Suppression of intestinal inflammation and inflammation-driven colon cancer in mice by dietary sphingomyelin: importance of peroxisome proliferator-activated receptor γ expression

Abstract

Inflammation of the gastrointestinal tract increases the risk of developing colon cancer especially in younger adults. Dietary compounds are not only associated with the etiology of inflammation and colon cancer but also in their prevention. Sphingolipid metabolites have been shown to play a role in the initiation and perpetuation of inflammatory responses. In the present study, we investigated the suppression of dextran sodium sulfate-induced colitis and azoxymethane-induced colon cancer by dietary sphingomyelin (SM) in mice that lack functional peroxisome proliferator-activated receptor γ (PPAR-γ) in intestinal epithelial and immune cells. Dietary SM decreased disease activity and colonic inflammatory lesions in mice of both genotypes but more efficiently in mice expressing PPAR-γ. The increased survival and suppression of tumor formation in the SM-fed mice appeared to be independent of PPAR-γ expression in immune and epithelial cells. Using a real-time polymerase chain reaction array, we detected an up-regulation in genes involved in Th1 (interferon γ) and Th17 (interleukin [IL]-17 and IL-23) responses despite the reduced inflammation scores. However, the genes involved in Th2 (IL-4, IL-13 and IL-13ra2) and Treg (IL-10rb) anti-inflammatory responses were up-regulated in a PPAR-γ-dependent manner. In line with the PPAR-γ dependency of our in vivo findings, treatment of RAW macrophages with sphingosine increased the PPAR-γ reporter activity. In conclusion, dietary SM modulated inflammatory responses at the early stages of the disease by activating PPAR-γ, but its anticarcinogenic effects followed a PPAR-γ-independent pattern.

Figure 1

Disease Activity Index (DAI) in PPAR-γ expressing (PPAR-γ^+/+) and tissue-specific knockout (PPAR-γ^−/−) mice lacking PPAR-γ in intestinal epithelial cells, macrophages and T cells. All mice were injected with a single dose of AOM, treatment with 2.0% DSS for 7 days, and fed either the AIN76A diet alone (circles), or supplemented with 0.1% sphingomyelin (triangles). n=10 per group

Figure 2

The survival of mice after DSS treatment (2% for 7 days) is enhanced in mice fed sphingomyelin independent of their genotype.

Figure 3

Changes in the colonic architecture, determined by H&E staining in colons from mice treated with a single dose of AOM, 2% DSS for 7 days and maintained on control AIN76A diet alone (upper panel) or supplemented with 0.1% sphingomyelin (80 days after begin of study; n=2/17 PPAR-γ^+/+ ctrl/SM fed, n=3/18 PPAR-γ^−/− ctrl/SM)

Figure 4

Tumor area PPAR-γ^+/+ and PPAR-γ^−/− mice 80 days after a single AOM injection. *p<0.05

Figure 5

Immune cell infiltration and progression of colon cancer in PPAR-γ^+/+ and PPAR-γ^−/− mice fed AIN76A diet alone or with sphingomyelin supplements. Means without common letter are different (p<0.05).

Figure 6

Changes in macrophage (left panels) and T-cell (right panels) populations in mesentery lymph nodes (MLN) of PPAR-γ^+/+ mice (black bars) and PPAR-γ^−/− mice (grey bars) 80 days after AOM injection. Mice were fed AIN76A diet alone (control) or supplemented with 0.1% sphingomyelin.

Figure 7

Representative images of H&E stained colonic sections from untreated PPAR-γ^+/+ or PPAR-γ^−/− mice (upper panels), at the peak of intestinal inflammation induced by 2.0% DSS. Images from mice fed the control AIN76A diet are depicted in the middle panels while the lower panels show representative images from mice fed SM supplements. Arrows: immune cell infiltration; arrowheads: typical colonic columnar tissue architecture.

Figure 8

Comparison of pro-and anti-inflammatory gene expression levels between PPAR-γ^+/+ and PPAR-γ^−/− mice (left panel), and after treatment with 2% DSS in the drinking water for 7 days (right panel) as determined by a real-time PCR array.

Figure 9

Changes in gene expression levels in the colons of PPAR-γ^+/+ (left panels) and PPAR-γ^−/− mice (right panels) in response to dietary SM. The mice were not treated (upper panels) or treated with 2% DSS for 7 days (lower panels).

Figure 10

Non-toxic concentrations of sphingosine activate PPAR-γ transcriptional activity in macrophages, determined by luciferase promoter activity assay, compared to the PPAR-γ agonist rosilitazone. *p<0.05; **p<0.01