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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

2015 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: Determine the mechanisms by which a diet rich in fruits affects inflammation and immune function by characterizing the effect of fruit-derived dietary polyphenols or their metabolites on cell surface receptor-mediated oxidation-reduction signaling. 3A: Determine whether a diet rich in fruit-derived polyphenols modulates activation of leukocyte receptor tyrosine kinases (RTKs). 3B: Determine whether individual fruit-derived polyphenols modulate activation of RTKs. 3C: Examine the relationships between leukocyte RTK activation and dietary and blood levels of fruit-derived polyphenols, antioxidant status and oxidative stress. 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 and 3 will also utilize samples from both of these studies. In addition, Objectives 2 and 3 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 and 3B) on monocyte activation and insulin receptor (IR) function (Objective 3B). 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
This project has been in place for approximately one year (with the exception of Objective 4, which was added on April 30, 2015). The first three objectives 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. The overall progress of this project has been inhibited by critical vacancies for two research scientists, particularly with regard to progress on Objective 3. These vacancies will be recruited and thus could be filled in the coming year. However, with that exception of these vacancies, progress this year has been excellent. Objective 4 is a new addition this year and, unlike the human studies in objectives 1-3, objective 4 is using mouse model systems to evaluate the effect of diet on immune function. Objective 1: This year’s principal goal for objective 1A involved initiating the WHNRC Phenotyping Study and this goal has been achieved. A study coordinator was hired in October, the protocol was developed through a series of planning meetings, approval of the Institutional Review Board overseeing human studies was received, the study was posted on in February 2015 (NCT02367287) and recruitment began in June. By the end of September we anticipate that 23 subjects will have completed the study. While this is short of the projected number, the study is running smoothly and we hope to increase our current throughput from 8 to 12 volunteers per month so that the study can be completed within the initially estimated four-year period. In addition to the study coordinator and ARS staff and scientists who are working on the study, we have hired additional staff to help support study visits (which involve about 12 hours of time over two days with the human volunteers conducting various tests and administering questionnaires) and to manage data entry and storage. In addition we are utilizing many student interns to help conduct this labor-intensive study. Laboratory aspects of this study under Objective 1 included the development and testing of immunology assays utilizing flow cytometry analysis. To test these assays, we conducted a pilot study in human volunteers to validate methods for assessing monocyte and neutrophil activation in whole blood and the activation phenotype of T and B lymphocytes from peripheral blood mononuclear cells. These assays are now established. In addition, we have conducted initial pilot tests to assess the level of systemic immune activation using whole blood transcriptomic analysis. This assay would be a new addition to the study and we will continue development work in the coming year. Its full implementation may require the filling of one of our critical vacancies. The principal aim for Objective 1B, this year, involved the initiation of the dietary guidelines intervention trial. We have completed the same initiation process as described for the phenotyping study (i.e., hiring a study coordinator, pursuing Institutional Review Board approval, etc.) and this study was posted on in November, 2014 (NCT02298725). Over 100 potential volunteers have been screened for inclusion in the study and we anticipate that 14 volunteers will be actively participating or will have completed the study by the end of this year. Recruitment for this intervention study has been quite challenging and while we have not reached our numerical target for this year the study is now running smoothly and we are satisfied with its progress. Objective 1 – Subordinate Projects: Five subordinate projects under Objective 1 have examined the effect of specific nutrients on immune function. One project (2032-53000-001-09T) with the World Health Organization evaluated the impact of vitamin A supplements on vaccine responses in Bangladeshi infants at risk of vitamin A deficiency. The study was completed in the previous year and results were presented this year at one scientific meeting and were incorporated into a manuscript. A second project (2032-53000-001-12A) from the Thrasher Research Fund is funding follow-up studies on these infants and is evaluating the effect of the vitamin A intervention on the intestinal microbiota composition as vitamin A directly affects the intestinal immune system and may thus affect bacterial colonization of the gut. Resulting effects on intestinal microbiota, particularly the relative abundance of bifidobacteria, may affect immune function. A third study (2032-53000-001-13T) will examine the effect on immune function of zinc supplementation of infants and children at risk of zinc deficiency in the Lao People’s Democratic Republic. The ARS scientist responsible for this project visited the collaborating laboratory at Khon Kaen University in Thailand and the study will begin in the coming year. Two other studies (58-5306-3-058 and 58-5306-1-474) with Tulane University and the University of Alabama at Birmingham are examining the effect of vitamin D supplementation or status on immune function and bone health in children, adolescents and young adults with HIV infection or at risk of HIV infection. Laboratory work and data analysis are in progress on these studies. Objective 2: Aspects of this objective involve use of samples from subjects in the phenotyping study, described above. In particular, we plan to conduct an assay in whole blood from a subset of subjects in the phenotyping study to assess the contribution of free fatty acids to the development of inflammation via the activation of monocytes in blood following a high-fat challenge meal. This assay is technically challenging, particularly with regard to the use of lipoprotein lipase to release fatty acids from lipoproteins found in whole blood, and the standardization of reagents is in progress and on schedule. In addition, this assay will be used for two subordinate projects (2032-53000-001-14I and 2032-53000-001-15H) Agriculture and Food Research Initiative (AFRI) and the U.S. Highbush Blueberry Commission that are evaluating the effect of inclusion of specific foods (blueberries) and fatty acids (docosahexaenoic acid) on the inflammatory response to the high-fat challenge meal. Also, the whole blood transcriptomic analysis that we are developing (as described under Objective 1A, above) is also being evaluated for use in assessing changes in inflammation in response to the high-fat challenge meal under Objective 2. Finally with regard to Objective 2, we are implementing an assay to be used in the phenotyping and related studies to assess endothelial function in human volunteers in response to the high-fat challenge meal. The assay uses the EndoPAT instrument (from Itamar Medical) which measures peripheral vascular responsiveness to ischemic stress. ARS staff members have been trained in the use of the instrument and it is now in routine use at the WHNRC. Objective 4: This objective was just approved in April and involves the conduct of studies in mouse model systems to evaluate the impact of polyphenol components of fruits on inflammation. Some aspects of these models are new to the laboratory and preparatory work is under way. For example, the biological use authorization for the use of Salmonella bacteria was submitted and has been obtained and the animal use protocol for these studies is currently being prepared. These studies are planned to begin early in the coming year. Other work has also been ongoing directly related to this objective (i.e., the effect of components of fruit on inflammation and health), including the analysis of data from two previous human studies as well as ex vivo studies examining the effects of fruit-derived phytochemicals in cell culture. (1) The first human study was conducted with another ARS scientist and involved evaluation of the effect of oral administration of citrus fruit-derived limonin glucoside to overweight and obese humans on markers of inflammation. A previous analysis showed that the intervention decreased markers of systemic inflammation. This new analysis evaluated the effect of the intervention on lymphocytes cultured ex vivo and found that the intervention did not alter lymphocyte function. (2) The second human study involved secondary analysis of dietary intake data from subjects involved in an intervention trial in which it was demonstrated that grape powder increased (relative to placebo) the production of pro-inflammatory cytokines, an unexpected result. The new analysis found that the background diet of the volunteers in the study did not account for this increased cytokine production, further supporting the conclusion that the grape powder itself increased cytokine production.(3) Finally with regard to objective 4, ex vivo studies were also conducted this year on the cellular effects of resveratrol, which is found in high abundance in grapes. This work has shown that resveratrol glucuronides, a principal metabolite found in the blood after consuming grapes, partially protects cellular DNA from oxidative damage. Such damage can impair cellular function and may increase the risk of cancer development, thus consumption of resveratrol-rich foods may have multiple health benefits.

