Impact of soyasaponin I on TLR2 and TLR4 induced inflammation in the MUTZ-3-cell model.

Previous studies have demonstrated that soyasaponin (SoSa) possesses anti-inflammatory properties in lipopolysaccharide (LPS)-stimulated immune cells by influencing the immune sensing of toll-like receptor (TLR) 4. The aim of this study was to investigate the immune modulatory effect of SoSa I on TLR2- and TLR4-induced inflammation within the monocytic MUTZ-3-cell model. MUTZ-3 cells were stimulated with gram-negative (Escherichia coli) or gram-positive (Staphylococcus aureus) bacteria or bacterial pathogen-associated molecular patterns (PAMPs) such as LPS or peptidoglycans (PGN) alone or in combination with SoSa I. Cell morphology was characterized by raster scanning and light microscopy. Cytokine production (IL-1ß, IL-6, TNF-α, IP-10, RANTES and IL-8) was measured by cytometric bead array and the expression of surface markers was assessed by flow cytometry. MUTZ-3 cells revealed a cell maturation-like alteration in morphology and increased expression of CD80, CD86, TLR2 and TLR4 after stimulation with either gram-negative and gram-positive bacteria or bacterial PAMPs. The addition of SoSa I suppressed pro-inflammatory cytokine and chemokine secretions in a dose-dependent manner regardless of TLR2 or TLR4 stimulation. Interestingly, E. coli- and S. aureus-induced inflammation was always inhibited better by SoSa I than that induced by LPS and PGN. Additionally, SoSa I reduced the expression of CD86 in PGN- or LPS-stimulated cells. This study demonstrated that the anti-inflammatory capacity of SoSa I is based on influencing both monocytic TLR2 and TLR4 and that SoSa I inhibits more effectively whole bacteria compared to solely LPS or PGN what points to a broader role of SoSa I in the down-regulation of inflammation.

Physiological properties of milk ingredients released by fermentation.

The demand for health-promoting food ingredients rises within an increasing market worldwide. Different milks fermented with bacteria, yeasts, moulds or enzymes from animal, plant and microbial sources offer a broad range of possibilities to cover different health aspects with new bioactive components. By the fermentation process interesting ingredients are enriched and released from the matrix, like lactoferrin, micro-nutrients, CLA and sphingolipids or synthesized, such as exo-polysaccharides and bioactive peptides. In particular, milk derived bioactive peptides exert several important health-promoting activities, such as anti-hypertensive, anti-microbial, anti-oxidative, immune-modulatory, opioid and mineral-binding properties. Milk-fermentation processes with probiotic bacteria synergistically combine health supporting bacterial and milk ingredient aspects which include new therapeutic solutions concerning hypercholesterolemia, carcinogenic intoxications, treatment of diarrhea, reduction of intestine pathogens, and supporting natural immune defense. Especially, milk-proteins and associated bioactive peptides released during microbial or enzymatic fermentation of milk offer a broad spectrum of new functional properties, for instance anti-hypertensive, anti-microbial, anti-oxidative, immuno-modulatory, opioid and mineral-binding properties. This review aimed at discussing recent research activities on physiological purposes and technical process aspects of functional components from fermented milk with a specific focus on biofunctional peptides released from fermented milk proteins.

(n-3) long-chain PUFA differentially affect resistance to Pseudomonas aeruginosa infection of male and female cftr-/- mice.

The aim of this study was to determine whether oral supplementation with EPA/DHA (10.5 and 5.1% of fat, respectively) could improve the outcome of pulmonary P. aeruginosa infection in cftr(-/-) mice compared with wild-type (Wt) mice similarly treated. Because gender could influence the susceptibility of cftr-deficient mice, results were analyzed by gender. Wt and (-/-) mice were randomized for 6 wk to consume a control or EPA/DHA diet, infected with endotracheal injection of 5 × 10(7) CFU/mouse of P. aeruginosa, and killed 24 h later. Cftr(-/-) mice were more susceptible to infection than were Wt mice; (-/-) males had more neutrophils (P < 0.01) and a higher keratinocyte-derived chemokine (KC) level (P < 0.05), and (-/-) females had greater lung injury and mortality (P < 0.05). Female (-/-) mice were more susceptible than (-/-) males with a higher mortality and lung injury (P < 0.05). The EPA/DHA diet reduced neutrophil numbers and KC and IL-6 levels (P < 0.05) in (-/-) males and reduced mortality rate (P < 0.001), lung permeability, and IL-6 level (P < 0.05) in (-/-) females compared with (-/-) mice fed the control diet. These results were associated with a reduction in the pulmonary bacterial load (P < 0.05), an increase in the EPA/DHA concentration in cell membranes of (-/-) males and females (P < 0.01), and an increased weight gain only in males compared with (-/-) mice fed the control diet (P < 0.01). In conclusion, EPA/DHA improves the host resistance of (-/-) mice, although the beneficial effect differed in males and females.

