Current and Expected Trends for the Marine Chitin/Chitosan and Collagen Value Chains.

Chitin/chitosan and collagen are two of the most important bioactive compounds, with applications in the pharmaceutical, veterinary, nutraceutical, cosmetic, biomaterials, and other industries. When extracted from non-edible parts of fish and shellfish, by-catches, and invasive species, their use contributes to a more sustainable and circular economy. The present article reviews the scientific knowledge and publication trends along the marine chitin/chitosan and collagen value chains and assesses how researchers, industry players, and end-users can bridge the gap between scientific understanding and industrial applications. Overall, research on chitin/chitosan remains focused on the compound itself rather than its market applications. Still, chitin/chitosan use is expected to increase in food and biomedical applications, while that of collagen is expected to increase in biomedical, cosmetic, pharmaceutical, and nutritional applications. Sustainable practices, such as the reuse of waste materials, contribute to strengthen both value chains; the identified weaknesses include the lack of studies considering market trends, social sustainability, and profitability, as well as insufficient examination of intellectual property rights. Government regulations, market demand, consumer preferences, technological advancements, environmental challenges, and legal frameworks play significant roles in shaping both value chains. Addressing these factors is crucial for seizing opportunities, fostering sustainability, complying with regulations, and maintaining competitiveness in these constantly evolving value chains.

Differential Effects of Iron Chelates vs. Iron Salts on Induction of Pro-Oncogenic Amphiregulin and Pro-Inflammatory COX-2 in Human Intestinal Adenocarcinoma Cell Lines.

We previously showed that two iron compounds that are orally ingested by humans, namely ferric EDTA and ferric citrate, can induce an oncogenic growth factor (amphiregulin) in human intestinal epithelial adenocarcinoma cell lines. Here, we further screened these iron compounds, plus four other iron chelates and six iron salts (i.e., 12 oral iron compounds in total), for their effects on biomarkers of cancer and inflammation. Ferric pyrophosphate and ferric EDTA were the main inducers of amphiregulin and its receptor monomer, IGFr1. Moreover, at the maximum iron concentrations investigated (500 µM), the highest levels of amphiregulin were induced by the six iron chelates, while four of these also increased IGfr1. In addition, we observed that ferric pyrophosphate promoted signaling via the JAK/STAT pathway by up-regulating the cytokine receptor subunit IFN-γr1 and IL-6. For pro-inflammatory cyclooxygenase-2 (COX-2), ferric pyrophosphate but not ferric EDTA elevated intracellular levels. This, however, did not drive the other biomarkers based on COX-2 inhibition studies and was probably downstream of IL-6. We conclude that of all oral iron compounds, iron chelates may particularly elevate intracellular amphiregulin. Ferric pyrophosphate additionally induced COX-2, probably because of the high IL-6 induction that was observed with this compound.

The Omega-3 Fatty Acids EPA and DHA, as a Part of a Murine High-Fat Diet, Reduced Lipid Accumulation in Brown and White Adipose Tissues.

Excess energy intake can trigger an uncontrolled inflammatory response, leading to systemic low-grade inflammation and metabolic disturbances that are hypothesised to contribute to cardiovascular disease and type 2 diabetes. The long chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are suggested to mitigate this inflammatory response, but the mechanisms are unclear, especially at the tissue level. Adipose tissues, the first tissues to give an inflammatory response, may be an important target site of action for EPA and DHA. To evaluate the effects of EPA and DHA in white and brown adipose tissues, we fed male C57Bl/6J mice either a high fat diet (HFD) with 5% corn oil, an HFD with 40% of the corn oil substituted for purified EPA and DHA triglycerides (HFD-ED), or normal chow, for 8 weeks. Fatty acid profiling and transcriptomics were used to study how EPA and DHA affect retroperitoneal white and brown adipose tissues. HFD-ED fed mice showed reduced lipid accumulation and levels of the pro-inflammatory fatty acid arachidonic acid in both white and brown adipose tissues, compared with HFD-corn oil fed animals. The transcriptomic analysis showed changes in ß-oxidation pathways, supporting the decreased lipid accumulation in the HFD-ED fed mice. Therefore, our data suggests that EPA and DHA supplementation of a high fat diet may be anti-inflammatory, as well as reduce lipid accumulation in adipose tissues.

Increased iron bioavailability from lactic-fermented vegetables is likely an effect of promoting the formation of ferric iron (Fe(3+)).

