Pluripotent stem cell models of Blau syndrome reveal an IFN-<gamma>-dependent inflammatory response in macrophages

Background: Blau syndrome, or early-onset sarcoidosis, is a juvenile-onset systemic granulomatosis associated with a mutation in Nucleotide-binding oligomerization domain 2 (NOD2). The underlying mechanisms of Blau syndrome leading to autoinflammation are still unclear, and there is currently no effective specific treatment for Blau syndrome.Objectives: To elucidate the mechanisms of autoinflammation in Blau syndrome, we sought to clarify the relation between disease associated-mutant NOD2 and the inflammatory response in human samples.Methods: Blau syndrome-specific induced pluripotent stem cells (iPSCs) lines were established. To precisely evaluate the in vitro phenotype of iPSC-derived cells, the disease-associated NOD2 mutation of iPSCs was corrected using a CRISPR/Cas9 system. We also introduced the same NOD2 mutation into a control iPSC line. These isogenic iPSCs were then differentiated into monocytic cell lineages, and the status of NF-κB pathway and proinflammatory cytokine secretion were investigated.Results: We focused on the signals that upregulate the expression of NOD2, especially IFN-γ signaling. IFN-γ treatment of NOD2-mutant macrophages induced ligand-independent NF-κB activation and proinflammatory cytokine production. IFN-γ treatment acted as a priming signal through the up-regulation of NOD2 protein and recruitment of NOD2 on the basement membrane. Conversely, the production of proinflammatory cytokines by MDP, a ligand of NOD2, was decreased in mutant macrophages.Conclusions: Our data support the significance of ligand-independent autoinflammation in the pathophysiology of Blau syndrome. Our comprehensive isogenic disease-specific iPSC panel provides a useful platform for probing therapeutic and diagnostic clues for the treatment of Blau syndrome patients.背景：布劳综合征（Blau syndrome），又称早发性结节病（early-onset sarcoidosis），是一种青少年起病的系统性肉芽肿性疾病，与核苷酸结合寡聚化结构域2（Nucleotide-binding oligomerization domain 2, NOD2）突变密切相关。目前，布劳综合征导致自身炎症的潜在分子机制尚未阐明，且尚无有效的特异性治疗方案。 目的：为阐明布劳综合征的自身炎症发病机制，本研究旨在明确疾病相关性突变型NOD2与人体样本中炎症反应之间的关联。 方法：本研究构建了布劳综合征特异性诱导多能干细胞（induced pluripotent stem cells, iPSCs）系。为精准评估iPSC衍生细胞的体外表型，采用CRISPR/Cas9系统对iPSCs携带的疾病相关性NOD2突变进行了校正。同时，将相同的NOD2突变引入对照iPSC系中。随后将这些同基因iPSC分化为单核细胞谱系，并对核因子κB（NF-κB）通路的激活状态及促炎细胞因子的分泌情况进行了检测分析。 结果：本研究聚焦于上调NOD2表达的信号通路，尤其是干扰素-γ（IFN-γ）信号通路。对携带NOD2突变的巨噬细胞施加IFN-γ处理后，可诱导配体非依赖性NF-κB激活及促炎细胞因子产生。IFN-γ处理通过上调NOD2蛋白表达并将NOD2招募至基底膜，发挥启动信号的作用。与之相反，NOD2的配体胞壁酰二肽（muramyl dipeptide, MDP）所诱导的促炎细胞因子产生，在突变型巨噬细胞中显著降低。 结论：本研究数据证实了配体非依赖性自身炎症在布劳综合征病理生理过程中的重要作用。本研究构建的综合性同基因疾病特异性iPSC文库，可为探索布劳综合征患者的治疗及诊断线索提供实用的研究平台。