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NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signaling and sepsis-induced mortality in Nrf2-deficient mice.

ABSTRACT: Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and LPS shock model; however, the underlying mechanisms are unclear. In the current study, compared with wild-type (Nrf2(+/+)) macrophages, we observed greater protein kinase C-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2(-/-)) macrophages that was modulated by glutathione levels. To address the NADPH oxidase-mediated hyperinflammatory response and sepsis-induced lung injury and mortality in Nrf2(-/-) mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91(phox) (Nrf2(-/-)//gp91(phox-/-)). Compared with Nrf2(+/+) macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MyD88 and Toll/IL-1R domain-containing adaptor in Nrf2(-/-) macrophages that was diminished by ablation of gp91(phox). Similarly, phosphorylation of IkappaB and IFN regulatory factor 3 as well as cytokine expression was markedly higher in Nrf2(-/-) macrophages; whereas, it was similar in Nrf2(+/+) and Nrf2(-/-)//gp91(phox-/-). In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2(-/-) mice that was significantly reduced by ablation of gp91(phox). Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2(-/-) mice; whereas, Nrf2(-/-)//gp91(phox-/-) showed prolonged survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis.

SUBMITTER: Kong X

PROVIDER: S-EPMC2913313 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signaling and sepsis-induced mortality in Nrf2-deficient mice.

Kong Xiaoni X Thimmulappa Rajesh R Kombairaju Ponvijay P Biswal Shyam S

Journal of immunology (Baltimore, Md. : 1950) 20100528 1

Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and LPS shock model; however, the underlying mechanisms are unclear. In the current study, compared wi ...[more]

PMID: 20511556

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Project description:NADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.We assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to canonical motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity, as verified by mutational analysis. The presence of these non-canonical, but highly conserved regions suggests that all Nox/Duox may possess a common biological function remained in a long history of Nox/Duox evolution.This report provides the first comprehensive analysis of the evolution and conserved functions of Nox and Duox family members, including identification of conserved amino acid residues. These results provide a guide for future structure-function studies and for understanding the evolution of biological functions of these enzymes.

Project description:Activation of microglia causes the production of proinflammatory factors and upregulation of NADPH oxidase (NOX) that form reactive oxygen species (ROS) that lead to neurodegeneration. Previously, we reported that 10 daily doses of ethanol treatment induced innate immune genes in brain. In the present study, we investigate the effects of chronic ethanol on activation of NOX and release of ROS, and their contribution to ethanol neurotoxicity.Male C57BL/6 and NF-?B enhanced GFP mice were treated intragastrically with water or ethanol (5 g/kg, i.g., 25% ethanol w/v) daily for 10 days. The effects of chronic ethanol on cell death markers (activated caspase-3 and Fluoro-Jade B), microglial morphology, NOX, ROS and NF-?B were examined using real-time PCR, immunohistochemistry and hydroethidine histochemistry. Also, Fluoro-Jade B staining and NOX gp91phox immunohistochemistry were performed in the orbitofrontal cortex (OFC) of human postmortem alcoholic brain and human moderate drinking control brain.Ethanol treatment of C57BL/6 mice showed increased markers of neuronal death: activated caspase-3 and Fluoro-Jade B positive staining with Neu-N (a neuronal marker) labeling in cortex and dentate gyrus. The OFC of human post-mortem alcoholic brain also showed significantly more Fluoro-Jade B positive cells colocalized with Neu-N, a neuronal marker, compared to the OFC of human moderate drinking control brain, suggesting increased neuronal death in the OFC of human alcoholic brain. Iba1 and GFAP immunohistochemistry showed activated morphology of microglia and astrocytes in ethanol-treated mouse brain. Ethanol treatment increased NF-?B transcription and increased NOX gp91phox at 24 hr after the last ethanol treatment that remained elevated at 1 week. The OFC of human postmortem alcoholic brain also had significant increases in the number of gp91phox + immunoreactive (IR) cells that are colocalized with neuronal, microglial and astrocyte markers. In mouse brain ethanol increased gp91phox expression coincided with increased production of O2- and O2- - derived oxidants. Diphenyleneiodonium (DPI), a NOX inhibitor, reduced markers of neurodegeneration, ROS and microglial activation.Ethanol activation of microglia and astrocytes, induction of NOX and production of ROS contribute to chronic ethanol-induced neurotoxicity. NOX-ROS and NF-?B signaling pathways play important roles in chronic ethanol-induced neuroinflammation and neurodegeneration.

Dataset Information

NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signaling and sepsis-induced mortality in Nrf2-deficient mice.

Publications

NADPH oxidase-dependent reactive oxygen species mediate amplified TLR4 signaling and sepsis-induced mortality in Nrf2-deficient mice.

Similar Datasets

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