Neuroscience Letters 584 (2015) 191–196
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-B pathways Junghyung Park a , Ju-Sik Min a , Bokyung Kim a , Un-Bin Chae a , Jong Won Yun b , Myung-Sook Choi c , Il-Keun Kong d , Kyu-Tae Chang e , Dong-Seok Lee a,∗ a
School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea Department of Biotechnology, Daegu University, Kyungsan, Republic of Korea c School of Life Science and Biotechnology, Center for Food and Nutritional Genomics Research, Kyungpook National University, Daegu, Republic of Korea d Department of Animal Science, Division of Applied Life Science (BK21plus) and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Republic of Korea e National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Chungcheongbuk-do, Republic of Korea b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Mito-TEMPO attenuates LPS-induced increase of mitochondrial ROS levels in microglia. • Mito-TEMPO prevents elevated levels of intracellular ROS in activated microglia cells. • Mito-TEMPO attenuates production of pro-inflammatory mediators by LPS. • Mito-TEMPO suppresses LPSinduced MAPKs and NF-B activation.
a r t i c l e
i n f o
Article history: Received 20 August 2014 Received in revised form 7 October 2014 Accepted 8 October 2014 Available online 22 October 2014 Keywords: Microglia Lipopolysaccharide Mitochondrial ROS Mito-TEMPO MAPKs NF-B
a b s t r a c t 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-␣, IL-1, 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 IB activation and NF-B nuclear localization. These results indicate that mitochondria constitute a major source of ROS generation in LPS-mediated activated microglia cells.
Abbreviations: cDNA, complementary DNA; Cox-2, cyclooxygenase-2; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; iNOS, inducible nitric oxide synthase; IB, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor; JNK, c-jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; Mito-TEMPO, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)2-oxoethyl) triphenylphosphonium chloride; NF-B, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, NLR family, pyrin domain containing 3; SOD, superoxide dismutase; TNF, tumor necrosis factor. ∗ Corresponding author. Tel.: +82 53 950 7366; fax: +82 53 943 6925. E-mail address: [email protected] (D.-S. Lee). http://dx.doi.org/10.1016/j.neulet.2014.10.016 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
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J. Park et al. / Neuroscience Letters 584 (2015) 191–196
Additionally, suppression of LPS-induced mitochondrial ROS plays a role in modulating the production of pro-inflammatory mediators by preventing MAPK and NF-B 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. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. 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-B [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-B-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 proinflammatory mediators in microglial cells stimulated with LPS. In addition, we characterized the LPS-stimulated activation of MAPK and NF-B 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. 2. Materials and methods
