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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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S-Mtb increases pro-inflammatory cytokine production in microglia. A-C) BV-2 cells were stimulated with 0.01, 0.1, or 1% s-Mtb or with 1 μg/ml LPS for the indicated times (0- 96 h). The supernatant was analyzed for cytokine production using ELISA. A significant increase in TNF-α (A), IL-6 (B), and IL-12p40 (C) production was observed. D) TNF-α, IL-6, and IL-12p40 protein levels in primary cultures of mixed glial cells were analyzed after 18 h of s-Mtb stimulation. Values are the mean ± SD of triplicate samples.
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Figure 2: S-Mtb increases pro-inflammatory cytokine production in microglia. A-C) BV-2 cells were stimulated with 0.01, 0.1, or 1% s-Mtb or with 1 μg/ml LPS for the indicated times (0- 96 h). The supernatant was analyzed for cytokine production using ELISA. A significant increase in TNF-α (A), IL-6 (B), and IL-12p40 (C) production was observed. D) TNF-α, IL-6, and IL-12p40 protein levels in primary cultures of mixed glial cells were analyzed after 18 h of s-Mtb stimulation. Values are the mean ± SD of triplicate samples.

Mentions: We examined the microglial production of pro-inflammatory cytokines in response to s-Mtb. Cell cultures were stimulated with different doses of s-Mtb (0.01, 0.1, or 1%), and the supernatant was collected at the indicated intervals for cytokine analysis. S-Mtb-stimulated BV-2 microglial cells produced robust amounts of TNF-α, IL-6, and IL-12p40 in a dose-dependent manner (Fig. 2A–C). Each cytokine had its peak response at 18 h or 48 h, which declined with prolonged treatment. LPS also induced cytokine production, although to a lesser extent than s-Mtb. Cytokine production in primary cultures of mixed glial cells was observed after 18 h of s-Mtb stimulation (Fig. 2D).


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)

S-Mtb increases pro-inflammatory cytokine production in microglia. A-C) BV-2 cells were stimulated with 0.01, 0.1, or 1% s-Mtb or with 1 μg/ml LPS for the indicated times (0- 96 h). The supernatant was analyzed for cytokine production using ELISA. A significant increase in TNF-α (A), IL-6 (B), and IL-12p40 (C) production was observed. D) TNF-α, IL-6, and IL-12p40 protein levels in primary cultures of mixed glial cells were analyzed after 18 h of s-Mtb stimulation. Values are the mean ± SD of triplicate samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2235845&req=5

Figure 2: S-Mtb increases pro-inflammatory cytokine production in microglia. A-C) BV-2 cells were stimulated with 0.01, 0.1, or 1% s-Mtb or with 1 μg/ml LPS for the indicated times (0- 96 h). The supernatant was analyzed for cytokine production using ELISA. A significant increase in TNF-α (A), IL-6 (B), and IL-12p40 (C) production was observed. D) TNF-α, IL-6, and IL-12p40 protein levels in primary cultures of mixed glial cells were analyzed after 18 h of s-Mtb stimulation. Values are the mean ± SD of triplicate samples.
Mentions: We examined the microglial production of pro-inflammatory cytokines in response to s-Mtb. Cell cultures were stimulated with different doses of s-Mtb (0.01, 0.1, or 1%), and the supernatant was collected at the indicated intervals for cytokine analysis. S-Mtb-stimulated BV-2 microglial cells produced robust amounts of TNF-α, IL-6, and IL-12p40 in a dose-dependent manner (Fig. 2A–C). Each cytokine had its peak response at 18 h or 48 h, which declined with prolonged treatment. LPS also induced cytokine production, although to a lesser extent than s-Mtb. Cytokine production in primary cultures of mixed glial cells was observed after 18 h of s-Mtb stimulation (Fig. 2D).

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