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Ethanol-Induced TLR4/NLRP3 Neuroinflammatory Response in Microglial Cells Promotes Leukocyte Infiltration Across the BBB

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Abstract

We reported that the ethanol-induced innate immune response by activating TLR4 signaling triggers gliosis and neuroinflammation. Ethanol also activates other immune receptors, such as NOD-like-receptors, and specifically NLRP3-inflammasome in astroglial cells, to stimulate caspase-1 cleavage and IL-1β and IL-18 cytokines production. Yet, whether microglia NLRs are also sensitive to the ethanol effects that contribute to neuroinflammation is uncertain. Using cerebral cortexes of the chronic alcohol-fed WT and TLR4−/− mice, we demonstrated that chronic ethanol treatment enhanced TLR4 mediated-NLRP3/Caspase-1 complex activation, and up-regulated pro-inflammatory cytokines and chemokines levels. Ethanol-induced NLRP3-inflammasome activation and mitochondria-ROS generation were also observed in cultured microglial cells. The up-regulation of CD45high/CD11b+ cell populations and matrix metalloproteinase-9 levels was also noted in the cortexes of the ethanol-treated WT mice. Notably, elimination of the TLR4 function abolished most ethanol-induced neuroinflammatory effects. Thus, our results demonstrate that ethanol triggers TLR4-mediated NLRP3-inflammasome activation in glial cells, and suggest that microglia stimulation may compromise the permeability of blood–brain barrier events to contribute to ethanol-induced neuroinflammation and brain damage.

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Acknowledgments

We would like to thank Alberto Hernandez and Alicia Martinez from the Confocal Microscopy and Flow Cytometry Services at Prince Felipe Research Center, respectively. We are thankful to Dr. S. Akira who provided us with the TLR4−/− knockout mice. This work has been supported by grants from the Spanish Ministry of Science and Innovation (SAF 2009-07503, SAF2012-33747), ERAB (EA 13 08), the Carlos III Institute and FEDER funds (RTA-Network, RD2012/0028/0007 G0/005), PNSD (2014I010), GV-Consellería de Educación (ACOMP2014, 2015) and PROMETEO/2014.

Author contributions

SAL designed, performed and analyzed the experimental work results. JUP participated in the immunohistochemistry and RT-PCR analyses. MJM helped with the RT-PCR analyses and western blot. UGP performed experiments and analyses of microglial activation. CG designed the experiments and corrected the final version of the manuscript. All the authors read and approved the final manuscript.

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Authors and Affiliations

  1. Molecular and Cellular Pathology of Alcohol, Prince Felipe Research Center, Eduardo Primo Yúfera 3, 46012, Valencia, Spain

    Silvia Alfonso-Loeches, Juan Ureña-Peralta, Mª José Morillo-Bargues & Consuelo Guerri

  2. Department of Regenerative Medicine/Neurology and Neurosurgery, Neuroscience Institute, Clinic San Carlos Hospital, IdISSC, Profesor Martín Lagos, S/N, 28040, Madrid, Spain

    Ulises Gómez-Pinedo

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  1. Silvia Alfonso-Loeches
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  2. Juan Ureña-Peralta
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Correspondence to Consuelo Guerri.

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Special Issue: 40th year of the Neurochemical Research.

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Alfonso-Loeches, S., Ureña-Peralta, J., Morillo-Bargues, M.J. et al. Ethanol-Induced TLR4/NLRP3 Neuroinflammatory Response in Microglial Cells Promotes Leukocyte Infiltration Across the BBB. Neurochem Res 41, 193–209 (2016). https://doi.org/10.1007/s11064-015-1760-5

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