ASK1 inhibition reduces cell death and hepatic fibrosis in an Nlrp3 mutant liver injury model
Susanne Schuster-Gaul, … , Hal M. Hoffman, Ariel E. Feldstein
Susanne Schuster-Gaul, … , Hal M. Hoffman, Ariel E. Feldstein
Published January 30, 2020
Citation Information: JCI Insight. 2020;5(2):e123294. https://doi.org/10.1172/jci.insight.123294.
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Research Article Hepatology Therapeutics Article has an altmetric score of 3

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 2

ASK1 inhibition reduced hepatic TNF-α protein expression and moderately improved inflammation score.

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ASK1 inhibition reduced hepatic TNF-α protein expression and moderately ...
(A) H&E staining of liver sections (thickness 5 μm) of WT and Nlrp3-KI treated with vehicle and GS-444217 (magnification, 10×; scale bar: 250 μm) (WT + vehicle n = 10, WT + ASK1i n = 7, Nlrp3-KI + vehicle n = 5, Nlrp3-KI + ASK1i n = 9). Arrows point to areas with inflammatory cell infiltration. Nlrp3 mutant mice treated with vehicle showed severe liver inflammation, while mice that received the ASK1i showed mild improvements in the grade of inflammation (P = 0.05 vs. Nlrp3-KI + vehicle; Mann-Whitney U test). (B) As a marker for neutrophil cell infiltration, we used Myeloperoxidase (MPO), which was significantly increased in Nlrp3 mutant mice (+ vehicle) and was reduced by GS-444217 (not significant) (magnification, 20×; scale bar: 100 μm) (WT + vehicle n = 2, WT + ASK1i n = 4, Nlrp3-KI + vehicle n = 5, Nlrp3-KI + ASK1i n = 9). (C) The same trend was observed on Mpo gene expression level (P = 0.08 vs. Nlrp3-KI + vehicle) (WT + vehicle n = 5, WT + ASK1i n = 4, Nlrp3-KI + vehicle n = 5, Nlrp3-KI + ASK1i n = 9). (D) Immunoblot analysis of liver lysates of WT and Nlrp3-KI mice treated with vehicle and ASK1i for detecting the active homotrimer (52 kDa) and the membrane-bound (26 kD) TNF-α (WT + vehicle n = 7, WT + ASK1i n = 7, Nlrp3-KI + vehicle n = 4, Nlrp3-KI + ASK1i n = 6). Data were normalized on GAPDH and are expressed as the mean ± SEM. WT + vehicle was set at 1. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (1-way ANOVA with Bonferroni post hoc test or otherwise stated).