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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

2016 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
The first two objectives of this project are linked largely to two human studies being conducted at the Western Human Nutrition Research Center (WHNRC) that involve multiple research scientists and two projects being conducted with appropriated funds, this project and the project “Improving Public Health by Understanding Diversity in Diet, Body, and Brain Interactions” (2032-51530-022-00D). Subordinate projects with extramural funding are also active under Objectives 1 and 2, as discussed below. In June of the current year a critical vacancy was filled with a new research scientist, a microbiologist, who will work on the intestinal microbiota aims described under Objective 1. A search to fill the one remaining vacancy for a research scientist on this project is in progress. Objective 3 was dropped from the project this year as part of a re-focusing of efforts with the recruitment goals for the two new research scientists, on Objectives 1, 2 and 4. Objective 4 focuses on how dietary phytochemicals affect immune function using mouse model systems. Objective 1A: The Western Human Nutrition Research Center (WHNRC) Phenotyping Study continued after its initiation last year, with the first 100 volunteers completing the study in July. Immunology, physiology, diet and other data collected in real time during the study are being stored in a secure online database established last year. Laboratory work began this year on stored serum samples to measure inflammation and immune-activation markers, and on stool samples to characterize the stool microbial community by DNA sequence analysis of the V4 region of the 16S rRNA gene. Descriptive statistical analysis was begun 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). In addition, diet and stool microbiota are being examined for associations between diet and microbial community structure. These descriptive analyses will help characterize the study population and confirm data quality and integrity. Objective 1B: The 30th volunteer in the Dietary Guidelines Intervention Trial (also called the iMAPS study) completed the study this year. Immune function data are being collected from independent cultures of peripheral blood mononuclear cells treated to activate cytokine production from monocytes and T lymphocytes. Laboratory analysis of these samples will begin in the coming year. Objective 1 – Subordinate Projects: Three subordinate projects under Objective 1 have examined the effect of specific nutrients on immune function. One project (2032-53000-001-11T) from the Thrasher Research Fund is evaluating the effect of vitamin A supplementation (vs placebo) at birth on the intestinal microbiota composition of Bangladeshi infants from birth through two years of age. Resulting effects on intestinal microbiota, particularly the relative abundance of bifidobacteria, may affect immune function which is also being evaluated, including responses to several childhood vaccines. Clinical work is completed and laboratory work and data analysis are in progress. A second study (2032-53000-001-13T with Micronutrients Initiative) is examining the effect of zinc supplementation on immune function of infants and children at risk of zinc deficiency in the Lao People’s Democratic Republic. Laboratory work is being carried out through collaboration with an immunology research group at Khon Kaen University in Thailand. Enrollment and follow-up of infants is nearly complete. Laboratory and data analysis will begin in the coming year. Two other studies funded via one agreement (2032-53000-001-02R with National Institutes of Health, in conjunction with University of Alabama, Birmingham) are focusing on vitamin D and bone health during HIV infection. One is examining the effect of vitamin D supplementation versus placebo on bone health in adolescents and young adults with HIV infection taking the antiretroviral drug tenofovir, which interferes specifically with vitamin D metabolism and bone mineralization. The second study is an observational study focusing on the association of vitamin D status with bone health in young men initiating tenofovir treatment. Laboratory work is in progress for the first study and was completed during the current year for the second. Objective 2: This objective is focusing on the effect of a high-fat diet, and of individual high-fat meals, on the activation of monocytes, which may cause inflammation that adversely affect health. Assays using whole blood to characterize monocyte activation for six hours following a challenge meal were implemented last year and ongoing work this year has collected data from most subjects in the Phenotyping Study. Plasma free fatty acids and cytokines are also being measured after the challenge meal and these analyses began this year. These data will provide a clue as to whether temporal change in plasma free fatty acids leads to a parallel change in cytokine concentration. Such an observation would suggest that the concentration of plasma free fatty acids is an important determinant of postprandial inflammation. Objective 2 – Subordinate Projects: Two extramurally funded studies are associated with this objective. The first (2032-53000-001-14I funded by National Institute of Food and Agriculture) will determine whether addition of blueberries or docosahexaenoic acid (DHA) to a high fat challenge meal will dampen the postprandial inflammatory responses using methods described above. This study is in progress and more than 50 test days (involving 18 adult volunteers) have been completed. Enrollment of the full cohort of 25 volunteers will be completed in the coming year. In addition, a new approach to evaluating postprandial inflammation is being evaluated. To determine whether DHA or blueberry intake affects the pattern of inflammatory gene expression in blood leukocytes, RNA sequencing with samples isolated from whole blood from five subjects is being performed with colleagues from the University of California, Davis. The reason for this analysis is that changes in gene expression measured via RNA sequencing in response to the high-fat challenge meal may be a more sensitive method than the measurement of cytokines in plasma or in monocyte cultures, as is also being pursued in this project. Thus the new method may be better able to detect beneficial effects of diet (e.g., blueberry intake) on postprandial inflammation. The second project (2032-53000-001-15H funded by U.S. Highbush Blueberry Council) is currently examining the effect of these same dietary interventions (the high-fat meal plus or minus the blueberries) in the same volunteers on peripheral vascular responsiveness. This measure of vascular health may be affected by postprandial inflammation and could be improved by inclusion of blueberries in the challenge meal. Data analysis will begin in the coming year. Objective 4: Obesity impairs immune function and increases the risk of infection by bacteria and viruses compared to normal weight. Monocytes are critical immune cells that provide a first line of defense against microbial infection. In previous 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 powders compared to the placebo group. Using mice as a model for diet-induced obesity, we are determining the differences in monocyte function after infection with Salmonella bacteria, and evaluating the function of Toll-like receptors, which are involved in the recognition of bacteria and activation of immune defense systems. Mice have been placed on low-fat and high-fat diets to compare the function of monocytes between lean and obese mice, and experiments have been performed to optimize assays for the isolation of monocytes from spleens, determine Toll-like receptor expression by flow cytometry, and measure phagocytosis. We are also determining how the polyphenol resveratrol and its metabolites, as well as other polyphenols, affect monocyte function using a cell culture system. In these experiments, we have pretreated the monocyte cells with resveratrol and its metabolites, and are currently evaluating phagocytosis, Toll-like receptor expression, and the production of pro-inflammatory cytokines in response to bacterial components. These experiments will provide information on how polyphenols and polyphenol-rich foods, such as grapes and strawberries, may increase immune cell activity in obesity and reduce the risk of infection in the obese population. Reduction of obesity-related infections by dietary intervention will have significant impact on health care costs.

