Ablation of Doublecortin-Like Kinase 1 in the Colonic Epithelium Exacerbates Dextran Sulfate Sodium-Induced Colitis

Abstract

Doublecortin-like kinase 1 (Dclk1), a microtubule-associated kinase, marks the fifth lineage of intestinal epithelial cells called tuft cells that function as tumor stem cells in Apc mutant models of colon cancer. In order to determine the role of Dclk1 in dextran sulfate sodium (DSS) induced colonic inflammation both intestinal epithelial specific Dclk1 deficient (VillinCre;Dclk1f/f) and control (Dclk1f/f) mice were fed 3% DSS in drinking water for 9 days, allowed to recover for 2 days, and killed. The clinical and histological features of DSS-induced colitis were scored and immunohistochemical, gene expression, pro-inflammatory cytokines/chemokines, and immunoblotting analyses were used to examine epithelial barrier integrity, inflammation, and stem and tuft cell features. In DSS-induced colitis, VillinCre;Dclk1f/f mice demonstrated exacerbated injury including higher clinical colitis scores, increased epithelial barrier permeability, higher levels of pro-inflammatory cytokines and chemokines, decreased levels of Lgr5, and dysregulated Wnt/b-Catenin pathway genes. These results suggest that Dclk1 plays an important role in regulating colonic inflammatory response and colonic epithelial integrity.

Fig 1. Characterization of intestinal epithelial deletion of Dclk1 under normal conditions.

A. There is no detectable Dclk1 in the colonic epithelia of Villin^Cre;Dclk1^f/f mice. Colonic tissues stained with anti-Dclk1 antibody (Brown) under normal conditions (200x magnification). B. Dclk1 mRNA levels were dramatically decreased in the Villin^Cre;Dclk1^f/f mice; C. The expression levels of Dclk1 and TJPs in Dclk1^f/f and Villin^Cre;Dclk1^f/f mice; D. Intestinal mucosal integrity was impaired in the Villin^Cre;Dclk1^f/f mice. FITC-Dextran level in the serum of each mouse was determined 4 h after gavage (n = 3 for each group, *p<0.02).

Fig 2. Deletion of Dclk1 exacerbates colonic barrier dysfunction following DSS treatment.

A. Experimental plan; B. H&E staining demonstrated normal histology in Villin^Cre;Dclk1^f/f and Dclk1^f/f mice at baseline and confirmed a significant inflammatory response following DSS treatment (200x magnification) C. Western blot analysis of tight junction and adherens junction proteins demonstrated a decrease in Claudin-1, Claudin-7, and E-cadherin in the Villin^Cre;Dclk1^f/f mice after DSS treatment; D. FITC-Dextran levels in the serum of each mouse were determined 4 h after gavage in baseline and post DSS treatment (n = 5 for post DSS groups, *p<0.02, **** p<0.0001 Villin^Cre;Dclk1^f/f vs Dclk1^f/f); E. Fold change of FITC-Dextran levels in the Villin^Cre;Dclk1^f/f mice post DSS treatment.

Fig 3. Deletion of Dclk1 increases epithelial apoptosis and decreases epithelial proliferation following DSS treatment.

A. Apoptotic cells (Green) identified by TUNEL assay; B. Proliferative cells identified by immunostaining with anti-Ki67 antibody.

Fig 4. Deletion of epithelial Dclk1 exacerbates DSS-induced inflammation of the colonic mucosa.

A. Colonic MPO activity was dramatically increased in the Villin^Cre;Dclk1^f/f mice following DSS treatment; B. The mRNA expression levels of Ptgs2, Il6, Il1β, Cxcl1, and Cxcl2 in colonic tissues were measured by quantitative real-time RT-PCR, and normalized against β-actin. Values in the bar graphs are given as mean ± SEM, *p<0.02, **** p<0.0001 Villin^Cre;Dclk1^f/f vs Dclk1^f/f; C. The protein expression level of RelA in colonic tissues was analyzed by Western blotting.

Fig 5. Deletion of Dclk1 increases NF-κB expression following DSS treatment.

The expression of NF-κB p65 (Brown) was identified by immunostaining with anti-p65 antibody. Occasional nuclear localization of p65 was indicated by arrow in Villin^Cre;Dclk1^f/f mice. Alcian blue was used for counterstaining.

Fig 6. Epithelial deletion of Dclk1 upregulates pro-inflammatory cytokines and chemokines after DSS treatment.

A. Heatmap of Bio-Plex Pro Mouse Cytokine 23-Plex Assay results. B. The individual expression levels of structural cytokines, IL-1α, IL-1β, IL-10, and IL17. C. The individual expression levels of chemokines, Cxcl1, Ccl2, Ccl3, and Ccl5.

Fig 7. Dclk1 epithelial deletion increases disease activity following DSS-induced colitis.

Both Villin^Cre;Dclk1^f/f and Dclk1^f/f mice were fed with 3% DSS in drinking water for 9 days followed by regular drinking water for 2 days to induce colitis (n = 8 for each group). A. Body weight. B. Diarrhea score. C. Fecal blood score. D. Disease activity index. E. Kaplan-Meier survival curve (dashed line = Villin^Cre;Dclk1^f/f, solid line = Dclk1^f/f). Data are presented as the mean ± S.E.M. *p<0.05 Villin^Cre;Dclk1^f/f vs Dclk1^f/f.

Fig 8. Deletion of epithelial Dclk1 alters the intestinal tuft and stem cell signature.

Total RNAs were isolated from colonic tissue. The mRNA expression levels of Dclk1, Ptgs1, Lgr5, and Bmi1 (A, B, C, D) were measured by quantitative real-time RT-PCR, and normalized against β-actin (dashed bar = Villin^Cre;Dclk1^f/f, open bar = Dclk1^f/f). Values in the bar graphs are given as mean ± SEM and *, **, ***, **** denote statistically significant differences (p<0.05, <0.005, <0.0005, 0.00005 respectively). E. Colonic tissues were stained with anti-Lgr5 and anti-Bmi1 antibodies (Brown) post DSS treatment. F. The expression levels of mouse Wnt/b-Catenin regulated genes in colonic tissues were determined using Mouse Wnt/b-Catenin Regulated cDNA Plate Array.

Fig 9. Immunohistochemical staining of β-Catenin in colon tissues following DSS treatment.

The expression of β-Catenin (Brown) was identified by immunostaining with anti- β-Catenin antibody. Increased expression and increased nuclear localization of β-Catenin were found in Villin^Cre;Dclk1^f/f relative to Dclk1^f/f mice. Alcian blue was used for counterstaining.