Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-κB pathways

doi:10.1016/j.neulet.2014.10.016

Neuroscience Letters

Volume 584, 1 January 2015, Pages 191-196

https://doi.org/10.1016/j.neulet.2014.10.016 Get rights and content

Highlights

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Mito-TEMPO attenuates LPS-induced increase of mitochondrial ROS levels in microglia.
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Mito-TEMPO prevents elevated levels of intracellular ROS in activated microglia cells.
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Mito-TEMPO attenuates production of pro-inflammatory mediators by LPS.
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Mito-TEMPO suppresses LPS-induced MAPKs and NF-κB activation.

Abstract

Activation of microglia cells in the brain contributes to neurodegenerative processes promoted by many neurotoxic factors such as pro-inflammatory cytokines and nitric oxide (NO). Reactive oxygen species (ROS) actively affect microglia-associated neurodegenerative diseases through their role as pro-inflammatory molecules and modulators of pro-inflammatory processes. Although the ROS which involved in microglia activation are thought to be generated primarily by NADPH oxidase (NOX) and involved in the immune response, mitochondrial ROS have also been proposed as important regulators of the inflammatory response in the innate immune system. However, the role of mitochondrial ROS in microglial activation has yet to be fully elucidated. In this study, we demonstrate that inhibition of mitochondrial ROS by treatment with Mito-TEMPO effectively suppressed the level of mitochondrial and intracellular ROS. Mito-TEMPO treatment also significantly prevented LPS-induced increase in the TNF-α, IL-1β, IL-6, iNOS and Cox-2 in BV-2 and primary microglia cells. Furthermore, LPS-induced suppression of mitochondrial ROS generation not only affected LPS-stimulated activation of MAPKs, including ERK, JNK, and p38, but also regulated IκB activation and NF-κB nuclear localization. These results indicate that mitochondria constitute a major source of ROS generation in LPS-mediated activated microglia cells. Additionally, suppression of LPS-induced mitochondrial ROS plays a role in modulating the production of pro-inflammatory mediators by preventing MAPK and NF-κB activation in microglia cells. Our findings suggest that a potential strategy in the development of therapy for inflammation-associated degenerative neurological diseases involves targeting the regulation of mitochondrial ROS in microglial cells.

Graphical abstract

Introduction

Microglia cells are considered the resident macrophage-like immune cells of the brain. Although microglia provide diverse beneficial functions for neuron cells, including cellular maintenance and innate immunity, constant activation of microglia can result in detrimental neurotoxic effects due to excessive production of cytotoxic mediators such as nitric oxide (NO) and pro-inflammatory cytokines. Therefore, activation of microglia has been regarded as a common and early indicator of various neurodegenerative diseases. Many studies have shown that preventing production of pro-inflammatory mediators from microglia may attenuate neuronal damage [2].

Reactive oxygen species (ROS) act as secondary messengers capable of modifying pro-inflammatory gene expression in microglia-mediated pathogenesis by altering kinase cascades and activating transcription factors, including MAPK and NF-κB [10], [15], [19]. ROS are mainly produced by members of the NADPH oxidase family in the plasma membrane and mitochondria [1], [5]. ROS generated by NADPH oxidase in activated microglia cell lines have been recognized as key modulators of immune signal transduction [4]. Increased activation of NADPH oxidase has also been implicated in elevated intracellular ROS accumulation, while inhibition of NADPH oxidase prevents NF-κB-dependent iNOS expression and NO production in LPS-stimulated macrophage [9]. Although NADPH oxidase-derived ROS are regarded as important molecules governing microglial phagocytosis and/or MAPK activation [18], recent studies have suggested that mitochondrial ROS play an important role in modulating immunoreactions as part of the innate immune system [6], [8], [25]. Therefore, it is likely that neutralization of mitochondrial ROS or suppression of the redox pathway can alleviate inflammation [6], [22]. However, the role of mitochondrial ROS production in microglial activation has yet to be fully elucidated.

Mito-TEMPO has been recently reported to function as a mitochondria-targeted SOD with low toxicity, making it a perfect candidate for mitochondrial ROS experiments [20]. Previous studies have demonstrated that Mito-TEMPO attenuates stress-induced apoptosis and necrosis by regulating mitochondrial ROS generation [11], [23].

In this study, we used Mito-TEMPO treatment to determine whether mitochondrial ROS are related to the generation of pro-inflammatory mediators in microglial cells stimulated with LPS. In addition, we characterized the LPS-stimulated activation of MAPK and NF-κB in microglial cells, and examined whether these activities are altered by regulation of mitochondrial ROS. Our results suggest that regulation of mitochondrial ROS may be essential for controlling the generation of pro-inflammatory mediators in activated microglial cells.

