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


OBJECTIVE
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.


CONCLUSIONS
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.
Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Dora
D
Lippai
Shashi
S
Bala
Timea
T
Csak
Evelyn A
EA
Kurt-Jones
Gyongyi
G
Szabo
D000437:Alcoholism / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D000818:Animals; D002531:Cerebellum / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D018932:Chemokine CCL2 / Q000235:genetics / Q000276:immunology; D002908:Chronic Disease; D000431:Ethanol / Q000009:adverse effects*; D005260:Female; D005786:Gene Expression Regulation; D007249:Inflammation / Q000139:chemically induced / Q000235:genetics / Q000276:immunology / Q000473:pathology; D053583:Interleukin-1beta / Q000235:genetics / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics* / Q000276:immunology; D017628:Microglia / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D061251:Primary Cell Culture; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology
C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D053583:Interleukin-1beta; D035683:MicroRNAs; C532623:Mirn155 microRNA, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D000431:Ethanol