BV-2 cells were pre-treated with Mito-TEMPO (Enzo Life Sciences, NY, USA) for 1 h, followed by stimulation with 1 g/mL LPS (Sigma, MO, USA). 2.2. RNA isolation and RT-PCR Total RNA was isolated by using TRI-Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized by using Reverse Transcription Premix (Bioneer, Korea). PCR was performed using gene-specific primers and PCR premix (Bioneer) with 27 cycles of application. The following primers were used for PCR amplification: 5 -TNF-␣, 5 -AGTTCTATGGCCCAGA CCCT-3 ; 3 -TNF-␣, 5 -GTGGGTGAGGAGCACGTAGT-3 ; 5 -IL-1, 5 -CGACAAAAACCTGTGGCCT-3 ; 3 -IL-1, 5 -AGGCCACAGGTATT TTGTCG-3; 5 -IL-6, 5 -AGTTGCCTTCTTGGGACTGA-3 ; 3 -IL-6, 5 TTCTGCAAGTGCATCATCGT-3 ; 5 -iNOS, 5 -CTGCAGCACTTGGAT CAGGAACCTG-3 ; 3 -iNOS, 5 -GGGAGTAGCCTGTGTGCACCTGGAA3 ; 5 -TCACAGCCCGAGGGTGTCCA-3 ; 5 -GAPDH, 5 -ACCACAGTC CATGCCATCAC-3 ; and 3 -GAPDH, 5 -TCCACCACCCTGTTGCTGTA3 . Band intensity was analyzed using Multi-Gauge software (Fujifilm, Japan). 2.3. ELISA assay BV-2 cells were cultured in 96-well plates (1 × 105 per well), and then added LPS in presence or absence of Mito-TEMPO. The amount of TNF-␣ and IL-6 was determined with specific ELISAs (eBioscience, CA, USA) as described in the manuals. 2.4. Western blot analysis Whole protein lysates were prepared using PRO-PREP protein extraction solution (Intron Biotechnology, Korea). Nuclear and cytoplasmic fractions were isolated using an NE-PER nuclear and cytoplasmic extraction reagent kit (Thermo Scientific, MA, USA), according to the manufacturer’s protocol. Equal amounts of protein were separated by electrophoresis on 10–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (BD Biosciences, NJ, USA). The membranes were incubated overnight at 4 ◦ C with either anti-ERK, anti-phosphorylated (p)-ERK, anti-JNK, anti-p-JNK, anti-p38, anti-p-p38, anti-p-IB (Cell Signaling, MA, USA), antiiNOS (Abcam, MA, USA), anti-Cox-2, anti--actin, anti-IB (Santa Cruz, TX, USA), anti-NF-B p65, or anti-lamin B (Ab Frontier, Korea) primary antibodies. 2.5. Cell viability (MTT) assay BV-2 cells were cultured in 96-well plates (1 × 104 per well), and then different concentrations of Mito-TEMPO tested were added to these wells. After 24 h, 0.5 mg/mL MTT (Sigma) was added, followed by 30-min incubation at 37 ◦ C; then, 100 L of DMSO were added to the cells. Absorbance was measured at 550 nm.
2.1. Cell culture and treatment 2.6. Measurement of mitochondrial and intracellular ROS levels 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
Generation of mitochondrial and intracellular ROS was assessed using MitoSOX (Invitrogen) and CM-H2 DCFDA (Invitrogen). Trypsinized BV-2 cells were incubated with 5 M MitoSOX
J. Park et al. / Neuroscience Letters 584 (2015) 191–196
Cell viability (% of control)
(A)
120
24h
100
48h
80 60 40 20 0
0
(B)
100
200
400
Mito-TEMPO (μM)
Relative level of mitochondrial ROS
** 2.0 1.5 1.0 0.5 0.0
-
(C)
+ -
+ + 100 200
+ LPS (1μg/mL) 400 Mito-TEMPO (μM)
**
*** ** 3.5
Relative level of intracellular ROS
were effectively reduced by treating BV-2 cells with Mito-TEMPO at concentrations of ≥200 M (Fig. 1B and C). Therefore, we selected 200 M of Mito-TEMPO because it did not influence cell viability and effectively suppressed LPS-induced both mitochondrial and intracellular ROS generation. Altogether, these results indicate that Mito-TEMPO can dramatically regulate both LPS-induced mitochondrial and intracellular ROS.
3.2. Mitochondrial ROS regulate production of pro-inflammatory mediators
***
***
193
3.0 2.5 2.0 1.5 1.0 0.5 0.0
-
+ -
+ + 100 200
+ LPS (1μg/mL) 400 Mito-TEMPO (μM)
Fig. 1. Inhibitory effect of Mito-TEMPO on LPS-stimulated mitochondrial and intracellular ROS induction. (A) The viability of BV-2 cells treated for 24 h and 48 h with the indicated concentrations of Mito-TEMPO was measured using the MTT assay. (B) BV-2 cells were stimulated with LPS for 12 h in the absence or presence of the indicated concentration of Mito-TEMPO, and then incubated with a mitochondrial ROS indicator, MitoSOX, after which mitochondrial ROS levels were analyzed by flow cytometry. (C) The level of intracellular ROS in LPS-treated cells in the presence or absence of Mito-TEMPO was examined using CM-H2 DCFDA and flow cytometry. Data are presented as the mean ± SD (n = 3). **p < 0.01, and ***p < 0.001.
