Chronic psychosocial stress increases the risk for inflammation-related colon carcinogenesis in male mice

Figures & data

Figure 1.  Schematic illustration of the experimental procedure. Following injection of AOM (10 mg/kg; i.p.) on day - 6, mice were either exposed to CSC housing (n = 12) or kept as SHCs (n = 11) from day 1 to 20. Subsequently, both SHC and CSC mice were treated with 1% DSS from day 50 to 57, with 0.5% DSS from day 121 to 131, and with 1% DSS from day 163 to 167. Importantly, three CSC mice died during first DSS cycle. Macroscopic changes were monitored by three colonoscopies performed on day 80, 117, and 160 in the same two narcotized mice of each group. On day 186, all mice were killed. Mice that underwent colonoscopy were not included in the analysis of any other readout parameter.

Table I.  Effects of chronic psychosocial stress on the development of macroscopic suspect lesions in the colon.

A Day of colonoscopy	Day 80		Day 117		Day 160
Treatment group	SHC	CSC	SHC	CSC	SHC	CSC
Granular mucosa	0	2	0	2	2	2
Flat polypoid lesions/polyps	0	2	0	2	0	2

Notes: Following injection of AOM (10 mg/kg; i.p.) on day - 6, mice were either exposed to CSC housing or kept as SHCs from day 1 to 20. Subsequently, both SHC and CSC mice were treated with three cycles of DSS, and macroscopic changes were monitored by three colonoscopies performed on day 80, 117, and 160 in the same two narcotized mice of each group (n = 2 per group). An experienced gastroenterologist assessed whether granular mucosa (indicated by black arrows in B) and/or flat polypoid lesions (indicated by black arrows in C)/polyps (indicated by black arrows in D) were present in none (0), one (1), or both (2) mice of each group (no statistical analysis was performed to the low number of mice per group). As these mice were exposed to the additional stressor of repeated anesthesia and colonoscopy, they were not included in the assessment of any other readout parameter in the current study.

Figure 2.  Effects of chronic psychosocial stress on colonic morphology and relative epithelial cell proliferation. Both SHC (n = 9, gray bars) and CSC housing (n = 7, black bars) mice were killed on day 186 following three cycles of DSS administration. (A) Hematoxylin–eosin stained paraffin sections of the distal colon were evaluated by an experienced pathologist with respect to the development of low-grade dysplasia (LGD; black arrows in B; section surrounded by the rectangle in (B) is shown in higher magnification in (D) and/or high-grade dysplasia (HGD; black arrows in (C); section surrounded by the rectangle in (C) is shown in higher magnification in (E). Data represent percentage of mice developing colonic dysplasia (HGD and/or LGD; ⁺p = 0.069 vs. respective SHC mice; Fisher's exact test). (F) Relative epithelial cell proliferation was assessed in Ki67-stained paraffin sections by determining the percentage of Ki67⁺ cells per cross-sectioned crypt in non-dysplastic tissue (normal, G&H) and LGD (indicated by arrows in G)/HGD (indicated by arrows in H). Data represent mean+SEM. ***p < 0.001 vs. respective non-dysplastic (normal) tissue mice (two-way ANOVA, factor treatment group and factor dysplasia).

Figure 3.  Effects of chronic psychosocial stress on colonic ß-catenin, COXII, and LRH-1 mRNA and protein expression patterns. Both SHC (n = 9) and CSC housing (n = 7) mice were killed on day 186 following three cycles of DSS administration. After killing, mRNA (A–C; tissue was collected proximal to tissue used for IHC/immunofluorescence) and protein (D–I; tissue was collected proximal to tissue used for qRT-PCR) expression levels of ß-catenin (SHC: n = 9, CSC: n = 7; A,D,G), COXII (SHC: n = 8, CSC: n = 6; B,E,H), and LRH-1 (SHC: n = 7, CSC: n = 6; C,F,I) were quantified by TaqMan^®-qPCR (relative to the expression of the housekeeping gene GAPDH) or by western blotting (relative to the expression of the housekeeping gene product ß-tubulin). Figures G–I show representative Western blot images of ß-catenin (G, upper bands; 92 kDa), COXII (H, upper bands; 61 kDa), LRH-1 (I, upper bands; 70 kDa), and the corresponding ß-tubulin (G–I, lower bands) protein expression of one SHC and one CSC mouse per treatment group. mRNA expression for each gene was quantified for each individual mouse in triplets and averaged per mouse. Data represent mean+SEM; * p < 0.05 vs. respective SHC mice (two-tailed Student's t-test).

