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

Abstract

INTRODUCTION: Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-alpha (TNFalpha), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1beta). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-kappaB (NF-kappaB) 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; TNFalpha, MCP1, IL-1beta mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-kappaB 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; TNFalpha, MCP1 protein and TNFalpha, MCP1, pro-IL-1beta and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-kappaB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1beta 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-kappaB 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 TNFalpha and MCP1 expression but not caspase-dependent IL-1beta increase in neuroinflammation.