Microglial-derived miRNA let-7 and HMGB1 contribute to ethanol-induced neurotoxicity via TLR7
© The Author(s). 2017
Received: 2 October 2016
Accepted: 16 January 2017
Published: 25 January 2017
Toll-like receptor (TLR) signaling is emerging as an important component of neurodegeneration. TLR7 senses viral RNA and certain endogenous miRNAs to initiate innate immune responses leading to neurodegeneration. Alcoholism is associated with hippocampal degeneration, with preclinical studies linking ethanol-induced neurodegeneration with central innate immune induction and TLR activation. The endogenous miRNA let-7b binds TLR7 to cause neurodegeneration.
TLR7 and other immune markers were assessed in postmortem human hippocampal tissue that was obtained from the New South Wales Tissue Bank. Rat hippocampal-entorhinal cortex (HEC) slice culture was used to assess specific effects of ethanol on TLR7, let-7b, and microvesicles.
We report here that hippocampal tissue from postmortem human alcoholic brains shows increased expression of TLR7 and increased microglial activation. Using HEC slice culture, we found that ethanol induces TLR7 and let-7b expression. Ethanol caused TLR7-associated neuroimmune gene induction and initiated the release let-7b in microvesicles (MVs), enhancing TLR7-mediated neurotoxicity. Further, ethanol increased let-7b binding to the danger signaling molecule high mobility group box-1 (HMGB1) in MVs, while reducing let-7 binding to classical chaperone protein argonaute (Ago2). Flow cytometric analysis of MVs from HEC media and analysis of MVs from brain cell culture lines found that microglia were the primary source of let-7b and HMGB1-containing MVs.
Our results identify that ethanol induces neuroimmune pathology involving the release of let-7b/HMGB1 complexes in microglia-derived microvesicles. This contributes to hippocampal neurodegeneration and may play a role in the pathology of alcoholism.
KeywordsAlcohol Neurodegeneration Toll-like receptor 7 Microvesicles Amphoterin Micro-RNA
The role of Toll-like receptors (TLRs) in innate immunity has recently been illuminated. TLRs recognize damage-associated molecular pattern molecules (DAMPs) to initiate innate immune signaling cascades. In the CNS, however, TLRs function not only as agents of immunity, but regulate neuronal morphology, pain, and neurodegeneration through recognition of endogenous agonists [1–3]. TLR7 is an endosomal TLR that recognizes endogenous micro-RNAs (miRs), single-stranded RNAs (ss), and short interfering (si) RNAs . TLR7 signaling can lead to activation of transcription factors IRF7 or NFκB and cause either neuroimmune responses or neurodegeneration [4–7]. The endogenous TLR7 agonist miR let-7 has been found to cause neurodegeneration . let-7b in particular has a GU-rich region that is readily recognized by TLR7. Studies utilizing miRNA profiling find increased expression of several let-7 isoforms in human and rodent brain after chronic alcohol [8, 9].
Both neuroimmune activation and neurodegeneration are inseparable in many CNS disorders including alcoholism , Parkinson’s [11, 12], and Alzheimer’s disease [13, 14]. Alcoholism is associated with progressive neurodegeneration throughout the brain including the hippocampus and cortex [15–17]. Neuroimmune activation precedes and exacerbates neurodegeneration [18–20]. Ethanol activates the neuroimmune system through TLR activation and the release of endogenous DAMPs, such as the TLR4 agonist high mobility group box-1 (HMGB1). Studies of postmortem human alcoholic brains, in vivo rodent studies, and in vitro hippocampal-entorhinal brain slice culture (HEC) show that ethanol increases the cortical expression of TLRs 2-4, HMGB1 [10, 21, 22]. Peripherally, ethanol causes the release of microvesicles (MVs) into the circulation that contain pro-inflammatory miRNAs [23, 24]. Since TLR7 activation causes both innate immune activation and neurodegeneration, we hypothesized that ethanol would activate TLR7 signaling leading to innate immune gene induction. We further hypothesized that ethanol would cause the release of the endogenous TLR7 agonist miR let-7b in MVs. HMGB1 is a nucleic acid binding protein that is released from neurons by ethanol [21, 22]. HMGB1 is required for immune responses to TLR7 agonists  and acts as a chaperone for DNA or cytokines potentiating their function through their native receptors [26–28]. HMGB1 is released in MVs and ethanol increases expression and active secretion of HMGB1 from the brain [22, 29]. Thus, we hypothesized that HMGB1 would bind miR let-7b in MVs to help facilitate its activity as an endogenous agonist of TLR7.
