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Chronic ethanol increases systemic TLR3 agonist-induced neuroinflammation and neurodegeneration.

Qin L, Crews FT - J Neuroinflammation (2012)

Bottom Line: Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment.Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina at Chapel Hill, NC 27599, USA.

ABSTRACT

Background: Increasing evidence links systemic inflammation to neuroinflammation and neurodegeneration. We previously found that systemic endotoxin, a TLR4 agonist or TNFα, increased blood TNFα that entered the brain activating microglia and persistent neuroinflammation. Further, we found that models of ethanol binge drinking sensitized blood and brain proinflammatory responses. We hypothesized that blood cytokines contribute to the magnitude of neuroinflammation and that ethanol primes proinflammatory responses. Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.

Methods: Polyinosine-polycytidylic acid (poly I:C) was used to induce inflammatory responses when sensitized with D-galactosamine (D-GalN). Male C57BL/6 mice were treated with water or ethanol (5 g/kg/day, i.g., 10 days) or poly I:C (250 μg/kg, i.p.) alone or sequentially 24 hours after ethanol exposure. Cytokines, chemokines, microglial morphology, NADPH oxidase (NOX), reactive oxygen species (ROS), high-mobility group box 1 (HMGB1), TLR3 and cell death markers were examined using real-time PCR, ELISA, immunohistochemistry and hydroethidine histochemistry.

Results: Poly I:C increased blood and brain TNFα that peaked at three hours. Blood levels returned within one day, whereas brain levels remained elevated for at least three days. Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment. Ethanol pretreatment potentiated poly I:C-induced brain TNFα (345%), IL-1β (331%), IL-6 (255%), and MCP-1(190%). Increased levels of brain cytokines coincided with increased microglial activation, NOX gp91phox, superoxide and markers of neurodegeneration (activated caspase-3 and Fluoro-Jade B). Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain. Minocycline and naltrexone blocked microglial activation and neurodegeneration.

Conclusions: Chronic ethanol potentiates poly I:C blood and brain proinflammatory responses. Poly I:C neuroinflammation persists after systemic responses subside. Increases in blood TNFα, IL-1β, IL-6, and MCP-1 parallel brain responses consistent with blood cytokines contributing to the magnitude of neuroinflammation. Ethanol potentiation of TLR3 agonist responses is consistent with priming microglia-monocytes and increased NOX, ROS, HMGB1-TLR3 and markers of neurodegeneration. These studies indicate that TLR3 agonists increase blood cytokines that contribute to neurodegeneration and that ethanol binge drinking potentiates these responses.

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Superoxide formation and oxidative stress in brain. Mice were injected with hydroethidine (dihydroethidium, 10 mg/kg, i.p.) 2.5 hours after poly I:C treatment and brains harvested 30 minutes later, frozen and sectioned (15 μm thickness) as described in the methods. The oxidation product, ethidium, is formed from dihydroethidium by superoxide resulting in ethidium accumulation within cells producing superoxide. Ethidium is detected as red nuclei by fluorescence microscopy. The level of fluorescence intensity of ethidium-positive cells was quantified by BioQuant image analysis software. (A) Quantitation of ethidium fluorescence indicates ethanol, poly I:C and ethanol + poly I:C treatment significantly increases O2- and O2--derived oxidant production in cortex. (B) Representative images of ethidium fluorescence. Ethanol and poly I:C alone increased O2- and O2--derived oxidant production compared with vehicle control. Ethanol pretreatment significantly potentiated poly I:C-induced O2- and O2--derived oxidant production. **P <0.01, compared with vehicle control group. ##P <0.01 compared with poly I:C group. Scale bar, 200 μm.
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Figure 7: Superoxide formation and oxidative stress in brain. Mice were injected with hydroethidine (dihydroethidium, 10 mg/kg, i.p.) 2.5 hours after poly I:C treatment and brains harvested 30 minutes later, frozen and sectioned (15 μm thickness) as described in the methods. The oxidation product, ethidium, is formed from dihydroethidium by superoxide resulting in ethidium accumulation within cells producing superoxide. Ethidium is detected as red nuclei by fluorescence microscopy. The level of fluorescence intensity of ethidium-positive cells was quantified by BioQuant image analysis software. (A) Quantitation of ethidium fluorescence indicates ethanol, poly I:C and ethanol + poly I:C treatment significantly increases O2- and O2--derived oxidant production in cortex. (B) Representative images of ethidium fluorescence. Ethanol and poly I:C alone increased O2- and O2--derived oxidant production compared with vehicle control. Ethanol pretreatment significantly potentiated poly I:C-induced O2- and O2--derived oxidant production. **P <0.01, compared with vehicle control group. ##P <0.01 compared with poly I:C group. Scale bar, 200 μm.

