Abstract

Background: During metabolic dysfunction-associated steatohepatitis (MASH), liver sinusoidal endothelial cells (LSECs) acquire a pro-inflammatory phenotype characterized by increased adhesion molecules expression and myeloid cells-associated liver inflammation culminating in disease progression. However, the exact molecular mediators of this phenomenon are unclear. Herein, we aim to uncover the pathogenic role and the transcriptional regulation of intercellular adhesion molecule (ICAM1), an upregulated LSEC adhesion molecule in MASH and lipotoxicity.

Methods: Chromatin immunoprecipitation (ChIP) assay was used to evaluate the enrichment at the ICAM1 promoter of the active epigenetic mark, H3K27ac in primary human hepatic sinusoidal endothelial cells (HHSECs). HHSECs were seeded in a flow chamber, and primary human neutrophils were infused using a flow-based adhesion assay. Wild-type C57BL/6J mice were fed fat, fructose, and cholesterol (FFC) diet or choline-deficient high-fat diet (CDHFD) to induce MASH. Paraffin embedded liver sections from MASH patients or normal subjects were subjected to immunostaining and multiplex imaging using the CODEX (Co-detection by indexing) system.

Results: Data mining of Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) in LSECs from MASH and control mice identified open chromatin regions at the promoter region of Icam1 that are significantly enriched in MASH mice. We validated the upregulation of ICAM1 expression in HHSECs with palmitate (PA) treatment. ChIP assay for H3K27ac showed H3K27ac enrichment is significantly induced by PA treatment at the ICAM1 promoter region. Using pharmacological and genetic inhibition approaches, we identified that ICAM1 upregulation in lipotoxicity is induced by glycogen synthase kinase 3 activation, and its downstream signaling pathway (JNK and c-Jun). Flow-based adhesion assay showed reduced neutrophil adhesion to PA treated HHSECs in the presence of ICAM1 Antibody (Ab). ICAM1 Ab-treated mice in both MASH models had reduced liver inflammation and fibrosis compared to controls, as assessed by decreased mRNA expressions of Mcp1 and Pdgfrb, reduced immunostaining of the neutrophil marker myeloperoxidase (MPO), and reduction of infiltrated proinflammatory monocytes and neutrophils by flowcytometry. ICAM1 liver immunostaining was increased in patients with MASH and correlated with disease severity (Figure 1A). Bioinformatic analysis of the CODEX multiplexed liver tissue showed distinct zonal distribution of 7 myeloid cells in steatosis versus MASH with adherent myeloid cells to the sinusoidal endothelium (Figure 1B).

Conclusion: Lipotoxic stress enhances the expression of LSECs ICAM1 via a c-Jun-associated histone modification. Inhibition of ICAM1 was salutary in murine MASH mainly secondary to reduced hepatic recruitment of proinflammatory myeloid cells and might serve as a potential therapeutic target for human MASH.