The differential effects of omega-6 and omega-3 polyunsaturated fatty acids on intestinal microbial ecology and host redox responses

The gastrointestinal tract harbors complex bacteria which plays an important role in health and disease. Gut microbial antigens, in conjunction with ingested dietary components, are important in intestinal immune homeostasis. High omega-6 polyunsaturated fatty acid (n6 PUFA) can induce oxidative stress and inflammation in the gut. In contrast, omega-3 (n3 PUFAs) supplementation can cure several inflammatory diseases. However, the relationship between dietary PUFAs and the intestinal microbiota remain unknown. Our study was to determine the effect of high fat diets with varying n6 and n3 PUFA on mice microbiota and host responses. We used 20% wt/wt corn oil (high n6 PUFA), corn + fish oil (19% wt/wt corn oil added to 1% wt/wt fish oil; high n6 PUFA + long chain n3 PUFA), 20% wt/wt canola oil (low n6 PUFA) as diets keeping 5% wt/wt corn oil as a chow control. After feeding mice the high fat diets for 5 weeks, Quantitative Polymerase Chain Reaction (qPCR) was used to examine the gut microbiota.Immunofluorescence was carried out to examine immune and redox responses. All high fat diets,regardless of composition, significantly reduced Bacteroides spp. and increased in intestinal epithelial cell death. Mice fed 20% corn oil had high levels of bacteria from the Clostridium and Enterobacteriaceae; associated with inflammatory bowel disease (IBD). In contrast, mice fed corn oil diets supplemented with fish oil, had enriched beneficial microbe Lactobacillus and lower levels of Enterobacteriaceae and Clostridia species. Fish oil also reduced neutrophil infiltration as well suggesting that n3 PUFA is anti-inflammatory. In addition, unexpectedly, fish oil supplementation induced oxidative stress in the colon evident by the increased presence of 4-hydroxy-2-nonenal, a lipid peroxide product, and dual oxidase 2, which generates H₂O₂. In addition, catalase, an antioxidant was also low in the fish oil group. Canola oil, which contains n3, n6 PUFAs and a monounsaturated fat oleic acid, alters the microbiota similar to the corn oil group. Overall, our research suggests that n6 PUFA alters the microbial composition, enriching it with detrimental microbes. Fish oil supplementation can reverse this effect. However, we also provide evidence of fish oil supplementation increasing oxidative stress.Arts and Sciences, Irving K. Barber School of (Okanagan)Biology, Department of (Okanagan)Graduat

Real-time polymerase chain reaction quantification of specific butyrate-producing bacteria, Desulfovibrio and Enterococcus faecalis in the feces of patients with colorectal cancer

Background and Aim: Bacterial metabolites produced in the bowel are potentially related to the genesis of colorectal cancer. Butyrate is protective against cancer, whereas hydrogen sulfide and oxygen free radicals can be toxic to the epithelium. The present study was designed to quantitate Eubacterium rectale, Faecalibacterium prausnitzii (both butyrate-producing bacteria), Desulfovibrio (sulfate-reducing bacteria), and Enterococcus faecalis (that produces extracellular superoxide) in the feces of patients with colorectal cancer. Methods: DNA was extracted from feces of 20 patients with colorectal cancer, nine patients with upper gastrointestinal cancer and 17 healthy volunteers. Real-time polymerase chain reaction using primers aimed at 16S rDNA was used to quantitate the above bacterial species or genus, and this was expressed relative to amplification of universal sequences conserved among all bacteria. Results: Levels of E. rectale and F. prausnitzii were decreased approximately fourfold (P = 0.0088 and 0.0028, respectively) in colorectal cancer patients compared to healthy control volunteers. Levels of Desulfovibrio were not significantly different between the three groups. E. faecalis populations were significantly higher in colorectal cancer patients compared to healthy volunteers (P = 0.0294). Conclusions: Butyrate producers were decreased and E. faecalis increased in the feces of colon cancer patients. These shifts in the colonic bacterial population could potentially lead to epithelial cell damage and increased turnover and may be a factor leading to colon cancer

Fish oil attenuates omega-6 polyunsaturated fatty acid-induced dysbiosis and infectious colitis but impairs LPS dephosphorylation activity causing sepsis.