Mentions: Activation of NADPH oxidase (NOX) produces superoxide in brain and superoxide formation was assessed in the present experiment by histochemistry using in situ visualization of reactive oxygen species, for example, O2- and O2--derived oxidant production of ethidium from hydroethidine [32,33]. In vehicle-treated mice, there was little to no detection of O2- and O2--derived oxidant production of ethidium (Figure 7). In mice that received either ethanol or poly I:C treatment, there was a significant increase in the production of O2- and O2--derived oxidants 27 hours after the last dose of ethanol or three hours after poly I:C administration. Pretreatment with ethanol increased poly I:C production of O2- and O2--derived oxidants 3 to 4 fold above poly I:C alone and more than 10 fold over controls (Figure 7). These findings indicate that ethanol increases expression of NOX gp91phox and the formation of reactive oxygen species by TLR3 agonist.


Chronic ethanol increases systemic TLR3 agonist-induced neuroinflammation and neurodegeneration.

Qin L, Crews FT - J Neuroinflammation (2012)

Superoxide formation and oxidative stress in brain. Mice were injected with hydroethidine (dihydroethidium, 10 mg/kg, i.p.) 2.5 hours after poly I:C treatment and brains harvested 30 minutes later, frozen and sectioned (15 μm thickness) as described in the methods. The oxidation product, ethidium, is formed from dihydroethidium by superoxide resulting in ethidium accumulation within cells producing superoxide. Ethidium is detected as red nuclei by fluorescence microscopy. The level of fluorescence intensity of ethidium-positive cells was quantified by BioQuant image analysis software. (A) Quantitation of ethidium fluorescence indicates ethanol, poly I:C and ethanol + poly I:C treatment significantly increases O2- and O2--derived oxidant production in cortex. (B) Representative images of ethidium fluorescence. Ethanol and poly I:C alone increased O2- and O2--derived oxidant production compared with vehicle control. Ethanol pretreatment significantly potentiated poly I:C-induced O2- and O2--derived oxidant production. **P <0.01, compared with vehicle control group. ##P <0.01 compared with poly I:C group. Scale bar, 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3412752&req=5

