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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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Activated caspase-3 + IR in brain. Brain sections were stained with polyclonal cleave caspase-3 (Asp175) antibody, a marker of cell death. (A) Quantitation of caspase-3 + IR in cortex. The number of caspase-3 + IR cells in cortex was increased by ethanol, poly I:C and sequential ethanol-poly I:C. (B) Quantitation of caspase-3 + IR in hippocampal dentate gyrus. The number of caspase-3 + IR cells in dentate gyrus was increased by ethanol, poly I:C and sequential ethanol-poly I:C. The results are the means ± SEM of two independent experiments performed with seven mice per group. *P <0.05, **P <0.01, compared with vehicle control. ##P <0.01, compared with poly I:C. (C and D) Representative images of caspase-3 + IR in cortex (C) and dentate gyrus (D) in vehicle control and ethanol-poly I:C groups. Scale bar, 200 μm. To determine if caspase-3 + IR was within neurons, brain sections were double-stained with NeuN (a neuronal marker). (E) Confocal microscopy images of cortex (upper panels) and dentate gyrus (lower panels) in ethanol-poly I:C group. Immunolabeling was visualized by using Alexa Fluor 488 and 555. Confocal microscopy indicates that caspase-3 + IR cells in green (left panels) are NeuN positive in red (middle panels), as shown in the merged images (right panels) with arrows indicating yellow co-labeling of caspase-3 and NeuN. Insets are higher magnification of the merged images. Scale bar, 30 μm; inset 5 μm.
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Figure 9: Activated caspase-3 + IR in brain. Brain sections were stained with polyclonal cleave caspase-3 (Asp175) antibody, a marker of cell death. (A) Quantitation of caspase-3 + IR in cortex. The number of caspase-3 + IR cells in cortex was increased by ethanol, poly I:C and sequential ethanol-poly I:C. (B) Quantitation of caspase-3 + IR in hippocampal dentate gyrus. The number of caspase-3 + IR cells in dentate gyrus was increased by ethanol, poly I:C and sequential ethanol-poly I:C. The results are the means ± SEM of two independent experiments performed with seven mice per group. *P <0.05, **P <0.01, compared with vehicle control. ##P <0.01, compared with poly I:C. (C and D) Representative images of caspase-3 + IR in cortex (C) and dentate gyrus (D) in vehicle control and ethanol-poly I:C groups. Scale bar, 200 μm. To determine if caspase-3 + IR was within neurons, brain sections were double-stained with NeuN (a neuronal marker). (E) Confocal microscopy images of cortex (upper panels) and dentate gyrus (lower panels) in ethanol-poly I:C group. Immunolabeling was visualized by using Alexa Fluor 488 and 555. Confocal microscopy indicates that caspase-3 + IR cells in green (left panels) are NeuN positive in red (middle panels), as shown in the merged images (right panels) with arrows indicating yellow co-labeling of caspase-3 and NeuN. Insets are higher magnification of the merged images. Scale bar, 30 μm; inset 5 μm.

Mentions: To investigate the relationship among proinflammatory gene induction, microglial activation, oxidative stress, and neuronal cell death, we assessed the cell death markers, activated caspase-3 and Fluoro-Jade B. Ethanol, poly I:C and sequential ethanol-poly I:C increased caspase-3 + IR cells in both cortex and hippocampus (Figure 9). Ethanol-poly I:C caused significantly greater increases in caspase-3 + IR cells than either alone. Double immunohistochemistry with confocal microscopy revealed that caspase-3 + IR was colocalized with NeuN, a neuronal marker, in both cortex and hippocampus (Figure 9E), suggesting neuronal cell death. Fluoro-Jade B, another cell death marker, was also increased by ethanol, poly I:C and sequential ethanol-poly I:C treatment in both cortex and hippocampus (Figure 10). Pretreatment with ethanol more than doubled poly I:C increases in Fluoro-Jade B staining, compared to poly I:C alone group (Figure 10A). Confocal microscopy indicated that most Fluoro-Jade B-positive cells were colocalized with NeuN + IR in both cortex and hippocampal dentate gyrus (Figure 10B). These results indicate that markers of neuronal death are increased by ethanol, poly I:C and sequential ethanol-poly I:C in parallel with induction of neuroinflammatory genes and oxidative stress.


