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Research Project: Assessing the Impact of Diet on Inflammation in Healthy and Obese Adults in a Cross-Sectional Phenotyping Study and a Longitudinal Intervention Trial

Location: Immunity and Disease Prevention Research

2017 Annual Report

Objective 1: Determine how diet quality (assessed using the Healthy Eating Index), nutritional status (assessed using biomarkers in a cross-sectional study) and adherence to a diet following Dietary Guidelines recommendations for intake of fat and fat-soluble vitamins affect immune function and inflammation. 1A: In the cross-sectional WHNRC Phenotyping Study (CSPS) determine if diet quality and intestinal dysbiosis are independently associated with systemic immune activation. 1B: In the WHNRC DGA Intervention Trial (IT) of adults with indicators of metabolic syndrome, determine if following the DGA diet improves markers of systemic and intestinal inflammation relative to a Typical American (TA) diet. Objective 2: Determine the degree of modulation and the mechanism of activation or inhibition of blood monocytes by different types of dietary fatty acids (including saturated fatty acids and docosahexaenoic acid [DHA]) and by fruit-derived dietary polyphenols or their metabolites. 2A: Determine (1) whether the high fat/sugar challenge meal administered during the CSPS induces postprandial monocyte activation; (2) whether this activation is mediated by saturated fatty acids; (3) whether and how the challenge meal-induced monocyte activation is suppressed by docosahexaenoic acid; and (4) in the DGA IT whether the diets affect challenge meal-induced monocyte activation. 2B: In subjects from the CSPS determine whether addition of DHA to the high fat/sugar challenge meal inhibits monocyte activation. 2C: In cell culture studies determine whether bioactive phytochemicals known to inhibit signaling pathways in monocytes, or their metabolites, also suppress SFA-induced monocyte activation. Objective 3: Removed per the PDRAM. Objective 4: Determine how diets enriched with polyphenol-rich fruits such as strawberries and grapes affect monocyte/macrophage function in obesity, determine possible chemical components of the fruits responsible for changes in function, and determine the mechanisms involved in changes in function. 4A: Determine if dietary strawberries and grapes affect monocyte/macrophage function, bacterial burden, morbidity and mortality in diet-induced obese mice infected with gram-negative bacteria. 4B: Determine if the polyphenols of strawberries and grapes are responsible for modulating monocytes in diet-induced obese mice infected with gram-negative bacteria. 4C: Determine mechanisms by which components of strawberries and grapes may modulate the function of monocytes isolated from diet-induced obese mice.

Objective 1 will utilize samples exclusively from the two human studies, the Western Human Nutrition Research Center (WHNRC) Cross-Sectional Phenotyping Study and the WHNRC Dietary Guidelines for Americans (DGA) Intervention Trial. Thus the designs of these studies are described under Objective 1 and the sample size calculations given relate to the goals of Objective 1. 1A: Such activation takes several forms and we will differentiate among pathways defined by the activity of pro-inflammatory T-helper (Th) cells (Th1, Th2 and Th17) and T-regulatory (Treg) cells. We hypothesize that those with low diet quality (including high solid fat and added sugar [SOFA] and low n-3 polyunsaturated fatty acids [PUFA]), or low intake (or status) of key nutrients (including vitamin D) will have greater immune activation after adjustment for appropriate covariates (e.g., age, BMI and sex). In addition, we hypothesize that dysbiosis of the gut microbiota (e.g., high levels of Proteobacteria) will be associated with gut inflammation that, in turn, will be associated with systemic immune activation. Microbiota will be assessed in stool using 16S rRNA gene sequence and inflammation by stool calprotectin and neopterin levels. 1B: DGA diet is optimized to minimize inflammation by decreasing SOFA, and increasing vitamin D, n-3 PUFA, fruit and vegetable intake. Objectives 2 will also utilize samples from both of these studies. In addition, Objectives will utilize cell culture methods to examine effects of dietary components on regulating cellular functions, including the effects of DHA (Objective 2B) and phytochemicals (Objectives 2C) on monocyte activation. Objective 4 will utilize a mouse model to examine the effect of diets rich in strawberry and grape preparations (freeze-dried whole fruit or fruit extracts) on monocyte/macrophage function in mice fed standard and high-fat diets and infected with gram-negative bacteria. Cell culture studies will also be used to examine the effect of fruit-derived phytochemicals on monocyte/macrophage function.