We report here in postmortem human alcoholic hippocampal brain tissue that TLR7, HMGB1, and the microglia activation marker CD11b are increased. Interestingly, TLR7 expression in human hippocampus correlated with lifetime alcohol intake, suggesting a role in the pathology of the disease. In rat HEC brain slice culture, we found that ethanol increases TLR7, HMGB1, IL-1β, TNFα, and let-7b consistent with findings in human alcoholics. Concomitant with the increase in TLR7, ethanol also increases the release of let-7b and HMGB1 in MVs and potentiates let-7b induced neurodegeneration via TLR7 activation. Using RNA immuno-purification followed by RT-PCR (RIP assay), we found that HMGB1 forms heterodimeric complexes with let-7b in MVs in response to ethanol. Using flow cytometry for MVs from HEC slice culture and analysis of SH-SY5Y neuronal and BV2 microglia cell lines, we found that the majority of microvesicular HMGB1 and let-7b are derived from microglia. Thus, we report here the identification of a novel inter-cellular communication mechanism in the pathology of alcohol abuse, whereby ethanol causes the release of HMGB1-let-7 complexes in MV from microglia.
The following reagents were purchased from Sigma-Aldrich (St Louis, USA): imiquimod (I5159) and glycyrrhizin (G2137). HMGB1 ELISA kit was purchased from IBL International (Hamburg, Germany); primary antibodies from Novus Biologicals: Na+/K+ ATPase α3 (NB300-540APC), GFAP (NBP2-34401 V2), CD11b (NBP2-34678PE), and HMGB1 (NB100-2322AF488); and primary antibodies from Abcam: CD11b for western blot (ab75476), argonaute 2 (ab32381).
Hippocampal-entorhinal cortex slice culture
All protocols followed in this study were approved by the Institutional Animal Care Use Committee at UNC and were in accordance with National Institute of Health regulation for the care and use of animal in research. Organotypic brain slice cultures are prepared as described previously . Briefly, the hippocampal entorhinal region is dissected and sliced transversely from post-natal day 7 rat pups. Slices are 375 μm thick. HEC slices were placed onto tissue insert membrane (10 slices/insert) and cultured with medium containing 75% MEM with 25 mM HEPES and Hank’s salts, 25% horse serum (HS), 5.5 g/L glucose, 2 mM L-glutamine in a humidified 5% CO2 incubator at 36.5 °C for 7 days in vitro (DIV), followed by 4 DIV in medium containing 12.5% HS and then 3 DIV in serum-free medium supplemented with N2. The cultures after 14 DIV were used for experiments and drug treatments with serum-free N2-supplemented medium. For ethanol exposures, slices were exposed to ethanol (25–100 mM) for 48 h. For let-7b mimic exposure studies, slices were treated with DOTAP, DOTAP plus let-7b mimic, or let-7b mimic plus ethanol for 48 h. For TLR7 agonist enhancement studies, slices were treated with either 500 ng/mL of imiquimod (IMQ) or vehicle for 48 h, followed by addition of either ethanol (100 mM) or vehicle for 96 h.
Immunofluorescent staining and analysis
HEC slice cultures were removed at the end of the experiment and fixed with 4% paraformaldehyde with 5% sucrose in 0.01 M PBS for 24 h at 4 °C. All primary antibodies were incubated for 48 h at 4 °C. Either Alexa Fluor 594 or Alexa Fluor 488 secondary antibodies (1:2000; Molecular Probes, Eugene, OR) were used for immunofluorescent staining and incubated for 1 h at room temperature. The slices were coverslipped with anti-fade mounting medium (pro-long; Molecular Probes). Images were obtained using a LeicaSP2 AOBS Upright Laser Scanning Confocal in Michael Hooker Microscopy Facility (University of North Carolina, Chapel Hill, NC). Fluorescent pixel density analysis was performed using ImageJTM software.
Neuronal and microglial cell line experiments
SH-SY5Y and BV2 cells were allowed to grow in culture as described previously in standard cell culture conditions [30, 31]. For BV2 microglia and SH-SY5Y neuronal experiments, 3 × 105 cells/well were plated on 6-well CorningTM cell culture plates. Cells were allowed to adhere overnight, and the next morning, they were treated with ethanol (100 mM) for 24 h.
