TLR4 Deficiency Affects the Microbiome and Reduces Intestinal Dysfunctions and Inflammation in Chronic Alcohol-Fed Mice.

Chronic alcohol abuse causes an inflammatory response in the intestinal tract with damage to the integrity of the mucosa and epithelium, as well as dysbiosis in the gut microbiome. However, the role of gut bacteria in ethanol effects and how these microorganisms interact with the immune system are not well understood. The aim of the present study was to evaluate if TLR4 alters the ethanol-induced intestinal inflammatory response, and whether the response of this receptor affects the gut microbiota profile. We analyzed the 16S rRNA sequence of the fecal samples from wild-type (WT) and TLR4-knockout (TLR4-KO) mice with and without ethanol intake for 3 months. The results demonstrated that chronic ethanol consumption reduces microbiota diversity and causes dysbiosis in WT mice. Likewise, ethanol upregulates several inflammatory genes (IL-1ß, iNOS, TNF-α) and miRNAs (miR-155-5p, miR-146a-5p) and alters structural and permeability genes (INTL1, CDH1, CFTR) in the colon of WT mice. Our results further demonstrated that TLR4-KO mice exhibit a different microbiota that can protect against the ethanol-induced activation of the immune system and colon integrity dysfunctions. In short, our results reveal that TLR4 is a key factor for determining the gut microbiota, which can participate in dysbiosis and the inflammatory response induced by alcohol consumption.

Role of mTOR-regulated autophagy in spine pruning defects and memory impairments induced by binge-like ethanol treatment in adolescent mice.

Adolescence is a brain maturation developmental period during which remodeling and changes in synaptic plasticity and neural connectivity take place in some brain regions. Different mechanism participates in adolescent brain maturation, including autophagy that plays a role in synaptic development and plasticity. Alcohol is a neurotoxic compound and its abuse in adolescence induces neuroinflammation, synaptic and myelin alterations, neural damage and behavioral impairments. Changes in synaptic plasticity and its regulation by mTOR have also been suggested to play a role in the behavioral dysfunction of binge ethanol drinking in adolescence. Therefore, by considering the critical role of mTOR in both autophagy and synaptic plasticity in the developing brain, the present study aims to evaluate whether binge ethanol treatment in adolescence would induce dysfunctions in synaptic plasticity and cognitive functions and if mTOR inhibition with rapamycin is capable of restoring both effects. Using C57BL/6 adolescent female and male mice (PND30) treated with ethanol (3 g/kg) on two consecutive days at 48-hour intervals over 2 weeks, we show that binge ethanol treatment alters the density and morphology of dendritic spines, effects that are associated with learning and memory impairments and changes in the levels of both transcription factor CREB phosphorylation and miRNAs. Rapamycin administration (3 mg/kg) prior to ethanol administration restores ethanol-induced changes in both plasticity and behavior dysfunctions in adolescent mice. These results support the critical role of mTOR/autophagy dysfunctions in the dendritic spines alterations and cognitive alterations induced by binge alcohol in adolescence.

Circulating MicroRNAs in Extracellular Vesicles as Potential Biomarkers of Alcohol-Induced Neuroinflammation in Adolescence: Gender Differences.

Current studies evidence the role of miRNAs in extracellular vesicles (EVs) as key regulators of pathological processes, including neuroinflammation and neurodegeneration. As EVs can cross the blood-brain barrier, and EV miRNAs are very stable in peripheral circulation, we evaluated the potential gender differences in inflammatory-regulated miRNAs levels in human and murine plasma EVs derived from alcohol-intoxicated female and male adolescents, and whether these miRNAs could be used as biomarkers of neuroinflammation. We demonstrated that while alcohol intoxication lowers anti-inflammatory miRNA (mir-146a-5p, mir-21-5p, mir-182-5p) levels in plasma EVs from human and mice female adolescents, these EV miRNAs increased in males. In mice brain cortices, ethanol treatment lowers mir-146a-5p and mir-21-5p levels, while triggering a higher expression of inflammatory target genes (Traf6, Stat3, and Camk2a) in adolescent female mice. These results indicate, for the first time, that female and male adolescents differ as regards the ethanol effects associated with the inflammatory-related plasma miRNAs EVs profile, and suggest that female adolescents are more vulnerable than males to the inflammatory effects of binge alcohol drinking. These findings also support the view that circulating miRNAs in EVs could be useful biomarkers for screening ethanol-induced neuroinflammation and brain damage in adolescence.

