Location: Immunity and Disease Prevention Research2019 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.
This is the final report for project 2032-53000-001-00D, which expired in January, 2019, and was replaced by new project 2032-51530-00D. In FY19, ARS researchers continued recruiting volunteers, in support of Objective 1, into the Nutritional Phenotyping Study (as described in Sub-Objective 1A), completing study visits for 22 volunteers, for an overall total of 304 volunteers completing all aspects of the study for the life of the project (plus an additional 31 who partially completed the study). Recruitment became more challenging this year as we sought to fill recruitment targets in two under-represented demographic categories, males and individuals with obesity. Changes in recruiting strategy improved our enrollment, particularly among men. Laboratory work to evaluate immune activation (using flow cytometric analysis of monocytes, neutrophils, eosinophils, T-cells and B-cells; and cytokine analysis of plasma samples) continues and is nearly complete for all 304 volunteers. Sample analysis related to gut health, intestinal inflammation (calprotectin, myeloperoxidase) and systemic exposure to endotoxin (Lipopolysaccharide (LPS)-binding protein) is also in progress. Fecal pH and calprotectin have been measured in 217 samples, myeloperoxidase in 108, and plasma LPS-binding protein in 232. Preliminary statistical analyses show that subjects consuming a low fiber diet have elevated calprotectin and these data were used to inform study design for the new project plan (2032-51530-026-00D). The gut microbial communities of 278 participants have been characterized by sequencing the 16S rRNA bacterial gene in stool samples, which have been prepared for metagenomics analysis of the microbiome, and for short-chain fatty acid analysis to characterize bacterial metabolism. A preliminary analysis of the relationship of these communities to systemic immune activation was presented at a scientific conference. In addition, assays to analyze the polyphenol and carbohydrate content of bacterial fermentation supernatants were optimized for the analysis of bacterial fermentations of a sample food containing high levels of fermentable carbohydrates. These optimized protocols will be applied to microbial communities from the Phenotyping Study in the ensuing project plan (2032-51530-026-00D). Volunteer enrollment will be completed in the first year of this ensuing project plan, thus data analysis and manuscript submission will occur during that project period. Overall, this study has progressed essentially as planned, recruiting 100 volunteers per year, on average, and is on track to complete enrollment in the coming project period. The study has been described in the literature in a study design manuscript, and two additional manuscripts are already submitted for publication using partial sets of data from this ongoing study. Under Sub-objective 1B, recruitment of study volunteers and laboratory analysis for the Dietary Guidelines for Americans intervention trial were both completed in previous years. Statistical analysis and manuscript preparation are in progress to determine if the intervention diet, which was based on the Dietary Guidelines for Americans, decreased markers of systemic inflammation relative to the control diet. Volunteers in this study were at high risk of chronic inflammatory diseases and this analysis will help determine if following the current Dietary Guidelines will improve health by decreasing chronic inflammation. The goal of Objective 2 was to evaluate the effect of saturated fat on postprandial activation of blood monocytes after a high-fat meal to determine if such activation contributes to chronic inflammation associated with high-fat diets. Activation of monocytes could come from direct action of the dietary saturated fats on monocytes, or by dietary fat increasing the absorption of bacterial endotoxin (which is known to activate monocytes) from the intestine during a high-fat meal. A second goal was to determine if two dietary components, the polyunsaturated fatty acid docosahexaenoic acid (DHA) and blueberry-derived polyphenols, could dampen such activation of monocytes. Samples for these analyses came from two sources: (1) a subset of volunteers from the Nutritional Phenotyping study; and (2) a randomized, double-blind, placebo-controlled crossover intervention trial funded from an extramural source (agreement 2032-53000-001-14I). In the intervention trial, volunteers received three dietary treatments (placebo, DHA and blueberry powder) with a four-week washout period between each treatment. The effects of the DHA and blueberry powder on monocyte activation are being evaluated separately. Sample collection and laboratory work for both studies was completed in previous years. Using these data, ARS scientists determined if these high-fat meals increased plasma endotoxin concentration and cytokine production from monocytes in postprandial human blood. Blood samples were probed from 98 volunteers from the cross-sectional Phenotyping study with a meal containing 60 percent kcal fat. Cytokine production in the postprandial blood cultured for 24 hours was not different from that of the fasting blood, indicating no activation of blood monocytes. In addition, plasma endotoxin concentrations in volunteers from the Phenotyping Study were mostly below the limit of detection of the standard Limulus assay for endotoxin. However, the endotoxin inhibitor polymyxin B (PMB) suppressed production of the cytokines IL-1beta, IL-6 and IL-8 in postprandial blood, suggesting a low-level presence of endotoxin (not detectable by the standard assay) did increase monocyte activation. Next, samples from 62 volunteers from the intervention study with a meal containing 36 percent kcal fat were evaluated. After culturing postprandial blood for 24 hours, cytokine production was not increased compared to fasting blood and was not inhibited by PMB, indicating the absence of biologically active endotoxin in these volunteers consuming a lower fat meal than