Limits...
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.

Show MeSH

Related in: MedlinePlus

Confocal microscopy with cell specific markers finds neuronal and microglial expression of NADPH oxidase subunit gp91phox. Brain sections from ethanol-poly I:C-treated mice were double-labeled for gp91phox in green with neuronal marker MAP-2, microglial marker Iba1, or astroglial marker GFAP in red. Co-labeling was investigated using a Leica SP2 LCS confocal microscope with associated software. The representative images shown are from dentate gyrus of mice treated with ethanol-poly I:C. The left panel of pictures shows gp91phox + IR. The middle panel shows cell specific markers, for example, neuronal MAP-2 (upper panel), microglial Iba1 (middle) and astrocyte GFAP (lower panel) pictures. Merged images are to the right. Merged yellow indicates red and green are combined and likely co-localized within the marked cell. Merged pictures on the right with enlarged cells suggest that gp91phox + IR is expressed in MAP-2 neurons (yellow) and Iba1 microglia (yellow), but not in astrocytes. Scale bar, 30 μm; inset 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3412752&req=5

Figure 6: Confocal microscopy with cell specific markers finds neuronal and microglial expression of NADPH oxidase subunit gp91phox. Brain sections from ethanol-poly I:C-treated mice were double-labeled for gp91phox in green with neuronal marker MAP-2, microglial marker Iba1, or astroglial marker GFAP in red. Co-labeling was investigated using a Leica SP2 LCS confocal microscope with associated software. The representative images shown are from dentate gyrus of mice treated with ethanol-poly I:C. The left panel of pictures shows gp91phox + IR. The middle panel shows cell specific markers, for example, neuronal MAP-2 (upper panel), microglial Iba1 (middle) and astrocyte GFAP (lower panel) pictures. Merged images are to the right. Merged yellow indicates red and green are combined and likely co-localized within the marked cell. Merged pictures on the right with enlarged cells suggest that gp91phox + IR is expressed in MAP-2 neurons (yellow) and Iba1 microglia (yellow), but not in astrocytes. Scale bar, 30 μm; inset 5 μm.

Mentions: NADPH oxidase (NOX) is a family of oxidases known to produce superoxide and NOX is thought to be involved in neurodegeneration [40]. To determine the role of NOX in TLR3 agonist proinflammatory responses, we investigated the expression of NOX gp91phox, the catalytic subunit of phagocytic oxidase commonly associated with proinflammatory responses. Poly I:C induced NOX gp91phox mRNA 2 to 3 fold (Figure 5A) and NOX gp91phox + IR by manyfold more in cortex and hippocampal dentate gyrus (Figure 5B). Ethanol treatment produced a non-significant trend toward an increase in NOX gp91 mRNA, but did increase gp91phox + IR above control levels to about half of that found with poly I:C alone. Ethanol potentiated the poly I:C TLR3 responses with both NOX gp91phox mRNA and NOX gp91phox + IR increased in cortex and hippocampus (Figure 5). Double antibody studies with cell specific markers and confocal microscopy indicate that ethanol-poly I:C-induced NOX gp91phox + IR is colocalized with neuronal marker (MAP-2) and a microglial marker (Iba1) but there is little colocalization with astrocytic marker (GFAP) (Figure 6). These results indicate that TLR3 activation increases brain NOX gp91phox.


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

Qin L, Crews FT - J Neuroinflammation (2012)

Confocal microscopy with cell specific markers finds neuronal and microglial expression of NADPH oxidase subunit gp91phox. Brain sections from ethanol-poly I:C-treated mice were double-labeled for gp91phox in green with neuronal marker MAP-2, microglial marker Iba1, or astroglial marker GFAP in red. Co-labeling was investigated using a Leica SP2 LCS confocal microscope with associated software. The representative images shown are from dentate gyrus of mice treated with ethanol-poly I:C. The left panel of pictures shows gp91phox + IR. The middle panel shows cell specific markers, for example, neuronal MAP-2 (upper panel), microglial Iba1 (middle) and astrocyte GFAP (lower panel) pictures. Merged images are to the right. Merged yellow indicates red and green are combined and likely co-localized within the marked cell. Merged pictures on the right with enlarged cells suggest that gp91phox + IR is expressed in MAP-2 neurons (yellow) and Iba1 microglia (yellow), but not in astrocytes. Scale bar, 30 μm; inset 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Confocal microscopy with cell specific markers finds neuronal and microglial expression of NADPH oxidase subunit gp91phox. Brain sections from ethanol-poly I:C-treated mice were double-labeled for gp91phox in green with neuronal marker MAP-2, microglial marker Iba1, or astroglial marker GFAP in red. Co-labeling was investigated using a Leica SP2 LCS confocal microscope with associated software. The representative images shown are from dentate gyrus of mice treated with ethanol-poly I:C. The left panel of pictures shows gp91phox + IR. The middle panel shows cell specific markers, for example, neuronal MAP-2 (upper panel), microglial Iba1 (middle) and astrocyte GFAP (lower panel) pictures. Merged images are to the right. Merged yellow indicates red and green are combined and likely co-localized within the marked cell. Merged pictures on the right with enlarged cells suggest that gp91phox + IR is expressed in MAP-2 neurons (yellow) and Iba1 microglia (yellow), but not in astrocytes. Scale bar, 30 μm; inset 5 μm.
Mentions: NADPH oxidase (NOX) is a family of oxidases known to produce superoxide and NOX is thought to be involved in neurodegeneration [40]. To determine the role of NOX in TLR3 agonist proinflammatory responses, we investigated the expression of NOX gp91phox, the catalytic subunit of phagocytic oxidase commonly associated with proinflammatory responses. Poly I:C induced NOX gp91phox mRNA 2 to 3 fold (Figure 5A) and NOX gp91phox + IR by manyfold more in cortex and hippocampal dentate gyrus (Figure 5B). Ethanol treatment produced a non-significant trend toward an increase in NOX gp91 mRNA, but did increase gp91phox + IR above control levels to about half of that found with poly I:C alone. Ethanol potentiated the poly I:C TLR3 responses with both NOX gp91phox mRNA and NOX gp91phox + IR increased in cortex and hippocampus (Figure 5). Double antibody studies with cell specific markers and confocal microscopy indicate that ethanol-poly I:C-induced NOX gp91phox + IR is colocalized with neuronal marker (MAP-2) and a microglial marker (Iba1) but there is little colocalization with astrocytic marker (GFAP) (Figure 6). These results indicate that TLR3 activation increases brain NOX gp91phox.

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