Postmortem human alcoholic analyses
Frozen postmortem human hippocampal tissue was obtained from the New South Wales Brain Tissue Bank in Sidney Australia as described previously . Approximately 100 mg of tissue was provided for each subject. Given the small fragments of tissue, morphological assessment was not possible. Individuals with comorbid liver cirrhosis or nutritional deficiencies were excluded. The leading common cause of death was cardiovascular disease for both groups (16/18). Postmortem intervals were also documented and did not correlate with mRNA measurements. Psychiatric and alcohol use disorder diagnoses were confirmed using the Diagnostic Instrument for Brain Studies, which is in compliance with the Diagnostic Statistical Manual of Mental Disorders . Details for each subject are provided in Table 2.
Isolation of mRNA, miRNA, and quantification via RT-PCR
Primers used for mRNA and miRNA quantification by RT-PCR
Forward (5′ to 3′)
Reverse (5′ to 3′)
MVs are isolated by sequential centrifugation as described previously . Briefly, media was centrifuged at 2000g for 20 min to remove cells. Supernatant was then centrifuged at 10,000g for 30 min to remove cellular debris. Remaining supernatant was then centrifuged at 21,000g for 1 h. The MV-containing pellet was washed in PBS and centrifuged again at 21,000g. The MV pellet was suspended in the appropriate buffer for analysis. This preparation results in MVs ranging between 100 nm and 1 μm in diameter.
ELISA measurements of HMGB1
Media HMGB1 levels were determined from undiluted media by ELISA (IBL, Germany) according to the manufacturer’s instruction. Tissue levels of HMGB1 from human hippocampus were measured by ELISA. Samples were first treated with perchloric acid (BioVision catalog #K808) to separate HMGB1 from its binding partners as previously described . Purified supernatant was then assessed by ELISA at a dilution of 1:25.
Flow cytometric analysis of microvesicle cellular origin
Composition and cell origin of MVs was done as described [35, 36]. Briefly, MVs were permeabilized with Fix/Perm buffer (Biolegend), incubated with Fc blocking buffer (Biolegend), and labeled using antibodies to HMGB1, GFAP (astrocytes), Na+/K+ ATPase α3 (neurons), and CD11b (microglia). Samples are incubated with fluorescent secondary antibodies when appropriate. The Stratedigm S1000Ex was used to assess the stained MVs at the UNC Flow Cytometry Core Facility. Size gates to identify MVs (0.1–1.0 μm) were set using MegaMixTM (BioCytex) size gating beads (Additional file 1: Figure S1A). Single color controls for each primary antibody, compared to unstained media, were used to develop the compensation matrix and distinguish background staining from specific staining using FloJo TM software version 10.0 (Additional file 1: Figure S1B). Approximately 5% of the MVs stained positive for lactadherin, which binds phosphotidyl-serine (PS). Of the PS+ MVs, ethanol increased HMGB1 in a dose-dependent fashion up to 123% of controls at 75 mM (not shown).
Media MVs were isolated by centrifugation as described above. The MV pellet was lysed in 0.1% Triton X-100 containing buffer. MV HMGB1 was immunopurified using Dynabeads® M-270 Epoxy (ThermoFisher 14321D) according to the manufacturer’s instructions as described . Briefly, Dynabeads were coupled overnight to either an anti-HMGB1 antibody (Abcam ab18256) or anti-Ago2 (ab32381). MV protein was incubated with anti-HMGB1 coupled Dynabeads overnight at 4 °C. HMGB1 was then eluted from the Dynabeads and incubated in TrizolTM buffer. Micro-RNA isolation was performed as above, with the same amount of total mRNA assessed for RT-PCR per sample. let-7b, miR-155, and miR181c were assessed as described above.
Human brain tissue was homogenized and sonicated in Tris lysis buffer containing 7.4% EDTA, 3.8% EGTA, and 1% Triton X-100. Lystates were centrifuged at 21,000g to remove the nuclear fraction. Samples were diluted in equal amounts of RIPA and DTT containing reducing buffer (Pierce TM catalog number 39000) to a final amount of either 30 or 40 μg protein per well. Samples were run on 4–15% Ready Gel Tris-HCL gel (BioRad) and transferred onto PVDF membranes (BioRad). Membranes were incubated overnight at 4 °C with primary antibody. Secondary incubation was performed the following day and membranes visualized and bands quantified using the LiCor Odyssey imaging systemTM. Values for proteins of interest were normalized to beta actin expression for each subject.
