Effect of a diet based on the dietary guidelines for americans on inflammation markers in women at risk for cardiometabolic disease: results of a randomized, controlled trial.

OBJECTIVE: To evaluate the effect of a diet pattern based on Dietary Guidelines for Americans (DGA), in a controlled feeding setting, on plasma markers of inflammation and on cytokine production by peripheral blood mononuclear cells (PBMC). DESIGN: Women (n = 44) with one or more risk factors of metabolic syndrome (and BMI: 25.2-39.8 kg/m2) completed an 8-wk controlled feeding study. They were randomized to either a group following a diet based on DGA 2010 (DGA), or a group given a 'typical American diet' (TAD), based largely on a Western diet pattern. By design, women maintained their body weight. Fasting plasma and PBMC were collected at wk. 0 (baseline) and at wk. 8 (post-intervention). Sixteen plasma markers of inflammation and eight PBMC cytokines were measured at both time points, to evaluate if the diet had a significant effect on concentrations of these inflammatory markers. Data were analyzed using ANCOVA, followed by multiple-comparison adjustment using Benjamini-Hochberg method. RESULTS: Significant changes observed in Serum Amyloid A (SAA) and Matrix Metalloproteinase 3 (MMP3) in plasma did not retain significance upon multiple comparison adjustment. SAA: p = 0.044, adj p = 0.450; DGA mean change [95% CI] = - 12.6[- 32.3 to 7.04]; TAD mean change [95% CI] = - 2.24 [- 9.99 to 5.51]. MMP3: p = 0.014, adj p = 0.35; DGA mean change [95% CI] = 2.72[- 4.16 to 9.59]; TAD mean change [95% CI] = - 0.98[- 16.7 to 14.7]). Other inflammation markers were not differently altered by DGA relative to TAD. Effect size of change (Cohens d) indicated a large/medium-large effect of intervention on MMP3 and CRP, and medium effect on IL-6. CONCLUSIONS: No statistically significant changes were observed in the immune markers examined in this study. The biological roles and magnitude of the non-significant differences seen with two variables, CRP and MMP3, suggest that they be examined in future studies. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT02298725.

Associations of microbial and indoleamine-2,3-dioxygenase-derived tryptophan metabolites with immune activation in healthy adults.

Background: Tryptophan (Trp) metabolites from intestinal bacteria (indole, indole acetic acid [IAA] and indole propionic acid [IPA]), and the Trp metabolite kynurenine (Kyn) from the indoleamine 2,3-dioxygenase (IDO) pathway, are aryl hydrocarbon receptor (AhR) agonists and thus, can regulate immune activity via the AhR pathway. We hypothesized that plasma concentrations of these metabolites would be associated with markers of immune activation in a cohort of healthy adults in a manner consistent with AhR-mediated immune-regulation. We also hypothesized that the plasma Kyn/Trp ratio, a marker of IDO activity, would be associated with immune markers reflecting IDO activation in innate immune cells. Finally, we hypothesized that some intestinal bacteria would be associated with plasma indole, IPA and IAA, and that these bacteria themselves would be associated with immune markers. Methods: A novel set of 88 immune markers, and plasma Trp metabolites, were measured in 362 healthy adults. Bacterial taxa from stool were identified by 16S rRNA gene analysis. Multiple linear regression analysis was used to identify significant associations with immune markers. Results: The sum of indole and IAA was positively associated with natural killer T-cells levels. Kyn and Kyn/Trp were positively associated with neopterin and IP-10, markers of type 1 immunity, and TNF-α and C-reactive protein (CRP), markers of the acute phase response, and the regulatory cytokine IL-10. Three bacteria negatively associated with Trp metabolites were associated with markers of immune activation: the family Lachnospiraceae with higher lymphocyte counts but lower level of activated CD4 T-cells, the genus Dorea with higher production of IFN-γ by T-cells in PBMC cultures, and the genus Ruminococcus with higher production IL-6 in PBMC cultures stimulated with bacterial lipopolysaccharide (LPS). Conclusions: In this cohort of healthy adults bacterial Trp metabolites were not strongly associated with immune markers. Conversely, the Kyn/Trp ratio was strongly associated with markers of systemic inflammation and the acute phase response, consistent with IDO activation in innate immune cells. Finally, commensal bacteria associated with lower plasma (and perhaps intestinal) levels of bacterial Trp metabolites were associated with greater immune activation, possibly reflecting decreased regulatory immune activity related to lower intestinal levels of bacterial indole metabolites.

Postprandial Inflammatory Responses and Free Fatty Acids in Plasma of Adults Who Consumed a Moderately High-Fat Breakfast with and without Blueberry Powder in a Randomized Placebo-Controlled Trial.