BACKGROUND: Lactic fermentation of foods increases the availability of iron as shown in a number of studies throughout the years. Several explanations have been provided such as decreased content of inhibitory phytate, increased solubility of iron, and increased content of lactic acid in the fermented product. However, to our knowledge, there are no data to support that the bioavailability of iron is affected by lactic fermentation. OBJECTIVES: The objective of the present study was to investigate whether the bioavailability of iron from a vegetable mix was affected by lactic fermentation and to propose a mechanism for such an event, by conducting human and cell (Caco-2, HepG2) studies and iron speciation measurements (voltammetry). We also investigated whether the absorption of zinc was affected by the lactic fermentation. RESULTS: In human subjects, we observed that lactic-fermented vegetables served with both a high-phytate and low-phytate meal increased the absorption of iron, but not zinc. In vitro digested fermented vegetables were able to provoke a greater hepcidin response per ng Fe than fresh vegetables, indicating that Fe in the fermented mixes was more bioavailable, independent on the soluble Fe content. We measured that hydrated Fe(3+) species were increased after the lactic fermentation, while there was no significant change in hydrated Fe(2+). Furthermore, lactate addition to Caco-2 cells did not affect ferritin formation in response to Fe nor did lactate affect the hepcidin response in the Caco-2/HepG2 cell system. CONCLUSIONS: The mechanism for the increased bioavailability of iron from lactic-fermented vegetables is likely an effect of the increase in ferric iron (Fe(3+)) species caused by the lactic fermentation. No effect on zinc bioavailability was observed.

Vitamin B12 as a potential compliance marker for fish intake.

OBJECTIVES: The aim of the present study was to investigate whether the following four markers: vitamin B12, selenium, vitamin D, and parvalbumin may be used as compliance markers for fish intake. METHODS: Blood samples from a randomized cross-over herring intervention study (n = 32) were analysed by HPLC and immunochemistry. The criteria were that plasma or serum concentrations of candidate compliance markers after the herring diet should increase significantly compared to starting concentrations. In addition, the reference meat diet should not yield an increase in plasma concentration of the candidate marker. RESULTS: Vitamin B12 and selenium met the set criteria for indicating a correlation between the marker and fish intake with significant increases in serum concentrations at 8.9% (p = 0.008) and 4.6% (p = 0.02), respectively, after a 6-week herring intervention (5 meals a week). Parvalbumin and 25-hydroxy vitamin D3 levels did not increase significantly after the herring interventions. CONCLUSIONS: Vitamin B12 may be suitable as a compliance marker for fish intake. Although selenium also met the criteria, the change in selenium serum concentrations was small compared to the change in vitamin B12 levels.

Development of celiac-safe foods: prevention of transglutaminase 2 (TG2) deamidation of gluten in healthy non-celiac volunteers.

In celiac disease, intestinal transglutaminase (TG2) produces immunogenic peptides by deamidation of gluten proteins. These products drive the celiac immune response. We have previously identified an interaction between gliadin and a food additive, E304i, which prevents gliadin processing (both deamidation and transamidation) by TG2, in vitro. In this study, we investigated if E304i could prevent TG2 processing of gluten in flours and if the effect was evident after simulated gastrointestinal digestion. We also confirmed the outcome in vivo in a human cross-over intervention study in healthy non-celiac participants. TG2 transamidation experiments (in vitro) of digested wheat and rye flours supplemented with E304i at 30 mg/g indicated full prevention of TG2 processing. In the intervention study, participant serum levels of deamidated gliadin peptides (dGDPs) increased after the intake of reference wheat rolls (80 g per day for a week; 41% ± 4% compared to washout), while the intake of the intervention E304i/zinc sulfate wheat rolls generated a modest response (80 g per day for a week; 8 ± 10% of control). The difference between the groups (32.8 ± 15.6%) was significant (p = 0.00003, n = 9), confirming that E304i /zinc addition to wheat rolls prevented TG2 deamidation of gluten. In conclusion, this study shows that E304i /zinc addition to wheat rolls prevents TG2 deamidation of gluten in non-celiac participants. Clinical trial registration: clinicaltrials.gov, identifier (NCT06005376).

Grains - a major source of sustainable protein for health.