1. Vitamin A supplementation of newborns at risk of vitamin A deficiency does not improve vaccine responses. Vitamin A deficiency impairs immune function and increases the risk of death from common childhood infections in areas of the world where vitamin A deficiency is a public health problem. While the World Health Organization (WHO) recommends that infants and young children from six to 59 months of age in such areas receive vitamin A supplements to decrease the risk of death, many studies also indicate that vitamin A supplementation at birth may have similar benefits, though results were not conclusive. As a result of this uncertainty, WHO organized a multi-study effort to determine if vitamin A supplementation at birth reduced mortality and improved immune function. As part of this effort, ARS scientists from Davis, California, worked with colleagues from the International Centre for Diarrhoeal Disease Research, Bangladesh, to conduct a randomized, placebo-controlled intervention trial among 306 infants to determine if providing vitamin A supplementation at birth improved immune function, including the responses to four vaccines (tuberculosis, polio, tetanus and hepatitis B). The study demonstrated that vitamin A supplementation did not significantly improve vaccine responses relative to the placebo. The results of this study clearly show that vitamin A supplementation at birth is not warranted based on this lack of effect on vaccine responses and these results will be directly considered in formulating future WHO guidelines on vitamin A supplementation for this age group.

Review Publications
Aguer, C., Mccoin, C.S., Knotts, T.A., Mcpherson, R., Dent, R., Hwang, D.H., Adams, S.H., Harper, M. 2014. Acylcarnitines: potential implications for skeletal muscle insulin resistance. Journal of Federation of American Societies for Experimental Biology. 29(1):336-345. doi: 10.1096/fj.14-255901.