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Chemogenomic screening in a patient-derived 3D model of metabolic dysfunction-associated steatohepatitis reveals the CHRM1-TRPM8 axis as a novel module for targeted intervention

ABSTRACT: Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of liver disease. A multitude of compounds have been developed in recent years but, due to lack of efficacy or safety concerns, receiving market approval has been challenging and restricted. These failures emphasize the need for robust pre-clinical data that can narrow the translational gap. We here present an organotypic 3D liver model established from primary human hepatocytes (PHH) and non-parenchymal cells derived from patients with clinical diagnosis of MASH. The model closely recapitulates disease relevant endpoints, such as steatosis, inflammation, hepatocyte ballooning and fibrosis for multiple weeks in culture. Using integrative multi-omics profiling, we moreover identify the underlying molecular networks of MASH at the transcriptomic, proteomic and lipidomic level. Importantly, by combining biochemical and high content imaging-based multiplexed chemogenomic screening using selective, highly validated chemical probes, we identified multiple novel targets with anti-steatotic, anti-inflammatory and anti-fibrotic effects. Among these, activation of the muscarinic receptor 1 (CHRM1) using a positive allosteric modulator and inhibition of the transient receptor potential cation channel subfamily M member 8 (TRPM8) ion channel resulted in strong anti-fibrotic effects, which could be confirmed using orthogonal genetic assays. Strikingly, using an array of bioluminescence resonance energy transfer (BRET) sensors and small molecule interference, we identify a novel signaling axis in which CHRM1 inhibits TRPM8 via Gq/11 and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate (PIP2). Combined, this study presents the first scalable patient-derived 3D liver model for NASH in which disease-relevant endpoints are recapitulated ex vivo, identified a novel CHRM1-TRPM8 signaling module with anti-fibrotic effects and highlights the potential of organotypic culture systems as phenotypic assays for comprehensive chemogenomic drug target identification.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Liver

DISEASE(S): Non-alcoholic Steatohepatitis

SUBMITTER: Pierre Sabatier

LAB HEAD: Jesper V. Olsen

PROVIDER: PXD052956 | Pride | 2024-11-28

REPOSITORIES: Pride

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			Action	DRS
	20230116_EXPL2_Evo5_PS__heptocyes_Phospho_TMT_frc-1.raw	Raw
	20230116_EXPL2_Evo5_PS__heptocyes_Phospho_TMT_frc-2.raw	Raw
	20230116_EXPL2_Evo5_PS__heptocyes_Phospho_TMT_frc-2_20230117195502.raw	Raw
	20230116_EXPL2_Evo5_PS__heptocyes_Phospho_TMT_frc-3.raw	Raw
	20230116_EXPL2_Evo5_PS__heptocyes_Phospho_TMT_frc-5.raw	Raw

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Project description:Metabolic Dysfunction Associated Steatohepatitis (MASH) is characterized by excess circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to the liver sinusoidal endothelial cells (LSEC) and transendothelial migration (TEM) are crucial in the inflammatory process. LSEC under lipotoxic stress develops a pro-inflammatory phenotype known as endotheliopathy. However, the mediators of endotheliopathy remain obscure. Primary mouse LSEC isolated from C57BL/6J mice on chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH, due to a choline-deficient high-fat diet (CDHFD) or FFC diet, were treated with two structurally distinct GSK3 inhibitors [LY2090314, elraglusib (9-ING-41)]. Integrated pathway analysis of mouse LSEC proteome and transcriptome indicates that leukocyte TEM and focal adhesion are major pathways altered in MASH. Kinome profiling of the LSEC phospho-proteome identified GSK3β as the major kinase hub in MASH. GSK3β activating phosphorylation was increased in primary human LSEC treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependant mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC and CDHFD-fed mice, respectively. Immunophenotyping of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib using cytometry by the time of flight showed reduced proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells infiltration.GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and may serve as a potential therapeutic strategy in human MASH.

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Chemogenomic screening in a patient-derived 3D model of metabolic dysfunction-associated steatohepatitis reveals the CHRM1-TRPM8 axis as a novel module for targeted intervention

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