Cereal grains are the main dietary source of energy, carbohydrates, and plant proteins world-wide. Currently, only 41% of grains are used for human consumption, and up to 35% are used for animal feed. Cereals have been overlooked as a source of environmentally sustainable and healthy plant proteins and could play a major role in transitioning towards a more sustainable food system for healthy diets. Cereal plant proteins are of good nutritional quality, but lysine is often the limiting amino acid. When consumed as whole grains, cereals provide health-protecting components such as dietary fiber and phytochemicals. Shifting grain use from feed to traditional foods and conceptually new foods and ingredients could improve protein security and alleviate climate change. Rapid development of new grain-based food ingredients and use of grains in new food contexts, such as dairy replacements and meat analogues, could accelerate the transition. This review discusses recent developments and outlines future perspectives for cereal grain use.

Iron regulates the uptake of ascorbic acid and the expression of sodium-dependent vitamin C transporter 1 (SVCT1) in human intestinal Caco-2 cells.

Ascorbic acid (vitamin C) has major effects on the intestinal uptake and utilisation of Fe in humans. The objective of the present study was to investigate the impact of Fe on the acquisition of ascorbic acid. The strategy was to study the cellular uptake and transport of ascorbic acid in the presence of Fe and also to observe the expression of the Na-dependent vitamin C transporter 1 (SVCT1) protein in human intestinal Caco-2 cells. SVCT1 is involved in the cellular uptake of ascorbic acid and is therefore a candidate for playing a role in the regulation of Fe utilisation. Caco-2 cells were cultured on transmembrane inserts in a three-compartment system followed by treatment with various combinations of FeCl2·4H2O (10-20 µmol/l) and sodium ascorbate (150 µmol/l). ELISA and Western blot analyses revealed that both SVCT1 and ferritin expressions were up-regulated in the presence of ascorbic acid in the basal compartment underneath the cells (10 and 22 %, respectively). Furthermore, when cells deficient in ascorbic acid were exposed to Fe, SVCT1 expression increased significantly (23·7 %). The increase in SVCT1 expression correlated with an increase in ascorbic acid uptake (285 %) in Fe-treated cells, as indicated by the SVCT1 inhibitor quercetin. We conclude that Fe plays an important role in regulating the uptake of ascorbic acid in human intestinal Caco-2 cells. This new angle could change the conceptual thinking of Fe and ascorbic acid utilisation and assist in the treatment and prevention of ascorbic acid-deficiency syndromes such as scurvy.

A LCMS Metabolomic Workflow to Investigate Metabolic Patterns in Human Intestinal Cells Exposed to Hydrolyzed Crab Waste Materials.

We have developed a LCMS metabolomic workflow to investigate metabolic patterns from human intestinal cells treated with simulated gastrointestinal-digested hydrolyzed crab waste materials. This workflow facilitates smart and reproducible comparisons of cell cultures exposed to different treatments. In this case the variable was the hydrolysis methods, also accounting for the GI digestion giving an output of direct correlation between cellular metabolic patterns caused by the treatments. In addition, we used the output from this workflow to select treatments for further evaluation of the Caco-2 cell response in terms of tentative anti-inflammatory activity in the hopes to find value in the crab waste materials to be used for food products. As hypothesized, the treatment identified to change the cellular metabolomic pattern most readily, was also found to cause the greatest effect in the cells, although the response was pro-inflammatory rather than anti-inflammatory, it proves that changes in cellular metabolic patterns are useful predictors of bioactivity. We conclude that the developed workflow allows for cost effective, rapid sample preparation as well as accurate and repeatable LCMS analysis and introduces a data pipeline specifically for probe the novel metabolite patterns created as a means to assess the performing treatments.

Brain foods - the role of diet in brain performance and health.

The performance of the human brain is based on an interplay between the inherited genotype and external environmental factors, including diet. Food and nutrition, essential in maintenance of brain performance, also aid in prevention and treatment of mental disorders. Both the overall composition of the human diet and specific dietary components have been shown to have an impact on brain function in various experimental models and epidemiological studies. This narrative review provides an overview of the role of diet in 5 key areas of brain function related to mental health and performance, including: (1) brain development, (2) signaling networks and neurotransmitters in the brain, (3) cognition and memory, (4) the balance between protein formation and degradation, and (5) deteriorative effects due to chronic inflammatory processes. Finally, the role of diet in epigenetic regulation of brain physiology is discussed.