Progress Report
Progress continued in FY 2017 under Objective 1 on the milk, health, and genetics study. The ability to digest lactose (a dominant sugar in milk) into adulthood is a trait known as lactase persistence. When this trait arose as a result of a DNA mutation in Europe, it conferred a survival advantage to those who could drink milk into adulthood. This advantageous mutation spread rapidly, carrying genetic neighbors which may confer additional health benefits or risks. The objective of this project is to understand how lactase persistence genotypes and dairy consumption interact to impact human health. In this phase of the project, we aim to understand the effect of the genetic neighborhood of lactase persistence on markers of disease risk. We have conducted meta-analyses of 32,965 subjects for bone mineral density, 66,240 individuals for cardiovascular disease biomarkers, and 11,013 individuals for prostate cancer biomarkers. In the second phase of the project we will investigate the interaction between genotype, dairy consumption, and numerous health outcomes in the Phenotyping Study cohort, where we have genotyped the first 99 subjects for lactase persistence. Work also continued on revealing the function of bovine milk oligosaccharides (BMOs) in the human gut using metatranscriptomes. BMOs are complex sugars that are found in cow’s milk. BMOs are safe and tolerable in healthy adults. In this study, we sequenced the microbial genes being expressed in the stool of healthy adults who had consumed a high dose of BMOs to understand which gut microbes are fermenting BMOs in the human colon and which enzymes the gut microbes are using to ferment BMOs. Data analysis is ongoing. Under Objective 1A, the Western Human Nutrition Center (WHNRC) Phenotyping Study will have enrolled 195 volunteers by the end of this year. Laboratory work continues this year to characterize markers of systemic immune activation and inflammation. Descriptive statistical analysis continues to characterize the distribution of inflammation and immune activation variables across the study population with regard to the principal recruitment characteristics: age, sex and body mass index (BMI). These descriptive analyses will help characterize the study population and confirm data quality and integrity. All data are entered into a secure database. One study goal related to gastrointestinal (GI) inflammation is to understand how diet affects GI inflammation. In the first 100 subjects, we have measured fecal calprotectin as an indicator of general GI inflammation, fecal pH as an indicator of microbial activity and resistance to enteric pathogens, and LPS-binding protein in plasma as an indicator of gut barrier function. We have also conducted preliminary analyses to determine the effect of diet on stool consistency and weight. Data analysis is ongoing. Gut microbiota lab work was performed, including a comparison of methods for extracting bacterial DNA from human stool samples to optimize methods. We used sterilized fecal material spiked with a defined community of 8 bacteria to create "mock" stool samples with known bacterial composition. DNA was extracted using 4 commercially available kits. The ZymoBIOMICS DNA Miniprep kit was determined to provide the closest representation of the "true" microbial community in stool sample and was selected for continued use. Work continued related to gut microbiota, immune activation and diet. Fecal DNA sequence analysis of the bacterial 16S rRNA gene V4 region from 44 subjects was analyzed to determine if specific gut bacteria were associated with markers of immune activation and habitual diet. Activated central memory CD4 and CD8 T cells and the percent Natural Killer (NK) cells were correlated with the overall composition of the bacterial communities. Additionally, the proportion of Actinobacteria was negatively correlated with the percent of NK cells. Results were presented at a conference. Soluble dietary fiber, whole fruit, and citrus, melons, and berries were among the dietary components significantly associated with gut bacterial community composition The clinical portion of the WHNRC Dietary Guidelines Intervention Trial was completed this year. The study enrolled 44 overweight and obese women with elevated blood lipids or insulin resistance. Half of the volunteers were randomized to a weight-maintenance diet based on the Dietary Guidelines for Americans and the other half to a “typical American diet”. Laboratory work for inflammation endpoints is in progress. Three subordinate projects have examined the effect of specific nutrients on immune function. One project (2032-53000-001-11T, "Newborn Vitamin A Supplementation, Gut Microbiota and Vaccine Response at 1 year in Bangladeshi Infants") is evaluating the effect of vitamin A supplementation at birth on intestinal microbiota composition of Bangladeshi infants because such changes may affect vaccine responses. Lab work is complete and