Data are presented as mean channel fluorescence (MCF) ± SD. All data presented are representative of three independent experiments. Data were analyzed by Student t test; †Significant compared with LysM-Keap1−/− group; *Significant compared with LPS alone; P < 0.05.
Figure 6.
Figure 6.
Nrf2–dependent regulation of TLR4–Nf-κB and TLR4-IRF3 signaling in macrophages. (A) Immunoblot analysis of MYD88, TRIF, and TLR4 in the macrophage cell lysates immunoprecipitated with anti-TLR4 antibody after LPS treatment. (B) Immunoblot analysis of phosphorylated IκB and IRF3 and total IκB and IRF3 in macrophage cell lysate after LPS challenge. Cell lysates were prepared from macrophages 0, 5, 15, and 45 minutes after LPS challenge. All immunoblots shown are representative of three independent experiments.
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