In vitro digestibility and Caco-2 cell bioavailability of sea lettuce (Ulva fenestrata) proteins extracted using pH-shift processing.

Seaweed is a promising sustainable source of vegan protein as its farming does not require arable land, pesticides/insecticides, nor freshwater supply. However, to be explored as a novel protein source the content and nutritional quality of protein in seaweed need to be improved. We assessed the influence of pH-shift processing on protein degree of hydrolysis (%DH), protein/peptide size distribution, accessibility, and cell bioavailability of Ulva fenestrata proteins after in vitro gastrointestinal digestion. pH-shift processing of Ulva, which concentrated its proteins 3.5-times, significantly improved the %DH from 27.7±2.6% to 35.7±2.1% and the amino acid accessibility from 56.9±4.1% to 72.7±0.6%. Due to the higher amino acid accessibility, the amount of most amino acids transported across the cell monolayers was higher in the protein extracts. Regarding bioavailability, both Ulva and protein extracts were as bioavailable as casein. The protein/peptide molecular size distribution after digestion did not disclose a clear association with bioavailability.

The development of a novel ferric phytate compound for iron fortification of bouillons (part I).

In a series of two studies, we report the development (this study) and evaluation (part II) of a novel ferric phytate compound designed as a condiment iron fortificant. Condiments are used as iron fortification vehicles to reduce the prevalence  of iron deficiency. The challenge for iron fortificants in e.g. a bouillon matrix is to avoid undesired sensory effects and to ensure a reasonable cost. We added phytic acid to chelate iron, and hydrolysed protein to counteract the inhibiting effect of phytic acid on iron bioaccessibility. We characterised four novel ferric phytate compounds, destabilised by hydrolysed plant protein or amino acids. Colour stability of fortified bouillons with ferric phytate compounds was superior to bouillons fortified with ferrous sulfate. The iron-phytate-hydrolysed corn protein compound (Fe-PA-HCP) resulted in highest cellular ferritin induction in Caco-2 cells, in both vegetable (36.1 ± 13.40 ng/mg protein) and chicken (73.9 ± 19.93 ng/mg protein) bouillon matrices as observed in the human Caco-2/HepG2 cell model. Iron uptake (as estimated by ferritin production) from the Fe-PA-HCP compound was about 55% (chicken bouillon) and 66% (vegetable bouillon) of the iron uptake from ferrous sulfate. Based on this study, the Fe-PA-HCP compound was chosen for further evaluation (part II).

Impaired uptake of beta-carotene by Caco-2 human intestinal cells in the presence of iron.

At present, there are conflicting data regarding whether or not beta-carotene has a positive effect on iron absorption. This study was undertaken to evaluate possible interactions involved in the uptake of beta-carotene and iron in a human intestinal cell model (Caco-2). The Caco-2 cells were incubated with test solutions containing different amounts of ferrous chloride (10-50 microM) and beta-carotene (0.3-2.5 microM) incorporated in synthetic micelles. In the absence of iron, cellular accumulation of beta-carotene from synthetic micelles was proportional (r (2)=0.97, P <0.001) to the beta-carotene concentration in the test solution. However, with addition of ferrous chloride (30 microM), the beta-carotene uptake was significantly reduced (P <0.05), on average by 22%. There was also an inverse relationship between the beta-carotene uptake and iron concentration in the test solution (r (2)=0.93, P <0.05). Iron provided in physiological amounts inhibited the uptake of beta-carotene in the in vitro Caco-2 cell system.

Ascorbic acid uptake affects ferritin, Dcytb and Nramp2 expression in Caco-2 cells.

