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Published on: April 21, 2015
DCLK1 isoforms and aberrant Notch signaling in the regulation of human and murine colitis
Badal C Roy1, Ishfaq Ahmed1, Jason Stubbs2
1Department of Surgery, University of Kansas Medical Center, Kansas City, KS, USA.
Abstract:
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 (Dclk1ΔIEC) or Dclk1ΔIEC;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.
Insights
The Notch-DCLK1 axis regulates intestinal immune responses, influencing colitis development in humans and mice. Blocking Notch signaling impacts doublecortin-like kinase-1 (DCLK1) expression and immune cell activity, suggesting a therapeutic target.
Area of Science:
- Immunology
- Gastroenterology
- Cell Biology
Background:
- Notch signaling is crucial for intestinal inflammation control.
- Alternative promoter usage generates doublecortin-like kinase-1 (DCLK1) long (DCLK1-L) and short (DCLK1-S) isoforms.
- The role of the Notch1-DCLK1 axis in mucosal immune responses to infection and colitis is unclear.
Purpose of the Study:
- To investigate the role of the Notch1-DCLK1 axis in regulating mucosal immune responses to infection.
- To determine if this axis is involved in the pathogenesis of human and murine colitis.
- To explore the therapeutic potential of targeting this axis.
Main Methods:
- Analysis of formalin-fixed paraffin-embedded colon sections from ulcerative colitis (UC) and immune-mediated colitis (IRAEC) patients.
- Immunohistochemical co-localization of DCLK1, Notch1, and immune cell markers (CD68, CD11c, neutrophil elastase).
- In vitro studies using human neutrophils treated with lipopolysaccharide (LPS) and Notch inhibitor (DBZ).
- In vivo studies using CR-infected Rag1-/- mice and intestinal epithelial cell-specific Dclk1-knockout mice treated with DBZ and/or antibiotics.
Main Results:
- DCLK1 isoform expression positively correlated with Notch1 and negatively with FoxD3 in human colitis samples.
- DCLK1 predominantly localized to the stroma, co-localizing with immune cells and neutrophil extracellular traps (NETs).
- Notch inhibition (DBZ) reduced DCLK1-S, CXCL-10, and neutrophil markers in LPS-treated human neutrophils.
- DBZ treatment in CR-infected mice reduced colonic DCLK1, induced dysbiosis, barrier disruption, and colitis, shifting DCLK1 to the stroma.
- Antibiotic treatment partially restored crypt DCLK1, reduced MPO activity, and improved survival.
- Dclk1-knockout mice treated with DBZ developed severe colitis with increased stromal DCLK1-S, neutrophil markers, and Notch1.
Conclusions:
- The Notch-DCLK1 axis plays a significant role in regulating mucosal immune responses during intestinal infection and inflammation.
- This axis is implicated in the development of both murine and human colitis.
- Targeting the Notch-DCLK1 pathway may offer a therapeutic strategy for inflammatory bowel diseases.
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