Section snippets

Cell culture and treatment

BV-2 murine microglial cells were kindly provided by Dr. Jau-Shyong Hong (NIEHS, NC, USA). BV-2 cells were propagated in DMEM (Welgene, Korea) containing 10% FBS (Gibco, NY, USA) and 1% penicillin/streptomycin (Welgene). Exponentially growing BV-2 cells were pre-treated with Mito-TEMPO (Enzo Life Sciences, NY, USA) for 1 h, followed by stimulation with 1 μg/mL LPS (Sigma, MO, USA).

RNA isolation and RT-PCR

Total RNA was isolated by using TRI-Reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. cDNA

Mito-TEMPO reduces levels of LPS-induced mitochondrial and intracellular ROS.

Prior to comparing the effect of Mito-TEMPO on the activation of microglia cells, we wished to evaluate the dose-dependent cytotoxic effect of Mito-TEMPO on BV-2 microglia cells by using MTT assay. Cell viability was not significantly affected by the Mito-TEMPO concentrations at 24 h, but it decreased in 400 μM of Mito-TEMPO at 48 h (Fig. 1A). We next confirmed the levels of LPS-induced mitochondrial and intracellular ROS by using MitoSOX and CM-H₂DCFDA, respectively. Our results showed that the

Discussion

Elevated levels of pro-inflammatory mediators in activated microglia play a central role in neurodegenerative disease, characterized by increased oxidative stress [2]. Apart from serving as an antimicrobial defense in microglia cells, ROS are key factors in immune cell signaling [15]. Mitochondrial ROS are implicated in the pro-inflammatory responses of microglia cells [7], [22]. In addition, various treatments that induce mitochondrial ROS result in enhanced activation of inflammation

Acknowledgments

This research was supported by grant No. NRF-2012R1A1A2008880 awarded by the National Research Foundation of Korea funded by the government of the Republic of Korea, grant No. 112020-03-2-SB020 awarded by the Korea Institute of Planning & Evaluation for Technology in Food, Agriculture, Forestry and Fisheries, and grant No. 2008-0062618 awarded by the SRC program, and by a grant from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM4611411).

References (26)

Z. Hu et al.
Subneurotoxic copper(II)-induced NF-kappaB-dependent microglial activation is associated with mitochondrial ROS
Toxicol. Appl. Pharmacol.
(2014)
E. Kasahara et al.
Mitochondrial density contributes to the immune response of macrophages to lipopolysaccharide via the MAPK pathway
FEBS Lett.
(2011)
J.H. Kim et al.
Desmethylanhydroicaritin inhibits NF-kappaB-regulated inflammatory gene expression by modulating the redox-sensitive PI3K/PTEN/Akt pathway
Eur. J. Pharmacol.
(2009)
J.H. Kim et al.
The non-provitamin A carotenoid, lutein, inhibits NF-kappaB-dependent gene expression through redox-based regulation of the phosphatidylinositol 3-kinase/PTEN/Akt and NF-kappaB-inducing kinase pathways: role of H(2)O(2) in NF-kappaB activation
Free Radic. Biol. Med.
(2008)
H.L. Liang et al.
SOD1 and MitoTEMPO partially prevent mitochondrial permeability transition pore opening, necrosis, and mitochondrial apoptosis after ATP depletion recovery
Free Radic. Biol. Med.
(2010)
L.A. Voloboueva et al.
Inflammatory response of microglial BV-2 cells includes a glycolytic shift and is modulated by mitochondrial glucose-regulated protein 75/mortalin
FEBS Lett.
(2013)
J.J. Watters et al.
A differential role for the mitogen-activated protein kinases in lipopolysaccharide signaling: the MEK/ERK pathway is not essential for nitric oxide and interleukin 1beta production
J. Biol. Chem.
(2002)
K. Bedard et al.
The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology
Physiol. Rev.
(2007)
M.L. Block et al.
Microglia-mediated neurotoxicity: uncovering the molecular mechanisms
Nat. Rev. Neurosci.
(2007)
A.C. Bulua et al.
Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS)
J. Exp. Med.
(2011)

C. Cheret et al.

Neurotoxic activation of microglia is promoted by a nox1-dependent NADPH oxidase

J. Neurosci.

(2008)

Y. Collins et al.

Mitochondrial redox signalling at a glance

J. Cell Sci.

(2012)

Y. Emre et al.

Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages

Biochem. J.

(2007)

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Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-κB pathways

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Cell culture and treatment

RNA isolation and RT-PCR

Mito-TEMPO reduces levels of LPS-induced mitochondrial and intracellular ROS.

Discussion

Acknowledgments

Toxicol. Appl. Pharmacol.

FEBS Lett.

Eur. J. Pharmacol.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

FEBS Lett.

J. Biol. Chem.

The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology

Physiol. Rev.

Microglia-mediated neurotoxicity: uncovering the molecular mechanisms

Nat. Rev. Neurosci.

Mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in TNFR1-associated periodic syndrome (TRAPS)

J. Exp. Med.