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

Abstract

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.

Figure 1

HA35 decreased LPS and PamCys3K-stimulated TNFα mRNA expression in primary cultures of rat Kupffer cells from ethanol and pair-fed rats, as well as PBMCs from patients with alcoholic hepatitis. Wistar rats were allowed free access to a Lieber-DeCarli ethanol diet or pair-fed control diet for 4 weeks. Kupffer cells were isolated and cultured overnight. Kupffer cells were then treated with 100 μg/ml HA35 for 5 h and then challenged with (A) 10 ng/ml LPS or (B) 10 ng/ml PamCys3K (PAM3) for an additional 1hr. Expression of TNFα mRNA was measured by qRT-PCR and normalized to 18 S rRNA. (C) PBMCs isolated from patients with AH and healthy controls, cultured overnight and then treated or not with 100 μg/ml HA35. PBMCs were then challenged with 10 ng/ml LPS for an additional 1hr. Expression of TNFα mRNA was measured by qRT-PCR and normalized to 18 S rRNA. (A/B) n = 4; (C) n = 10 for AH and 6 for healthy controls. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different from each other, p < 0.05.

Figure 2

siRNA knockdown of HA receptors in Kupffer cells from pair- and ethanol-fed rats. Wistar rats were allowed free access to a Lieber-DeCarli ethanol diet or pair-fed control diet for 4 weeks. Kupffer cells were isolated and nucleofected with 25 nM of scrambled siRNA or siRNA targeted to (A) TLR4, (B) TLR2, (C) RHAMM and (D) CD44. 24 h post-nucleofection, expression of (A) TLR4 (green), (B) TLR2 (red), (C) RHAMM (red) and (D) CD44 (red) was visualized by immunostaining with specific antibodies. Nuclei were labelled with DAPI (blue). All images were acquired using 20x objectives and are representative of 3 independent experiments.

Figure 3

siRNA knockdown of HA receptors in Kupffer cells from pair- and ethanol-fed rats. Wistar rats were allowed free access to a Lieber-DeCarli ethanol diet or pair-fed control diet for 4 weeks. Kupffer cells were isolated and nucleofected with 25 nM of scrambled siRNA or siRNA targeted to (A) TLR4, (B) TLR2, (C) RHAMM and (D) CD44. 18 h post-nucleofection, Kupffer cells were treated with or without 100 µg/ml HA35 for 5 h and then challenged with 10 ng/ml (A) PamCys3K or (B,C,D) LPS for 1hr and TNFα mRNA measured by qRT-PCR and normalized to 18 S rRNA. Values represent means ± SEM, n = 4, *p < 0.05 compared to pair-fed within a treatment group.

Figure 4

Next Generation Sequencing of miRNAs in Kupffer cells from ethanol- and pair-fed rats after treatment with or without HA35. (A–D) Wistar rats were allowed free access to a Lieber-DeCarli ethanol diet or pair-fed control diet for 4 weeks. Kupffer cells were isolated and cultured overnight. Kupffer cells were then treated with or without 100 μg/ml HA35 for 5 h. Total miRNAs were isolated and analyzed by NGS. (A) Heat maps illustrate the changes in expression for the 11 miRNAs identified whose expression was increased by more than 2-fold by ethanol feeding. (B) Of these eleven miRNA, heat maps are shown for the 4 miRNAs whose expression was decreased by treatment with HA35. Fold changes are shown in the column to the left of the heat maps. (C) Predicted binding site for miR291b in the 3′UTR of Tollip (miRDB). Sequence alignment between miR291b and Tollip is illustrated. (D) Kupffer cells isolated from ethanol- and pair-fed rats were treated or not with HA35 or HA7 for 5 h and expression of miR291b measured by qRT-PCR and normalized to Hs-SNORD68-11. (E) Isolated Kupffer cells were nucleofected with scrambled siRNA or siRNA targeted against CD44. 18 h post-nucleofection, Kupffer cells were treated or not with 100 μg/ml HA35 for 5 h and expression of miR291b measured by qRT-PCR and normalized to Hs-SNORD68-11. (F) C57BL/6 J mice were allowed free access to an ethanol containing diet for 4 days (2 days at 1% (v/v) ethanol, followed by 2 days 6% (v/v) ethanol) or pair-fed an isocaloric control diet. Mice were gavaged once daily for the last 3 days of ethanol feeding with HA35 at 15 mg/kg body weight or saline as a vehicle control the last three days of the study. Paraffin-embedded livers were de-paraffinized followed by in situ hybridization for miR291b using locked nucleic acid probes tagged with digoxigenin at both the 5′and 3′ ends. All images were captured using 40X objectives. miR291b –positive cells were counted using Image Pro-Plus software and analyzed. Zoomed images illustrate positive staining in cells identified by morphology as hepatocytes (pink) and non-parenchymal cells (white). (A,B) n = 3 (C) n = 5 (D) n = 4 (E) n = 3–6. (C,D,E) Values represent means ± SEM. Values with different alphabetical superscripts are significantly different from each other, p < 0.05.

Figure 5

Regulation of Tollip expression by ethanol, HA35 and miR291b in primary cultures of Kupffer cells. (A) Kupffer cells isolated from ethanol- and pair-fed rats were cultured overnight and then treated with 100 µg/ml HA35 for 5 h and then challenged with 10 ng/ml LPS for an additional 60 min. (B,C) Kupffer cells isolated from ethanol- and pair-fed rats were nucleofected with either control hairpin or miR291b hairpin inhibitor 18 h post-nucleofection, Kupffer cells were treated or not with 100 µg/ml HA35 for 5 h and then challenged with 10 ng/ml LPS for an additional 1 hr. (A,B) Kupffer cells were lysed and Tollip expression measured by Western blot. HSC70 was used as a loading control. Images of the blots were cropped. Full size blots are shown in Supplemental Information Fig. 2. (C) Expression of TNFα mRNA was determined by qRT-PCR and normalized to 18 S. n = 4–5. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different from each other, p < 0.05.

Figure 6

Model illustrating the interactions between HA35 on expression of miR291b and Tollip in Kupffer cells from ethanol-fed rats. Chronic ethanol feeding increases the expression of miR291b which in turn results in decreased expression of Tollip. This decreased expression of Tollip contributes to enhanced TNFα expression in response to activation of TLR2/TLR4. Treatment of Kupffer cells from ethanol-fed rats with HA35 ex vivo decreased the expression of miR291b and increased Tollip, reducing the sensitivity of Kupffer cells to TLR2/TLR4-mediated TNFα expression. Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography© 2016. All Rights Reserved.