BET protein inhibition regulates macrophage chromatin accessibility and microbiota-dependent colitis [ATAC-seq]

In colitis, macrophage functionality is altered compared to normal homeostatic conditions. Loss of IL-10 signaling results in an inappropriate chronic inflammatory response to bacterial stimulation. It remains unknown if inhibition of bromodomain and extra-terminal domain (BET) proteins alters usage of DNA regulatory elements responsible for driving inflammatory gene expression. We determined if the BET inhibitor, (+)-JQ1, could suppress inflammatory activation of macrophages in Il10-/- mice. We performed ATAC-seq and RNA-seq on Il10-/- bone marrow-derived macrophages (BMDMs) cultured in the presence and absence of lipopolysaccharide (LPS) with and without treatment with (+)-JQ1 and evaluated changes in chromatin accessibility and gene expression. Treatment with (+)-JQ1 suppressed LPS-induced changes in chromatin at distal regulatory elements associated with inflammatory genes, particularly in regions that contain motifs for AP-1 and IRF transcription factors. Overall design: Mature bone marrow derived macrophages were cultured and biologically matched samples (n=3 biological replicates/condition) were generated for the following conditions: 1) unstimulated and no treatment, 2) unstimulated and (+)-JQ1 treatment (500nM), 3) unstimulated and IL-10 treatment (10ng/mL), 4) lipopolysaccharide (LPS) stimulation (50ng/mL) and no treatment, 5) LPS stimulation and (+)-JQ1 treatment, and 6) LPS stimulation and IL-10 treatment. Samples were treated with (+)-JQ1 or IL-10 or remained untreated in culture for 12 hours followed by LPS stimulation for 4 hours in for appropriate conditions (with PBS serving as a control).

结肠炎状态下，巨噬细胞功能与正常生理稳态条件相比发生改变。白细胞介素10（IL-10）信号通路的缺失会导致机体对细菌刺激产生不当的慢性炎症反应。目前尚不明确溴结构域与额外末端结构域（BET）蛋白的抑制作用是否会改变驱动炎症基因表达的DNA调控元件的使用模式。本研究旨在探究BET抑制剂(+)-JQ1能否抑制Il10-/-小鼠巨噬细胞的炎症活化。我们对在添加/不添加脂多糖（LPS）、且经(+)-JQ1处理或未经处理的培养体系中培养的Il10-/-小鼠骨髓来源巨噬细胞（BMDMs）进行了ATAC-seq与RNA-seq检测，并评估了其染色质开放性与基因表达的变化情况。(+)-JQ1处理可抑制炎症基因相关远端调控元件处LPS诱导的染色质开放性改变，尤其针对含有AP-1与IRF转录因子结合基序的区域。整体实验设计：将成熟骨髓来源巨噬细胞进行体外培养，针对以下6组实验条件分别设置生物学重复样本（每组含3个生物学重复）：1）未刺激且未处理组；2）未刺激且经(+)-JQ1处理组（浓度为500nM）；3）未刺激且经IL-10处理组（浓度为10ng/mL）；4）经脂多糖（LPS，浓度为50ng/mL）刺激且未处理组；5）经LPS刺激且经(+)-JQ1处理组；6）经LPS刺激且经IL-10处理组。所有样本先在培养液中经(+)-JQ1、IL-10处理或不予处理培养12小时，随后对对应组别添加LPS刺激4小时（以磷酸盐缓冲液PBS作为空白对照）。