Hepatocyte TLR4 triggers inter-hepatocyte Jagged1/Notch signaling to determine NASH-induced fibrosis

Abstract

Aberrant hepatocyte Notch activity is critical to the development of nonalcoholic steatohepatitis (NASH)-induced liver fibrosis, but mechanisms underlying Notch reactivation in developed liver are unclear. Here, we identified that increased expression of the Notch ligand Jagged1 (JAG1) tracked with Notch activation and nonalcoholic fatty liver disease (NAFLD) activity score (NAS) in human liver biopsy specimens and mouse NASH models. The increase in Jag1 was mediated by hepatocyte Toll-like receptor 4 (TLR4)-nuclear factor κB (NF-κB) signaling in pericentral hepatocytes. Hepatocyte-specific Jag1 overexpression exacerbated fibrosis in mice fed a high-fat diet or a NASH-provoking diet rich in palmitate, cholesterol, and sucrose and reversed the protection afforded by hepatocyte-specific TLR4 deletion, whereas hepatocyte-specific Jag1 knockout mice were protected from NASH-induced liver fibrosis. To test therapeutic potential of this biology, we designed a Jag1-directed antisense oligonucleotide (ASO) and a hepatocyte-specific N-acetylgalactosamine (GalNAc)-modified siRNA, both of which reduced NASH diet-induced liver fibrosis in mice. Overall, these data demonstrate that increased hepatocyte Jagged1 is the proximal hit for Notch-induced liver fibrosis in mice and suggest translational potential of Jagged1 inhibitors in patients with NASH.

Fig. 1.. JAG1 expression tracks with liver Notch activity and NASH severity in patients.

(A) NOTCH receptor and (B) ligand expression in end-of-treatment biopsies in nonresponders (n = 49) and responders (n = 69), regardless of treatment group, from the PIVENS trial. In 157 consecutive patients undergoing liver biopsy for suspected NASH or severe obesity, (C) correlation between liver expression of JAG1 and Notch target HES1, and (D and E) expression of JAG1 and Notch targets in patients with increased NAS and fibrosis scores. (F) Representative image of Jagged1 (red) and HNF4α (green) staining. In a subset of patients with available paired baseline and 96-week end-of-treatment liver biopsy specimens from PIVENS patients (n = 10 to 11 per group), (G) DLL4 and JAG1 expression, and (H) association between change in JAG1 (ΔJAG1) and ΔNAS. *P < 0.05 as compared to the indicated controls by two-tailed t tests (two groups) or one-way ANOVA followed by post hoc t tests (multiple groups). AU, arbitrary unit. All data are shown as means ± SEM.

Fig. 2.. Pericentral hepatocyte Jag1 expression is increased in livers of mice fed a NASH-provoking diet.

(A) Expression of Notch receptors, (B) ligands, and (C) liver Jagged1 protein in livers from C57BL/6J WT mice fed normal chow diet or NASH diet for 8 weeks (n = 5 to 7 per group). (D) Jag1 expression in hepatocytes and NPC in livers of mice fed either chow or NASH diet for 16 weeks (n = 9 per group). (E) Representative image of Jagged1 (red) and HNF4α (green) staining, and quantitation of HNF4α/Jagged1 double-positive cells, (F) representative image of Jagged1 (red) and GS (green) staining, and quantitation of Jagged1⁺ cells among GS⁺ and GS⁻ cells in WT mice fed normal chow or NASH diet for 16 weeks (n = 3 to 5 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to chow-fed mice by two-tailed t tests (two groups) or one-way ANOVA followed by post hoc t tests (multiple groups). All data are shown as means ± SEM.

Fig. 3.. Hepatocyte-specific Jag1 KO mice are protected from NASH-induced liver fibrosis.

(A) Adult Jag1^flox/flox mice were transduced with either AAV8-Tbg-GFP (Cre⁻) or AAV8-Tbg-CRE (L-Jag1^KO) and then fed NASH diet for 16 weeks before sacrifice. (B) Liver Jag1 mRNA and (C) protein expression, (D) representative image of Jagged1 (red) and HNF4α (green) staining, (E and F) Jag1 and Notch target expression in hepatocytes and NPC, and (G) gene expression markers of HSC activation. (H) Sirius red staining. n = 5 to 7 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to Cre⁻ mice by two-tailed t tests (two groups). All data are shown as means ± SEM.