Supplementation with fatty acids influences the airway nitric oxide and inflammatory markers in patients with cystic fibrosis.

OBJECTIVES: To obtain a balance in the fatty acid (FA) metabolism is important for the inflammatory response and of special importance in cystic fibrosis (CF), which is characterized by impaired FA metabolism, chronic inflammation, and infection in the airways. Nitric oxide (NO) has antimicrobial properties and low nasal (nNO) and exhaled NO (FENO), commonly reported in CF that may affect bacterial status. The present study investigates the effect of different FA blends on nNO and FENO and immunological markers in patients with CF. PATIENTS AND METHODS: Forty-three patients with CF and "severe" mutations were consecutively enrolled in a randomized double-blind placebo-controlled study with 3 FA blends containing mainly n-3 or n-6 FA or saturated FA acting as placebo. FENO, nNO, serum phospholipid concentrations of FA, and biomarkers of inflammation were measured before and after 3 months of supplementation. RESULTS: Thirty-five patients in clinically stable condition completed the study. The serum phospholipid FA pattern changed significantly in all 3 groups. An increase of the n-6 FA, arachidonic acid, was associated with a decrease of FENO and nNO. The inflammatory biomarkers, erythrocyte sedimentation rate, and interleukin-8 decreased after supplementation with n-3 FA and erythrocyte sedimentation rate increased after supplementation with n-6 FA. CONCLUSIONS: This small pilot study indicated that the composition of dietary n-3 and n-6 FA influenced the inflammatory markers in CF. FENO and nNO were influenced by changes in the arachidonic acid concentration, supporting previous studies suggesting that both the lipid abnormality and the colonization with Pseudomonas influenced NO in the airways.

Reactive oxygen species abrogate the anticarcinogenic effect of eicosapentaenoic acid in Atm-deficient mice.

Recent studies have demonstrated that n-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) are able to suppress cell proliferation and inhibit tumor growth. The objective of our study was to investigate the influence of a high dose EPA on the development of the tumor phenotype in ataxia-telangiectasia mutated (Atm)-deficient mice, a genetic cancer model that is associated with increased levels of oxidative stress. We analyzed toxicity, proliferation, cell-cycle progression, and apoptosis of EPA in vitro and latency to tumorigenesis in vivo. Because of the impact of reactive oxygen species (ROS) on the tumor incidence in ataxia telangiectasia (AT), we further analyzed the effect of EPA on the generation of ROS and oxidative DNA damage (ODD). EPA effectively inhibited proliferation, altered cell-cycle progression, and induced apoptosis of tumor cells (AT-4). EPA showed no effect on the latency to tumorigenesis in Atm-deficient mice. EPA treatment was accompanied by a significant increase of ROS and ODD. Our results demonstrate the antiproliferative effect of EPA on tumor cells by alteration of cell-cycle progression and induction of apoptosis in vitro. On the other hand, EPA treatment of Atm-deficient mice led to the formation of ROS and accumulation of ODD that might have abrogated the anticarcinogenic effect caused by EPA.

Dietary (n-3) polyunsaturated fatty acids affect the kinetics of pro- and antiinflammatory responses in mice with Pseudomonas aeruginosa lung infection.

