Hyaluronic acid 35 normalizes TLR4 signaling in Kupffer cells from ethanol-fed rats via regulation of microRNA291b and its target Tollip.

TLR4 signaling in hepatic macrophages is increased after chronic ethanol feeding. Treatment of hepatic macrophages after chronic ethanol feeding with small-specific sized hyaluronic acid 35 (HA35) normalizes TLR4 signaling; however, the mechanisms for HA35 action are not completely understood. Here we used Next Generation Sequencing of microRNAs to identify negative regulators of TLR4 signaling reciprocally modulated by ethanol and HA35 in hepatic macrophages. Eleven microRNAs were up-regulated by ethanol; only 4 microRNAs, including miR291b, were decreased by HA35. Bioinformatics analysis identified Tollip, a negative regulator of TLR4, as a target of miR291b. Tollip expression was decreased in hepatic macrophages from ethanol-fed rats, but treatment with HA35 or transfection with a miR291b hairpin inhibitor restored Tollip expression and normalized TLR4-stimulated TNFα expression. In peripheral blood monocytes isolated from patients with alcoholic hepatitis, expression of TNFα mRNA was robustly increased in response to challenge with lipopolysaccharide. Importantly, pre-treatment with HA35 reduced TNFα expression by more than 50%. Taken together, we have identified miR291b as a critical miRNA up-regulated by ethanol. Normalization of the miR291b → Tollip pathway by HA35 ameliorated ethanol-induced sensitization of TLR4 signaling in macrophages/monocytes, suggesting that HA35 may be a novel therapeutic agent in the treatment of ALD.