Clinically, excessive ω-6 polyunsaturated fatty acid (PUFA) and inadequate ω-3 PUFA have been associated with enhanced risks for developing ulcerative colitis. In rodent models, ω-3 PUFAs have been shown to either attenuate or exacerbate colitis in different studies. We hypothesized that a high ω-6: ω-3 PUFA ratio would increase colitis susceptibility through the microbe-immunity nexus. To address this, we fed post-weaned mice diets rich in ω-6 PUFA (corn oil) and diets supplemented with ω-3 PUFA (corn oil+fish oil) for 5 weeks. We evaluated the intestinal microbiota, induced colitis with Citrobacter rodentium and followed disease progression. We found that ω-6 PUFA enriched the microbiota with Enterobacteriaceae, Segmented Filamentous Bacteria and Clostridia spp., all known to induce inflammation. During infection-induced colitis, ω-6 PUFA fed mice had exacerbated intestinal damage, immune cell infiltration, prostaglandin E2 expression and C. rodentium translocation across the intestinal mucosae. Addition of ω-3 PUFA on a high ω-6 PUFA diet, reversed inflammatory-inducing microbial blooms and enriched beneficial microbes like Lactobacillus and Bifidobacteria, reduced immune cell infiltration and impaired cytokine/chemokine induction during infection. While, ω-3 PUFA supplementation protected against severe colitis, these mice suffered greater mortality associated with sepsis-related serum factors such as LPS binding protein, IL-15 and TNF-α. These mice also demonstrated decreased expression of intestinal alkaline phosphatase and an inability to dephosphorylate LPS. Thus, the colonic microbiota is altered differentially through varying PUFA composition, conferring altered susceptibility to colitis. Overall, ω-6 PUFA enriches pro-inflammatory microbes and augments colitis; but prevents infection-induced systemic inflammation. In contrast, ω-3 PUFA supplementation reverses the effects of the ω-6 PUFA diet but impairs infection-induced responses resulting in sepsis. We conclude that as an anti-inflammatory agent, ω-3 PUFA supplementation during infection may prove detrimental when host inflammatory responses are critical for survival

Major fatty acid compositions of dietary oil used in preparing high-fat diets.

<p>Major fatty acid compositions of dietary oil used in preparing high-fat diets.</p

A combination of single nucleotide polymorphisms is associated with the interindividual variability in the blood lipid response to dietary fatty acid consumption in a randomized clinical trial

International audienceABSTRACT Background Blood lipid concentrations display high interindividual variability in response to dietary interventions, partly due to genetic factors. Existing studies have focused on single nucleotide polymorphisms (SNPs) analyzed individually, which only explain a limited fraction of the variability of these complex phenotypes. Objective We aimed to identify combinations of SNPs associated with the variability in LDL cholesterol and triglyceride (TG) concentration changes following 5 dietary interventions. Design In a multicenter randomized crossover trial, 92 participants with elevated waist circumference and low HDL cholesterol concentrations consumed 5 isoenergetic diets for 4 wk: a diet rich in saturated fatty acids (SFAs) from cheese, SFA from butter, monounsaturated fatty acids (MUFAs), n–6 polyunsaturated fatty acids (PUFAs), and a diet higher in carbohydrates (CHO). The association between 22 candidate SNPs in genes involved in lipid and bile acid metabolism and transport and changes in LDL cholesterol and TG concentrations was assessed with univariate statistics followed by partial least squares regression. Results Endpoint LDL cholesterol concentrations were significantly different (cheese: 3.18 ± 0.04, butter: 3.31 ± 0.04, MUFA: 3.00 ± 0.04, PUFA: 2.81 ± 0.04, CHO: 3.11 ± 0.04 mmol/L; P &lt; 0.001) while endpoint TG concentrations were not (P = 0.117). Both displayed consistently elevated interindividual variability following the dietary interventions (CVs of 34.5 ± 2.2% and 55.8 ± 1.8%, respectively). Among the 22 candidate SNPs, only ABCA1-rs2066714 and apolipoprotein E (APOE) isoforms exhibited consistent significant effects, namely on LDL cholesterol concentrations. However, several SNPs were significantly associated with changes in LDL cholesterol and TG concentrations in a diet-specific fashion. Generated multivariate models explained from 16.0 to 33.6% of the interindividual variability in LDL cholesterol concentration changes and from 17.5 to 32.0% of that in TG concentration changes. Conclusions We report combinations of SNPs associated with a significant part of the variability in LDL cholesterol and TG concentrations following dietary interventions differing in their fatty acid profiles

Composition of high-fat diets.

