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.

Individuals vary in their resistance to enteric infections. The role of the intestinal microbiota in altering susceptibility to enteric infection is relatively unknown. Previous studies have identified that C3H/HeOuJ mice suffer 100% mortality during Citrobacter rodentium-induced colitis, whereas C57BL/6 mice recover from infection. The basis for their differences in susceptibility is unclear and has been mainly attributed to differences in host genetics. This study investigated the role of the intestinal microbiota in altering susceptibility to C. rodentium-induced colitis. When the feces of C57BL/6 mice were gavaged into antibiotic treated C3H/HeOuJ mice, the C57BL/6 microflora led to a complete reversal in mortality patterns where 100% of the C3H/HeOuJ mice survived infection. This protection corresponded with reduced colonic pathology and less systemic pathogen load and was associated with increased inflammatory and redox responses with reduced epithelial cell death. C3H/HeOuJ mice are normally susceptible to infection-induced dehydration due to defective expression of colonic ion transporters such as Dra, CA IV, and CA I; expression of these genes was normalized when C3H/HeOuJ mice were colonized with the C57BL/6 microflora. Together, these data reveal that the colonic microbiota play a critical role in protecting against intestinal infection by inducing proinflammatory and prooxidant responses that control pathogen load as well as ion transporter gene expression previously shown to prevent fatal dehydration. Protection of mice from lethal colitis was associated with higher levels of bacteria from Bacteroidetes. This study reveals that the microbiota is sufficient to overcome inherent genetic susceptibility patterns in C3H/HeOuJ mice that cause mortality during C. rodentium infection.

Inflammatory Bowel Diseases (IBD), including Crohn's Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48 hr and rats fed SO by 72 hr. Weight gain resumed after 72 hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5 days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD. Video LinkThe video component of this article can be found at

Attaching and effacing bacterial pathogens attach to the apical surface of epithelial cells and disrupt epithelial barrier function, increasing permeability and allowing luminal contents access to the underlying milieu. Previous in vitro studies demonstrated that the neuropeptide vasoactive intestinal peptide (VIP) regulates epithelial paracellular permeability, and the high concentrations and close proximity of VIP-containing nerve fibers to intestinal epithelial cells would support such a function in vivo. The aim of this study was to examine whether VIP treatment modulated Citrobacter rodentium-induced disruption of intestinal barrier integrity and to identify potential mechanisms of action. Administration of VIP had no effect on bacterial attachment although histopathological scoring demonstrated a VIP-induced amelioration of colitis-induced epithelial damage compared with controls. VIP treatment prevented the infection-induced increase in mannitol flux a measure of paracellular permeability, resulting in levels similar to control mice, and immunohistochemical studies demonstrated that VIP prevented the translocation of tight junction proteins: zonula occludens-1, occludin, and claudin-3. Enteropathogenic Escherichia coli (EPEC) infection of Caco-2 monolayers confirmed a protective role for VIP on epithelial barrier function. VIP prevented EPEC-induced increase in long myosin light chain kinase (MLCK) expression and myosin light chain phosphorylation (p-MLC). Furthermore, MLCK inhibition significantly attenuated bacterial-induced epithelial damage both in vivo and in vitro. In conclusion, our results indicate that VIP protects the colonic epithelial barrier by minimizing bacterial-induced redistribution of tight junction proteins in part through actions on MLCK and MLC phosphorylation.

The long-chain polyunsaturated n-6 and n-3 fatty acids are essential nutrients in membrane biogenesis and regulate gene expression via their eicosanoid metabolites. We investigated whether the n-6 and n-3 fatty acid supply as determined by maternal diet alters colonic phospholipid fatty acids, intestinal morphology, and epithelial barrier permeability during milk feeding with lasting effect on mucosal responsiveness to dinitrobenzene sulfonic acid (DNBS)-induced colitis in young adulthood. Female rats were fed diets with 20% energy from safflower oil (SO) or canola oil (CO), or 8% fish oil (FO) plus 2% SO (10% FO) or 18% FO plus 2% SO (20% FO) throughout gestation and lactation and offspring weaned to a standard diet at 21 days of age. At 15 days of age, pups in the 20% and 10% FO groups had lower 20:4n-6 and higher 20:5n-3 and 22:6n-3 in colon phospholipids (P Ͻ 0.01), shorter crypts (P Ͻ 0.05), and higher paracellular permeability than SO or CO groups. At 3 mo of age, male offspring in the FO groups showed lasting reduction of crypt depth and a heightened inflammatory response to DNBS. We demonstrate that early decreased colon 20: 4n-6 with increased n-3 fatty acids impairs intestinal barrier development and sensitizes the colon response to inflammatory insults later in life.n-6 fatty acids; n-3 fatty acids; arachidonic acid; milk fatty acids; intestinal programming; inflammatory bowel disease INFLAMMATORY BOWEL DISEASE (IBD), including Crohn disease and ulcerative colitis, are chronic recurrent inflammatory disorders thought to be precipitated by interplay of environmental and host immune factors in genetically susceptible individuals (40). A pivotal role of environmental factors is clear from the increasing incidence of IBD in recent decades in Western countries (4, 23, 34) and among migrant populations (37,38,43) and the emergence of IBD in populations with previously low prevalence (28).

Dietary lipids modulate immunity, yet the means by which specific fatty acids affect infectious disease susceptibility remains unclear. Deciphering lipid-induced immunity is critical to understanding the balance required for protecting against pathogens while avoiding chronic inflammatory diseases. To understand how specific lipids alter susceptibility to enteric infection, we fed mice isocaloric, high-fat diets composed of corn oil (rich in n-6 polyunsaturated fatty acids [n-6 PUFAs]), olive oil (rich in monounsaturated fatty acids), or milk fat (rich in saturated fatty acids) with or without fish oil (rich in n-3 PUFAs). After 5 weeks of dietary intervention, mice were challenged with Citrobacter rodentium, and pathological responses were assessed. Olive oil diets resulted in little colonic pathology associated with intestinal alkaline phosphatase, a mucosal defense factor that detoxifies lipopolysaccharide. In contrast, while both corn oil and milk fat diets resulted in inflammation-induced colonic damage, only milk fat induced compensatory protective responses, including short chain fatty acid production. Fish oil combined with milk fat, unlike unsaturated lipid diets, had a protective effect associated with intestinal alkaline phosphatase activity. Overall, these results reveal that dietary lipid type, independent of the total number of calories associated with the dietary lipid, influences the susceptibility to enteric damage and the benefits of fish oil during infection.

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