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. 2013 Aug 9;8(8):e70945.
doi: 10.1371/journal.pone.0070945. eCollection 2013.

Chronic alcohol-induced microRNA-155 contributes to neuroinflammation in a TLR4-dependent manner in mice

Dora Lippai  1 , Shashi Bala, Timea Csak, Evelyn A Kurt-Jones, Gyongyi Szabo
Affiliations

Chronic alcohol-induced microRNA-155 contributes to neuroinflammation in a TLR4-dependent manner in mice

Dora Lippai et al. PLoS One. .

Abstract

Introduction: Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation.

Aim: To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation.

Methods: Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation.

Results: Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155.

Conclusion: Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Pro-inflammatory cytokines and microRNAs are increased in alcohol-induced brain injury.
WT mice were fed with control (PF, n = 7) or EtOH (n = 8) diet for 5 weeks. Inflammatory cytokines, TNFα (A), MCP1 (B) and IL-1β (C) were measured by specific ELISAs on whole cerebellar lysates, and corrected with total protein. Various microRNAs (125b, 132, 146a, 155) were measured by real-time PCR on whole cerebellar miRNA extract and corrected with snoRNA202 (D). Bars represent mean±SEM (*: p value<0.05 relative to appropriate PF controls by Kruskal-Wallis non-parametric test).
Figure 2
Figure 2. MicroRNA-155 deficiency protects from alcohol-induced TNFα and MCP1 in mouse cerebellum.
WT (n = 6 or 7) or miR-155-KO (n = 5 or 10) mice were fed with control (PF) or EtOH diet for 5 weeks, respectively. Pro-inflammatory cytokines, TNFα (A) and MCP1 (C) mRNAs were assessed by real-time PCR from whole cerebellar RNA extract and corrected with 18S. TNFα (B) and MCP1 (D) proteins of whole cerebellar lysates were measured by specific ELISAs and corrected with total protein. Bars represent mean±SEM (*, #: p value<0.05 relative to appropriate PF or WT controls, respectively, by Kruskal-Wallis non-parametric test).
Figure 3
Figure 3. MicroRNA-155 KO mice are not protected from alcohol-induced IL-1β increase in the cerebellum.
WT (n = 6 or 7) or miR-155-KO (n = 5 or 10) mice were fed with control (PF) or EtOH diet for 5 weeks, respectively. Inflammatory cytokine, IL-1β was measured by specific ELISA on whole cerebellar lysates and corrected with total protein (A). Mature IL-1β protein of whole cerebellar lysates was assessed by Western blot using β-actin as loading control (B), and further quantified by densitometry (C) which represents six to ten samples per group. The inflammasome activity was measured by caspase-1 colorimetric assay from whole cerebellar lysates and corrected with total protein (D). Pro-IL-1β mRNA was assessed by real-time PCR from whole cerebellar RNA extract, corrected with 18S (E). Bars represent mean±SEM (*, #: p value<0.05 relative to appropriate PF or WT controls, respectively, by Kruskal-Wallis non-parametric test).
Figure 4
Figure 4. MicroRNA-155 deficiency protects from alcohol-induced NFκB activation in mouse cerebellum.
WT (n = 6 or 7) or miR-155-KO (n = 5 or 10) mice were fed with control (PF) or EtOH diet for 5 weeks, respectively. NF-κB activity of whole cerebellar lysates was assessed by EMSA for NF-κB (A–B) and supershift with anti-p65 antibody (C–D), loading equal amounts of protein, using EtOH-fed cerebellar sample for cold competition control (ctr), and further quantified by densitometry. Phosphorylated-p65 (E–F) and total-p65 (G–H) protein of whole cerebellar lysates was assessed by Western blot, using β-actin as loading control, and further quantified by densitometry which represents six to ten samples per group. Bars represent mean±SEM (*, #: p value<0.05 relative to appropriate PF or WT controls, respectively, by Kruskal-Wallis non-parametric test).
Figure 5
Figure 5. Induction of microRNA-155 is TLR4 dependent in alcohol-fed mouse cerebellum.
WT (n = 8 or 7) and TLR4-KO (n = 8 or 13) mice were fed with control (PF) or EtOH diet for 5 weeks, respectively. MiR-155 (A) was assessed by real-time PCR from whole cerebellar miRNA extract, corrected with snoRNA202. Phosphorylated-p65 (B–C) and total-p65 (D–E) protein of whole cerebellar lysates was assessed by Western blot, using β-actin as loading control, and further quantified by densitometry which represents six to twelve samples per group. Bars represent mean±SEM (*: p value<0.05 relative to appropriate PF or WT controls, by Kruskal-Wallis non-parametric test).
Figure 6
Figure 6. Induction of microRNA-155 is TLR4-dependent in microglia.
WT (n = 8 or 7) and TLR4-KO (n = 8 or 13) mice were fed with control (PF) or EtOH diet for 5 weeks, respectively. Microglia markers, Iba1 (A) and CD-68 (B), were assessed by real-time PCR from whole cerebellar RNA extract, and corrected with 18S. WT mouse immortalized microglia cells incubated with or without 50 mM ethanol for 6 days were stimulated with 100 ng/ml LPS for 18 hours. MiR-155 was assessed by real-time PCR of cellular miRNA extracts, corrected with snoRNA202 (C). Primary microglia cells were isolated from WT (n = 10 or 9) mice, fed with control (PF) or EtOH diet for 5 weeks, respectively. Prior to plating, cells were pooled from two brains. Mouse primary microglia cells were stimulated with 100 ng/ml LPS for 18 hours. Microglia from pair-fed mice was also challenged with 50 mM ethanol in vitro for 18 hours. MiR-155 was assessed by real-time PCR of cellular miRNA extracts, corrected with snoRNA202 (D). Bars represent mean±SEM (*: p value<0.05 relative to appropriate PF or WT controls, by Kruskal-Wallis non-parametric test).
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