data analysis is ongoing. A second study (2032-53000-001-13T, "Effect of Zinc Intervention on Innate and Adaptive Immunity") is examining the effect of zinc supplementation on immune function of children at risk of zinc deficiency in the Lao People’s Democratic Republic. Laboratory work is being carried out at the WHNRC (2030-53000-001-16A, "Immunology Component of Lao Zinc Trial at Khon Kaen University"). Enrollment and follow-up of infants was completed this year and laboratory work is ongoing. Two other studies are focusing on vitamin D and bone health during HIV infection and antiretroviral treatment. Both studies are now complete, some data analysis continues, and two manuscripts have been submitted for publication. Progress continued on Objective 2 related to Metabolic endotoxemia. Studies using the Limulus Amebocyte Lysate (LAL) assay have measured bacterial endotoxin in human blood following high-fat meals as a possible explanation of the systemic inflammation that results from high fat diets. These studies suggest that such meals cause metabolic endotoxemia by facilitating absorption of endotoxin from intestinal bacteria. However, the accuracy of the LAL test when used in human blood has been questioned due to the presence of blood factors that sequester or metabolize endotoxin, thus raising doubts about the endotoxemia hypothesis. To address this question, we have assessed plasma endotoxin concentrations by a second method, in addition to LAL, in Phenotyping Study volunteers following a high-fat meal. Results from 24 subjects show endotoxin to be undetectable by the LAL assay. The second assay, the endotoxin neutralizing capacity (ENC) assay, showed no increase in endotoxin. Thus, our results do not support studies in the literature reporting bacterial endotoxemia following a high-fat meal and we propose that the high fat meal itself induces inflammation, rather than endotoxemia. Work is ongoing to characterize this alternative mechanism. Work continued on two projects related to postprandial inflammation (2032-53000-001-14I, "Suppression of Postprandial Monocyte Activation by Fruits Rich in Anti-Inflammatory Polyphenols or Docosahexaenoic Acid (DHA) in Humans," and 2032-53000-001-15H, "Effects of Blueberry Supplementation on High Fat Diet-induced Postprandial Inflammation and Endothelial Vasodilator Function in Humans"). We have taken the novel approach of using whole blood transcriptomic analysis to identify postprandial changes in gene expression by white blood cells. This method measures changes in all expressed genes and is thus more inclusive than targeted assays measuring a limited selection of inflammatory genes. Five subjects were randomly selected from our ongoing, crossover study examining the ability of two dietary components, docosahexaenoic acid (DHA) and freeze-dried blueberry powder, to dampen inflammation following a high-fat meal, relative to a placebo control. The results showed that the genes involved in innate immune responses, particularly those for pattern recognition receptors and their downstream signaling components, are differentially expressed 3 and 6 hours after the meal relative to the fasting sample. Effects of DHA and blueberry were not seen. The study demonstrates a new approach to assessing the pro- and anti-inflammatory effects of foods. A manuscript is in preparation. We have established an informal research project with colleagues at the Food Research Institute (NARO) in Japan to analyze plasma polyphenols and their metabolites in the plasma samples from the subjects who participated in the Blueberry/DHA study. Strawberries, grapes and monocyte function are being studied in Objective 4. Obesity impairs immune function and increases the risk of infection compared to normal weight. In previous human studies with dietary grape and strawberry powders, we observed an increase in the production of pro-inflammatory cytokines from stimulated monocytes derived from obese subjects fed the fruit powders compared to the placebo group. Monocytes are critical immune cells that provide a first line of defense against infection. Using mice as a model for diet-induced obesity, we determined that monocyte function was enhanced and Toll-like receptor expression in monocytes, which is involved in the recognition of bacteria, was increased in obese compared to lean animals. We are currently using the diet-induced obese mouse model to determine if dietary fruit reduces infection with Salmonella, an infection controlled, in part, by monocytes. In separate experiments, we determined that the polyphenol resveratrol increased monocyte function in a cell culture system, and we are extending these experiments to determine if resveratrol increases the elimination of Salmonella by monocytes in an intestinal cell culture system. These experiments will provide information on how polyphenols and/or polyphenol-rich foods may enhance immune cell activity and reduce the risk of infection.