Lack of TLR4 modifies the miRNAs profile and attenuates inflammatory signaling pathways.

TLR4 is a member of the toll-like receptors (TLR) immune family, which are activated by lipopolysaccharide, ethanol or damaged tissue, among others, by triggering proinflammatory cytokines release and inflammation. Lack of TLR4 protects against inflammatory processes and neuroinflammation linked with several neuropathologies. By considering that miRNAs are key post-transcriptional regulators of the proteins involved in distinct cellular processes, including inflammation, this study aimed to assess the impact of the miRNAs profile in mice cortices lacking the TLR4 response. Using mice cerebral cortices and next-generation sequencing (NGS), the findings showed that lack of TLR4 significantly reduced the quantity and diversity of the miRNAs expressed in WT mice cortices. The results also revealed a significant down-regulation of the miR-200 family, while cluster miR-99b/let-7e/miR-125a was up-regulated in TLR4-KO vs. WT. The bioinformatics and functional analyses demonstrated that TLR4-KO presented the systematic depletion of many pathways closely related to the immune system response, such as cytokine and interleukin signaling, MAPK and ion Channels routes, MyD88 pathways, NF-κß and TLR7/8 pathways. Our results provide new insights into the molecular and biological processes associated with the protective effects of TLR-KO against inflammatory damage and neuroinflammation, and reveal the relevance of the TLR4 receptors response in many neuropathologies.

TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles.

BACKGROUND: Current evidence indicates that extracellular vesicles (EVs) participate in intercellular signaling, and in the regulation and amplification of neuroinflammation. We have previously shown that ethanol activates glial cells through Toll-like receptor 4 (TLR4) by triggering neuroinflammation. Here, we evaluate if ethanol and the TLR4 response change the release and inflammatory content of astrocyte-derived EVs, and whether these vesicles are capable of communicating with neurons by spreading neuroinflammation. METHODS: Cortical neurons and astrocytes in culture were used. EVs were isolated from the extracellular medium of the primary culture of the WT and TLR4-KO astrocytes treated with or without ethanol (40 mM) for 24 h. Flow cytometry, nanoparticle tracking analysis technology, combined with exosomal molecular markers (tetraspanins) along with electron microscopy, were used to characterize and quantify EVs. The content of EVs in inflammatory proteins, mRNA, and miRNAs was analyzed by Western blot and RT-PCR in both astrocyte-derived EVs and the neurons incubated or not with these EVs. Functional analyses of miRNAs were also performed. RESULTS: We show that ethanol increases the number of secreted nanovesicles and their content by raising the levels of both inflammatory-related proteins (TLR4, NFκB-p65, IL-1R, caspase-1, NLRP3) and by changing miRNAs (mir-146a, mir-182, and mir-200b) in the EVs from the WT-astrocytes compared with those from the untreated WT cells. No changes were observed in either the number of isolated EVs or their content between the untreated and ethanol-treated TLR4-KO astrocytes. We also show that astrocyte-derived EVs could be internalized by naïve cortical neurons to increase the neuronal levels of inflammatory protein (COX-2) and miRNAs (e.g., mir-146a) and to compromise their survival. The functional analysis of miRNAs revealed the regulatory role of the expressed miRNAs in some genes involved in several inflammatory pathways. CONCLUSIONS: These results suggest that astrocyte-derived EVs could act as cellular transmitters of inflammation signaling by spreading and amplifying the neuroinflammatory response induced by ethanol through TLR4 activation.

Disturbed Glucose Metabolism in Rat Neurons Exposed to Cerebrospinal Fluid Obtained from Multiple Sclerosis Subjects.

Axonal damage is widely accepted as a major cause of permanent functional disability in Multiple Sclerosis (MS). In relapsing-remitting MS, there is a possibility of remyelination by myelin producing cells and restoration of neurological function. The purpose of this study was to delineate the pathophysiological mechanisms underpinning axonal injury through hitherto unknown factors present in cerebrospinal fluid (CSF) that may regulate axonal damage, remyelinate the axon and make functional recovery possible. We employed primary cultures of rat unmyelinated cerebellar granule neurons and treated them with CSF obtained from MS and Neuromyelitis optica (NMO) patients. We performed microarray gene expression profiling to study changes in gene expression in treated neurons as compared to controls. Additionally, we determined the influence of gene-gene interaction upon the whole metabolic network in our experimental conditions using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) program. Our findings revealed the downregulated expression of genes involved in glucose metabolism in MS-derived CSF-treated neurons and upregulated expression of genes in NMO-derived CSF-treated neurons. We conclude that factors in the CSF of these patients caused a perturbation in metabolic gene(s) expression and suggest that MS appears to be linked with metabolic deformity.