Fig. 4.. Forced hepatocyte Jag1 expression exacerbates diet-induced liver fibrosis.

(A) Eight- to 10-week-old Jag1^teto/−/rtTA^flox/− or rtTA^flox/− mice were transduced with AAV8-Tbg-CRE to generate Control (CON) or hepatocyte-specific Jag1 transgenic (L-Jag1^TG) mice and then fed NASH diet with ad libitum access to doxycycline-containing drinking water for 16 weeks (n = 5 to 11 per group). (B) Representative image of Jagged1 (red) and HNF4α (green) staining, (C) Jagged1 protein, (D) expression of Notch targets, and (E) markers of HSC activation. (F) Liver Sirius red staining (n = 4 per group). (G) Control and L-Jag1^TG mice were fed HFD with ad libitum access to doxycycline-containing drinking water for 16 weeks (n = 7 to 8 per group). (H) Gene expression of markers of HSC activation. (I) Sirius red staining (n = 6 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to control mice by two-tailed t tests or nonparametric Mann-Whitney U test (two groups). All data are shown as means ± SEM.

Fig. 5.. TLR4–NF-κB signaling drives hepatocyte Jag1 expression.

(A) Serum LPS, (B) liver Tlr4 mRNA, and (C) liver p65 protein expression in WT mice fed chow or NASH diet for 16 weeks (n = 3 to 6 per group). (D) Jag1 promoter luciferase activity in WT primary hepatocytes cotransfected with p65 (n = 4 per group), and (E) ChIP assay for NF-κB occupancy at the Jag1 promoter in livers from mice fed normal chow or NASH diet for 16 weeks (n = 4 per group). (F) Eight- to 10-week-old Tlr4^flox/flox mice were transduced with AAV8-Tbg-GFP (Cre⁻) or AAV8-Tbg-CRE (L-Tlr4) and then fed NASH diet for 16 weeks (n = 12 to 13 per group). (G) Jagged1 protein expression, (H) expression of Notch targets, and (I) markers of HSC activation. (J) Liver Sirius red staining (n = 4 to 9 per group). (K) Control (pLive-con) or Jag1 expression (pLive-Jagged1) vectors were hydrodynamically administered to adult Tlr4^flox/flox mice. Two weeks later, all mice were transduced with AAV8-Tbg-CRE and then fed NASH diet for 16 weeks (n = 8 per group). (L) Expression of Jag1 mRNA and (M) markers of HSC activity. (N) Sirius red staining (n = 5 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to the indicated controls by two-tailed t tests (two groups). All data are presented as means ± SEM.

Fig. 6.. Jag1 inhibitors protect from NASH-induced liver fibrosis.

(A) NASH diet–fed WT mice were administered weekly intraperitoneal injections of control (Ctrl ASO) or Jag1-directed (Jag1 ASO) antisense oligonucleotides (ASOs), (B) Jag1 and Notch target gene expression, (C) Jagged1 protein expression, (D) representative image of Jagged1 (red) and HNF4α (green) staining, (E) expression of markers of liver HSC activation, and (F) Sirius red staining (n = 5 per group). (G) NASH diet–fed WT mice were administered weekly subcutaneous injections of control (siCtrl) or Jag1-directed (siJag1) GalNAc-modified siRNA, (H) Jag1 and Notch target gene expression, (I) liver Jagged1 protein, (J) representative image of Jagged1 (red) and HNF4α (green) staining, (K) expression of markers of liver HSC activation, and (L) Sirius red staining (n = 5 to 7 per group). (M) NASH diet–fed WT mice were allowed to develop liver fibrosis and then administered weekly subcutaneous injections of siCtrl or siJag1. (N) Liver Jag1 and Notch target gene expression, (O) expression of markers of liver HSC activation, and (P) Sirius red staining (n = 5 to 6 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to the indicated controls by two-tailed t tests (two groups). Data are presented as means ± SEM.