ASK1 inhibition reduces cell death and hepatic fibrosis in an Nlrp3 mutant liver injury model
Susanne Schuster-Gaul, Lukas Jonathan Geisler, Matthew D. McGeough, Casey D. Johnson, Anna Zagorska, Li Li, Alexander Wree, Vivian Barry, Igor Mikaelian, Lily J. Jih, Bettina G. Papouchado, Grant Budas, Hal M. Hoffman, Ariel E. Feldstein
Susanne Schuster-Gaul, Lukas Jonathan Geisler, Matthew D. McGeough, Casey D. Johnson, Anna Zagorska, Li Li, Alexander Wree, Vivian Barry, Igor Mikaelian, Lily J. Jih, Bettina G. Papouchado, Grant Budas, Hal M. Hoffman, Ariel E. Feldstein
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Research Article Hepatology Therapeutics

ASK1 inhibition reduces cell death and hepatic fibrosis in an Nlrp3 mutant liver injury model

Abstract

Hepatic inflammasome activation is considered a major contributor to liver fibrosis in NASH. Apoptosis signal–regulating kinase 1 (ASK1) is an apical mitogen-activated protein kinase that activates hepatic JNK and p38 to promote apoptosis, inflammation, and fibrosis. The aim of the current study was to investigate whether pharmacologic inhibition of ASK1 could attenuate hepatic fibrosis driven by inflammasome activation using gain-of-function NOD-like receptor protein 3 (Nlrp3) mutant mice. Tamoxifen-inducible Nlrp3 knock-in (Nlrp3A350V/+CreT-KI) mice and WT mice were administered either control chow diet or diet containing the selective ASK1 inhibitor GS-444217 for 6 weeks. Livers of Nlrp3-KI mice had increased inflammation, cell death, and fibrosis and increased phosphorylation of ASK1, p38, and c-Jun. GS-444217 reduced ASK1 pathway activation, liver cell death, and liver fibrosis. ASK1 inhibition resulted in a significant downregulation of genes involved in collagen production and extracellular matrix deposition, as well as in a reduced hepatic TNF-α expression. ASK1 inhibition also directly reduced LPS-induced gene expression of Collagen 1A1 (Col1a1) in hepatic stellate cells isolated from Nlrp3-KI mice. In conclusion, ASK1 inhibition reduced liver cell death and fibrosis downstream of inflammatory signaling induced by NLRP3. These data provide mechanistic insight into the antifibrotic mechanisms of ASK1 inhibition.

Authors

Susanne Schuster-Gaul, Lukas Jonathan Geisler, Matthew D. McGeough, Casey D. Johnson, Anna Zagorska, Li Li, Alexander Wree, Vivian Barry, Igor Mikaelian, Lily J. Jih, Bettina G. Papouchado, Grant Budas, Hal M. Hoffman, Ariel E. Feldstein

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Figure 1

GS-444217 inhibits hepatic ASK1 signaling in Nlrp3-KI mice.

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GS-444217 inhibits hepatic ASK1 signaling in Nlrp3-KI mice. (A) Schemati...
(A) Schematic overview of the experimental set up. Nlrp3-KI mice and WT mice were injected i.p. with tamoxifen daily for 4 days, followed by a once-weekly injection. Mice were administered control chow (NC) diet (+ vehicle) or diet containing the ASK1 inhibitor (ASK1i) GS-444217 (0.2% in chow) starting 2 days before tamoxifen injection and continuing for 6 weeks. (B–D) Body weight [g] (B), total liver weight [g] (C), and liver/body weight [mg/g body weight] (D) of WT and Nlrp3-KI mice treated with vehicle (white, WT n = 10, Nlrp3-KI n = 5) or ASK1i (black, WT n = 8, Nlrp3-KI n = 10). (E) Immunoblot analysis of liver lysates from WT and Nlrp3-KI mice treated with vehicle (white, WT n = 5, Nlrp3-KI n = 5) or ASK1i (black, WT n = 5, Nlrp3-KI n = 6) for phosphorylated ASK1 (P-ASK1) (WT + vehicle n = 6, WT + ASK1i n = 5, Nlrp3-KI + vehicle n = 5, Nlrp3-KI + ASK1i n = 8), P-p38, and p38 (WT + vehicle n = 8, WT + ASK1i n = 8, Nlrp3-KI + vehicle n = 4, Nlrp3-KI + ASK1i n = 6). Western blot of P-ASK1 and P-p38 run on 2 different gels indicated by the black horizontal line. Therefore, 2 loading controls (GAPDH) are shown. In the lower blot, a dashed black line is used to indicate splicing of noncontiguous lanes of the same blot. Densitometric analysis was performed on background-substracted blots and was normalized on GAPDH. WT + vehicle was used as reference control and was set at 1. (F and G) Representative immunohistochemical staining of P–c-Jun (magnification, 40×; scale bar: 100 μm; WT + vehicle n = 10; WT + ASK1i n = 8; Nlrp3-KI + vehicle n = 5; Nlrp3-KI + ASK1i n = 10) (F) and P-p38 (magnification, 10×; scale bar: 500 μm; WT + vehicle n = 10; WT + ASK1i n = 8; Nlrp3-KI + vehicle n = 8; Nlrp3-KI + ASK1i n = 5) (G) on formalin-fixed paraffin-embedded liver tissue slides. Staining grade of P–c-Jun and percentage of area of P-p38 was calculated by using whole tissue slide. Treatment with ASK1i significantly reduced P–c-Jun+ and P-p38+ cells in Nlrp3 mutant mice. P–c-Jun was not detectable in WT mice (vehicle and ASK1i). Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001 (1-way ANOVA with Bonferroni post hoc test).