The underlying mechanisms by which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affect host resistance to Pseudomonas aeruginosa are unclear. The aim of this study was to determine their role on the kinetic of pro- and antiinflammatory response in lung infection. Mice fed either a control diet or a diet enriched with EPA and DHA were infected intratracheally and we studied lung expression of proinflammatory markers [CXCL1, interleukin (IL)-6, tumor necrosis factor-alpha], antiinflammatory markers (IL-10, A20, and IkappaB alpha), and PPARalpha and PPARgamma. The inflammatory response was assessed using recruitment of neutrophils and macrophages into bronchoalveolar lavage fluid, bacterial clearance from the lung, pulmonary injury, and 7-d survival rate. Compared with the control group, EPA and DHA delayed the expression of proinflammatory markers during the first 2 h (P < 0.05), upregulated proinflammatory marker expression (P < 0.05), and induced overexpression of antiinflammatory markers at 8 h (P < 0.05), enhanced recruitment of neutrophils at 16 h (P < 0.05), and induced PPARalpha and PPARgamma overexpression at 4 and 8 h (P < 0.01), respectively. Pulmonary bacterial load decreased and pulmonary injury and mortality were reduced during the first 24 h (P < 0.05). In conclusion, EPA and DHA modulate the balance between pro- and antiinflammatory cytokines, alter the early response of the host to P. aeruginosa infection, and affect the early outcome of infection.

Polyunsaturated fatty acids support epithelial barrier integrity and reduce IL-4 mediated permeability in vitro.

BACKGROUND: The intestinal mucosa functions as a barrier against harmful dietary and microbial antigens. An intact gut barrier forms a prerequisite for protection against infection and allergy. Both allergic and inflammatory mediators (e.g. IL-4, IFN-gamma) are known to compromise the epithelial barrier integrity by enhancing permeability. Breast milk provides protection against infection and allergy and contains polyunsaturated fatty acids (PUFA). AIM OF THE STUDY: Although PUFA are commonly used in infant formulas their effect on intestinal barrier is still poorly understood. Therefore the effects of distinct PUFA (n-6: LA, GLA, DGLA, AA; n-3: ALA, EPA, DHA) and a fat blend with PUFA composition similar to that of the human breast milk fat fraction, on barrier integrity were investigated. METHODS: Human intestinal epithelial cells (T84) were pre-incubated with individual PUFA or a lipase treated fat blend, with or without subsequent IL-4 exposure. Barrier integrity was evaluated by measuring transepithelial resistance and permeability. Membrane phospholipid composition was determined by capillary gas chromatography. RESULTS: DGLA, AA, EPA, DHA and to a lesser extend GLA enhanced basal TER and strongly reduced IL-4 mediated permeability, while LA and ALA were ineffective. Furthermore, the lipase treated fat blend effectively supported barrier function. PUFA were incorporated in the membrane phospholipid fraction of T84 cells. CONCLUSIONS: Long chain PUFA DGLA, AA, EPA and DHA were particularly effective in supporting barrier integrity by improving resistance and reducing IL-4 mediated permeability. Fat blends that release specific PUFA upon digestion in the gastrointestinal tract may support natural resistance.

Influence of low-dose polyunsaturated fatty acids supplementation on the inflammatory response of healthy adults.

OBJECTIVE: The aim of the present study was to examine the immune-modulating effect of two different fat blends enriched with a low dose of anti- or proinflammatory polyunsaturated fatty acids on the fatty acid status and subsequently on the immune response of healthy volunteers. METHODS: Thirty healthy volunteers were randomly assigned to group A (anti-inflammatory blend rich in polyunsaturated fatty acids: alpha-linolenic acid, 240 mg/d; eicosapentaenoic acid, 120 mg/d; stearidonic acid, 49 mg/d; and gamma-linolenic acid, 73 mg/d) or group B (arachidonic acid, 40 mg/d; containing an inflammatory fat blend) for a 2-wk dietary supplementation period. Concentrations of interleukin-8, interleukin-10, tumor necrosis factor-alpha, prostaglandins E(1) and E(2), and leukotriene B(4) were investigated before, after 2 wk of supplementation, and 2 wk after stopping supplementation using a whole blood ex vivo lipopolysaccharide-stimulation assay. RESULTS: Plasma concentrations of alpha-linolenic acid and eicosapentaenoic acid were significantly increased in group A. In addition, dietary fat blends influenced eicosapentaenoic acid concentration in erythrocyte membranes. Supplementation of the fat blends resulted in contrasting effects on the expression of lipid mediators and cytokines after ex vivo lipopolysaccharide stimulation. Release of prostaglandin E(1) and leukotriene B(4) were significantly decreased in group A, whereas prostaglandin E(2) and interleukin-10 concentrations were significantly increased in group B. No effect on interleukin-8 or tumor necrosis factor-alpha release was found after supplementation with either fat blend. CONCLUSIONS: These results show an immune-modulating effect of a low-dose dietary polyunsaturated fatty acid supplementation. However, further studies regarding fat-blend composition and period of supplementation in patients with inflammatory conditions are required.

