Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn’s Disease. Eicosatetraynoic Acid Regulates Pro-Fibrotic Pathways in an Induced Pluripotent Stem Cell Derived Macrophage:Human Intestinal Organoid Model of Crohn’s Disease

Background: We previously reported Crohn’s Disease (CD) ileal macrophage and fibroblast gene expression modules linked to future strictures, and identified small molecules which may reverse this gene signature. Here we developed a model system to test a lead candidate, eicosatetraynoic acid (ETYA), a Peroxisome Proliferator-Activated Receptor (PPAR) agonist. Methods: CD patient induced pluripotent stem cells (iPSC) were differentiated into macrophages and human intestinal organoids (HIOs). Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. 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 pro-fibrotic gene expression and collagen content. Results: iPSC-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 pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation fibroblast activation genes including ACTA2 and COL1A1, and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. Conclusions: ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model system may be used to screen personalized effects of candidate small molecules upon inflammatory and fibrotic pathways implicated in refractory CD patients. Overall design: CD patient-induced pluripotent stem cells (iPSC) were differentiated into macrophages. These macrophages were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Overall there were 6 macrophages controls with no treatment, 6

研究背景：本团队此前曾报道与克罗恩病（Crohn’s Disease, CD）回肠狭窄相关的巨噬细胞及成纤维细胞基因表达模块，并筛选出可逆转该基因特征的小分子化合物。本研究构建了一套模型体系，用于测试候选先导化合物二十碳四炔酸（eicosatetraynoic acid, ETYA）——一种过氧化物酶体增殖物激活受体（Peroxisome Proliferator-Activated Receptor, PPAR）激动剂。
实验方法：将克罗恩病患者诱导多能干细胞（induced pluripotent stem cells, iPSC）诱导分化为巨噬细胞及人类肠道类器官（human intestinal organoids, HIOs）。分别对巨噬细胞及巨噬细胞与HIO共培养体系施加脂多糖（lipopolysaccharide, LPS）刺激，同时设置ETYA预处理组与未预处理对照组。采用细胞离心涂片法与流式细胞术表征巨噬细胞形态与活化标志物，通过RNA测序获取巨噬细胞基因表达的全局图谱。运用TaqMan低密度芯片、Luminex多重检测法、免疫组织化学染色及天狼星红偏振光显微镜术，检测巨噬细胞细胞因子分泌情况、HIO的促纤维化基因表达水平及胶原含量。
实验结果：诱导多能干细胞分化获得的巨噬细胞形态与原代巨噬细胞相似，且表达CD64、CD68等炎性巨噬细胞表面标志物。经LPS刺激的巨噬细胞基因表达全局图谱富集于克罗恩病回肠炎性巨噬细胞及基质组（matrisome）分泌蛋白，同时分泌CCL2、IL1B、OSM等与难治性疾病相关的细胞因子与趋化因子。ETYA可抑制经LPS刺激的巨噬细胞中CD64的表达丰度及促纤维化基因表达通路。将LPS致敏的巨噬细胞与HIO共培养，可上调ACTA2、COL1A1等成纤维细胞活化基因，并增加HIO的胶原含量。ETYA预处理可阻断LPS致敏巨噬细胞的促纤维化效应。
研究结论：ETYA可抑制LPS致敏巨噬细胞对共培养HIO的促纤维化效应。该模型体系可用于筛选候选小分子化合物对难治性克罗恩病患者相关炎性及纤维化通路的个体化调控效应。
总体实验设计：将克罗恩病患者诱导多能干细胞诱导分化为巨噬细胞，分别经脂多糖刺激，同时设置ETYA预处理组与未预处理对照组。本研究共设置6例未处理的巨噬细胞对照组，6例