Graduate Research Assistant Univ. of Cincinnati Col. of Med. Cincinnati, Ohio, United States
Disclosure(s):
Amelia M. Pearson: No financial relationships to disclose
Introduction/Rationale: Metabolic dysfunction-associated steatohepatitis (MASH) is an inflammatory liver disease linked to type 2 diabetes and cardiovascular disease. During MASH, Kupffer cells and other liver macrophages exacerbate liver inflammation in response to elevated lipids and immune stimuli. In a mouse model, Kupffer cells had increased expression and genomic occupancy of activating transcription factor 3 (ATF3)—a known transcriptional repressor of macrophage inflammation.
Methods: Kupffer cells and other liver macrophages from Clec4f-Cre Atf3-fl/fl or Atf3-fl/wt controls were analyzed by flow cytometry and RNA-seq. Bone marrow derived macrophages from Lyz2-Cre Atf3-fl/fl mice or Cre negative controls were treated with oxidized low-density lipoprotein, lipopolysaccharide, and/or methyl-beta-cyclodextrin. Genotype-dependent effects were assessed by ELISA, RNA-seq and cholesterol assays.
Results: ATF3 binds cholesterol-associated genes, and its loss in Clec4f+ Kupffer cells increased neutral lipid levels with limited transcriptional differences compared to wild type controls. In vitro, ATF3 restricted baseline cellular cholesterol, while its deficiency elevated basal interferon-stimulated genes (ISG) expression and exaggerated type 1 interferon and cytokine responses to TLR stimulation. Lipid loading reduced ISG expression, potentially explaining limited in vivo differences. However, inflammatory stimuli in lipid loaded cells amplified gene expression changes, suggesting ATF3 function is both lipid- and inflammation-dependent. ATF3-deficient macrophages upregulated Gramdb1 (Aster B), a membrane to ER cholesterol transporter, and downregulated Cav1 (Caveolin-1), a potential efflux mediator. These changes may increase membrane cholesterol. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin negated the genotype-dependent cytokine response.
Conclusion: Together, our data suggest ATF3 supports cholesterol trafficking from the plasma membrane to preserve membrane homeostasis and limit inflammation.