1<p>Mineral mix (mg/g): di calcium phosphate 500, magnesium oxide 24; potassium citrate 220, potassium sulfate 52; sodium chloride 74, chromium KSO₄ 12H₂0 0.55; cupric carbonate 0.3, potassium iodate 0.01; ferric citrate 6, manganous carbonate 3.5, sodium selenite 0.01, zinc carbonate 1.6; sucrose 118.03.</p>2<p>Vitamin Mix (mg/g): vitamin A 0.8; vitamin D₃ 1; vitamin E 10; menadione sodium bisulfite 0.08; nicotinic acid 3; calcium pantothenate 1.6; pyridoxine HCl 0.7; riboflavin 0.6; thiamin 0.6; sucrose 978.42.</p>3<p>Added to meet essential fatty acid requirements for all groups.</p

While ω-3 PUFA supplementation prevents ω-6 PUFA induced histopathologic severity, these mice suffered increased mortality and morbidity during <i>C. rodentium</i> infection.

<p>A) C57BL/6 mice fed ω-6 PUFA rich diets supplemented with ω-3 PUFA suffered increased mortality during infection with <i>C. rodentium</i>. 30% of mice fed ω-3 PUFA required euthanization by 6–8 days p.i., in contrast to the low or high-fat ω-6 PUFA groups. B) While both low or high-fat ω-6 PUFA diets result in similar weight changes during infection with <i>C. rodentium</i>, the ω-3 PUFA supplemented group suffered significantly increased cachexia throughout days 5–10 p.i. C) Mice fed ω-6 PUFA rich diets had the highest induction of histopathologic severity during <i>C. rodentium</i>-induced colitis while ω-3 PUFA supplementation reduced this. D) Representative colon sections from diet groups were taken at 100× magnification and stitched together using Metamorph software (black scale bar = 46.5 µm) or 200× magnification (white scale bar = 20.7 µm). E) Colonic tissue sections were stained for TUNEL-positive cells. Colons from mice fed low and high-fat ω-6 PUFA diets showed increased cell death compared to mice fed diets supplemented with ω-3 PUFA. (*, <i>P</i><0.05).</p

While ω-6 PUFA rich diets resulted in the induction of LPS binding protein prior to infection, it was the ω-3 PUFA supplemented diet that induced infection-induced septic responses.

<p>LPS binding protein, TNF-α and IL-15 were measured in sera from in mice that had been fed the various diets both before and after infection with <i>C. rodentium</i>. (*, <i>P</i><0.05).</p

ω-6 PUFA rich diets resulted in increased translocation of microbes from Enterobacteriacea across the intestinal mucosae.

<p>A) Colonic tissue sections were hybridized with a γ-Proteobacteria probe (green) and the nuclei stained with DAPI (blue) to examine the locations of Enterobacteriaceae. These Gram-negative microbes were found in the submucosae (600× magnification scale bar = 14.2 µm) prior to infection in the colons of mice fed ω-6 PUFA rich diets. During infection with <i>C. rodentium</i>, a member of Enterobacteriaceae, the pathogen was found deep into the crypts (100× magnification, scale bar = 85.4 µm; upper panel) and in the submucosae (600× magnification scale bar = 14.2 µm) in the colons of mice fed ω-6 PUFA rich diets. B) Colony forming units (CFU) recovered from the spleen and mesenteric lymph nodes (MLN) were highest from mice fed ω-6 PUFA rich diets after 10 days of <i>C. rodentium</i> infection. CFU were enumerated from tissues removed from mice fed various diets and were homogenized followed by plating in serial dilutions on Lb agar. (*, <i>P</i><0.05).</p

ω-6 PUFA rich diets induce dysbiosis and ω-3 PUFA supplementation reverses these blooms while enriching beneficial microbes.

<p>A) ω-6 PUFA rich diets promote a microbiota enriched with Enterobacteriacea, Segmented Filamentous Bacteria (SFB) and microbes from the <i>Clostridia coccoides</i> group and ω-3 supplementation reverses this and B) enriches <i>Bifidobacteria</i> spp., <i>Lactobacillus</i> spp. and <i>Enterococcus faecium</i>. C) High-fat diet groups resulted in decreased <i>Bacteroides</i> spp. and <i>Enterococcus faecalis</i>. Expression is relative to the low ω-6 PUFA group. (*, <i>P</i><0.05).</p