BACKGROUND: Ascorbic acid (vitamin C) enhances iron uptake in human intestinal cells. It is commonly believed that the enhancement is due to the capacity of ascorbic acid to reduce ferric iron to ferrous iron. Other suggestions have recently been made about the effects of ascorbic acid on the cellular metabolism of iron. These effects must be investigated for several reasons. One important issue is to study whether ascorbic acid has effects on iron metabolism in the absence of extracellular iron in the intestinal lumen. AIM OF THE STUDY: The aim of this investigation was to determine whether cellular uptake of ascorbic acid affects iron acquisition in the Caco-2 cell line. The possible event was investigated by studying the expression of the iron storage protein ferritin, the iron uptake protein Nramp2 and a duodenal ferric reductase Dcytb after incubating ascorbic acid deficient or ascorbic acid fed cells with iron and/or ascorbic acid. METHODS: The above stated interactions were studied in the human Caco-2 cell model. Cell lysates were collected and subjected to SDS-PAGE and Western blotting. The blotted samples were stained with specific antibodies (Rabbit alpha-human-Nramp2 and Goat alpha-human Dcytb) against the respective proteins and the bands achieved were analysed by reflective density measurements. The cellular ferritin content was analysed with a commercial kit and the intracellular ascorbic acid concentration was measured by HPLC. RESULTS: The results indicate that ascorbic acid uptake induces both iron independent and iron dependent ferritin formation, but the effect on iron dependent ferritin expression was significantly greater (470% compared to 19%). Western Blot analyses revealed a long term down-regulating effect of ascorbic acid on iron independent and iron dependent Nramp2 and Dcytb expression. However, the down-regulation of Dcytb was in general more extensive than that of Nramp2 (31-50% compared to 8-29%). In a second study of short term Nramp2 and Dcytb expression, the results suggested that both proteins were significantly up-regulated by ascorbic acid, regardless of intracellular ascorbic acid status. However, the impact of iron alone on Nramp2 up-regulation seems to be greater in the absence of ascorbic acid. CONCLUSIONS: The influence of intracellular ascorbic acid status on ferritin formation must be considered in iron uptake studies in Caco-2 cells. This could be a cause of diverging inter-laboratory results. The long term down-regulation of Nramp2 and Dcytb seems to correlate with results of human studies, where long term ascorbic acid supplementation does not affect iron status. Similarly, the short term up-regulation of Nramp2 and Dcytb seems to agree with the improvement in iron uptake shown in humans when single doses of ascorbic acid were administrated. These results are important for the understanding of the impact of ascorbic acid on iron status and will hopefully lead to further investigations on the matter.

Iron Supplements Containing Lactobacillus plantarum 299v Increase Ferric Iron and Up-regulate the Ferric Reductase DCYTB in Human Caco-2/HT29 MTX Co-Cultures.

Several human interventions have indicated that Lactobacillus plantarum 299v (L. plantarum 299v) increases intestinal iron absorption. The aim of the present study was to investigate possible effects of L. plantarum 299v on the mechanisms of iron absorption on the cellular level. We have previously shown that lactic fermentation of vegetables increased iron absorption in humans. It was revealed that the level of ferric iron [Fe (H2O)5]2+ was increased after fermentation. Therefore, we used voltammetry to measure the oxidation state of iron in simulated gastrointestinal digested oat and mango drinks and capsule meals containing L. plantarum 299v. We also exposed human intestinal co-cultures of enterocytes and goblet cells (Caco-2/HT29 MTX) to the supplements in order to study the effect on proteins possibly involved (MUC5AC, DCYTB, DMT1, and ferritin). We detected an increase in ferric iron in the digested meals and drinks containing L. plantarum 299v. In the intestinal cell model, we observed that the ferric reductase DCYTB increased in the presence of L. plantarum 299v, while the production of mucin (MUC5AC) decreased independently of L. plantarum 299v. In conclusion, the data suggest that the effect of L. plantarum 299v on iron metabolism is mediated through driving the Fe3+/DCYTB axis.

Ferric citrate and ferric EDTA but not ferrous sulfate drive amphiregulin-mediated activation of the MAP kinase ERK in gut epithelial cancer cells.

Ferric chelates may be used as oral iron supplements or phosphate binders but both ferric citrate and ferric EDTA have been shown to promote tumor burden in murine models of colon cancer. Here we studied their effects on cancer cell growth, at typical supplemental iron levels encountered in the gastrointestinal tract (0.01-0.2 mM). Caco-2 and/or Hutu-80 cells were exposed to these forms of chelated iron or to ferrous sulfate and outcomes were assessed using cell proliferation assays, proteome profiler arrays, western blot, and ELISA. Ferric EDTA and ferric citrate increased cellular levels of the onco-protein amphiregulin and its receptor (EGFr) which in turn stimulated the activation of the MAP kinase ERK. Simultaneously, the expression of the negative Wnt regulator, DKK-1, increased suggesting that cell proliferation through the Wnt pathway may be less pronounced in the presence of ferric EDTA and ferric citrate, unlike for ferrous sulfate. Moreover, ferrous sulfate did not increase levels of cellular amphiregulin or EGFr. We conclude that specific iron compounds affect cell signaling differently and some may increase the risk of colon cancer advancement in an amphiregulin-dependent fashion. Further scrutiny of safe oral iron use is merited.