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

Qin L, Crews FT - J Neuroinflammation (2012)

Activated caspase-3 + IR in brain. Brain sections were stained with polyclonal cleave caspase-3 (Asp175) antibody, a marker of cell death. (A) Quantitation of caspase-3 + IR in cortex. The number of caspase-3 + IR cells in cortex was increased by ethanol, poly I:C and sequential ethanol-poly I:C. (B) Quantitation of caspase-3 + IR in hippocampal dentate gyrus. The number of caspase-3 + IR cells in dentate gyrus was increased by ethanol, poly I:C and sequential ethanol-poly I:C. The results are the means ± SEM of two independent experiments performed with seven mice per group. *P <0.05, **P <0.01, compared with vehicle control. ##P <0.01, compared with poly I:C. (C and D) Representative images of caspase-3 + IR in cortex (C) and dentate gyrus (D) in vehicle control and ethanol-poly I:C groups. Scale bar, 200 μm. To determine if caspase-3 + IR was within neurons, brain sections were double-stained with NeuN (a neuronal marker). (E) Confocal microscopy images of cortex (upper panels) and dentate gyrus (lower panels) in ethanol-poly I:C group. Immunolabeling was visualized by using Alexa Fluor 488 and 555. Confocal microscopy indicates that caspase-3 + IR cells in green (left panels) are NeuN positive in red (middle panels), as shown in the merged images (right panels) with arrows indicating yellow co-labeling of caspase-3 and NeuN. Insets are higher magnification of the merged images. Scale bar, 30 μm; inset 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 9: Activated caspase-3 + IR in brain. Brain sections were stained with polyclonal cleave caspase-3 (Asp175) antibody, a marker of cell death. (A) Quantitation of caspase-3 + IR in cortex. The number of caspase-3 + IR cells in cortex was increased by ethanol, poly I:C and sequential ethanol-poly I:C. (B) Quantitation of caspase-3 + IR in hippocampal dentate gyrus. The number of caspase-3 + IR cells in dentate gyrus was increased by ethanol, poly I:C and sequential ethanol-poly I:C. The results are the means ± SEM of two independent experiments performed with seven mice per group. *P <0.05, **P <0.01, compared with vehicle control. ##P <0.01, compared with poly I:C. (C and D) Representative images of caspase-3 + IR in cortex (C) and dentate gyrus (D) in vehicle control and ethanol-poly I:C groups. Scale bar, 200 μm. To determine if caspase-3 + IR was within neurons, brain sections were double-stained with NeuN (a neuronal marker). (E) Confocal microscopy images of cortex (upper panels) and dentate gyrus (lower panels) in ethanol-poly I:C group. Immunolabeling was visualized by using Alexa Fluor 488 and 555. Confocal microscopy indicates that caspase-3 + IR cells in green (left panels) are NeuN positive in red (middle panels), as shown in the merged images (right panels) with arrows indicating yellow co-labeling of caspase-3 and NeuN. Insets are higher magnification of the merged images. Scale bar, 30 μm; inset 5 μm.
Mentions: To investigate the relationship among proinflammatory gene induction, microglial activation, oxidative stress, and neuronal cell death, we assessed the cell death markers, activated caspase-3 and Fluoro-Jade B. Ethanol, poly I:C and sequential ethanol-poly I:C increased caspase-3 + IR cells in both cortex and hippocampus (Figure 9). Ethanol-poly I:C caused significantly greater increases in caspase-3 + IR cells than either alone. Double immunohistochemistry with confocal microscopy revealed that caspase-3 + IR was colocalized with NeuN, a neuronal marker, in both cortex and hippocampus (Figure 9E), suggesting neuronal cell death. Fluoro-Jade B, another cell death marker, was also increased by ethanol, poly I:C and sequential ethanol-poly I:C treatment in both cortex and hippocampus (Figure 10). Pretreatment with ethanol more than doubled poly I:C increases in Fluoro-Jade B staining, compared to poly I:C alone group (Figure 10A). Confocal microscopy indicated that most Fluoro-Jade B-positive cells were colocalized with NeuN + IR in both cortex and hippocampal dentate gyrus (Figure 10B). These results indicate that markers of neuronal death are increased by ethanol, poly I:C and sequential ethanol-poly I:C in parallel with induction of neuroinflammatory genes and oxidative stress.

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