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Reactive oxygen species and p47phox activation are essential for the Mycobacterium tuberculosis-induced pro-inflammatory response in murine microglia.

Yang CS, Lee HM, Lee JY, Kim JA, Lee SJ, Shin DM, Lee YH, Lee DS, El-Benna J, Jo EK - J Neuroinflammation (2007)

Bottom Line: Furthermore, the activation of cytosolic NADPH oxidase p47phox and MAPKs (p38 and ERK1/2) is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia.Neither TLR2 nor dectin-1 was involved in s-Mtb-induced inflammatory responses in murine microglia.These data collectively demonstrate that s-Mtb actively induces the pro-inflammatory response in microglia through NADPH oxidase-dependent ROS generation, although the specific pattern-recognition receptors involved in these responses remain to be identified.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, College of Medicine, Chungnam National University, Daejeon 301-747, S. Korea. ironwater514@gmail.com

ABSTRACT

Background: Activated microglia elicits a robust amount of pro-inflammatory cytokines, which are implicated in the pathogenesis of tuberculosis in the central nervous system (CNS). However, little is known about the intracellular signaling mechanisms governing these inflammatory responses in microglia in response to Mycobacterium tuberculosis (Mtb).

Methods: Murine microglial BV-2 cells and primary mixed glial cells were stimulated with sonicated Mtb (s-Mtb). Intracellular ROS levels were measured by staining with oxidative fluorescent dyes [2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE)]. NADPH oxidase activities were measured by lucigenin chemiluminescence assay. S-Mtb-induced MAPK activation and pro-inflammatory cytokine release in microglial cells were measured using by Western blot analysis and enzyme-linked immunosorbent assay, respectively.

Results: We demonstrate that s-Mtb promotes the up-regulation of reactive oxygen species (ROS) and the rapid activation of mitogen-activated protein kinases (MAPKs), including p38 and extracellular signal-regulated kinase (ERK) 1/2, as well as the secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-12p40 in murine microglial BV-2 cells and primary mixed glial cells. Both NADPH oxidase and mitochondrial electron transfer chain subunit I play an indispensable role in s-Mtb-induced MAPK activation and pro-inflammatory cytokine production in BV-2 cells and mixed glial cells. Furthermore, the activation of cytosolic NADPH oxidase p47phox and MAPKs (p38 and ERK1/2) is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia. Neither TLR2 nor dectin-1 was involved in s-Mtb-induced inflammatory responses in murine microglia.

Conclusion: These data collectively demonstrate that s-Mtb actively induces the pro-inflammatory response in microglia through NADPH oxidase-dependent ROS generation, although the specific pattern-recognition receptors involved in these responses remain to be identified.

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The cytosolic NADPH oxidase subunit p47phox and MAPK activation is mutually dependent on the ROS generation and cytokine production by s-Mtb-stimulated microglia. A) p47phox is required for s-Mtb-induced MAPK activation. BV-2 cells were transfected with wild-type p47phox (WT), dominant-negative p47phox (DN), or empty vector. The cells were then stimulated with 1% s-Mtb for 30 min, harvested, and subjected to Western blot analysis to detect total and phosphorylated ERK1/2 and p38. B) p47phox phosphorylation at Ser345 is required for s-Mtb-induced cytokine production. BV-2 cells were pre-treated with TAT-Ser345 peptide (20 or 40 μM) or TAT-scramble peptide (20 or 40 μM) and stimulated with 1% s-Mtb for 18 h. The supernatants were analyzed for TNF-α, IL-6, and IL-12p40 production by ELISA. Data are presented as the percentage of the control. Significant differences compared to cultures incubated with s-Mtb alone: **, P < 0.01; ***, P < 0.001. C) MAPK activation is essential for p47phox activation. After pretreatment for 30 min with inhibitors of either MEK1 (U0126; 5, 10, or 20 μM) or p38 (SB203580; 1, 5, or 10 μM), BV-2 cells were stimulated with 1% s-Mtb for 30 min. The cells were then harvested and subjected to Western blotting to detect phosphorylated pSer345 and p47phox. D) MAPK activity is required for s-Mtb-induced superoxide production. BV-2 cells were pretreated with U0126 (10 μM) or SB203580 (5 μM) for 30 min and then incubated with DHE (for superoxide detection) after stimulation with 1% s-Mtb for 30 min. The live cells were washed with serum-free medium and imaged using confocal microscopy. The images are representative of three independent experiments. M, medium only; D, 0.1% DMSO as a solvent control.
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Figure 5: The cytosolic NADPH oxidase subunit p47phox and MAPK activation is mutually dependent on the ROS generation and cytokine production by s-Mtb-stimulated microglia. A) p47phox is required for s-Mtb-induced MAPK activation. BV-2 cells were transfected with wild-type p47phox (WT), dominant-negative p47phox (DN), or empty vector. The cells were then stimulated with 1% s-Mtb for 30 min, harvested, and subjected to Western blot analysis to detect total and phosphorylated ERK1/2 and p38. B) p47phox phosphorylation at Ser345 is required for s-Mtb-induced cytokine production. BV-2 cells were pre-treated with TAT-Ser345 peptide (20 or 40 μM) or TAT-scramble peptide (20 or 40 μM) and stimulated with 1% s-Mtb for 18 h. The supernatants were analyzed for TNF-α, IL-6, and IL-12p40 production by ELISA. Data are presented as the percentage of the control. Significant differences compared to cultures incubated with s-Mtb alone: **, P < 0.01; ***, P < 0.001. C) MAPK activation is essential for p47phox activation. After pretreatment for 30 min with inhibitors of either MEK1 (U0126; 5, 10, or 20 μM) or p38 (SB203580; 1, 5, or 10 μM), BV-2 cells were stimulated with 1% s-Mtb for 30 min. The cells were then harvested and subjected to Western blotting to detect phosphorylated pSer345 and p47phox. D) MAPK activity is required for s-Mtb-induced superoxide production. BV-2 cells were pretreated with U0126 (10 μM) or SB203580 (5 μM) for 30 min and then incubated with DHE (for superoxide detection) after stimulation with 1% s-Mtb for 30 min. The live cells were washed with serum-free medium and imaged using confocal microscopy. The images are representative of three independent experiments. M, medium only; D, 0.1% DMSO as a solvent control.

