Roy Badal C, Ahmed Ishfaq, Stubbs Jason, Zhang Jun, Attard Thomas, Septer Seth, Welch Danny, Anant Shrikant, Sampath Venkatesh, Umar Shahid
Department of Surgery, University of Kansas Medical Center, Kansas City, KS, USA.
Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS, USA.
Cell Death Discov. 2021 Jun 17;7(1):169. doi: 10.1038/s41420-021-00526-9.
Alternative promoter usage generates long and short isoforms (DCLK1-L and DCLK1-S) of doublecortin-like kinase-1 (DCLK1). Tight control of Notch signaling is important to prevent and restitute inflammation in the intestine. Our aim was to investigate whether Notch1-DCLK1 axis regulates the mucosal immune responses to infection and whether this is phenocopied in human models of colitis. In the FFPE (formalin-fixed paraffin-embedded) sections prepared from the colons of ulcerative colitis (UC) and immune-mediated colitis (IRAEC) patients, expression of DCLK1 isoforms correlated positively with Notch1 and negatively with a transcriptional repressor, FoxD3 (Forkhead Box D3). DCLK1 protein staining in these sections was predominantly sub-epithelial (stromal) wherein DCLK1 co-localized with NICD, CD68, CD11c, and neutrophil elastase (NE). NE also co-stained with Citrullinated-H3 indicating the presence of neutrophil extracellular traps. In human neutrophils, elevated levels of DCLK1-S, CXCL-10, Ly6G, MPO, NE, and Notch1/2 in LPS-treated cells were inhibited when LPS was added in conjunction with Notch blocker dibenzazepine (DBZ; LPS + DBZ group). In CR-infected Rag1 mice, higher levels of DCLK1 in the colonic crypts were inhibited when mice received DBZ for 10 days coincident with significant dysbiosis, barrier disruption, and colitis. Concurrently, DCLK1 immunoreactivity shifted toward the stroma in CR + DBZ mice with predominance of DCLK1-S that coincided with higher Notch1 levels. Upon antibiotic treatment, partial restoration of crypt DCLK1, reduction in MPO activity, and increased survival followed. When intestinal epithelial cell-specific Dclk1-knockout (Dclk1) or Dclk1;Rag1 double knockout (DKO) mice were infected with CR and given a single dose of DBZ, they developed barrier defect and severe colitis with higher levels of stromal DCLK1-S, Ly6G, NE, and Notch1. We therefore propose that, by regulating the mucosal immune responses, the Notch-DCLK1 axis may be integral to the development of murine or human colitis.
可变启动子的使用产生了双皮质素样激酶-1（DCLK1）的长亚型和短亚型（DCLK1-L和DCLK1-S）。严格控制Notch信号对于预防和恢复肠道炎症很重要。我们的目的是研究Notch1-DCLK1轴是否调节对感染的黏膜免疫反应，以及在人类结肠炎模型中是否也有类似情况。在从溃疡性结肠炎（UC）和免疫介导性结肠炎（IRAEC）患者的结肠制备的福尔马林固定石蜡包埋（FFPE）切片中，DCLK1亚型的表达与Notch1呈正相关，与转录抑制因子FoxD3（叉头框D3）呈负相关。这些切片中的DCLK1蛋白染色主要位于上皮下（基质），其中DCLK1与NICD、CD68、CD11c和中性粒细胞弹性蛋白酶（NE）共定位。NE也与瓜氨酸化-H3共染色，表明存在中性粒细胞胞外陷阱。在人中性粒细胞中，当脂多糖（LPS）与Notch阻滞剂二苯并氮杂卓（DBZ；LPS + DBZ组）联合添加时，LPS处理的细胞中DCLK1-S、CXCL-10、Ly6G、MPO、NE和Notch1/2水平的升高受到抑制。在柠檬酸杆菌（CR）感染的Rag1小鼠中，当小鼠连续10天接受DBZ时，结肠隐窝中较高水平的DCLK1受到抑制，同时伴有明显的菌群失调、屏障破坏和结肠炎。同时，CR + DBZ小鼠中DCLK1免疫反应性向基质转移，以DCLK1-S为主，这与较高的Notch1水平一致。抗生素治疗后，隐窝DCLK1部分恢复，MPO活性降低，存活率提高。当肠道上皮细胞特异性Dclk1基因敲除（Dclk1）或Dclk1;Rag1双基因敲除（DKO）小鼠感染CR并给予单剂量DBZ时，它们出现屏障缺陷和严重结肠炎，基质中DCLK1-S、Ly6G、NE和Notch1水平升高。因此，我们提出，通过调节黏膜免疫反应，Notch-DCLK1轴可能是小鼠或人类结肠炎发生所必需的。