Pro-inflammatory cytokines such as TNF-␣, IL-1, and IL-6 are considered markers of activated microglia cells [2]. We, therefore, examined whether mitochondrial ROS involved in LPS stimulation increase the mRNA expression of pro-inflammatory cytokines and iNOS in BV-2 and primary microglia cells using RT-PCR analysis. Interestingly, our results show that the upregulated mRNA levels of TNF-␣, IL-1, IL-6, and iNOS were clearly suppressed by pre-treatment with Mito-TEMPO in both BV-2 (Fig. 2A) and primary microglia cells (Supplementary Fig. 1). In addition, enhanced release of TNF-␣ and IL-6 protein by LPS was reduced by MitoTEMPO. Various pathological conditions lead to the production of high levels of NO in the brain because of iNOS expression in activated microglia cells, thereby exerting toxic effects [16]. Additionally, Cox-2 expression causes secondary damage to neurons [21]. We, therefore, sought to confirm the role of mitochondrial ROS in LPS-induced production of pro-inflammatory responses in microglia cells by establishing the protein expression levels of iNOS and Cox-2 in LPS-induced BV-2 cells. Our findings indicate that increased protein expression of iNOS and Cox-2 in BV-2 cells due to LPS treatment was significantly inhibited by the addition of MitoTEMPO (Fig. 2B). Consequently, our data suggest that mitochondrial ROS play an important part in the regulation of pro-inflammatory mediator production and microglia activation.
3.3. Mitochondrial ROS control microglia activation through the regulation of MAPK and CM-H2 DCFDA at 37 ◦ C for 15 min, washed with PBS, and then analyzed with a flow cytometer (BD Biosciences). 2.7. Statistical analysis The data represent the mean and SD of three independent experiments (n = 3). Experimental differences were tested for statistical significance using two-way ANOVA using the GraphPad Prism 5 software. A p-value of
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Mitochondrial ROS govern the LPS-induced pro-inflammatory response in microglia cells by regulating MAPK and NF-B pathways Junghyung Park a , Ju-Sik Min a , Bokyung Kim a , Un-Bin Chae a , Jong Won Yun b , Myung-Sook Choi c , Il-Keun Kong d , Kyu-Tae Chang e , Dong-Seok Lee a,∗ a
School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, Republic of Korea Department of Biotechnology, Daegu University, Kyungsan, Republic of Korea c School of Life Science and Biotechnology, Center for Food and Nutritional Genomics Research, Kyungpook National University, Daegu, Republic of Korea d Department of Animal Science, Division of Applied Life Science (BK21plus) and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Republic of Korea e National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Chungcheongbuk-do, Republic of Korea b
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• Mito-TEMPO attenuates LPS-induced increase of mitochondrial ROS levels in microglia. • Mito-TEMPO prevents elevated levels of intracellular ROS in activated microglia cells. • Mito-TEMPO attenuates production of pro-inflammatory mediators by LPS. • Mito-TEMPO suppresses LPSinduced MAPKs and NF-B activation.
a r t i c l e
i n f o
Article history: Received 20 August 2014 Received in revised form 7 October 2014 Accepted 8 October 2014 Available online 22 October 2014 Keywords: Microglia Lipopolysaccharide Mitochondrial ROS Mito-TEMPO MAPKs NF-B
a b s t r a c t 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-␣, IL-1, 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 IB activation and NF-B nuclear localization. These results indicate that mitochondria constitute a major source of ROS generation in LPS-mediated activated microglia cells.
