Chemical inhibition of immune signaling rescues defects during heart development in Down syndrome via regulating canonical Wnt pathway

Congenital heart defects (CHDs) are very frequent in children with Down syndrome (DS), the genetic condition caused by trisomy of chromosome 21 (T21). However, the mechanisms by which T21 causes susceptibility to CHDs are poorly understood. Here, using a combination of human induced pluripotent stem cell (iPSC)-based model and Dp(16)1Yey/+ (Dp16) a mouse model of DS, we identified downregulation of canonical Wnt signaling that is caused by increased dosage of interferon (IFN) receptors encoded on chromosome 21 (HSA21) as a causative factor of CHDs in DS. We differentiated human iPSCs derived from individuals with DS as well as CHDs (DS/CHD iPSCs), and controls (control iPSCs) into cardiac cells. We observed that T21 upregulates IFN signaling, downregulates the canonical WNT pathway, and impairs cardiac differentiation. Furthermore, genetic and pharmacological normalization of IFN pathways restored canonical WNT signaling and rescued defects during cardiac differentiation of DS/CHD iPSCs. Strikingly, treatment with an inhibitor of Janus kinase, which is activated by IFN receptor engagement normalized the canonical Wnt pathway and ameliorated CHDs in Dp16 embryos. Our findings provide new mechanisms underlying CHDs in DS, ultimately aiding the development of novel therapeutic strategies. Overall design: Comparative gene expression profiling analysis of RNA-seq data for: 1) human cells derived from healty controls, patients with Down syndrome as well as congenital heart defects, with or without Wnt signaling activator (CHIR99021) and/or JAK inhibitor (JAKi, Tofacitinib) treatments by using the indicated dosage, harvested at different day points during cardiac differentiation as indicated; 2) E15.5 mouse hearts from WT, Dp16 with or without 10 mg/kg bodyweight/day of JAK inhibitor (JAKi, Tofacitinib) treatments from E6.5 to E14.5, low p-STAT1 indicated the p-STAT1 levels are comparable to WT without JAKi treatment, high p-STAT1 indicated the p-STAT1 levels are significantly higher than WT without JAKi treatment.

先天性心脏缺陷（Congenital heart defects, CHDs）在唐氏综合征（Down syndrome, DS）患儿中极为常见；唐氏综合征是由21号染色体三体（trisomy of chromosome 21, T21）引发的遗传疾病。然而，目前对于21号染色体三体如何导致先天性心脏缺陷易感性的分子机制仍知之甚少。本研究结合基于人类诱导多能干细胞（induced pluripotent stem cell, iPSC）的模型与唐氏综合征小鼠模型Dp(16)1Yey/+（Dp16），发现人类21号染色体（Human chromosome 21, HSA21）编码的干扰素（interferon, IFN）受体剂量升高会引发经典Wnt信号通路下调，这一现象是唐氏综合征患者并发先天性心脏缺陷的致病因素。我们分别将源自唐氏综合征患者、并发先天性心脏缺陷的唐氏综合征患者的诱导多能干细胞（DS/CHD iPSCs），以及健康对照诱导多能干细胞（control iPSCs）定向分化为心肌细胞。实验结果显示，21号染色体三体可上调干扰素信号通路、下调经典Wnt信号通路，并损害心肌分化过程。此外，通过遗传学与药理学手段干预干扰素通路使其恢复正常水平，可修复经典Wnt信号通路，并挽救DS/CHD诱导多能干细胞在心肌分化过程中的缺陷。值得注意的是，使用贾纳斯激酶（Janus kinase, JAK）抑制剂——该激酶可通过干扰素受体结合被激活——进行干预，可使Dp16胚胎的经典Wnt通路恢复正常，并改善其先天性心脏缺陷表型。本研究揭示了唐氏综合征并发先天性心脏缺陷的全新分子机制，可为新型治疗策略的开发提供理论依据。实验整体设计：对以下两组RNA测序（RNA-seq）数据进行比较基因表达谱分析：1. 心肌分化过程中不同时间点收集的细胞样本，来源包括健康对照、唐氏综合征患者以及并发先天性心脏缺陷的唐氏综合征患者；部分样本经指定剂量的Wnt信号通路激活剂（CHIR99021）、贾纳斯激酶抑制剂（JAKi，托法替布，Tofacitinib）或二者联合处理；2. 野生型（wild type, WT）与Dp16小鼠的E15.5心脏样本：其中部分Dp16小鼠自E6.5至E14.5每日以10 mg/kg体重的剂量接受贾纳斯激酶抑制剂（JAKi，托法替布）处理。低p-STAT1水平指其磷酸化STAT1水平与未接受JAKi处理的野生型小鼠相当，高p-STAT1水平指其磷酸化STAT1水平显著高于未接受JAKi处理的野生型小鼠。