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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.

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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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Immunocytochemical analysis of microglia. Mice were treated as described above. (A) Levels of immunoreactive density of Iba1, a marker of microglia, in cortex and hippocampal dentate gyrus were quantified using BioQuant image analysis software and presented as mean ± SEM in pixel/mm2. Ethanol alone, poly I:C alone and ethanol-poly I:C treated groups all show increased Iba1 + IR in both brain regions. (B) Representative images of Iba1 + IR cells in cortex and dentate gyrus from control and ethanol-poly I:C-treated groups. In water control group, microglia showed a resting morphological shape. In either ethanol or poly I:C alone-treated groups, some of the microglia are enlarged (images not shown). Iba1 + IR cells in EtOH-poly I:C-treated mouse brains have increased cell size, irregular shape, and intensified Iba1 staining consistent with morphological changes in activated microglia. Scale bar, 200 μm.
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Figure 4: Immunocytochemical analysis of microglia. Mice were treated as described above. (A) Levels of immunoreactive density of Iba1, a marker of microglia, in cortex and hippocampal dentate gyrus were quantified using BioQuant image analysis software and presented as mean ± SEM in pixel/mm2. Ethanol alone, poly I:C alone and ethanol-poly I:C treated groups all show increased Iba1 + IR in both brain regions. (B) Representative images of Iba1 + IR cells in cortex and dentate gyrus from control and ethanol-poly I:C-treated groups. In water control group, microglia showed a resting morphological shape. In either ethanol or poly I:C alone-treated groups, some of the microglia are enlarged (images not shown). Iba1 + IR cells in EtOH-poly I:C-treated mouse brains have increased cell size, irregular shape, and intensified Iba1 staining consistent with morphological changes in activated microglia. Scale bar, 200 μm.

Mentions: Microglia, the resident innate immune cells in the brain, produce proinflammatory factors that contribute to neurodegeneration through increased proinflammatory superoxide and other toxic agents [6]. In previous studies, we found that the postmortem human alcoholic brain showed increased Iba1 + IR, a microglial marker [25]. We investigated microglial Iba1 + IR to evaluate size and morphological changes of microglia. Control subjects showed resting microglial morphology (Figure 4), with mild increases in Iba1 + IR staining with ethanol or poly I:C alone treatment (images not shown). Ethanol pretreatment further potentiated poly I:C increased Iba1 + IR. Multiple brain regions, including cortex and dentate gyrus, showed increased Iba1 + IR (Figure 4). These studies indicate that ethanol primes microglia and potentiates TLR3 agonist poly I:C activation of microglia.


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

Qin L, Crews FT - J Neuroinflammation (2012)

Immunocytochemical analysis of microglia. Mice were treated as described above. (A) Levels of immunoreactive density of Iba1, a marker of microglia, in cortex and hippocampal dentate gyrus were quantified using BioQuant image analysis software and presented as mean ± SEM in pixel/mm2. Ethanol alone, poly I:C alone and ethanol-poly I:C treated groups all show increased Iba1 + IR in both brain regions. (B) Representative images of Iba1 + IR cells in cortex and dentate gyrus from control and ethanol-poly I:C-treated groups. In water control group, microglia showed a resting morphological shape. In either ethanol or poly I:C alone-treated groups, some of the microglia are enlarged (images not shown). Iba1 + IR cells in EtOH-poly I:C-treated mouse brains have increased cell size, irregular shape, and intensified Iba1 staining consistent with morphological changes in activated microglia. Scale bar, 200 μm.
© Copyright Policy - open-access
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Figure 4: Immunocytochemical analysis of microglia. Mice were treated as described above. (A) Levels of immunoreactive density of Iba1, a marker of microglia, in cortex and hippocampal dentate gyrus were quantified using BioQuant image analysis software and presented as mean ± SEM in pixel/mm2. Ethanol alone, poly I:C alone and ethanol-poly I:C treated groups all show increased Iba1 + IR in both brain regions. (B) Representative images of Iba1 + IR cells in cortex and dentate gyrus from control and ethanol-poly I:C-treated groups. In water control group, microglia showed a resting morphological shape. In either ethanol or poly I:C alone-treated groups, some of the microglia are enlarged (images not shown). Iba1 + IR cells in EtOH-poly I:C-treated mouse brains have increased cell size, irregular shape, and intensified Iba1 staining consistent with morphological changes in activated microglia. Scale bar, 200 μm.
Mentions: Microglia, the resident innate immune cells in the brain, produce proinflammatory factors that contribute to neurodegeneration through increased proinflammatory superoxide and other toxic agents [6]. In previous studies, we found that the postmortem human alcoholic brain showed increased Iba1 + IR, a microglial marker [25]. We investigated microglial Iba1 + IR to evaluate size and morphological changes of microglia. Control subjects showed resting microglial morphology (Figure 4), with mild increases in Iba1 + IR staining with ethanol or poly I:C alone treatment (images not shown). Ethanol pretreatment further potentiated poly I:C increased Iba1 + IR. Multiple brain regions, including cortex and dentate gyrus, showed increased Iba1 + IR (Figure 4). These studies indicate that ethanol primes microglia and potentiates TLR3 agonist poly I:C activation of microglia.

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