Abbreviations: cDNA, complementary DNA; Cox-2, cyclooxygenase-2; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; iNOS, inducible nitric oxide synthase; IB, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor; JNK, c-jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; Mito-TEMPO, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)2-oxoethyl) triphenylphosphonium chloride; NF-B, nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3, NLR family, pyrin domain containing 3; SOD, superoxide dismutase; TNF, tumor necrosis factor. ∗ Corresponding author. Tel.: +82 53 950 7366; fax: +82 53 943 6925. E-mail address: [email protected] (D.-S. Lee). http://dx.doi.org/10.1016/j.neulet.2014.10.016 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
192
J. Park et al. / Neuroscience Letters 584 (2015) 191–196
Additionally, suppression of LPS-induced mitochondrial ROS plays a role in modulating the production of pro-inflammatory mediators by preventing MAPK and NF-B 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. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. 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-B [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-B-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 proinflammatory mediators in microglial cells stimulated with LPS. In addition, we characterized the LPS-stimulated activation of MAPK and NF-B 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. 2. Materials and methods
BV-2 cells were pre-treated with Mito-TEMPO (Enzo Life Sciences, NY, USA) for 1 h, followed by stimulation with 1 g/mL LPS (Sigma, MO, USA). 2.2. RNA isolation and RT-PCR Total RNA was isolated by using TRI-Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized by using Reverse Transcription Premix (Bioneer, Korea). PCR was performed using gene-specific primers and PCR premix (Bioneer) with 27 cycles of application. The following primers were used for PCR amplification: 5 -TNF-␣, 5 -AGTTCTATGGCCCAGA CCCT-3 ; 3 -TNF-␣, 5 -GTGGGTGAGGAGCACGTAGT-3 ; 5 -IL-1, 5 -CGACAAAAACCTGTGGCCT-3 ; 3 -IL-1, 5 -AGGCCACAGGTATT TTGTCG-3; 5 -IL-6, 5 -AGTTGCCTTCTTGGGACTGA-3 ; 3 -IL-6, 5 TTCTGCAAGTGCATCATCGT-3 ; 5 -iNOS, 5 -CTGCAGCACTTGGAT CAGGAACCTG-3 ; 3 -iNOS, 5 -GGGAGTAGCCTGTGTGCACCTGGAA3 ; 5 -TCACAGCCCGAGGGTGTCCA-3 ; 5 -GAPDH, 5 -ACCACAGTC CATGCCATCAC-3 ; and 3 -GAPDH, 5 -TCCACCACCCTGTTGCTGTA3 . Band intensity was analyzed using Multi-Gauge software (Fujifilm, Japan). 2.3. ELISA assay BV-2 cells were cultured in 96-well plates (1 × 105 per well), and then added LPS in presence or absence of Mito-TEMPO. The amount of TNF-␣ and IL-6 was determined with specific ELISAs (eBioscience, CA, USA) as described in the manuals. 2.4. Western blot analysis Whole protein lysates were prepared using PRO-PREP protein extraction solution (Intron Biotechnology, Korea). Nuclear and cytoplasmic fractions were isolated using an NE-PER nuclear and cytoplasmic extraction reagent kit (Thermo Scientific, MA, USA), according to the manufacturer’s protocol. Equal amounts of protein were separated by electrophoresis on 10–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (BD Biosciences, NJ, USA). The membranes were incubated overnight at 4 ◦ C with either anti-ERK, anti-phosphorylated (p)-ERK, anti-JNK, anti-p-JNK, anti-p38, anti-p-p38, anti-p-IB (Cell Signaling, MA, USA), antiiNOS (Abcam, MA, USA), anti-Cox-2, anti--actin, anti-IB (Santa Cruz, TX, USA), anti-NF-B p65, or anti-lamin B (Ab Frontier, Korea) primary antibodies. 2.5. Cell viability (MTT) assay BV-2 cells were cultured in 96-well plates (1 × 104 per well), and then different concentrations of Mito-TEMPO tested were added to these wells. After 24 h, 0.5 mg/mL MTT (Sigma) was added, followed by 30-min incubation at 37 ◦ C; then, 100 L of DMSO were added to the cells. Absorbance was measured at 550 nm.
