{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T02:13:49Z","timestamp":1791512029495,"version":"4.3.4"},"reference-count":53,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2023,8,21]],"date-time":"2023-08-21T00:00:00Z","timestamp":1692576000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000027","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 AA024695"],"award-info":[{"award-number":["R01 AA024695"]}],"id":[{"id":"10.13039\/100000027","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000027","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 AA026665"],"award-info":[{"award-number":["R01 AA026665"]}],"id":[{"id":"10.13039\/100000027","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000027","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 AA027111"],"award-info":[{"award-number":["R01 AA027111"]}],"id":[{"id":"10.13039\/100000027","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Ethanol induces neuroinflammation, which is believed to contribute to the pathogenesis of alcohol use disorder (AUD). Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) expressed on both immune cells, including microglia and astrocytes, and non-immune cells in the central nervous system (CNS). Studies have shown that alcohol activates TLR4 signaling, resulting in the induction of pro-inflammatory cytokines and chemokines in the CNS. However, the effect of alcohol on signaling pathways downstream of TLR4, such as MyD88 and TRIF (TICAM) signaling, has not been evaluated extensively. In the current study, we treated male wild-type, TLR4-, MyD88-, and TRIF-deficient mice using a chronic plus binge mouse model of AUD. Evaluation of mRNA expression by qRT-PCR revealed that ethanol increased IL-1\u03b2, TNF-\u03b1, CCL2, COX2, FosB, and JunB in the cerebellum in wild-type and TRIF-deficient mice, while ethanol generally did not increase the expression of these molecules in TLR4- and MyD88-deficient mice. Furthermore, IRF3, IRF7, and IFN-\u03b21, which are associated with the TRIF-dependent signaling cascade, were largely unaffected by alcohol. Collectively, these results suggest that the TLR4 and downstream MyD88-dependent signaling pathways are essential in ethanol-induced neuroinflammation in this mouse model of AUD.<\/jats:p>","DOI":"10.3390\/cells12162109","type":"journal-article","created":{"date-parts":[[2023,8,21]],"date-time":"2023-08-21T01:38:31Z","timestamp":1692581911000},"page":"2109","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":32,"title":["Ethanol Induces Neuroinflammation in a Chronic Plus Binge Mouse Model of Alcohol Use Disorder via TLR4 and MyD88-Dependent Signaling"],"prefix":"10.3390","volume":"12","author":[{"given":"Kalee N.","family":"Holloway","sequence":"first","affiliation":[{"name":"Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James C.","family":"Douglas","sequence":"additional","affiliation":[{"name":"Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tonya M.","family":"Rafferty","sequence":"additional","affiliation":[{"name":"Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cynthia J. M.","family":"Kane","sequence":"additional","affiliation":[{"name":"Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8984-8099","authenticated-orcid":false,"given":"Paul D.","family":"Drew","sequence":"additional","affiliation":[{"name":"Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA"},{"name":"Department of Neurology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e73","DOI":"10.1016\/j.amepre.2015.05.031","article-title":"2010 National and State Costs of Excessive Alcohol Consumption","volume":"49","author":"Sacks","year":"2015","journal-title":"Am. J. Prev. Med."},{"key":"ref_2","unstructured":"Substance Abuse and Mental Health Services Administration (SAMHSA) CfBHSaQ (2023, May 17). National Survey on Drug Use and Health. Table 5.4A\/B\u2014Alcohol Use Disorder in Past Year among Persons Aged 12 or Older, by Age Group and Demographic Characteristics: Numbers in Thousands, 2018 and 2019, Available online: https:\/\/www.samhsa.gov\/."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"19","DOI":"10.3109\/10408363.2011.580567","article-title":"Molecular and behavioral aspects of the actions of alcohol on the adult and developing brain","volume":"48","author":"Guerri","year":"2011","journal-title":"Crit. Rev. Clin. Lab. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"S80","DOI":"10.1016\/j.bbi.2011.02.012","article-title":"Impact of TLR4 on behavioral and cognitive dysfunctions associated with alcohol-induced neuroinflammatory damage","volume":"25","author":"Pascual","year":"2011","journal-title":"Brain Behav. Immun."