Psychological Stress Exacerbates Inflammation of the Ileum via the Corticotropin-Releasing Hormone-Mast Cell Axis in a Mouse Model of Eosinophilic Enteritis

Abstract

The effects of psychological stress on eosinophilic gastrointestinal disorders have not been elucidated. This study investigated the effects of psychological stress in a mouse model of eosinophilic enteritis (EoN). BALB/c mice were treated with ovalbumin (OVA) to create an EoN model and subjected to either water avoidance stress (WAS) or sham stress (SS). Microscopic inflammation, eosinophil and mast cell counts, mRNA expression, and protein levels of type 2 helper T cell (Th2) cytokines in the ileum were compared between groups. We evaluated ex vivo intestinal permeability using an Ussing chamber. A corticotropin-releasing hormone type 1 receptor (CRH-R1) antagonist was administered before WAS, and its effects were analyzed. WAS significantly increased diarrhea occurrence and, eosinophil and mast cell counts, and decreased the villus/crypt ratio compared to those in the SS group. The mRNA expression of CRH, interleukin IL-4, IL-5, IL-13, eotaxin-1, and mast cell tryptase β2 significantly increased, and the protein levels of IL-5, IL-13, and OVA-specific immunoglobulin E (IgE) also significantly increased in the WAS group. Moreover, WAS significantly increased the intestinal permeability. The CRH-R1 antagonist significantly inhibited all changes induced by WAS. Psychological stress exacerbated ileal inflammation via the CRH-mast cell axis in an EoN mouse model.

Keywords: corticotropin-releasing hormone; eosinophilic enteritis; eosinophilic gastrointestinal disorders; eosinophils; intestinal permeability; mast cells; psychological stress.

Figure 1

Experimental protocol and diarrhea occurrence. (A) The mice were intraperitoneally sensitized on days 0 and 14, and intragastrically challenged using OVA on days 30, 32, 35, 37, and 39. From day 32 to day 39, mice were subjected to WAS or SS for 1 h before OVA or PBS challenge for eight consecutive days. In OVA+WAS-treated mice, CRH-R1 antagonist or vehicle were intraperitoneally administered 1.5 h before each OVA challenge. The mice were sacrificed 1 h after the final OVA challenge, and their ileums were analyzed. (B) The comparison of diarrhea occurrence among the four groups of mice: SS + PBS, SS + OVA, WAS + PBS, and WAS + OVA. * p < 0.05 and ** p < 0.01 between SS + PBS and SS + OVA, † p < 0.05 and †† p < 0.01 between SS + OVA and WAS + OVA. CRH-R1, corticotropin-releasing hormone type 1 receptor; OVA, ovalbumin; PBS, phosphate buffered saline; SS, sham stress; WAS, water avoidance stress.

Figure 2

Microscopic findings and mRNA expressions in the ileum. (A) Histological images of HE staining. Scale bar = 50 μm. (B) The comparison of villus/crypt ratio representing microscopic damage. (C) The mRNA expression of CRH. (D) DFS staining for eosinophils. Scale bar = 50 μm. (E) Eosinophil counts. (F) The mRNA expression of eotaxin-1. (G) Chloroacetate esterase staining for mast cells. Scale bar = 50 μm. (H) Mast cell counts. (I) The mRNA expression of tpsb2. (J) Histological image of HE staining. (K) Mast cell tryptase staining (green). (L) CRH-R1 staining (red). (M) The colocalization of mast cell tryptase and CRH-R1 is represented in yellow (merge) with blue nuclear counterstain. Each scale bar in Figure 2J–M is 20 μm. * p < 0.05, ** p < 0.01. CRH, corticotropin-releasing hormone; CRH-R1, corticotropin-releasing hormone type 1 receptor; DFS, direct fast scarlet 4BS; HE, hematoxylin–eosin; tpsb2, tryptase beta 2.

Figure 3

The expressions of Th2 cytokines and OVA-specific IgE in the ileum, and ileal permeability. The mRNA expressions of IL-5 (A), IL-13 (B), and IL-4 (C). Protein levels of IL-5 (D), IL-13 (E), and OVA-specific IgE (F). (G) Ex vivo ileal permeability evaluated using TEER. (H) Ex vivo ileal permeability evaluated using the FITC-dextran flux. (I) mRNA expression of zonulin. * p < 0.05, ** p < 0.01. FITC, fluorescein isothiocyanate; IgE, immunoglobulin E; IL, interleukin; OVA, ovalbumin; TEER, transepithelial electrical resistance; Th2, type 2 helper T cell.

Figure 4

Effects of CRH-R1 antagonist on diarrhea occurrence, microscopic findings, and mRNA expressions in the ileum. Effects of the CRH-R1 antagonist on diarrhea occurrence (A), villus/crypt ratio (B), number of eosinophils (C), and mast cells (D) in the ileal mucosa of WAS + OVA-treated mice. Effects of the CRH-R1 antagonist on ileal mRNA expressions of eotaxin-1 (E) and tpsb2 (F) in WAS + OVA-treated mice. * p < 0.05, ** p < 0.01. CRH-R1, corticotropin-releasing hormone type 1 receptor; OVA, ovalbumin; tpsb2, tryptase beta 2; WAS, water avoidance stress.

Figure 5

Effects of the CRH-R1 antagonist on Th2 cytokines and OVA-specific IgE in the ileum, and ileal permeability. Effects of the CRH-R1 antagonist on ileal mRNA expressions of IL-5 (A), IL-13 (B), and IL-4 (C) in WAS + OVA-treated mice. Effects of the CRH-R1 antagonist on ileal protein levels of IL-5 (D), IL-13 (E), and OVA-specific IgE (F) in WAS + OVA-treated mice. Effects of CRH-R1 antagonist on TEER (G), FITC-dextran flux (H), and mRNA expression of zonulin (I) in the ileum of WAS + OVA-treated mice. * p < 0.05, ** p < 0.01. CRH-R1, corticotropin-releasing hormone type 1 receptor; FITC, fluorescein isothiocyanate; IgE, immunoglobulin E; IL, interleukin; OVA, ovalbumin; TEER, transepithelial electrical resistance; Th2, type 2 helper T cell; WAS, water avoidance stress.