Abundant fish protein inhibits α-synuclein amyloid formation.

The most common allergen in fish, the highly-abundant protein ß-parvalbumin, forms amyloid structures as a way to avoid gastrointestinal degradation and transit to the blood. In humans, the same amyloid structures are mostly associated with neurodegenerative disorders such as Alzheimer's and Parkinson's. We here assessed a putative connection between these amyloids using recombinant Atlantic cod ß-parvalbumin and the key amyloidogenic protein in Parkinson's disease, α-synuclein. Using a set of in vitro biophysical methods, we discovered that ß-parvalbumin readily inhibits amyloid formation of α-synuclein. The underlying mechanism was found to involve α-synuclein binding to the surface of ß-parvalbumin amyloid fibers. In addition to being a new amyloid inhibition mechanism, the data suggest that health benefits of fish may be explained in part by cross-reaction of ß-parvalbumin with human amyloidogenic proteins.

Effects of Marine Oils, Digested with Human Fluids, on Cellular Viability and Stress Protein Expression in Human Intestinal Caco-2 Cells.

In vitro digestion of marine oils has been reported to promote lipid oxidation, including the formation of reactive aldehydes (e.g., malondialdehyde (MDA) and 4-hydroxy-2-hexenal (HHE)). We aimed to investigate if human in vitro digestion of supplemental levels of oils from algae, cod liver, and krill, in addition to pure MDA and HHE, affect intestinal Caco-2 cell survival and oxidative stress. Cell viability was not significantly affected by the digests of marine oils or by pure MDA and HHE (0-90 µM). Cellular levels of HSP-70, a chaperone involved in the prevention of stress-induced protein unfolding was significantly decreased (14%, 28%, and 14% of control for algae, cod and krill oil, respectively; p ≤ 0.05). The oxidoreductase thioredoxin-1 (Trx-1) involved in reducing oxidative stress was also lower after incubation with the digested oils (26%, 53%, and 22% of control for algae, cod, and krill oil, respectively; p ≤ 0.001). The aldehydes MDA and HHE did not affect HSP-70 or Trx-1 at low levels (8.3 and 1.4 µM, respectively), whilst a mixture of MDA and HHE lowered Trx-1 at high levels (45 µM), indicating less exposure to oxidative stress. We conclude that human digests of the investigated marine oils and their content of MDA and HHE did not cause a stress response in human intestinal Caco-2 cells.

Correction: Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models.

Correction for 'Formation of reactive aldehydes (MDA, HHE, HNE) during the digestion of cod liver oil: comparison of human and porcine in vitro digestion models' by Cecilia Tullberg et al., Food Funct., 2016, 7, 1401-1412.

Splenic Immune Response Is Down-Regulated in C57BL/6J Mice Fed Eicosapentaenoic Acid and Docosahexaenoic Acid Enriched High Fat Diet.

Dietary n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are associated with reduction of inflammation, although the mechanisms are poorly understood, especially how the spleen, as a secondary lymphoid organ, is involved. To investigate the effects of EPA and DHA on spleen gene expression, male C57BL/6J mice were fed high fat diets (HFD) differing in fatty acid composition, either based on corn oil (HFD-CO), or CO enriched with 2 g/100 g EPA and DHA (HFD-ED), for eight weeks. Spleen tissue was analyzed using transcriptomics and for fatty acids profiling. Biological processes (BPs) related to the immune response, including T-cell receptor signaling pathway, T-cell differentiation and co-stimulation, myeloid dendritic cell differentiation, antigen presentation and processing, and the toll like receptor pathway were downregulated by HFD-ED compared with control and HFD-CO. These findings were supported by the down-regulation of NF-κB in HFD-ED compared with HFD-CO fed mice. Lower phospholipid arachidonic acid levels in HFD-ED compared with HFD-CO, and control mice suggest attenuation of pathways via prostaglandins and leukotrienes. The HFD-ED also upregulated BPs related to erythropoiesis and hematopoiesis compared with control and HFD-CO fed mice. Our findings suggest that EPA and DHA down-regulate the splenic immune response induced by HFD-CO, supporting earlier work that the spleen is a target organ for the anti-inflammatory effects of these n-3 fatty acids.