Kaiso-induced intestinal inflammation is preceded by diminished E-cadherin expression and intestinal integrity

Abstract

Chronic intestinal inflammation contributes to pathologies such as inflammatory bowel disease (IBD) and colon cancer. While the precise etiology remains controversial, IBD is believed to manifest as a result of various factors. We previously reported that intestinal-specific overexpression of the transcription factor Kaiso results in an intestinal inflammatory response; however, the cause of this inflammation is unknown. To elucidate the underlying mechanism(s) of the Kaiso-mediated intestinal inflammatory phenotype, we evaluated two independent transgenic mouse lines that express varying levels of Kaiso (KaisoTg). Histological analyses of KaisoTg mice revealed intestinal damage including thickening of the mucosa, intestinal "lesions" and crypt abscesses, which are reminiscent of IBD pathology. Additionally, higher Kaiso levels induced intestinal neutrophilia as early as 12 weeks, which worsened as the mice aged. Notably, the Kaiso-induced intestinal inflammation correlated with a leaky intestinal barrier and mis-regulation of E-cadherin expression and localization. Interestingly, Kaiso overexpression resulted in reduced proliferation but enhanced migration of intestinal epithelial cells prior to the onset of inflammation. Collectively, these data suggest that Kaiso plays a role in regulating intestinal epithelial cell integrity and function, dysregulation of which contributes to a chronic inflammatory phenotype as mice age.

Fig 1. Kaiso^Tg mice display hallmarks of chronic inflammation.

(A) Inflammation in Crohn’s disease (CD) patients correlates with higher Kaiso expression levels. (B) Kaiso^Tg mice display discontinuous lesions through the small intestine (red asterisks), blunted villi and thickening of the muscularis externa (black asterisks) compared to the NonTg siblings at 8-months of age. Insets are enlarged images of the areas outlined in yellow. (C) Line A (VKA) shows fewer crypt abscesses at 8 months of age compared to Line E (VKE) (n = 3 mice/phenotype). Statistical significance was determined by student’s t-test, and error bars are SEM *p <0.05. (D) Early age MPO analysis on age matched VKA and VKE mice show an increase in inflammation in VKE mice compared to VKA and NonTg mice at 14 weeks in SPF (n = 5–7 mice/genotype per age). Statistical significance was determined by two-way ANOVA, and error bars are SEM, ****p <0.0001. (E) At 14 weeks, VKE mice show a significant increase in neutrophils compared to age-matched NonTg mice and VKA mice. (n = 7–9 mice/genotype). Statistical significance was determined by one-way ANOVA, and error bars are SEM, *p <0.05.

Fig 2. Kaiso^Tg mice develop inflammation with age.

(A) Kaiso^Tg mice do not show inflammation upon weaning (~3 wk.). However, Line E (VKE) mice develop inflammation faster than Line A (VKA) mice (12- vs. 24-wks of age; n = 5–9 mice/genotype/time point). Statistical significance was determined by two-way ANOVA, and error bars are SEM; * p<0.05; ****p <0.0001. (B) mRNA expression levels of the neutrophil attracting cytokine, MIP-2, are increased after, but not before, inflammation in VKA mice compared to NonTg mice, at 24- and 12-weeks of age, respectively. qPCR data are of pooled biological replicates from IECs isolated from the entire small intestine of 6 mice/genotype for each time point, run in experimental and technical triplicate. Statistical significance was determined by student’s t-test, and error bars are SEM; ***p <0.001.

Fig 3. Pre-symptomatic VKA mice exhibit reduced E-cadherin expression prior to inflammation onset.

(A) Inflamed intestinal tissues of 14-week old VKE mice exhibit significantly increased permeability to 4 kDa FITC-dextran, compared to the non-inflamed tissues of age-matched VKA and NonTg mice (n = 10 mice/genotype). Statistical significance was determined by one-way ANOVA, ****p<0.0001, error bars represent SEM. (B) IHC of ZO-1 and Claudin-2 show similar localization to the apical membrane (red arrowheads) in both 12-week old NonTg and VKA mice. (C) IHC of E-Cadherin, p120^ctn and β-catenin reveals localization along the lateral membrane (red arrowheads) in both 12-week old NonTg and VKA mice. (C-i) 12-week old VKA mice display a modest reduction in laterally-localized E-cadherin in both villi and crypts, compared to NonTg mice prior to inflammation onset, however Kaiso overexpression did not alter the expression or subcellular localization of either p120^ctn or β-catenin (C-ii, iii). (D) Immunoblot analysis of adherens junction proteins from isolated IECs reveals decreased levels of E-cadherin, but not p120^ctn or β-catenin, in 12-week old VKA mice compared to NonTg siblings.

Fig 4. Intestinal epithelial cell repair mechanisms are altered prior to inflammation onset in 12-week old VKA mice.

(A-i) VKA mice do not demonstrate a change in number of Ki67-positive cells. Yellow arrowheads denote crypt base cells. (A-ii) Ki67-positive cells from 100 open crypts (n = 5 mice/genotype) were quantified. Crypt base cells were scored by counting the number of Ki67-positive cells in the bottom third of the crypt. The remaining cells were scored as progenitor cells. (B) Proliferation marker Cyclin D1 and apoptotic marker cleaved (c-) Caspase 3 are both decreased in VKA mice. (C, D) VKA mice show increased migration of epithelial cells up the crypt-villus axis relative to their NonTg counterparts, as demonstrated by IHC (C), and quantified by measuring the distance of the furthest-migrated BrdU-positive cell (~ 100 villi from 3 mice/genotype) (D). (D-i, -iii) Distance migrated was quantified by measuring the distance from the base of the crypt, to the furthest positively BrdU-labelled cell. (D-ii, iv) To correct for possible differences in villus height, the average distance migrated was normalized to the average villus height. Representative plots of at least three blind measurements are shown. Statistical significance was determined by unpaired student’s t-test. ***p<0.001, error bars represent SEM.

Fig 5. Working model of kaiso-mediated intestinal inflammation.

Pre-symptomatic Kaiso^Tg mice exhibit attenuated E-cadherin expression, which is accompanied by accelerated IEC migration along the crypt-villus axis. Together with diminished Cyclin D1 expression, this suggests that IECs migrate faster than they are turned over. Poor intestinal epithelial integrity renders Kaiso^Tg mice susceptible to microbial infection, which triggers a neutrophil-specific response. Pathogenic neutrophil recruitment results in continued intestinal tissue damage (e.g. blunted villi, crypt abscesses etc.), and thus exacerbates inflammation. EC–enterocyte, GC–goblet cell, EEC–enteroendocrine cell, PC–Paneth cell, TA–transit amplifying cell, ISC–intestinal stem cell.