Immunomodulatory action of dietary fish oil and targeted deletion of intestinal epithelial cell PPARδ in inflammation-induced colon carcinogenesis

Abstract

The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR)-δ is highly expressed in colonic epithelial cells; however, the role of PPARδ ligands, such as fatty acids, in mucosal inflammation and malignant transformation has not been clarified. Recent evidence suggests that the anti-inflammatory/chemoprotective properties of fish oil (FO)-derived n-3 polyunsaturated fatty acids (PUFAs) may be partly mediated by PPARδ. Therefore, we assessed the role of PPARδ in modulating the effects of dietary n-3 PUFAs by targeted deletion of intestinal epithelial cell PPARδ (PPARδ(ΔIEpC)). Subsequently, we documented changes in colon tumorigenesis and the inflammatory microenvironment, i.e., local [mesenteric lymph node (MLN)] and systemic (spleen) T cell activation. Animals were fed chemopromotive [corn oil (CO)] or chemoprotective (FO) diets during the induction of chronic inflammation/carcinogenesis. Tumor incidence was similar in control and PPARδ(ΔIEpC) mice. FO reduced mucosal injury, tumor incidence, colonic STAT3 activation, and inflammatory cytokine gene expression, independent of PPARδ genotype. CD8(+) T cell recruitment into MLNs was suppressed in PPARδ(ΔIEpC) mice. Similarly, FO reduced CD8(+) T cell numbers in the MLN. Dietary FO independently modulated MLN CD4(+) T cell activation status by decreasing CD44 expression. CD11a expression by MLN CD4(+) T cells was downregulated in PPARδ(ΔIEpC) mice. Lastly, splenic CD62L expression was downregulated in PPARδ(ΔIEpC) CD4(+) and CD8(+) T cells. These data demonstrate that expression of intestinal epithelial cell PPARδ does not influence azoxymethane/dextran sodium sulfate-induced colon tumor incidence. Moreover, we provide new evidence that dietary n-3 PUFAs attenuate intestinal inflammation in an intestinal epithelial cell PPARδ-independent manner.

Fig. 1.

Experimental dosing regimen. Wild-type (PPAR^F/F) mice and intestinal epithelial cell (IEpC) peroxisome proliferator-activated receptor (PPAR)-δ null (PPARδ^ΔIEpC) mice consuming a 5% corn oil (CO) or a 1% corn oil + 4% fish oil (FO) diet were acclimated to experimental diets for 14 days prior to injection of azoxymethane (AOM, 7.5 mg/kg body wt ip). Subsequently, mice were exposed to 3 cycles of dextran sodium sulfate (DSS, 1% wt/wt via drinking water). Mice were euthanized 12 wk after completion of the final DSS cycle.

Fig. 2.

PPARδ deletion and genotyping strategy. A: DNA gel showing mouse genotypes that were assessed using 2 primer sets. Even-numbered lanes (2–20) detected loxP sites at 400 bp (arrow); odd-numbered lanes (3–21) detected Cre recombinase at 380 bp. B: primer set 1, used to detect specific deletion of PPARδ exon 4 by Cre recombinase, results in a nonfunctional form of PPARδ; primer set 2, used to detect PPARδ exon 7–8, indicates that the remainder of PPARδ is still intact.

Fig. 3.

Confirmation of an intestine-specific PPARδ knockout mouse. RNA was extracted from scraped colonic mucosa, duodenum, and kidney. A: deletion of exon 4 of PPARδ in PPARδ^ΔIEpC mice, which was confirmed by detection of PPARδ mRNA expression using primer sets detecting exon boundaries 4–5 (n = 4–6 mice/genotype at each tissue site). B: intact PPARδ exons 7 and 8 (primer set detecting exon boundaries 7–8), indicating expression of a partial, nonfunctional protein (n = 4–6 mice/genotype at each tissue site). Values are means ± SE. C and D: confirmation of PPARδ deletion within colonic mucosa following completion of AOM/DSS treatment regimen. C: mRNA (n = 4–8 mice per experimental group). Values are means ± SE. D: protein expression level. Blots represent results from 8 PPARδ^ΔIEpC and 3 PPAR^F/F mice. Protein expression was normalized to the housekeeping gene lactate dehydrogenase (LDH), and positive control (+) was a cell lysate from COS1 cells transfected with a mouse PPARδ expression vector. PPAR^F/F mice have a band at 52 kDa (arrow) that is absent in PPARδ^ΔIEpC mice.