Mentions: Phosphorylation of the cytosolic subunit p47phox is necessary for NADPH oxidase activation and regulation [39]. Although p47phox has been detected in cultured microglia [26], its role in MAPK activation and cytokine production in microglia has not been investigated. To examine whether ERK1/2 or p38 activation is dependent on p47phox activation, we examined the effect of wild-type (WT) or dominant-negative (DN) p47phox constructs on p38 and ERK1/2 phosphorylation. Our results showed that ERK1/2 and p38 phosphorylation increased substantially in BV-2 microglia transfected with WT p47phox, whereas phosphorylation was abolished in cells expressing DN p47phox (Fig. 5A). In addition, we pre-treated cells with an inhibitory cell-permeable peptide (TAT-Ser345 peptide) that corresponds to amino acids 339–350 (ARPGPQSPGSPL) of p47phox [40]. In cells treated with the TAT-Ser345 peptide, TNF-α, IL-6, and IL-12p40 production decreased significantly in a dose-dependent manner, whereas the TAT-scramble peptide had little or no inhibitory effect on cytokine production (Fig. 5B). These results suggest that p47phox activation is necessary for MAPK activation and the pro-inflammatory response in microglial cells.


Reactive oxygen species and p47phox activation are essential for the Mycobacterium tuberculosis-induced pro-inflammatory response in murine microglia.

Yang CS, Lee HM, Lee JY, Kim JA, Lee SJ, Shin DM, Lee YH, Lee DS, El-Benna J, Jo EK - J Neuroinflammation (2007)

