DDX5 RNA interactome in cultured T cells. DDX5 RNA interactome in cultured T cells

RAR-related orphan receptor gamma (ROR t)-expressing regulatory T (ROR t+ Treg) cells play pivotal roles in preventing T cell hyperactivation and maintaining tissue homeostasis, in part, by secreting the anti-inflammation cytokine interleukin 10 (IL-10). Here, we report that Hypoxia Induced Factor 1a (HIF1alpha) is the master transcription factor for *Il10* in ROR t+ Tregs. Interestingly, this critical anti-inflammatory pathway is negatively regulated by an RNA-binding protein DEAD box helicase 5 (DDX5). As a transcriptional corepressor, DDX5 restricts the expression of HIF1 and its downstream target gene *Il10* in ROR t+ Tregs. T cell specific *Ddx5* knockout (DDX5 T) mice have augmented ROR t+ Treg suppressor activities and are better protected from intestinal inflammation. Genetic ablation or pharmacologic inhibition of HIF1a restores enteropathy susceptibility in DDX5 T mice. The DDX5-HIF1 -IL-10 pathway is conserved in mice and humans. These findings reveal potential therapeutic targets for intestinal inflammatory diseases. Overall design: Primary cultured Th17 from two 8-10 weeks old wild-type (C57BL/6) female mice were subjected to UV- mediated crosslinking, lysis, and treatment with RNases, followed by immunoprecipitation (IP) of the DDX5-containing RNA complexes.

视黄酸相关孤儿受体γ（RAR-related orphan receptor gamma, RORγt）阳性调节性T细胞（RORγt+ Treg）在抑制T细胞过度活化、维持组织稳态中发挥关键作用，其部分功能依赖于分泌抗炎细胞因子白细胞介素10（interleukin 10, IL-10）。本研究发现，缺氧诱导因子1α（Hypoxia Induced Factor 1a, HIF1α）是RORγt+ Treg中*Il10*基因的核心转录因子。有趣的是，这一关键抗炎通路可被RNA结合蛋白DEAD盒解旋酶5（RNA-binding protein DEAD box helicase 5, DDX5）负向调控。作为转录共抑制因子，DDX5可抑制RORγt+ Treg中HIF1α及其下游靶基因*Il10*的表达。T细胞特异性*Ddx5*基因敲除（DDX5 T）小鼠的RORγt+ Treg抑制活性增强，且能更有效地抵御肠道炎症。敲除HIF1α或采用药物抑制HIF1α，可恢复DDX5 T小鼠的肠病易感性。DDX5-HIF1α-IL-10通路在小鼠与人类中均保守存在。本研究结果为肠道炎症性疾病提供了潜在治疗靶点。整体实验设计：从2只8~10周龄野生型（C57BL/6）雌性小鼠中分离原代培养的辅助性T细胞17（Th17），经紫外介导交联、细胞裂解及核糖核酸酶处理后，对含有DDX5的RNA复合物进行免疫沉淀（IP）实验。