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"8285","DOI":"10.1523\/JNEUROSCI.0976-10.2010","article-title":"Pivotal role of TLR4 receptors in alcohol-induced neuroinflammation and brain damage","volume":"30","author":"Blanco","year":"2010","journal-title":"J. Neurosci."},{"key":"ref_6","first-page":"345","article-title":"Effects of chronic and binge ethanol administration on mouse cerebellar and hippocampal neuroinflammation","volume":"49","author":"Douglas","year":"2022","journal-title":"Am. J. Drug Alcohol. Abuse."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"745","DOI":"10.3390\/cells12050745","article-title":"Cerebellar Transcriptomic Analysis in a Chronic plus Binge Mouse Model of Alcohol Use Disorder Demonstrates Ethanol-Induced Neuroinflammation and Altered Glial Gene Expression","volume":"12","author":"Douglas","year":"2023","journal-title":"Cells"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/j.physbeh.2015.02.032","article-title":"Male adolescent rats display blunted cytokine responses in the CNS after acute ethanol or lipopolysaccharide exposure","volume":"148","author":"Gano","year":"2015","journal-title":"Physiol. Behav."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1186\/1742-2094-5-10","article-title":"Increased systemic and brain cytokine production and neuroinflammation by endotoxin following ethanol treatment","volume":"5","author":"Qin","year":"2008","journal-title":"J. Neuroinflamm."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1111\/acer.12244","article-title":"Effects of ethanol on immune response in the brain: Region-specific changes in adolescent versus adult mice","volume":"38","author":"Kane","year":"2014","journal-title":"Alcohol. Clin. Exp. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1016\/j.nbd.2012.12.016","article-title":"Microglial activation is not equivalent to neuroinflammation in alcohol-induced neurodegeneration: The importance of microglia phenotype","volume":"54","author":"Marshall","year":"2013","journal-title":"Neurobiol. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"173390","DOI":"10.1016\/j.pbb.2022.173390","article-title":"Prenatal and adolescent alcohol exposure programs immunity across the lifespan: CNS-mediated regulation","volume":"216","author":"Deak","year":"2022","journal-title":"Pharmacol. Biochem. Behav."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1016\/j.biopsych.2012.09.030","article-title":"High mobility group box 1\/Toll-like receptor danger signaling increases brain neuroimmune activation in alcohol dependence","volume":"73","author":"Crews","year":"2013","journal-title":"Biol. Psychiatry"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3391","DOI":"10.3892\/mmr.2016.4948","article-title":"Role of neuroinflammation in neurodegenerative diseases (Review)","volume":"13","author":"Chen","year":"2016","journal-title":"Mol. Med. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1038\/npp.2016.121","article-title":"Glial and Neuroimmune Mechanisms as Critical Modulators of Drug Use and Abuse","volume":"42","author":"Lacagnina","year":"2017","journal-title":"Neuropsychopharmacology"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1016\/j.conb.2013.01.024","article-title":"Neuroimmune signaling: A key component of alcohol abuse","volume":"23","author":"Mayfield","year":"2013","journal-title":"Curr. Opin. Neurobiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1483","DOI":"10.1007\/s00213-017-4560-6","article-title":"Toll-like receptor signaling and stages of addiction","volume":"234","author":"Crews","year":"2017","journal-title":"Psychopharmacology"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e839","DOI":"10.1002\/iid3.839","article-title":"Toll-like receptors in pathogenesis of neurodegenerative diseases and their therapeutic potential","volume":"11","author":"Dabi","year":"2023","journal-title":"Immun. Inflamm. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"672395","DOI":"10.1155\/2010\/672395","article-title":"DAMPening inflammation by modulating TLR signalling","volume":"2010","author":"Piccinini","year":"2010","journal-title":"Mediat. Inflamm."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1128\/CMR.00046-08","article-title":"Pathogen recognition and inflammatory signaling in innate immune defenses","volume":"22","author":"Mogensen","year":"2009","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.nbd.2013.07.002","article-title":"Increased receptor for advanced glycation end product expression in the human alcoholic prefrontal cortex is linked to adolescent drinking","volume":"59","author":"Vetreno","year":"2013","journal-title":"Neurobiol. Dis."