Figure 7: Superoxide formation and oxidative stress in brain. Mice were injected with hydroethidine (dihydroethidium, 10 mg/kg, i.p.) 2.5 hours after poly I:C treatment and brains harvested 30 minutes later, frozen and sectioned (15 μm thickness) as described in the methods. The oxidation product, ethidium, is formed from dihydroethidium by superoxide resulting in ethidium accumulation within cells producing superoxide. Ethidium is detected as red nuclei by fluorescence microscopy. The level of fluorescence intensity of ethidium-positive cells was quantified by BioQuant image analysis software. (A) Quantitation of ethidium fluorescence indicates ethanol, poly I:C and ethanol + poly I:C treatment significantly increases O2- and O2--derived oxidant production in cortex. (B) Representative images of ethidium fluorescence. Ethanol and poly I:C alone increased O2- and O2--derived oxidant production compared with vehicle control. Ethanol pretreatment significantly potentiated poly I:C-induced O2- and O2--derived oxidant production. **P <0.01, compared with vehicle control group. ##P <0.01 compared with poly I:C group. Scale bar, 200 μm.
Mentions: Activation of NADPH oxidase (NOX) produces superoxide in brain and superoxide formation was assessed in the present experiment by histochemistry using in situ visualization of reactive oxygen species, for example, O2- and O2--derived oxidant production of ethidium from hydroethidine [32,33]. In vehicle-treated mice, there was little to no detection of O2- and O2--derived oxidant production of ethidium (Figure 7). In mice that received either ethanol or poly I:C treatment, there was a significant increase in the production of O2- and O2--derived oxidants 27 hours after the last dose of ethanol or three hours after poly I:C administration. Pretreatment with ethanol increased poly I:C production of O2- and O2--derived oxidants 3 to 4 fold above poly I:C alone and more than 10 fold over controls (Figure 7). These findings indicate that ethanol increases expression of NOX gp91phox and the formation of reactive oxygen species by TLR3 agonist.

Bottom Line: Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment.Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bowles Center for Alcohol Studies, School of Medicine, University of North Carolina at Chapel Hill, NC 27599, USA.

ABSTRACT

Background: Increasing evidence links systemic inflammation to neuroinflammation and neurodegeneration. We previously found that systemic endotoxin, a TLR4 agonist or TNFα, increased blood TNFα that entered the brain activating microglia and persistent neuroinflammation. Further, we found that models of ethanol binge drinking sensitized blood and brain proinflammatory responses. We hypothesized that blood cytokines contribute to the magnitude of neuroinflammation and that ethanol primes proinflammatory responses. Here, we investigate the effects of chronic ethanol on neuroinflammation and neurodegeneration triggered by toll-like receptor 3 (TLR3) agonist poly I:C.

Methods: Polyinosine-polycytidylic acid (poly I:C) was used to induce inflammatory responses when sensitized with D-galactosamine (D-GalN). Male C57BL/6 mice were treated with water or ethanol (5 g/kg/day, i.g., 10 days) or poly I:C (250 μg/kg, i.p.) alone or sequentially 24 hours after ethanol exposure. Cytokines, chemokines, microglial morphology, NADPH oxidase (NOX), reactive oxygen species (ROS), high-mobility group box 1 (HMGB1), TLR3 and cell death markers were examined using real-time PCR, ELISA, immunohistochemistry and hydroethidine histochemistry.

Results: Poly I:C increased blood and brain TNFα that peaked at three hours. Blood levels returned within one day, whereas brain levels remained elevated for at least three days. Escalating blood and brain proinflammatory responses were found with ethanol, poly I:C, and ethanol-poly I:C treatment. Ethanol pretreatment potentiated poly I:C-induced brain TNFα (345%), IL-1β (331%), IL-6 (255%), and MCP-1(190%). Increased levels of brain cytokines coincided with increased microglial activation, NOX gp91phox, superoxide and markers of neurodegeneration (activated caspase-3 and Fluoro-Jade B). Ethanol potentiation of poly I:C was associated with ethanol-increased expression of TLR3 and endogenous agonist HMGB1 in the brain. Minocycline and naltrexone blocked microglial activation and neurodegeneration.

Conclusions: Chronic ethanol potentiates poly I:C blood and brain proinflammatory responses. Poly I:C neuroinflammation persists after systemic responses subside. Increases in blood TNFα, IL-1β, IL-6, and MCP-1 parallel brain responses consistent with blood cytokines contributing to the magnitude of neuroinflammation. Ethanol potentiation of TLR3 agonist responses is consistent with priming microglia-monocytes and increased NOX, ROS, HMGB1-TLR3 and markers of neurodegeneration. These studies indicate that TLR3 agonists increase blood cytokines that contribute to neurodegeneration and that ethanol binge drinking potentiates these responses.

Show MeSH
Related in: MedlinePlus