Assessment of neuronal cell death
The uptake of the fluorescent exclusion dye propidium iodide (PI) was used for determination of neuronal cell death. PI is a polar compound that is impermeable to a cell with an intact cell membrane but penetrates damaged cell membranes. Inside the cells, it binds nuclear DNA to generate the brightly red fluorescence. This method has been well characterized as accurately measuring neuronal degeneration in organotypic slice cultures . For each experiment, PI was added into the culture medium at the beginning of treatment at a concentration of 5 μg/ml and PI fluorescence images were captured at indicated time points. PI fluorescent intensity was measured and analyzed with the AxioVision 3.1 software. Mean fluorescent density was quantified using ImageJTM software.
Data are expressed as a mean values ± standard error of mean from the indicated number of slices or experiments. Student’s t tests were performed for two-group analyses. For concentration-response curves, a one-way ANOVA followed by Dunnett’s multiple comparisons test was utilized. Differences were considered to be statistically significant if p value of <0.05. For human brain tissue analyses, paired t tests were performed between alcoholic subjects and their matched controls. Pearson’s correlation test was performed to assess for correlations of normally distributed data.
Postmortem human alcoholic brains have increased TLR7, HMGB1, and microglial activation
Demographics of alcoholics and control subjects from New South Wales Brain Tissue Bank
DSM V alcohol classification
Lifetime alcohol (kg)
Cause of death
Agonal state/mode of death
Mean ± SEM
48 ± 3
33 ± 3
6.56 ± 0.1
7.28 ± 1
35 ± 13
CO and EtOH
Mean ± SEM
48 ± 3
31 ± 5
6.63 ± 0.1
7.2 ± 1
2743 ± 829
Ethanol increases TLR7 expression and its ligand let-7b and causes neuroimmune gene induction in rat HEC slice culture
Ethanol releases miR let-7b and HMGB1 complexes in MVs from microglia
Consistent with previous findings, ethanol stimulated the release of HMGB1 in the absence of detectable cell death as assessed by propidium iodine uptake (Fig. 7b, c), consistent with active release from cells, rather than passive release from necrotic cells . Therefore, we assessed ethanol-treated HEC slice media MVs to determine which cell types were secreting HMGB1. Using flow cytometry of HEC media, we were able to identify the cellular origin of HMGB1+ MVs. Media MVs were labeled with fluorescent antibodies to HMGB1, GFAP (astrocytes), CD11b (microglia), and Na+/K+ ATPase α3 (neurons). Flow cytometric analysis revealed that 73% of the HMGB1-positive MVs were CD11b positive, indicating microglial origin. Approximately 15.3 and 11.8% were positive for GFAP and Na+/K+ ATPase α3 indicating astroglial and neuronal origins, respectively (Fig. 4e, f). Thus, ethanol causes secretion of HMGB1 in MVs primarily from microglia, as well as increasing let7b secretion in MVs.
Ethanol enhances TLR7 mediated neurodegeneration, a requirement of HMGB1
We also found that HMGB1 was required for ethanol withdrawal-induced neurotoxicity. Studying withdrawal in the HEC slice culture model is critical, since ethanol exposure itself does not cause cell death. Consistent with previous studies using hippocampal slice culture, ethanol-induced neurotoxicity was seen during the ethanol withdrawal phase [45–49]. This withdrawal toxicity is known to involve glutamate release . However, we found that inhibition of HMGB1 prevented ethanol withdrawal neurotoxicity. We have found previously that glutamate toxicity in the absence of ethanol also involves HMGB1 release, with glycyrrhizin preventing death . The exact role of HMGB1 in withdrawal toxicity is not clear. However, TLR4 responses have been shown to be inhibited during the neurotoxic withdrawal phase in HEC cultures . We found, however, that TLR7 responses during ethanol withdrawal are enhanced and require HMGB1 (Additional file 3: Figure S3). This also suggests that the requirement for HMGB1 during ethanol withdrawal might not involve its actions at TLR4, but might rather involve TLR7. We also found that let-7b is released during withdrawal. Future studies should investigate the role of HMGB1, let-7, and TLR7 in ethanol withdrawal-induced toxicity. Human alcoholism involves frequent exposures of chronic alcohol followed by withdrawal. Our findings suggest that recurrent TLR7 activation by ethanol-induced microglial let-7 and HMGB1 release contributes to the progressive neurodegeneration associated with alcoholism. This sensitization of microglia to release pro-inflammatory MVs may be similar in Parkinson’s, Alzheimer’s, and other neurodegenerative diseases.
