NanoString Immunology Panel, fluorescence assisted cell sorting, immunoblotting, immunofluorescence, and quantitative real-time polymerase chain reaction assays were used in wild-type and DRA knockout (KO) mice. Interleukin (IL)-33 blocking was used to determine specific changes in immune cells and co-housing/broad spectrum antibiotics administration, and ex vivo studies in colonoids were conducted to rule out the involvement of microbiota. Colonoid-derived monolayers from healthy and UC patient biopsies were analyzed for translatability.
There was a marked induction of Th2 (>2-fold), CD4+ Th2 cells (∼8-fold), RORγt+ Th17, and FOXP3+ regulatory T cells (Tregs). DRA KO colons also exhibited a robust induction of IL-33 (>8-fold). In vivo studies using blocking of IL-33 established that T2 immune dysregulation (alterations in ILC2, Th2, and GATA3+ iTregs) in response to loss of DRA was due to altered epithelial-immune cell crosstalk via IL-33.
Loss of DRA in colonocytes triggers the release of IL-33 to drive a type 2 immune response. These observations emphasize the critical importance of DRA in mucosal immune homeostasis and its implications in the pathogenesis of UC.
方法:NanoString免疫学小组,荧光辅助细胞分选,免疫印迹,免疫荧光,在野生型和DRA敲除(KO)小鼠中使用定量实时聚合酶链反应测定。白细胞介素(IL)-33阻断用于确定免疫细胞和共住房/广谱抗生素给药的特异性变化,在结肠样中进行了离体研究,以排除微生物群的参与。分析了来自健康和UC患者活检的结肠样来源的单层的可翻译性。
结果:Th2有明显的诱导(p2倍),CD4+Th2细胞(~8倍),RORγt+Th17和FOXP3+调节性T细胞(Tregs)。DRA-KO结肠也表现出IL-33的强诱导(>8倍)。使用IL-33阻断的体内研究确定,响应于DRA损失的T2免疫失调(ILC2、Th2和GATA3+iTregs的改变)是由于通过IL-33改变的上皮-免疫细胞串扰。
结论:结肠细胞中DRA的丢失触发IL-33的释放以驱动T2免疫应答。这些观察结果强调了DRA在粘膜免疫稳态中的至关重要性及其在UC发病机理中的意义。