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4733","DOI":"10.4049\/jimmunol.0803590","article-title":"Critical role of TLR4 response in the activation of microglia induced by ethanol","volume":"183","author":"Pascual","year":"2009","journal-title":"J. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"889","DOI":"10.1111\/adb.12539","article-title":"Chronic ethanol consumption: Role of TLR3\/TRIF-dependent signaling","volume":"23","author":"McCarthy","year":"2018","journal-title":"Addict. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/j.bbi.2018.12.004","article-title":"Toll-like receptor 3 activation increases voluntary alcohol intake in C57BL\/6J male mice","volume":"77","author":"Warden","year":"2019","journal-title":"Brain Behav. Immun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1747","DOI":"10.1111\/acer.14669","article-title":"Increased Toll-like Receptor-MyD88-NFkappaB-Proinflammatory neuroimmune signaling in the orbitofrontal cortex of humans with alcohol use disorder","volume":"45","author":"Vetreno","year":"2021","journal-title":"Alcohol. Clin. Exp. Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e12932","DOI":"10.1111\/adb.12932","article-title":"Deletion of Tlr3 reduces acute tolerance to alcohol and alcohol consumption in the intermittent access procedure in male mice","volume":"26","author":"Blednov","year":"2021","journal-title":"Addict. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"173379","DOI":"10.1016\/j.pbb.2022.173379","article-title":"Increased alcohol self-administration following repeated Toll-like receptor 3 agonist treatment in male and female rats","volume":"216","author":"Lovelock","year":"2022","journal-title":"Pharmacol. Biochem. Behav."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1038\/ni.1863","article-title":"The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors","volume":"11","author":"Kawai","year":"2010","journal-title":"Nat. Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"900","DOI":"10.1002\/iub.2510","article-title":"Toll-like receptors in neuroinflammation, neurodegeneration, and alcohol-induced brain damage","volume":"73","author":"Pascual","year":"2021","journal-title":"IUBMB Life"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2085","DOI":"10.1126\/science.282.5396.2085","article-title":"Defective LPS signaling in C3H\/HeJ and C57BL\/10ScCr mice: Mutations in Tlr4 gene","volume":"282","author":"Poltorak","year":"1998","journal-title":"Science"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"619","DOI":"10.4049\/jimmunol.122.2.619","article-title":"Characterization of a congenitally LPS-resistant, athymic mouse strain","volume":"122","author":"Vogel","year":"1979","journal-title":"J. Immunol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1016\/j.immuni.2008.05.016","article-title":"Toll-like receptors activate innate and adaptive immunity by using dendritic cell-intrinsic and -extrinsic mechanisms","volume":"29","author":"Hou","year":"2008","journal-title":"Immunity"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1038\/nature01889","article-title":"Identification of Lps2 as a key transducer of MyD88-independent TIR signalling","volume":"424","author":"Hoebe","year":"2003","journal-title":"Nature"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"948","DOI":"10.1002\/glia.22327","article-title":"Toll-like receptor 4 participates in the myelin disruptions associated with chronic alcohol abuse","volume":"60","author":"Pascual","year":"2012","journal-title":"Glia"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1016\/j.alcohol.2014.08.003","article-title":"TNF-\u03b1 and IL-6 serum levels: Neurobiological markers of alcohol consumption in alcohol-dependent patients?","volume":"48","author":"Heberlein","year":"2014","journal-title":"Alcohol"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.neuropharm.2017.01.031","article-title":"The role of neuroimmune signaling in alcoholism","volume":"122","author":"Crews","year":"2017","journal-title":"Neuropharmacology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1016\/j.biopsych.2014.02.003","article-title":"Role of inflammatory pathways, blood mononuclear cells, and gut-derived bacterial products in alcohol dependence","volume":"76","author":"Leclercq","year":"2014","journal-title":"Biol. Psychiatry"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1016\/j.expneurol.2007.11.017","article-title":"Increased MCP-1 and microglia in various regions of the human alcoholic brain","volume":"210","author":"He","year":"2008","journal-title":"Exp. Neurol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"475","DOI":"10.1016\/j.neuroscience.2012.08.046","article-title":"Adolescent binge drinking increases expression