1. Vitamin D supplementation decreases bone loss in HIV-infected patients. Scientists in Davis, California have previously shown that providing a single, monthly dose of 50,000 IU vitamin D3, in addition to multivitamins containing 400 IU/day, improved biochemical markers of bone turnover (relative to the daily dose alone) in patients taking the antiretroviral drug Tenofovir Disoproxil Fumarate (TDF) for human immunodeficiency virus (HIV) infections. This finding suggested that this dosing regimen could decrease the loss of bone mineral density (BMD) that is known to occur with this commonly used drug that is prescribed worldwide. In a collaborative research project with colleagues from the Adolescent Trial Network, ARS scientists from Davis, California showed that this regimen did significantly decrease loss of BMD in those using TDF, while the low-dose regimen did not. This finding suggests that such a regimen should be considered as adjunct therapy when TDF is used as a component of antiretroviral therapy in HIV infection.

2. Obesity increases peripheral monocyte function in mice. Obesity has been reported to increase the risk of infections compared to normal weight individuals. Monocytes are critical components of the immune system that are a first line of defense against pathogenic organisms. Using a mouse model, ARS researchers in Davis, California found that isolated monocytes from obese mice had an increased ability to engulf bacteria and increased production of inflammatory molecules after stimulation with a bacterial component compared to lean mice. These data suggest that the monocyte population is not compromised in obesity and actually has increased sensitivity to bacterial pathogens in mice.

3. Resveratrol increases monocyte function. Resveratrol is a phytochemical found in grapes, wine, and peanuts, and has antioxidant and anti-cancer activities. Little is known about the effects of resveratrol on the immune system. Using a cell culture system, ARS researchers in Davis, California discovered that resveratrol increased the function of monocytes, a white blood cell important in fighting bacterial and viral infections. Resveratrol increased engulfment of bacteria by monocytes and also increased production of protective, pro-inflammatory molecules, with increasing doses of resveratrol providing increasing benefit. These data suggest that dietary resveratrol may increase immune responses to pathogenic organisms and decrease risk of infections.

Review Publications
Zunino, S.J., Keim, N.L., Kelley, D.S., Bonnel, E.L., Souza, E.C., Peerson, J.M. 2017. Increased cytokine production by monocytes from human subjects who consumed grape powder was not mediated by differences in dietary intake patterns. Nutrition Research. 40:32-39.
Adkins, Y.C., Belda, B.J., Pedersen, T.L., Mackey, B.E., Newman, J.W., Kelley, D.S. 2017. Dietary docosahexaenoic acid and trans-10, cis-12-conjugated linoleic acid alter oxylipins profiles in mouse adipose tissue. Lipids. 52(5):399-413.
Zunino, S.J., Storms, D.H. 2017. Resveratrol-3-O-glucuronide and resveratrol-4’-O-glucuronide reduce DNA strand breakage but not apoptosis in Jurkat T cells treated with camptothecin. Oncology Letters. 14:2517-2522.
Zunino, S.J., Storms, D.H., Freytag, T.L., Adkins, Y.C., Bonnel, E.L., Woodhouse, L.R., Breksa III, A.P., Manners, G.D., Mackey, B.E., Kelley, D.S. 2016. Dietary supplementation with purified citrus limonin glucoside does not alter ex vivo functions of circulating T lymphocytes or monocytes in overweight/obese human adults. Nutrition Research. 36(1):24-30.
Rubin, L.P., Ross, C.A., Stephensen, C.B., Bohn, T., Tanumihardjo, S.A. 2017. Metabolic effects of inflammation on vitamin A and carotenoids in humans and animal models. Advances in Nutrition. 8:197-212. doi: 10.39945:an.116.014167.
Kable, M.E., Hansen, L.M., Styer, C.M., Gideonsson, P., Deck, S.L., Shevtsova, A., Rakhimova, O., Eaton, K.A., Martin, M.E., Boren, T., Solnick, J.V. 2017. Host determinants of expression of the helicobacter pylori BabA adhesin. Scientific Reports. 7:46499.