Effects of increase in fish oil intake on intestinal eicosanoids and inflammation in a mouse model of colitis

Abstract

Background: Inflammatory bowel diseases (IBD) are chronic intestinal inflammatory diseases affecting about 1% of western populations. New eating behaviors might contribute to the global emergence of IBD. Although the immunoregulatory effects of omega-3 fatty acids have been well characterized in vitro, their role in IBD is controversial.

Methods: The aim of this study was to assess the impact of increased fish oil intake on colonic gene expression, eicosanoid metabolism and development of colitis in a mouse model of IBD. Rag-2 deficient mice were fed fish oil (FO) enriched in omega-3 fatty acids i.e. EPA and DHA or control diet for 4 weeks before colitis induction by adoptive transfer of naïve T cells and maintained in the same diet for 4 additional weeks. Onset of colitis was monitored by colonoscopy and further confirmed by immunological examinations. Whole genome expression profiling was made and eicosanoids were measured by HPLC-MS/MS in colonic samples.

Results: A significant reduction of colonic proinflammatory eicosanoids in FO fed mice compared to control was observed. However, neither alteration of colonic gene expression signature nor reduction in IBD scores was observed under FO diet.

Conclusion: Thus, increased intake of dietary FO did not prevent experimental colitis.

Figure 1

Study design.

Figure 2

Measures of colitic outcomes 4 weeks after AT. Shown are body weight measures in grams at sacrifice and in inset shown is relative body composition with% lean mass in left panel and% fat mass in right panel (A). Endoscopic score is shown (B). Colon weight/length (W/L) ratio is shown (C). MPO content measured by ELISA is depicted (D). Black bars are median values and each dot represents the value obtained with an individual mouse. Statistics are shown as * when P <0.05, ** when P <0.01 and *** when P <0.001.

Figure 3

Cytokine measurements in colon tissue. IL-1β; IL-12p70; IFNγ; IL-6; KC; IL-10 and TNFα are shown (from A-G respectively). Black bars are median values and each dot represents the value obtained with an individual mouse. Statistics are shown as * when P <0.05, ** when P <0.01 and *** when P <0.001.

Figure 4

Phenotypic analysis of T-helper cells in mesenteric lymph nodes at 4 weeks after AT. Ex-vivo T cell stimulation, fixation and staining were performed then analyzed by flow-cytometry. Representative flow-cytometry dot plot pictures and gating strategy to quantify% of Th1, Th17 and regulatory T cells (A). Note the clear differences in each subset frequency between a healthy mice and colitic mice irrespectively of the diet used.% of Th1, Th17 cells and regulatory T cells are shown in colitic mice (tRag2 and tRag2 + FO) (B). Black bars are median values and each dot represents the value obtained with an individual mouse.

Figure 5

Colonic free FA levels of AA, DHA and EPA. AA, DHA and EPA levels are shown for each experimental group (from A-C respectively). Black bars are median value and each dot represents the value obtained with an individual mouse. Statistics are shown as * when P <0.05, ** when P <0.01 and *** when P <0.001.

Figure 6

Levels of EPA and AA-derived metabolites in colon. Free fatty acid levels of EPA-derived metabolites prostaglandin E3 (PGE3); thromboxane B3 (TxB3); leukotriene B5 (LTB5); 5-hydroxyeicosapentaenoic acid (5-HEPE); 17,18-epoxyeicosatetraenoic acid (17,18-EEP); and the AA-derived metabolites prostaglandin J2 (PGJ2); 8,9-epoxyeicosatrienoic acid (8,9-EET); prostaglandin E2 (PGE2) (from A-H respectively) are shown. Black bars are median values and each dot represents the value obtained with an individual mouse where dark circles are tRag2 and open circles are tRag2 + FO. Statistics are shown as * when P <0.05, ** when P <0.01 and *** when P <0.001. Of note whole set of metabolites measured and corresponding values are given in Additional files 1 and 2.

Figure 7

Transcriptomic analysis of colon preparation in healthy and colitic mice. Shown is Venn diagram made with significantly regulated colonic genes (P < 0.001) showing the relative impact of diet (treatment) vs. colitis (induction) and interaction between diet and colitis (A). Two-dimensional visualization of unsupervised PCA analysis constructed with the whole set of gene significantly differentially expressed in the colon upon colitis induction (B). Each dot represents the value obtained with an individual mouse (n = 6-9 mice per group). Of note top 40 colonic genes up- and down regulated in colitis mice and corresponding value are given in Additional files 3 and 4.