BACKGROUND: Saturated fatty acids (FAs) released from triglyceride-rich lipoproteins (TGRLs) activate Toll-like receptor 2 (TLR-2) and induce the expression of proinflammatory cytokines in monocytes. Certain plant polyphenols inhibit TLR-mediated signaling pathways. OBJECTIVE: We determined whether plasma free FAs (FFAs) after a moderately high-fat (MHF, 40% kcal from fat) breakfast modulate the inflammatory status of postprandial blood, and whether blueberry intake suppresses FFA-induced inflammatory responses in healthy humans. METHODS: Twenty-three volunteers with a mean ± SEM age and body mass index (in kg/m(2)) of 30 ± 3 y and 21.9 ± 0.4, respectively, consumed an MHF breakfast with either a placebo powder or 2 or 4 servings of blueberry powder in a randomized crossover design. The placebo powder was provided on the first test day and the blueberry powder doses were randomized with a 2-wk washout period. Plasma concentrations of lipids, glucose, and cytokines were determined. To determine whether FFAs derived from TGRL stimulate monocyte activation, and whether this is inhibited by blueberry intake, whole blood was treated with lipoprotein lipase (LPL). RESULTS: The median concentrations of FFAs and cytokines [tumor necrosis factor-α, interleukin (IL)-6 and IL-8] in postprandial plasma (3.5 h) decreased compared with fasting plasma regardless of the blueberry intake (P < 0.001 for FFAs and P < 0.05 for cytokines). However, concentrations of FFAs and cytokines including IL-1ß increased in LPL-treated whole blood compared with untreated blood samples from participants who consumed the placebo powder. Blueberry intake suppressed IL-1ß and IL-6 production in LPL-treated postprandial blood compared with the placebo control when fasting changes were used as a covariate. CONCLUSIONS: The plasma FFA concentration may be an important determinant affecting inflammatory cytokine production in blood. Supplementation with blueberry powder did not affect plasma FFA and cytokine concentrations; however, it attenuated the cytokine production induced by ex vivo treatment of whole blood with LPL. This trial was registered at clinicaltrials.gov as NCT01594008.

Dietary supplementation with purified citrus limonin glucoside does not alter ex vivo functions of circulating T lymphocytes or monocytes in overweight/obese human adults.

Overweight/obesity is associated with chronic inflammation and impairs both innate and adaptive immune responses. Limonoids found in citrus fruits decreased cell proliferation and inflammation in animal studies. We hypothesized that limonin glucoside (LG) supplementation in vivo will decrease the ex vivo proliferation of T cells and the production of inflammatory cytokines by monocytes and T cells. In a double-blind, randomized, cross-over study, 10 overweight/obese human subjects were served purified LG or placebo drinks for 56 days each to determine the effects of LG on immune cell functions. The percentage of CD14+CD36+ cells in whole blood was analyzed by flow cytometry. Peripheral blood mononuclear cells were isolated and activated with CD3 plus CD28 antibodies (T-lymphocyte activation) or lipopolysaccharide (monocyte activation). Interferon γ, tumor necrosis factor α, interleukin (IL) 2, IL-4, and IL-10 were measured in supernatants from activated T cells. Supernatants from activated monocytes were analyzed for the production of tumor necrosis factor α, IL-1ß, and IL-6. Peripheral blood mononuclear cells were prestained with PKH dye and activated with CD3 plus CD28 antibodies to determine the proliferative responses of CD4+ and CD8+ T lymphocytes by flow cytometry. No differences were observed for CD14+CD36+ monocyte populations, T-cell proliferation, or the production of T cell and monocyte cytokines between the 2 treatments. Thus, LG supplementation in vivo did not affect ex vivo functions of T cells and monocytes, whereas it decreased several circulating markers of hepatic inflammation as we previously reported.

Dietary grape powder increases IL-1ß and IL-6 production by lipopolysaccharide-activated monocytes and reduces plasma concentrations of large LDL and large LDL-cholesterol particles in obese humans.

Obese individuals are at an increased risk of developing CVD, hypertension, type 2 diabetes, and bacterial and viral infections when compared with the normal-weight population. In a 9-week randomised, double-blind, cross-over study, twenty-four obese subjects aged between 20 and 60 years and with a BMI between 30 and 45 kg/m2 were fed grape or placebo powder for 3-week intervals to determine the effects of dietary grapes on blood lipid profiles, plasma inflammatory marker concentrations and immune cell function. Blood samples were collected on days 1 and 8 for obtaining baseline information and at weeks 3, 4, 8 and 9. Comprehensive chemistry panels, lipid profile analyses by NMR, measurement of plasma inflammatory marker concentrations, and analyses of cytokine production by activated T lymphocytes and monocytes were performed for each blood draw. Dietary grape powder reduced the plasma concentrations of large LDL-cholesterol and large LDL particles compared with the placebo powder (P< 0·05). The concentrations of interferon-γ, TNF-α, IL-4 and IL-10 were measured in supernatants from peripheral blood mononuclear cells (PBMC) activated with anti-CD3/CD28 antibodies and those of TNF-α, IL-1ß, IL-6 and IL-8 were measured in supernatants from PBMC activated with lipopolysaccharide (LPS). No difference in the production of T-cell cytokines was observed between the two intervention groups. The production of IL-1ß and IL-6 was increased in supernatants from LPS-activated PBMC in the grape powder group compared with the placebo powder group (P< 0·05). These data suggest that dietary grapes may decrease atherogenic lipid fractions in obese individuals and increase the sensitivity of monocytes in a population at a greater risk of developing infections.