1. High fat meals cause inflammation. People suffering from obesity and type 2 diabetes have high levels of inflammation that can have adverse effects on health. The factors causing this inflammation are not well understood. ARS scientists working in Davis, California conducted human studies and laboratory experiments to determine if high dietary fat levels, which also occur in obesity and type 2 diabetes, might be the cause of this inflammation. Results showed that elevated blood lipids, particularly saturated fats, following a high-fat meal act directly on a specific type of white blood cell (monocytes) to increase production of mediators that cause inflammation. These results indicate that a single, high-fat meal can increase inflammation and that such a dietary pattern may have adverse health effects due to this effect on inflammation. Future studies will evaluate how inclusion of other foods (e.g., blueberries) in high-fat meals may dampen this inflammation and thus could improve health.

2. Vitamin D supplementation during pregnancy improves immune health and may thus decrease the risk of preeclampsia. Vitamin D deficiency in pregnancy is associated with an increased risk of preeclampsia, a complication that results in hypertension and damage to the kidneys and other organs of the mother. Preeclampsia may be caused by an over-active inflammatory response that appears to be made worse by vitamin D deficiency. ARS scientists working in Davis, California (along with University of California, Davis colleagues) conducted a randomized, controlled trial of high-level vitamin D supplementation during pregnancy to determine if this treatment would dampen inflammation by increasing the abundance of regulatory immune cells. The study found that vitamin D supplementation did increase the abundance of regulatory immune cells, as predicted, and that higher blood vitamin D levels were also associated with lower levels for markers of inflammation just before delivery. These results indicate that vitamin D supplementation at 2,000 international units per day (or greater), higher than currently recommended during pregnancy, may be beneficial in promoting immune health and preventing inflammation, and thus should be further evaluated as a means to prevent the development of preeclampsia.

African Americans are at increased risk of both vitamin D deficiency and a complication of pregnancy known as preeclampsia. ARS scientists working in Davis, California showed that supplementation with vitamin D has benefits during pregnancy that may decrease the risk of this complication. This work may thus have special implications for nutritional recommendations for African American women at risk of vitamin D deficiency.