MicroRNA-155 deficiency protects from alcohol-induced TNFα and MCP1 in mouse cerebellum.

<p>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 (*, #: <i>p</i> value<0.05 relative to appropriate PF or WT controls, respectively, by Kruskal-Wallis non-parametric test).</p

Real-Time PCR Primers.

<p>The following forward and reverse sequences of primers were used in real-time PCR. CD68: cluster of differentiation 68; Iba1: ionized calcium binding adaptor molecule-1; MCP1: monocyte chemoattractant protein 1; pro-IL-1β: pro-interleukin-1β; TNFα: tumor necrosis factor-α.</p

Induction of microRNA-155 is TLR4-dependent in microglia.

<p>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 (*: <i>p</i> value<0.05 relative to appropriate PF or WT controls, by Kruskal-Wallis non-parametric test).</p

Pro-inflammatory cytokines and microRNAs are increased in alcohol-induced brain injury.

<p>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 (*: <i>p</i> value<0.05 relative to appropriate PF controls by Kruskal-Wallis non-parametric test).</p

Induction of microRNA-155 is TLR4 dependent in alcohol-fed mouse cerebellum.

<p>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 (*: <i>p</i> value<0.05 relative to appropriate PF or WT controls, by Kruskal-Wallis non-parametric test).</p

MicroRNA-155 deficiency protects from alcohol-induced NFκB activation in mouse cerebellum.

<p>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 (*, #: <i>p</i> value<0.05 relative to appropriate PF or WT controls, respectively, by Kruskal-Wallis non-parametric test).</p

MCD diet-induced steatohepatitis is associated with increased miR-155 expression in parenchymal and non-parenchymal cells.

<p>C57Bl/6 mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 3, 6 and 8 weeks. Serum alanine aminotransferase levels (A) and TNFα mRNA expression (B) were determined (n = 5-8/group). Liver fibrosis was assessed by Sirius Red staining (100x; n = 3-6/group), representative slides are shown (C). miR-155 expression was detected by qPCR in total livers (n = 5-8/group) (D). Primary murine hepatocytes (n = 7/group), Kupffer cells (n = 6, pooled data, 2 datapoints/group) and liver mononuclear cells (LMNC; n = 3-4/group) were isolated from a subset of mice after 6 weeks of MCS or MCD diet feeding and cell-specific miR-155 expression was determined and represented as 1/dCt (E). miR-155* expression was determined in total livers (F). (*) indicates p<0.05 MCS vs. corresponding MCD group. Statistics was performed on fold change data.</p

miR-155 deficiency does not attenuate hepatic inflammation in MCD-steatohepatitis.

<p>Wild type (WT) and miR-155 deficient (KO) mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 5 weeks. Liver TNFα (A top panel: mRNA, A bottom panel: protein), MCP1 (B top panel: mRNA, B bottom panel: protein) and IL1-β (C top panel: mRNA, C bottom panel: protein) mRNA and protein levels were measured by qPCR and ELISA, respectively (n = 6-8/group). NF-κB nuclear binding was evaluated by EMSA using liver nuclear extracts (D, top panel: representative blot, bottom panel: densitometry showing cumulative data of n = 6/group). (*) indicates p<0.05 MCS vs. corresponding MCD group.</p

miR-155 deficiency alters expression of genes in the lipid metabolism.

<p>Wild type (WT) and miR-155 deficient (KO) mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 5 weeks. mRNA expression of Adrp (A), Dgat2 (B), Acc1 (C), Fasn (D), Cpt1a (E), Fabp4 (F), Ldlr (G), Hmgcr (H), Nr1h3 (I) and Ppara (J) was measured in the livers (n = 6-8/group). (*) indicates p<0.05 MCS vs. corresponding MCD group.</p

miR-155 deficiency does not prevent liver injury, but attenuates liver steatosis in MCD-steatohepatitis.

<p>Wild type (WT) and miR-155 deficient (KO) mice were fed with methionine-choline deficient (MCD) or supplemented (MCS) control diet for 5 weeks. Liver histology was evaluated by hematoxilin-eosin staining (200x, inserts 100x; n = 5/group), representative slides are shown (A). Steatosis, necrosis, and lobular inflammation, were scored by a pathology expert (A). Liver triglyceride (B) and serum ALT (C) levels were determined (n = 6-8/group). (*) indicates p<0.05 MCS vs. corresponding MCD group. N.D. = not detectable or score = 0.</p