Immune-restricted epigenetic reader SP140 maintains macrophage identity and activation states critical to intestinal homeostasis [RNA-seq]. Homo sapiens

Introduction: SP140 is a bromodomain/plant homeo domain (PHD)-containing reader with immune-restricted expression, and single nucleotide polymorphisms (SNPs) within SP140 associate with Crohn’s disease (CD). However, the function of SP140 and the consequences of disease-associated SP140 SNPs have remained unknown. Results: Individuals carrying CD-associated SNPs within SP140 had defective SP140 mRNA splicing, diminished SP140 protein, and severely suppressed innate immune signatures that stratified them from other CD patients. Conclusion: SP140 is a critical orchestrator of macrophage identity, and a loss of SP140 due to genetic variation contributes to a molecularly defined subset of CD characterized by suppressed innate immunity. Overall design: RNA-seq of 8 samples of peripheral blood derived mononuclear cells from CD patients having T/T or C/C genotype for rs6716753 SNP, with and without LPS treatment. 4 samples from healthy individuals with and without LPS treatment. RNA-seq of human macrophages (total 8 samples from 2 independent donors) stimulated with IFNg for 24h, and LPS (100ng/mL) for 4 hours: siRNA mediated knockdown of SP140 versus controls. RNA-seq of human macrophages (total 8 samples from 2 independent donors) stimulated with IFNg for 24h, and LPS (100ng/mL) for 4 hours for each condition: siRNA mediated knockdown of SP140 versus controls.

引言：SP140是一种携带溴结构域/植物同源结构域 (plant homeo domain, PHD) 的组蛋白阅读器蛋白，其表达具有免疫组织限制性；SP140基因内的单核苷酸多态性 (Single Nucleotide Polymorphism, SNPs) 与克罗恩病 (Crohn’s disease, CD) 存在关联。然而，SP140的具体功能以及疾病相关SP140 SNPs所引发的生物学效应，此前始终未被阐明。
结果：携带CD相关SP140 SNPs的个体，其SP140 mRNA剪接存在缺陷，SP140蛋白水平显著降低，且先天免疫特征谱受到严重抑制，该特征可将此类患者与其他CD患者明确区分。
结论：SP140是巨噬细胞身份认同的核心协调因子，由遗传变异导致的SP140功能缺失，会促成一类以先天免疫受抑为特征的CD分子亚型。
实验设计：
1. 针对rs6716753 SNP位点为T/T或C/C基因型的CD患者，采集其外周血单个核细胞 (Peripheral Blood Mononuclear Cells, PBMCs)，设置脂多糖 (Lipopolysaccharide, LPS) 处理与未处理两组，共获得8个样本进行RNA测序；同时纳入4名健康个体，同样设置LPS处理与未处理组进行RNA测序。
2. 对来自2名独立供体的人类巨噬细胞，先以干扰素γ (Interferon gamma, IFNG) 刺激24小时，再以100ng/mL脂多糖 (LPS) 刺激4小时，设置SP140小干扰RNA (Small interfering RNA, siRNA) 介导的敲低组与阴性对照组，进行RNA测序。
3. 对来自2名独立供体的人类巨噬细胞，在每种刺激条件下（即IFNG预处理后加LPS刺激），分别设置SP140小干扰RNA (siRNA) 介导的敲低组与阴性对照组，进行RNA测序。