Role of peripheral corticotropin-releasing factor and urocortin II in intestinal inflammation and motility in terminal ileum

Abstract

Corticotropin-releasing factor (CRF) and the closely related family of neuropeptides urocortins (Ucns) are ancient paracrine-signaling peptides secreted in both the central and peripheral neural circuits. CRF and Ucns released from the CNS (central) regulate a plethora of physiological processes that include food intake, inflammation, and bowel motility and permeability. In the gastrointestinal tract, CRF actions are largely proinflammatory, whereas the effects of the Ucn subtypes can be either pro- or antiinflammatory. Central (intracerebroventricular) or peripheral (i.p.) administration of CRF or Ucns inhibits gastric emptying and promotes colonic motility. To ascertain the role of peripherally expressed CRF and UcnII in gastrointestinal inflammation and motility, we generated ileum-specific phenotypic knockouts of these peptides by using RNA interference. Long dsRNA effectively silenced basal expression of CRF and UcnII in ileum. Control dsRNA or saline treatment did not affect CRF or UcnII expression. In an experimental model of toxin-induced intestinal inflammation, inhibition of CRF ablated the inflammatory response (measured by epithelial damage, mucosal edema, and neutrophil infiltration). UcnII dsRNA treatment did not alter the inflammatory response to toxin. Furthermore, ileal motility was increased after site-specific inhibition of both CRF and UcnII. Thus, we demonstrate that ileal-specific CRF promotes inflammation and both CRF and UcnII modulate bowel motility.

Fig. 1.

CRF and UcnII inhibition by RNAi in rat ileum. Groups of rats (n = 4–6) were treated with long dsRNA for CRF (dsCRF), UcnII (dsUcnII), or nonspecific control (dsGFP) for 5–7 days. (A) CRF peptide is undetectable by IHC in the nerve fibers (arrows) of the submucosal (SM) plexus of rats treated with dsRNA for CRF, whereas other treatment groups continued to express CRF in the ileum. (B) Results from RT-PCR reveal that mRNA for CRF or UcnII is significantly inhibited by RNAi treatment, with no UcnII cDNA product amplifiable in dsUcnII groups or very low levels of CRF cDNA product in dsCRF group. Cyclophilin, a housekeeping gene, was detectable in all three groups, and its expression was unaffected by RNAi treatment. (A and C) CRF expression is induced 3 h after TxA treatment in the submucosa and lamina propria (LP). This induction is specifically blocked by 6 days of pretreatment with long dsRNA against CRF. CRF, but not UcnII, is proinflammatory. (D and E) TxA treatment markedly destroys tissue morphology in control or UcnII dsRNA groups (n = 4–6) as assessed by epithelial damage, edema, and neutrophil infiltration. Inhibition of CRF by dsRNA resulted in significant protection of tissue damage that appeared similar to buffer-treated naïve animals. ANOVA indicated a global significant effect [F (3, 12) = 5.5; P < 0.02]. (F) Reduced tissue damage also resulted in reduced fluid secretion in dsCRF animals as a measured loop ratio (weight of the loop/length). ANOVA indicated a global effect [F (4, 17) = 8; P < 0.001]. Different letters denote statistically significant differences among groups (P < 0.01). [Magnification: ×10 (A and D) and ×40 (C).]

Fig. 2.

CRF and UcnII inhibition modulate intestinal motility. (A) Inhibition of CRF, and not UcnII, results in cumulative increase in fecal output over 24 h. Fecal pellets were counted daily for up to 4 days after dsRNA treatment. In contrast to CRF, UcnII displays circadian rhythm in exerting effects on ileal motility. (B) Assessment of the number of times red mucus or red-tinted pellets were expelled within a 5-h time frame (soon after lights on and just before the circadian rise in endogenous corticosteroid levels) indicates that ileal motility is enhanced by inhibition of both CRF and UcnII in this period.

Fig. 3.

Effect of down-regulation of ligands on their (CRF) receptors. After TxA administration, CRF-R1 protein expression is specifically up-regulated in rats pretreated with dsRNA against CRF, whereas that of CRF-R2 is not different (as determined by Western blot analysis; n = 4–6 per group).

Fig. 4.

CRF or UcnII dsRNA differentially alters TxA-mediated rise in corticosterone (B) concentrations. Basal prefasting or pre-TxA B concentrations were not significantly different among the groups. TxA treatment, however, increased B concentrations significantly in UcnII and control dsRNA-treated rats [ANOVA: F (3, 14) = 7.58; P < 0.01]. B levels in CRF dsRNA-treated rats were not significantly elevated or different from naïve buffer-treated controls. Different letters denotes statistically significant differences.