Bypassing hazard of housekeeping genes: their evaluation in rat granule neurons treated with cerebrospinal fluid of multiple sclerosis subjects.

Gene expression studies employing real-time PCR has become an intrinsic part of biomedical research. Appropriate normalization of target gene transcript(s) based on stably expressed housekeeping genes is crucial in individual experimental conditions to obtain accurate results. In multiple sclerosis (MS), several gene expression studies have been undertaken, however, the suitability of housekeeping genes to express stably in this disease is not yet explored. Recent research suggests that their expression level may vary under different experimental conditions. Hence it is indispensible to evaluate their expression stability to accurately normalize target gene transcripts. The present study aims to evaluate the expression stability of seven housekeeping genes in rat granule neurons treated with cerebrospinal fluid of MS patients. The selected reference genes were quantified by real time PCR and their expression stability was assessed using GeNorm and NormFinder algorithms. GeNorm identified transferrin receptor (Tfrc) and microglobulin beta-2 (B2m) the most stable genes followed by ribosomal protein L19 (Rpl19) whereas ß-actin (ActB) and glyceraldehyde-3-phosphate-dehydrogenase (Gapdh) the most fluctuated ones in these neurons. NormFinder identified Tfrc as the best invariable gene followed by B2m and Rpl19. ActB and Gapdh were the least stable genes as analyzed by NormFinder algorithm. Both methods reported Tfrc and B2m the most stably expressed genes and Gapdh the least stable one. Altogether our data demonstrate the significance of pre-validation of housekeeping genes for accurate normalization and indicates Tfrc and B2m as best endogenous controls in MS. ActB and Gapdh are not recommended in gene expression studies related to current one.

Role of mitochondria ROS generation in ethanol-induced NLRP3 inflammasome activation and cell death in astroglial cells.

Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are innate immunity sensors that provide an early/effective response to pathogenic or injury conditions. We have reported that ethanol-induced TLR4 activation triggers signaling inflammatory responses in glial cells, causing neuroinflammation and brain damage. However, it is uncertain if ethanol is able to activate NLRs/inflammasome in astroglial cells, which is the mechanism of activation, and whether there is crosstalk between both immune sensors in glial cells. Here we show that chronic ethanol treatment increases the co-localization of caspase-1 with GFAP(+) cells, and up-regulates IL-1ß and IL-18 in the frontal medial cortex in WT, but not in TLR4 knockout mice. We further show that cultured cortical astrocytes expressed several inflammasomes (NLRP3, AIM2, NLRP1, and IPAF), although NLRP3 mRNA is the predominant form. Ethanol, as ATP and LPS treatments, up-regulates NLRP3 expression, and causes caspase-1 cleavage and the release of IL-1ß and IL-18 in astrocytes supernatant. Ethanol-induced NLRP3/caspase-1 activation is mediated by mitochondrial (m) reactive oxygen species (ROS) generation because when using a specific mitochondria ROS scavenger, the mito-TEMPO (500 µM) or NLRP3 blocking peptide (4 µg/ml) or a specific caspase-1 inhibitor, Z-YVAD-FMK (10 µM), abrogates mROS release and reduces the up-regulation of IL-1ß and IL-18 induced by ethanol or LPS or ATP. Confocal microscopy studies further confirm that ethanol, ATP or LPS promotes NLRP3/caspase-1 complex recruitment within the mitochondria to promote cell death by caspase-1-mediated pyroptosis, which accounts for ≈73% of total cell death (≈22%) and the remaining (≈25%) die by caspase-3-dependent apoptosis. Suppression of the TLR4 function abrogates most ethanol effects on NLRP3 activation and reduces cell death. These findings suggest that NLRP3 participates, in ethanol-induced neuroinflammation and highlight the NLRP3/TLR4 crosstalk in ethanol-induced brain injury.