The cytosolic NADPH oxidase subunit p47phox and MAPK activation is mutually dependent on the ROS generation and cytokine production by s-Mtb-stimulated microglia. A) p47phox is required for s-Mtb-induced MAPK activation. BV-2 cells were transfected with wild-type p47phox (WT), dominant-negative p47phox (DN), or empty vector. The cells were then stimulated with 1% s-Mtb for 30 min, harvested, and subjected to Western blot analysis to detect total and phosphorylated ERK1/2 and p38. B) p47phox phosphorylation at Ser345 is required for s-Mtb-induced cytokine production. BV-2 cells were pre-treated with TAT-Ser345 peptide (20 or 40 μM) or TAT-scramble peptide (20 or 40 μM) and stimulated with 1% s-Mtb for 18 h. The supernatants were analyzed for TNF-α, IL-6, and IL-12p40 production by ELISA. Data are presented as the percentage of the control. Significant differences compared to cultures incubated with s-Mtb alone: **, P < 0.01; ***, P < 0.001. C) MAPK activation is essential for p47phox activation. After pretreatment for 30 min with inhibitors of either MEK1 (U0126; 5, 10, or 20 μM) or p38 (SB203580; 1, 5, or 10 μM), BV-2 cells were stimulated with 1% s-Mtb for 30 min. The cells were then harvested and subjected to Western blotting to detect phosphorylated pSer345 and p47phox. D) MAPK activity is required for s-Mtb-induced superoxide production. BV-2 cells were pretreated with U0126 (10 μM) or SB203580 (5 μM) for 30 min and then incubated with DHE (for superoxide detection) after stimulation with 1% s-Mtb for 30 min. The live cells were washed with serum-free medium and imaged using confocal microscopy. The images are representative of three independent experiments. M, medium only; D, 0.1% DMSO as a solvent control.
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Figure 5: The cytosolic NADPH oxidase subunit p47phox and MAPK activation is mutually dependent on the ROS generation and cytokine production by s-Mtb-stimulated microglia. A) p47phox is required for s-Mtb-induced MAPK activation. BV-2 cells were transfected with wild-type p47phox (WT), dominant-negative p47phox (DN), or empty vector. The cells were then stimulated with 1% s-Mtb for 30 min, harvested, and subjected to Western blot analysis to detect total and phosphorylated ERK1/2 and p38. B) p47phox phosphorylation at Ser345 is required for s-Mtb-induced cytokine production. BV-2 cells were pre-treated with TAT-Ser345 peptide (20 or 40 μM) or TAT-scramble peptide (20 or 40 μM) and stimulated with 1% s-Mtb for 18 h. The supernatants were analyzed for TNF-α, IL-6, and IL-12p40 production by ELISA. Data are presented as the percentage of the control. Significant differences compared to cultures incubated with s-Mtb alone: **, P < 0.01; ***, P < 0.001. C) MAPK activation is essential for p47phox activation. After pretreatment for 30 min with inhibitors of either MEK1 (U0126; 5, 10, or 20 μM) or p38 (SB203580; 1, 5, or 10 μM), BV-2 cells were stimulated with 1% s-Mtb for 30 min. The cells were then harvested and subjected to Western blotting to detect phosphorylated pSer345 and p47phox. D) MAPK activity is required for s-Mtb-induced superoxide production. BV-2 cells were pretreated with U0126 (10 μM) or SB203580 (5 μM) for 30 min and then incubated with DHE (for superoxide detection) after stimulation with 1% s-Mtb for 30 min. The live cells were washed with serum-free medium and imaged using confocal microscopy. The images are representative of three independent experiments. M, medium only; D, 0.1% DMSO as a solvent control.
Mentions: Phosphorylation of the cytosolic subunit p47phox is necessary for NADPH oxidase activation and regulation [39]. Although p47phox has been detected in cultured microglia [26], its role in MAPK activation and cytokine production in microglia has not been investigated. To examine whether ERK1/2 or p38 activation is dependent on p47phox activation, we examined the effect of wild-type (WT) or dominant-negative (DN) p47phox constructs on p38 and ERK1/2 phosphorylation. Our results showed that ERK1/2 and p38 phosphorylation increased substantially in BV-2 microglia transfected with WT p47phox, whereas phosphorylation was abolished in cells expressing DN p47phox (Fig. 5A). In addition, we pre-treated cells with an inhibitory cell-permeable peptide (TAT-Ser345 peptide) that corresponds to amino acids 339–350 (ARPGPQSPGSPL) of p47phox [40]. In cells treated with the TAT-Ser345 peptide, TNF-α, IL-6, and IL-12p40 production decreased significantly in a dose-dependent manner, whereas the TAT-scramble peptide had little or no inhibitory effect on cytokine production (Fig. 5B). These results suggest that p47phox activation is necessary for MAPK activation and the pro-inflammatory response in microglial cells.

Bottom Line: Furthermore, the activation of cytosolic NADPH oxidase p47phox and MAPKs (p38 and ERK1/2) is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia.Neither TLR2 nor dectin-1 was involved in s-Mtb-induced inflammatory responses in murine microglia.These data collectively demonstrate that s-Mtb actively induces the pro-inflammatory response in microglia through NADPH oxidase-dependent ROS generation, although the specific pattern-recognition receptors involved in these responses remain to be identified.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, College of Medicine, Chungnam National University, Daejeon 301-747, S. Korea. ironwater514@gmail.com

ABSTRACT

Background: Activated microglia elicits a robust amount of pro-inflammatory cytokines, which are implicated in the pathogenesis of tuberculosis in the central nervous system (CNS). However, little is known about the intracellular signaling mechanisms governing these inflammatory responses in microglia in response to Mycobacterium tuberculosis (Mtb).

Methods: Murine microglial BV-2 cells and primary mixed glial cells were stimulated with sonicated Mtb (s-Mtb). Intracellular ROS levels were measured by staining with oxidative fluorescent dyes [2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE)]. NADPH oxidase activities were measured by lucigenin chemiluminescence assay. S-Mtb-induced MAPK activation and pro-inflammatory cytokine release in microglial cells were measured using by Western blot analysis and enzyme-linked immunosorbent assay, respectively.

Results: We demonstrate that s-Mtb promotes the up-regulation of reactive oxygen species (ROS) and the rapid activation of mitogen-activated protein kinases (MAPKs), including p38 and extracellular signal-regulated kinase (ERK) 1/2, as well as the secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-12p40 in murine microglial BV-2 cells and primary mixed glial cells. Both NADPH oxidase and mitochondrial electron transfer chain subunit I play an indispensable role in s-Mtb-induced MAPK activation and pro-inflammatory cytokine production in BV-2 cells and mixed glial cells. Furthermore, the activation of cytosolic NADPH oxidase p47phox and MAPKs (p38 and ERK1/2) is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia. Neither TLR2 nor dectin-1 was involved in s-Mtb-induced inflammatory responses in murine microglia.

Conclusion: These data collectively demonstrate that s-Mtb actively induces the pro-inflammatory response in microglia through NADPH oxidase-dependent ROS generation, although the specific pattern-recognition receptors involved in these responses remain to be identified.

Show MeSH
Related in: MedlinePlus