Dietary n-3 fatty acids have suppressive effects on mucin upregulation in mice infected with Pseudomonas aeruginosa.

BACKGROUND: Mucin hypersecretion and mucus plugging in the airways are characteristic features of chronic respiratory diseases like cystic fibrosis (CF) and contribute to morbidity and mortality. In CF, Pseudomonas aeruginosa superinfections in the lung exacerbate inflammation and alter mucus properties. There is increasing evidence that n-3 polyunsaturated fatty acids (PUFAs) exhibit anti-inflammatory properties in many inflammatory diseases while n-6 PUFA arachidonic acid (AA) favors inflammatory mediators such as eicosanoids prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) that may enhance inflammatory reactions. This suggests that n-3 PUFAs may have a protective effect against mucus over-production in airway diseases. Therefore, we hypothesized that n-3 PUFAs may downregulate mucins expression. METHODS: We designed an absolute real-time PCR assay to assess the effect of a 5-week diet enriched either with n-3 or n-6 PUFAs on the expression of large mucins in the lungs of mice infected by P. aeruginosa. RESULTS: Dietary fatty acids did not influence mucin gene expression in healthy mice. Lung infection induced an increase of the secreted gel-forming mucin Muc5b and a decrease of the membrane bound mucin Muc4. These deregulations are modulated by dietary fatty acids with a suppressive effect of n-3 PUFAs on mucin (increase of Muc5b from 19-fold up to 3.6 x 10(5)-fold for the n-3 PUFAs treated group and the control groups, respectively, 4 days post-infection and decrease of Muc4 from 15-fold up to 3.2 x 10(4)-fold for the control and the n-3 PUFAs treated groups, respectively, 4 days post-infection). CONCLUSION: Our data suggest that n-3 PUFAs enriched diet represents an inexpensive strategy to prevent or treat mucin overproduction in pulmonary bacterial colonization.

Comparison of the structures of triacylglycerols from native and transgenic medium-chain fatty acid-enriched rape seed oil by liquid chromatography--atmospheric pressure chemical ionization ion-trap mass spectrometry (LC-APCI-ITMS).

The sn position of fatty acids in seed oil lipids affects physiological function in pharmaceutical and dietary applications. In this study the composition of acyl-chain substituents in the sn positions of glycerol backbones in triacylglycerols (TAG) have been compared. TAG from native and transgenic medium-chain fatty acid-enriched rape seed oil were analyzed by reversed-phase high performance liquid chromatography coupled with online atmospheric-pressure chemical ionization ion-trap mass spectrometry. The transformation of summer rape with thioesterase and 3-ketoacyl-[ACP]-synthase genes of Cuphea lanceolata led to increased expression of 1.5% (w/w) caprylic acid (8:0), 6.7% (w/w) capric acid (10:0), 0.9% (w/w) lauric acid (12:0), and 0.2% (w/w) myristic acid (14:0). In contrast, linoleic (18:2n6) and alpha-linolenic acid (18:3n3) levels decreased compared with the original seed oil. The TAG sn position distribution of fatty acids was also modified. The original oil included eleven unique TAG species whereas the transgenic oil contained sixty. Twenty species were common to both oils. The transgenic oil included trioctadecenoyl-glycerol (18:1/18:1/18:1) and trioctadecatrienoyl-glycerol (18:3/18:3/18:3) whereas the native oil included only the latter. The transgenic TAG were dominated by combinations of caprylic, capric, lauric, myrisitic, palmitic (16:0), stearic (18:0), oleic (18:1n9), linoleic, arachidic (20:0), behenic (22:0), and lignoceric acids (24:0), which accounted for 52% of the total fat. In the original TAG palmitic, stearic, oleic, and linoleic acids accounted for 50% of the total fat. Medium-chain triacylglycerols with capric and lauric acids combined with stearic, oleic, linoleic, alpha-linolenic, arachidic, and gondoic acids (20:1n9) accounted for 25% of the transgenic oil. The medium-chain fatty acids were mainly integrated into the sn-1/3 position combined with the essential linoleic and alpha-linolenic acids at the sn-2 position. Eight species contained caprylic, capric, and lauric acids in the sn-2 position. The appearance of new TAG in the transgenic oil illustrates the extensive effect of genetic modification on fat metabolism by transformed plants and offers interesting possibilities for improved enteral applications.

