Eicosatetraynoic Acid Regulates Profibrotic Pathways in an Induced Pluripotent Stem Cell–Derived Macrophage-Human Intestinal Organoid Model of Crohn’s Disease

Abstract

Abstract Background and Aims We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn’s Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). Methods Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage-HIO cocultures were exposed to lipopolysaccharide (LPS) with and without ETYA pretreatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan low-density array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO profibrotic gene expression and collagen content. Results Induced PSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome-secreted products and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and profibrotic gene expression pathways in LPS-stimulated macrophages. Coculture of LPS-primed macrophages with HIO led to upregulation of fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content. ETYA pretreatment prevented profibrotic effects of LPS-primed macrophages. Conclusions ETYA inhibits profibrotic effects of LPS-primed macrophages upon cocultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.