of the danger signal receptor agonist HMGB1 and Toll-like receptors in the adult prefrontal cortex","volume":"226","author":"Vetreno","year":"2012","journal-title":"Neuroscience"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1543","DOI":"10.1007\/s00213-015-3906-1","article-title":"Mechanisms of neuroimmune gene induction in alcoholism","volume":"233","author":"Crews","year":"2016","journal-title":"Psychopharmacology"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"625","DOI":"10.1111\/j.1471-4159.2008.05425.x","article-title":"Ethanol mimics ligand-mediated activation and endocytosis of IL-1RI\/TLR4 receptors via lipid rafts caveolae in astroglial cells","volume":"106","author":"Blanco","year":"2008","journal-title":"J. Neurochem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/j.bbi.2014.11.015","article-title":"TLR4 elimination prevents synaptic and myelin alterations and long-term cognitive dysfunctions in adolescent mice with intermittent ethanol treatment","volume":"45","author":"Montesinos","year":"2015","journal-title":"Brain Behav. Immun."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1016\/j.neuropharm.2014.10.014","article-title":"Cytokines and chemokines as biomarkers of ethanol-induced neuroinflammation and anxiety-related behavior: Role of TLR4 and TLR2","volume":"89","author":"Pascual","year":"2015","journal-title":"Neuropharmacology"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1021\/acschemneuro.2c00413","article-title":"Differential Effects of Nicotine, Alcohol, and Coexposure on Neuroimmune-Related Protein and Gene Expression in Corticolimbic Brain Regions of Rats","volume":"14","author":"Randall","year":"2023","journal-title":"ACS Chem. Neurosci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"60","DOI":"10.31083\/j.jin2203060","article-title":"Salvianolic Acids Alleviate Chronic Mild Stress-Induced Depressive-Like Behaviors in Rats","volume":"22","author":"Zhen","year":"2023","journal-title":"J. Integr. Neurosci."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Teng, T., Yang, H., Xu, T., Sun, G., Song, X., Bai, G., and Shi, B. (2022). Activation of Inflammatory Networks in the Lungs Caused by Chronic Cold Stress Is Moderately Attenuated by Glucose Supplementation. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms231810697"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/j.intimp.2018.03.002","article-title":"Toll-like receptors in immunity and inflammatory diseases: Past, present, and future","volume":"59","author":"Vijay","year":"2018","journal-title":"Int. Immunopharmacol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.lfs.2019.03.030","article-title":"Rosmarinic acid attenuates inflammatory responses through inhibiting HMGB1\/TLR4\/NF-\u03baB signaling pathway in a mouse model of Parkinson's disease","volume":"223","author":"Lv","year":"2019","journal-title":"Life Sci."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1007\/s12975-020-00884-z","article-title":"Attenuation of the Induction of TLRs 2 and 4 Mitigates Inflammation and Promotes Neurological Recovery After Focal Cerebral Ischemia","volume":"12","author":"Nalamolu","year":"2021","journal-title":"Transl. Stroke Res."},{"key":"ref_50","first-page":"3043","article-title":"MicroRNA-451 relieves inflammation in cerebral ischemia-reperfusion via the Toll-like receptor 4\/MyD88\/NF-\u03baB signaling pathway","volume":"20","author":"Li","year":"2019","journal-title":"Mol. Med. Rep."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"107918","DOI":"10.1016\/j.pharmthera.2021.107918","article-title":"TLR4 biased small molecule modulators","volume":"228","author":"Lin","year":"2021","journal-title":"Pharmacol. Ther."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"14246","DOI":"10.1038\/srep14246","article-title":"Discovery of small molecule inhibitors of MyD88-dependent signaling pathways using a computational screen","volume":"5","author":"Olson","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1016\/j.molimm.2014.06.033","article-title":"Toll-like receptor signalling through macromolecular protein complexes","volume":"63","author":"Bryant","year":"2015","journal-title":"Mol. Immunol."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/16\/2109\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:38:13Z","timestamp":1760128693000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/16\/2109"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,21]]},"references-count":53,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2023,8]]}},"alternative-id":["cells12162109"],"URL":"https:\/\/doi.org\/10.3390\/cells12162109","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,8,21]]}}}