Dietary strawberries increase the proliferative response of CD3/CD28-activated CD8⁺ T cells and the production of TNF-α in lipopolysaccharide-stimulated monocytes from obese human subjects.

Obesity increases the risk of developing bacterial and viral infections compared with normal weight. In a 7-week double-blind, randomised, cross-over trial, twenty obese volunteers (BMI between 30 and 40 kg/m²) were fed freeze-dried strawberry powder or strawberry-flavoured placebo preparations to determine the effects of dietary strawberries on immune function. Blood was collected at six time points during the study and peripheral blood mononuclear cells (PBMC) were isolated at each time point and activated with CD3 plus CD28 antibodies (T-lymphocyte activation) or lipopolysaccharide (LPS, monocyte activation). Interferon-γ, TNF-α, IL-4 and IL-10 were measured in supernatants from the activated T cells. Supernatants from the activated monocytes were analysed for the production of TNF-α, IL-1ß, IL-6 and IL-8. PBMC were pre-stained with PKH (Paul Karl Horan) dye and activated with CD3 plus CD28 antibodies to determine the proliferative responses of CD4⁺ and CD8⁺ T-lymphocytes by flow cytometry. To detect global changes in gene expression, microarray analysis was performed on LPS- and vehicle-treated PBMC from two subjects before and after the strawberry intervention. No difference was observed for the production of T-cell cytokines between the intervention groups. The production of TNF-α was increased in the supernatants from LPS-activated PBMC in the group consuming strawberries compared with the placebo. A modest increase in the proliferation of the CD8⁺ T-lymphocyte population was observed at 24 h post-activation. These data suggest that dietary strawberries may increase the immunological response of T-lymphocytes and monocytes in obese people who are at greater risk for developing infections.

Effects of dietary strawberry powder on blood lipids and inflammatory markers in obese human subjects.

Obesity is a strong risk factor for the development of CVD, hypertension and type 2 diabetes. The overall goal of the present pilot study was to feed strawberries, in the form of freeze-dried powder, to obese subjects to determine whether dietary strawberries beneficially altered lipid profiles and reduced blood markers of inflammation compared with a control intervention. A total of twenty healthy subjects (thirteen females and seven males) aged between 20 and 50 years with a BMI between 30 and 40 kg/m2 completed the present 7-week double-blind, randomised, cross-over trial. Each subject received a prepared diet 7 d/week for 7 weeks consisting of approximately 35 % of energy from fat, 20 % protein, 45 % carbohydrate and 14 g fibre. Blood was collected on days 1 and 8 for baseline information. After the first week, subjects were randomly assigned to the strawberry powder (equivalent to four servings of frozen strawberries) or control (strawberry-flavoured) intervention for 3 weeks. For the remaining 3 weeks, subjects crossed over to the opposite intervention. Blood was collected again at the end of weeks 3, 4, 6 and 7. A comprehensive chemistry panel, lipid profile analyses and measurement of inflammatory mediators were performed for each blood draw. A 3-week dietary intervention with strawberry powder reduced plasma concentrations of cholesterol and small HDL-cholesterol particles, and increased LDL particle size in obese subjects (P < 0·05). Dietary strawberry powder reduced risk factors for CVD, stroke and diabetes in obese volunteers, suggesting a potential role for strawberries as a dietary means to decrease obesity-related disease.

Vitamin A deficiency increases the in vivo development of IL-10-positive Th2 cells and decreases development of Th1 cells in mice.

Vitamin A deficiency impairs both T helper type 1 (Th1)- and type 2 (Th2)-mediated immune responses, although Th2 responses seem to be principally affected. Multiple mechanisms are involved in this immune suppression, but the hypothesis that deficiency affects development of Th1/Th2 memory cell phenotype has not been tested directly in vivo. To do so, lymphocytes from DO11.10 T cell receptor (TCR)-transgenic mice were transferred to vitamin A-deficient or control BALB/c recipients. Recipients were then immunized with the cognate peptide antigen for the TCR-transgenic DO11.10 T cells (OVA(323-339)). After 2-5 wk, the transferred OVA(323-339)-specific T cells were identified from draining lymph nodes with the TCR-clonotypic antibody KJ1-26, and their Th1/Th2 phenotype was characterized by intracellular cytokine staining after in vitro stimulation with phorbol myristate acetate and ionomycin. The percentage of CD4(+)KJ1-26(+) cells positive for IL-10 was 100% greater in vitamin A-deficient mice (3.49 +/- 0.41%; mean +/- SE) than in control mice (1.74 +/- 0.37%). IL-4 did not differ between groups. In addition, the percentages of CD4(+)KJ1-26(+) cells from vitamin A-deficient mice that were positive for interferon (IFN)-gamma (8.8 +/- 0.73%) and interleukin (IL)-2 (39.5 +/- 3.1%) were both lower than the percentages in control mice (11.4 +/- 0.67 and 47.0 +/- 2.8%, respectively). Thus vitamin A deficiency, at the time of initial antigen exposure, enhances the development of IL-10-producing Th2 or T regulatory cells and diminishes the development of Th1 memory cells.