Fig. 4.

Identification of colonic phenotype in AOM/DSS-treated mice. PPAR^F/F and PPARδ^ΔIEpC mice were fed CO (n = 11–12 per genotype) or FO (n = 13–14 per genotype) diet (n = 11–14 mice/treatment group) and euthanized 12 wk after completion of the final DSS cycle. Histological scoring (0–3) of colon epithelial injury and typing of tumor entities (total adenomas and adenocarcinomas) were carried out in a blinded manner by a board-certified pathologist (B. Weeks). A and B: independent effect of diet and genotype (IEpC PPARδ status) on colon injury in the middle region of the colon. Data were analyzed by Kruskal-Wallis test, and bars represent median values. *Statistical significance (P ≤ 0.05). C and D: effect of diet and genotype on tumor incidence in the middle region of the colon. Dot plots identify tumor distribution among treatment groups; solid black line denotes median value in each group.

Fig. 5.

Ratio of phosphorylated to total STAT3 expression in murine colonic mucosa as assessed by immunoblotting. Within each genotype (PPARδ^F/F and PPARδ^ΔIEpC), mice were fed 5% corn oil (CO) diet and treated with AOM/DSS (n = 4) or received an equal volume of saline intraperitoneally without DSS (control) in drinking water (n = 2). Ratio of phosphorylated (i.e., activated) to total STAT3 protein expression (pSTAT3/STAT3) was assessed by 2-way ANOVA. Values are means ± SE. *P ≤ 0.05. A: effect of AOM/DSS treatment (P = 0.0002). B: effect of AOM/DSS in mice from each genotype fed CO (n = 4) or FO (n = 4) diet (P = 0.01). C: representative immunoblots from PPARδ^F/F mice (top) and phosphorylated and total STAT3 (bottom). Samples are from AOM/DSS-treated FO-fed PPARδ^F/F mice (lane 1), AOM/DSS-treated CO-fed PPARδ^F/F mice (lane 2), and saline-treated (no DSS treatment) CO-fed PPARδ^F/F mice (lane 3).

Fig. 6.

Colonic mucosal cytokine mRNA expression. RNA was isolated from colonic mucosal scrapings from mice following exposure to carcinogen and 3 subsequent cycles of DSS. mRNA levels were determined by quantitative RT-PCR, and expression of each gene of interest was normalized to ribosomal 18S expression. Relative expression levels were analyzed for IL-6 (A), IFN-γ (B), IL-17A (C), IL-17F (D), IL-23 (E), and IL-23R (F). Data were analyzed by 2-way ANOVA (main effects: diet and genotype), and significance was at the level of P < 0.05. For all data sets, P (interaction) was not significant (P > 0.05); however, the outcome from each main effect is shown. Values are means ± SE.

Fig. 7.

Characterization of mesenteric lymph node (MLN) cells by surface staining followed by flow cytometry. A and B: representative plots of CD3 vs. CD4 and CD3 vs. CD8 quadrants from MLN cells of CO-fed PPARδ^F/F mice. C and D: percentages of CD3⁺, CD4⁺/CD3⁺, and CD8⁺/CD3⁺ T cells in MLN and spleen calculated by summing the number of events in each quadrant (n = 5–9 mice per treatment). Within each separate surface marker analysis, resultant P values from a 2-way ANOVA are listed in tables below each graph. Values are means ± SE. *Statistical significance (P ≤ 0.05). E–G: significant main effects from 2-way analysis conducted on MLN and spleen data.

Fig. 8.

Activation assessment of MLN T lymphocytes by surface staining. CD3⁺CD4⁺ or CD3⁺CD8⁺ double-positive cells were gated, and representative histograms of CD11a, CD44, and CD62L costaining are shown. PE, phycoerythrin.

Fig. 9.

A: quantitative analysis of T lymphocyte activation markers in MLN calculated by mean fluorescence intensity (MFI). Values are means ± SE. *Statistical significance (P ≤ 0.05). P values (main effects) were determined by 2-way ANOVA. B and C: significant main effects.

Fig. 10.

A: quantitative analysis of T lymphocyte activation markers in the spleen calculated as mean fluorescence intensity. Values are means ± SE. *Statistical significance (P ≤ 0.05). P values (main effects) were determined by 2-way ANOVA. B and C: significant main effects.