2.1. Cell culture and treatment 2.6. Measurement of mitochondrial and intracellular ROS levels 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
Generation of mitochondrial and intracellular ROS was assessed using MitoSOX (Invitrogen) and CM-H2 DCFDA (Invitrogen). Trypsinized BV-2 cells were incubated with 5 M MitoSOX
J. Park et al. / Neuroscience Letters 584 (2015) 191–196
Cell viability (% of control)
(A)
120
24h
100
48h
80 60 40 20 0
0
(B)
100
200
400
Mito-TEMPO (μM)
Relative level of mitochondrial ROS
** 2.0 1.5 1.0 0.5 0.0
-
(C)
+ -
+ + 100 200
+ LPS (1μg/mL) 400 Mito-TEMPO (μM)
**
*** ** 3.5
Relative level of intracellular ROS
were effectively reduced by treating BV-2 cells with Mito-TEMPO at concentrations of ≥200 M (Fig. 1B and C). Therefore, we selected 200 M of Mito-TEMPO because it did not influence cell viability and effectively suppressed LPS-induced both mitochondrial and intracellular ROS generation. Altogether, these results indicate that Mito-TEMPO can dramatically regulate both LPS-induced mitochondrial and intracellular ROS.
3.2. Mitochondrial ROS regulate production of pro-inflammatory mediators
***
***
193
3.0 2.5 2.0 1.5 1.0 0.5 0.0
-
+ -
+ + 100 200
+ LPS (1μg/mL) 400 Mito-TEMPO (μM)
Fig. 1. Inhibitory effect of Mito-TEMPO on LPS-stimulated mitochondrial and intracellular ROS induction. (A) The viability of BV-2 cells treated for 24 h and 48 h with the indicated concentrations of Mito-TEMPO was measured using the MTT assay. (B) BV-2 cells were stimulated with LPS for 12 h in the absence or presence of the indicated concentration of Mito-TEMPO, and then incubated with a mitochondrial ROS indicator, MitoSOX, after which mitochondrial ROS levels were analyzed by flow cytometry. (C) The level of intracellular ROS in LPS-treated cells in the presence or absence of Mito-TEMPO was examined using CM-H2 DCFDA and flow cytometry. Data are presented as the mean ± SD (n = 3). **p < 0.01, and ***p < 0.001.
Pro-inflammatory cytokines such as TNF-␣, IL-1, and IL-6 are considered markers of activated microglia cells [2]. We, therefore, examined whether mitochondrial ROS involved in LPS stimulation increase the mRNA expression of pro-inflammatory cytokines and iNOS in BV-2 and primary microglia cells using RT-PCR analysis. Interestingly, our results show that the upregulated mRNA levels of TNF-␣, IL-1, IL-6, and iNOS were clearly suppressed by pre-treatment with Mito-TEMPO in both BV-2 (Fig. 2A) and primary microglia cells (Supplementary Fig. 1). In addition, enhanced release of TNF-␣ and IL-6 protein by LPS was reduced by MitoTEMPO. Various pathological conditions lead to the production of high levels of NO in the brain because of iNOS expression in activated microglia cells, thereby exerting toxic effects [16]. Additionally, Cox-2 expression causes secondary damage to neurons [21]. We, therefore, sought to confirm the role of mitochondrial ROS in LPS-induced production of pro-inflammatory responses in microglia cells by establishing the protein expression levels of iNOS and Cox-2 in LPS-induced BV-2 cells. Our findings indicate that increased protein expression of iNOS and Cox-2 in BV-2 cells due to LPS treatment was significantly inhibited by the addition of MitoTEMPO (Fig. 2B). Consequently, our data suggest that mitochondrial ROS play an important part in the regulation of pro-inflammatory mediator production and microglia activation.
3.3. Mitochondrial ROS control microglia activation through the regulation of MAPK and CM-H2 DCFDA at 37 ◦ C for 15 min, washed with PBS, and then analyzed with a flow cytometer (BD Biosciences). 2.7. Statistical analysis The data represent the mean and SD of three independent experiments (n = 3). Experimental differences were tested for statistical significance using two-way ANOVA using the GraphPad Prism 5 software. A p-value of