It is important to note that high ethanol concentrations (>75 mM) were used in HEC slice culture and in vitro cell line experiments. However, human alcoholics reach very high blood alcohol concentrations (BACs). A report of 117 alcoholics showed that >57% had BACs from 43 to 125 mM , with alcoholics remaining functional at higher BACs . Further, the in vitro findings of increased HMGB1 and TLR7 are seen in postmortem human alcoholics and are of similar magnitude. Thus, the concentrations used in our in vitro studies appropriately model the human condition. Also, it is important to note that different cell lines might have different features. We employed in vitro models from a variety of sources including rat hippocampal slice culture, mouse BV2 microglia, and human SY-SY5Y neurons. The rat hippocampal slice culture has all brain cell types present in their native configurations making it the best in vitro system we have employed. The combination of these tools shows that microglia are the primary source of let-7b in MVs in response to ethanol. Though the use of multiple systems strengthens our conclusions, other neuronal or microglial cell lines might show different responses and should be investigated in future studies.
Our findings elucidate a novel mechanism of inter-cellular communication in neuroimmune pathology (Fig. 8). Flow cytometric and cell line studies identified microglia as the primary source of the MV-secreted HMGB1 and let-7b. These findings are consistent with our previous in vivo findings of activated microglia following ethanol [19, 64, 65], evidence of microglial sensitization in postmortem human alcoholic brain , and the in vitro observation that the microglia are required for neuronal death due to agonists to TLR2, 4, and 9 . Our findings further emphasize the need to develop microglia-targeted therapies for neuroimmune diseases. Also, the observation that ethanol increases the formation of HMGB1-let-7b complexes, and that HMGB1 inhibition prevents TLR7 induced neurotoxicity, uncovers a new potential for HMGB1 inhibition in preventing alcohol-induced and other neuroimmune pathologies. The investigation of the mechanisms of MV packaging of contents and secretion might also produce additional therapeutic targets. Further, several let-7 family members and other miRNAs were altered by ethanol in MVs (Additional file 4: Table S1), warranting further future investigation. The method of MV isolation used in this study yields vesicles between 0.1 and 1.0 μm in diameter. The origin of these particles may include exocytosis of multivesicular bodies, budding of the plasma membrane, or autophagy-associated secretory vesicles from living cells [66–68]. Identification of the mechanisms underlying this MV secretion may produce novel pharmacological targets for alcoholism or other conditions involving neuroimmune activation. In summary, we identify a novel inter-cellular neuroimmune mechanism involved in the pathology of alcoholism that provides multiple potential therapeutic targets.
We find increased TLR7 in alcoholic hippocampus and with ethanol treatment of slice cultures. Ethanol increases TLR7 activation and releases of HMGB1-miR-let-7 complexes in microglia-derived vesicles that cause neurotoxicity via TLR7 activation. TLR7 activation by alcohol in humans may contribute to the neuropathology of alcoholism.
Damage-associated molecular pattern molecules
High mobility group box 1 protein
New South Wales
We would like to thank the New South Wales Brain Bank for their provision of the human alcoholic brain samples. Flow cytometry research reported in this publication was supported by the Office of the Director, National Institutes of Health under award number S10OD012052. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
NIAAA AA019767, AA11605, AA007573, and AA021040
Availability of data and materials
The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request.
FTC is the senior author and provided oversight and experimental direction. LGC and JZ performed the experiments presented and collaborated on experimental direction. LGC authored the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
For animal studies, the Institutional Animal Care and Use Committee approved our experiments under the protocol number: 16-023.0. Human brain tissue was acquired from the New South Wales Brain Bank. This study is supported by the National Health and Medical Research Council of Australia-Schizophrenia Research Institute and the National Institute of Alcohol Abuse and Alcoholism (NIH [NIAAA] R24AA012725).
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