Figure 4.  Effects of chronic psychosocial stress on colonic IFN-γ, FoxP3, and TNF mRNA expression patterns. Both SHC (n = 9) and CSC (n = 7) mice were killed on day 186 following three cycles of DSS administration. After killing, the expression levels of IFN-γ (SHC: n = 7, CSC: n = 6; A), FoxP3 (SHC: n = 7, CSC: n = 6; B), TNF (SHC: n = 9, CSC: n = 6; C) were quantified then by TaqMan^®-qPCR, relative to the expression of the housekeeping gene (GAPDH). mRNA expression for each gene was quantified for each individual mouse in triplets and averaged per mouse. Data represent mean+SEM; ⁺p = 0.063, *p < 0.05 vs. respective SHC mice (two-tailed Student's t-test).

Figure 5.  Effects of chronic psychosocial stress on epithelial apoptosis, absolute epithelial cell proliferation, and the number of CD4⁺ and F4/80⁺ cells in the colonic tissue. Both SHC (n = 9) and CSC housing (n = 7) mice were killed on day 186 following three cycles of DSS administration. After killing, the colon was removed, mechanically cleaned, rinsed, embedded in histological tissue-freezing medium, snap-frozen, and cut on a cryostat. Three 6 μm cross sections taken from 100 μm apart were acetone fixated and afterwards stained for epithelial apoptosis (TUNEL; SHC: n = 8, CSC: n = 5; A), absolute epithelial proliferation (Ki67; SHC: n = 9, CSC: n = 7; B), and the number of CD4⁺ (SHC: n = 7, CSC: n = 5; C) or F4/80⁺ (SHC: n = 8, CSC: n = 6; D) cells. Respective positive cells were counted and averaged from the three cross-sectioned slices per mouse (Ki67, CD4, F4/80) or counted per mm² of colon tissue in three fields of views in each cross section and averaged per mouse (TUNEL). Data represent mean+SEM; *p < 0.05, ***p ≤ 0.001 vs. respective SHC controls (two-tailed Student's t-test).

Figure 6.  Effects of chronic psychosocial stress on the percentage of CD3⁺ mesLNC, the in vitro IFN-γ secretion from isolated mesLNC, and the number of CD3⁺/FoxP3⁺ mesLNC. Both SHC (n = 9) and CSC housing (n = 7) mice were killed on day 186 following three cycles of DSS administration. After killing, mesLNC were either double-stained for CD3⁺ (red)/FoxP3⁺ (green) using immunofluorescence (C; SHC, D; CSC, E; white arrows indicate five representative double-positive cells in each slice) or mesLNC were isolated and pooled (three pools per treatment group containing 2–3 mice each) for assessment of anti-CD3-induced IFN-γ secretion (B). Remaining cells of all three pools were again pooled and surface stained with FITC-conjugated rat IgG2a anti-CD3 and respective isotype control (rat IgG2a) antibodies to quantify the percentage of CD3⁺ cells within all gated cells (A; no statistical analysis was done as all mice per group were pooled) using flow cytometry. IFN-γ data represent mean+SEM of three pools (2–4 mice per pool; Mann–Whitney U test) per treatment group. Numbers of CD3⁺/FoxP3⁺ mesLNC represent mean+SEM of individual mice (*p < 0.05; two-tailed Student's t-test). Scale bars represent 50 μm (SHC, D; CSC, E).