Omega-3 polyunsaturated fatty acids improve host response in chronic Pseudomonas aeruginosa lung infection in mice.

Pseudomonas aeruginosa is a gram-negative bacilli frequently encountered in human pathology. This pathogen is involved in a large number of nosocomial infections and chronic diseases. Herein we investigated the effects of polyunsaturated fatty acids (PUFA) in chronic Pseudomonas aeruginosa lung infection. C57BL/6 mice were fed for 5 wk with specifically designed diets with high contents in either omega-3 (omega-3) or omega-6 PUFA and compared to a control diet. P. aeruginosa included in agarose beads was then instilled intratracheally, and the animals were studied for 7 days. On the 4th day, the mice fed with the omega-3 diet had a higher lean body mass gain and a lower omega-6:omega-3 ratio of fatty acids extracted from the lung tissue compared with the other groups (P < 0.05). The omega-3 group had the lowest mortality. Distal alveolar fluid clearance (DAFC) as well as the inflammatory response and the cellular recruitment were higher in the omega-3 group on the 4th day. The effect on DAFC was independent of alpha-epithelial Na(+) channels (alpha-ENaC), beta-ENaC, and alpha(1)-Na-K-ATPase mRNA expressions, which were not altered by the different diets. In conclusion, a diet enriched in omega-3 PUFA can change lung membrane composition and improve survival in chronic pneumonia. This effect on survival is probably multifactorial involving the increased DAFC capacity as well as the optimization of the initial inflammatory response. This work suggests that a better control of the omega-6/omega-3 PUFA balance may represent an interesting target in the prevention and/or control of P. aeruginosa infection in patients.

Human milk oligosaccharides affect P-selectin binding capacities: in vitro investigation.

OBJECTIVE: In the initial phase of cellular immune response, selectins mediate the emigration of leukocytes from the blood stream into inflammatory regions. Human milk oligosaccharides (HMOs) possess binding epitopes of selectin ligands such as sialyl Lewis(x) and sialyl Lewis(a) and therefore might impair the interaction of selectins with cellular ligands. Neutral, acidic, sialylated, or fucosylated HMO fractions with polymerization degrees of 3 to 50 were investigated regarding this interaction in a dynamic flow chamber model that considers physiologic shear stress conditions. METHODS: Human milk oligosaccharides were compared with kappa-carrageenans and pectin oligosaccharides to deduce structure-activity relations. Fucoidan and sialyl Lewis(x) served as positive controls. RESULTS: All HMO fractions affected P-selectin ligand binding capacity but were not comparable to fucoidan. The activity of the acidic HMO fraction resembled sialyl Lewis(x) in decreasing the binding of the ligand to P-selectin. CONCLUSION: Human milk oligosaccharides modulate rather than block the function of P-selectin.

Long-chain polyunsaturated fatty acids modulate lung inflammatory response induced by Pseudomonas aeruginosa in mice.

Polyunsaturated fatty acid (PUFA) immunomodulatory properties have been studied extensively in chronic infections. Few studies have focused on acute infection; thus, PUFA effects in a mouse model of Pseudomonas aeruginosa (PA)-induced lung injury were evaluated. C57BL/6 mice were randomized to be fed for 3 wk with an eicosapentaenoic acid (EPA) diet, an arachidonic acid (AA) diet, or a control diet [saturated fatty acids]. Lung injury was induced by intratracheal instillation of 10(7) CFU of PA per mouse. In each diet group, animals were studied either without or after PA-inducing lung injury. Evaluation criteria were early mortality; inflammatory response assessed with tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6 and IL-10 levels in bronchoalveolar lavage; lung injury evaluation; and extravascular lung water, assessed 24 h after the injury. After PA-induced lung injury, no difference in early mortality was observed; TNF-alpha level was significantly higher in the EPA diet than in the other two diet groups. No difference for the other cytokines was found among the groups. Lung edema was also more important in the EPA group, consistent with the variations of TNF-alpha levels. Our study clearly shows that in PA-induced acute lung injury, n-3 PUFA induces differences in the inflammatory response with a higher level of lung edema. Modulation of the inflammatory response with n-3 PUFA can influence the response to a bacterial challenge.