was used in the Phenotyping Study. Treatment of whole blood with lipoprotein lipase increased the concentrations of palmitic and stearic acids and of IL-1beta, IL-6 and IL-8 in both studies, showing a direct effect of meal-derived fatty acids on monocyte activation. Contrary to the current assumption in the field that endotoxin is a significant cause of postprandial inflammation, these studies demonstrate that endotoxin may not be a significant cause of postprandial inflammation in healthy adults consuming the typical American diet (34% kcal fat). Plasma free fatty acids, however, may be an important cause of postprandial inflammation with such a diet, suggesting that decreasing the intake of saturated fat from high-fat diets will decrease meal-related inflammation. An additional approach was also taken to characterize this postprandial inflammation. RNA sequencing was used to observe gene expression in whole blood samples in five participants of the intervention trial who were each fed a high fat meal on three separate days. Genes involved in inflammation were expressed at three and/or six hours after the high-fat meal. The expression of specific target genes from the RNA sequencing study was then examined in 20 participants from the same study. Some participants had a stronger response than others and the timing of the peak response differed between participants, suggesting individual responsiveness to a high fat meal. Further, several genes were identified as candidate biomarkers to determine individual responsiveness to a high-fat meal. This work has been accepted for publication. Research under Objective 4 was completed in previous project years. The goal of this objective was to determine how diets enriched with polyphenol-rich fruits affected monocyte and macrophage activation in a mouse model of obesity and in cell culture studies, to determine if consumption of fruit-rich diets may dampen such inflammation, and thus promote health, in humans. Using the human THP-1 monocyte cell line, ARS scientists evaluated the effects of physiological levels of metabolites of the polyphenol resveratrol (resveratrol aglycone, resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide, and resveratrol-3-O-sulfate) on phagocytosis, production of pro-inflammatory cytokines (IL-1beta, IL-1alpha, and IL-18), cell viability, and expression of innate immune receptors that allow monocyte activation. In brief, THP-1 cells were treated with several concentrations of resveratrol and its metabolites. These metabolites, selected because they are found in blood after consumption of resveratrol in food, had no effect on the functional parameters tested. Resveratrol aglycone (the component found in food) increased phagocytosis and increased IL-1beta production. These data suggest that resveratrol may be effective in modulating monocyte function in an environment where there is direct exposure to the aglycone, such as in the intestinal immune tissue near the site of absorption. Such tissues are rich in monocytes and macrophages and may represent a mechanism by which resveratrol-rich foods could affect intestinal inflammation. Under a project with University of California, Davis (2032-53000-001-11T) researchers evaluated the effect of vitamin A supplementation at birth on immune function and the intestinal microbiome composition of Bangladeshi infants. Project work is complete and one manuscript was published in FY19. The manuscript reports that a high abundance of intestinal bacteria from the genus Bifidobacterium is associated with better vaccine responses. A project with Micronutrients Initiative (2032-53000-001-13T) examined the effect of zinc supplementation on immune function of children at risk of zinc deficiency. The project is complete and two manuscripts are in progress describing the effect of the zinc interventions on immune function.
1. A healthy microbiome in infants predicts better vaccine response. Vaccination is the surest approach to prevent infectious diseases, but many vaccines do not provide long-lived immunologic memory, particularly when given in early infancy. ARS scientists from Davis, California, conducted a study in 306 infants from birth to three years of age to determine if colonization with the commensal intestinal bacterium Bifidobacterium infantis in early infancy (between birth and four months of age) was associated with better responses to four vaccines also given in early infancy: tuberculosis, polio, hepatitis B, and tetanus. Bifidobacterium abundance in early infancy was positively associated with higher responses to the tuberculosis, tetanus and hepatitis B vaccines when the responses were measured in early infancy, and was also associated with higher responses to the tuberculosis, tetanus, and polio vaccines when the responses were measured between two and three years of age. This study is the first to demonstrate that bifidobacteria, which are abundant in the infant gut as a result of breastfeeding, may enhance long-term immunologic memory, a novel observation demonstrating that early life nutrition can improve health by enhancing vaccine memory responses.
2. Individual response to a high-fat meal. ARS researchers in Davis, California, conducted a study in humans to understand how the blood, which contains immune cells, responds in the hours after eating a meal that is moderately high in fat. Genes involved in inflammation were “turned on” at three and/or six hours after the high-fat meal. Some participants had a stronger response than others and the timing of the peak response differed between participants. This discovery implies that the concept of personalized nutrition (dietary guidance for individual people) includes responsiveness to a high-fat meal.
Sanctuary, M., Kain, J., Chen, S., Kalentra, K., Lemay, D.G., Rose, D., Yang, H., Tancredi, D.J., German, B.J., Slupsky, C., Ashwood, P., Mills, D., Smilowtz, J.T., Angkustsiri, K. 2019. Pilot study of probiotic/colostrum supplementation on gut function in children with autism and gastrointestinal symptoms. PLoS One. 14(1):1-30. https://doi.org/10.1371/journal.pone.0210064.