Condition-specific role of colonic inflammatory molecules in persistent functional colorectal hypersensitivity in the mouse

Abstract

Background: A low-level inflammation has been hypothesized to mediate visceral hypersensitivity in functional bowel disorders that persist after or even in the absence of gut inflammation. We aimed to test the efficacy of a steroidal anti-inflammatory treatment, and identify local inflammatory molecules mediating post- and non-inflammatory colorectal hypersensitivity using two mouse models.

Methods: Visceromotor responses to colorectal distension were quantified as a measure of colorectal sensitivity. On day 1, mice received intracolonic saline (control), trinitrobenzenesulfonic acid (postinflammatory on day 15), or acidified hypertonic saline (non-inflammatory). Colorectal sensitivity before (day 10) and after (day 15) 4-day dexamethasone (Dex) treatment was compared, and colonic gene expression of inflammatory molecules was quantified.

Key results: Dexamethasone effectively inhibited gene expression of inflammatory molecules such as interleukin (IL)-1β and mast cell protease-1 in the colon, but did not attenuate colorectal hypersensitivity in either model. Gene expression of inflammatory molecules in the colon did not differ between control and the non-inflammatory model, but the postinflammatory model showed increased IL-10 and tight junction protein 2, and decreased IL-6, transforming growth factor (TGF)-β, a precursor of β-endorphin, occludin, and mucin 2. While no common molecule explained colorectal hypersensitivity in these models, hypersensitivity was positively correlated with TGF-β2 mRNA in control, and with IL-1β, inhibin βA, and prostaglandin E2 synthase in the Dex-treated postinflammatory model. In the non-inflammatory model, cyclooxygenase-2 mRNA was negatively correlated with colorectal sensitivity.

Conclusions & inferences: These results suggest that persistent functional colorectal hypersensitivity is mediated by condition-specific mediators whose gene expression in the colon is not inevitably sensitive to steroidal anti-inflammatory treatment.

Keywords: colorectal hypersensitivity; cytokines; inflammation; inflammatory molecules.

Fig 1

Effect of dexamethasone (Dex) on post-inflammatory (post-TNBS) and non-inflammatory (AHS) colorectal hypersensitivity in the mouse. (A) Scatter plot showing colorectal sensitivity at 60 mmHg colorectal distension (CRD) measured on day 10 in individual mouse (% of maximum baseline responses). Note the outlier in the control group (in a dotted box). Excluding the outlier revealed significantly greater colorectal sensitivity in post-TNBS and AHS groups than in control (* p<0.05 by 1-way ANOVA). (B-D) Dex (5 mg kg-¹), administered once daily for four days (days 11-14) did not alleviate colorectal hypersensitivity in any groups (before, day 10; after, day 15). Veh, saline vehicle. Mice with colorectal sensitivity greater than 125% (above shaded area) were considered hypersensitive to CRD.

Fig 2

Colonic gene expression of pro-inflammatory cytokines and mast cell protease. Gene expression data were subgrouped by model (control, post-TNBS, or AHS), treatment (Veh or Dex), and colorectal sensitivity (normo- or hyper-sensitive). Numbers in each horizontal bar in A represent the number of mice in each subgroup throughout. Gene expression of pro-inflammatory cytokines interleukin (IL)-1β (A) and mast cell protease (MCPT)-1 (B) was significantly reduced by Dex (*** p<0.001 and * p<0.05 by 3-way ANOVA). (C) IL-6 gene expression was lower in post-TNBS mice than in control (* p<0.05 by 3-way ANOVA followed by Tukey's multiple comparison tests).

Fig 3

Colonic gene expression of IL-1β and colorectal hypersensitivity in mice with active colitis. (A) Dex effectively inhibited up-regulation of IL-1β gene expression in the colons from TNBS-treated mice on day 3. (B) However, Dex did not prevent colorectal hypersensitivity in these mice. Numbers in each bar indicate the number of mice in each group.

Fig 4

Colonic gene expression of IL-10 and its receptor. (A) IL-10 gene transcript was more abundant in post-TNBS mice than in control and AHS-treated mice (* p<0.05 by 3-way ANOVA followed by Tukey's multiple comparison tests). (B) Gene expression of IL-10 receptor A (RA) was attenuated by Dex (* p<0.05 by 3-way ANOVA).

Fig 5

Colonic gene expression of prostaglandin synthases and β-endorphin precursor, proopiomelanocortin (POMC). (A) Gene expression of cyclooxygenase (COX)-2 was lower in post-TNBS mice than in control (* p<0.05 by 3-way ANOVA followed by Tukey's multiple comparison tests). (B) Dex tended to increase the gene expression of prostaglandin E synthase (PGES, p=0.057 by 3 way ANOVA). (C) Gene expression of POMC was lower in post-TNBS mice than in control (p<0.05 by 3-way ANOVA followed by Tukey's multiple comparison tests).

Fig 6

Colonic gene expression of transforming growth factor (TGF)-β cytokine family and their receptors. Gene expression of TGF-β1 (A) and TGF-β2 (B) was significantly lower in post-TNBS mice than in control or AHS-treated mice (** p<0.01 by 3-way ANOVA followed by Tukey's multiple comparison tests). (C) Gene expression of inhibin βA showed a tendency to a decrease in post-TNBS mice (p=0.074 vs. control). Its gene expression also tended to be higher in hypersensitive mice (p=0.056). (D) Gene expression of the mock receptor for TGF-β, BAMBI, tended to be higher in AHS-treated mice than in post-TNBS mice (p=0.07).

Fig 7

Colonic gene expression of tight junction proteins and mucins. (A) Gene expression of occludin was decreased in post-TNBS mice, and increased by Dex (p=0.052 by 3-way ANOVA). (B) Gene expression of TJP1 was increased by Dex in AHS-treated mice (p<0.05 by Bonferroni's t-test). (C) Post-TNBS mice showed an increase in the gene expression of TJP2. (D) Gene expression of mucin 2 was lower in post-TNBS mice than in control. ** p<0.01 and * p<0.05 by 3-way ANOVA.

Fig 8

Condition-specific correlation between gene expression of inflammatory molecules and colorectal sensitivity. Colorectal sensitivity was positively correlated with TGF-β2 (A) in control, and with IL-1β (B), inhibin βA (C) and PGES (D) in Dex-treated post-TNBS mice. Broken lines in B-D indicate statistically insignificant linear regressions in Veh-treated post-TNBS mice. (E) Gene expression of COX-2 was negatively correlated with colorectal sensitivity in AHS-treated mice.