Research Project: Impact of Diet on Intestinal Microbiota, Gut Health and Immune Function

Location: Immunity and Disease Prevention Research

2023 Annual Report

Objectives
Objective 1 Define associations between diet and gut microbiota composition and function. Sub-objective 1A (Phenotyping Study): Examine the association between dietary features (e.g. fiber intake), gut microbial composition (bacterial taxa) and gut microbial functional capacity. Sub-objective 1B (Phenotyping Study): Use ex vivo culture models to examine the difference between high and low fiber groups in gut microbial functional capacity and colonization resistance to a food-borne pathogen. Sub-objective 1C (Longitudinal Study): Determine which bacterial taxa are consistently present over time and which bacterial taxa vary and correlate with dietary patterns for each subject. Sub-objective 1D (Intervention Study): Examine the specific effects of an inulin intervention on short term changes in composition and functional capacity of the gut microbial community. Objective 2 – Assess the association of diet and microbiota with gut health. Sub-Objective 2A (Phenotyping Study): Determine how intake of dietary fiber is associated with markers of gut health in a cross-sectional study. Sub-Objective 2B (Phenotyping Study): Determine whether dietary fiber intake and gut microbiome functional capacity are correlated with markers of gut health. Sub-Objective 2C (Longitudinal Study): Determine whether a long-term habitual low fiber diet is associated with markers of chronic gut inflammation relative to high fiber-consuming controls in a longitudinal study. Sub-Objective 2D (Intervention Study): Determine if consumption of dietary inulin reduces gut inflammation and impairs intestinal permeability when perturbed by an oral typhoid fever vaccine. Objective 3 – Determine if dietary patterns that promote gut health also promote systemic immune health. Sub-Objective 3A (Phenotyping Study): Determine if dietary features or nutritional status, gut microbial composition or functional capacity, and gut inflammation markers are associated with markers of systemic inflammation, specific immune cell types, or their level of activation. Sub-Objective 3B (Longitudinal Study): Determine if the associations identified in 3A are also seen in the baseline samples from the Longitudinal Study and determine if these associations are constant across time. Sub-Objective 3C (Intervention Study): Determine if consumption of 12 g/d inulin for 10 wk (for 4 wk before, 1 wk during and 1 wk after administration of the Vivotif® vaccine) will increase the vaccine-specific ALS IgG and IgA responses (primary endpoints), the plasma antibody, and stool IgA and T-cell responses (secondary endpoints) to the vaccine, relative to 12 g/d maltodextrin. Objective 4: Investigate the immunological properties of peanuts, other nuts and alternative proteins. Objective 5 - Investigate whether peanut consumption affects immune cell function and inflammation in healthy adults, including those at risk for immune dysfunction due to underlying conditions such as intestinal dysbiosis, obesity or chronic stress.

Approach
Our central hypothesis is that immunological health is a function of both dietary intake and the functional capability of gut microbes to respond to that diet. We will use three human studies to examine our central hypothesis: a cross-sectional Phenotyping Study, a Longitudinal Study, and a Fiber Intervention Study. The Western Human Nutrition Research Center (WHNRC) Nutritional Phenotyping Study is a cross-sectional study of healthy adults balanced by sex, age and body mass index with the recruitment phase to be completed in 2019. We will use stool samples from this project in ex vivo culture models—stool fermentations, pathogen challenge, and intestinal cell response—to address how the microbial environment interacts with substrate and how it affects physiology. The WHNRC Longitudinal Study is an observational cohort of middle-aged non-obese human participants selected at baseline to have adequate or low fiber intake. This cohort will be followed for up to 20 years, subject to renewal, with baseline and year 1 occurring in the current project cycle. Primary outcomes are measures of gastrointestinal and systemic inflammation. The WHNRC Fiber Intervention Study is a randomized controlled trial designed to test whether dietary inulin improves response to an oral vaccine that includes a live attenuated enteric pathogen. To address the hypothesis that dietary fiber consumption is associated with altered gut microbiome composition and function, stool samples from the studies will be sequenced for DNA content. Stool samples from the Phenotyping Study will additionally be assessed for fermentation capability, and pathogen resistance. To address the hypothesis that dietary fiber consumption is associated with altered gastrointestinal health, stool samples from the studies will be assessed for markers of inflammation and tested in an in vitro culture model of intestinal epithelial cells. In the intervention trial, intestinal permeability will be measured by quantifying the permeability of non-metabolizable sugar molecules. To address the hypothesis that dietary fiber consumption is associated with altered systemic immunity, blood samples from the studies will be assessed for measures of innate and adaptive immunity. These include plasma markers and complete blood count (CBC) in all trials as well as flow cytometry and ex vivo cytokine production by PBMC in the Phenotyping Study and measurement of vaccine-specific lymphocyte and antibody responses in the Intervention Study. Both gastrointestinal and systemic response will also be analyzed with gut microbiota as a mediator to determine whether these responses are microbiota-dependent. The most challenging aspect of all of these studies is the recruitment and retention of human participants, particularly for the Longitudinal Study. If we are unable to recruit enough participants, we may pursue new partnerships (e.g. UC Davis alumni association) or open a second study site (e.g. Sacramento). If we are unable to retain enough participants, we could consider the subset of outcomes that can be assessed remotely or backfill by recruiting more participants.

Progress Report
In support of Sub-objective 1A, ARS researchers in Davis, California, mapped dietary data from the USDA Nutritional Phenotyping Study to the Davis Food Glycopedia to determine the monosaccharide content of the diet and found that abundance of some dietary monosaccharides positively correlate with the abundances of certain microbes. Mapping of dietary data from the same study to polyphenols in FooDB is underway. A new tablet-based tool for recording mixed dishes in digital food records or recalls was designed and programmed. A study protocol for validating this tool against an existing food record tool and true nutrient contents of test meals has been written and a study coordinator has been hired. To determine the contribution of microbial taxa to outcomes of interest, researchers at Davis, California, developed software, called TaxaHFE, that reduces the feature set based on known taxonomic relationships of microbes and their information content relative to the outcome. Application of TaxaHFE to data from six different studies demonstrates superior performance and feature set reduction. In support of Sub-objective 1B, to determine microbial function and its relation to diet, fermentation studies are being conducted using participant stool. After pilot studies showed that a 13-day sequence was required to complete one set of continuous flow fermentations with four carbohydrates (mucin, pectin, arabinoxylan, and resistant starch type 3) for each participant stool, the decision was made to perform fermentations with 10 participant stools total. Five stools were selected from subjects who habitually consumed low amounts of dietary fiber and five from subjects with diets high in dietary fiber. The fermentations have all been completed, although, one will need to be repeated due to potential oxygen contamination. The microbial community dynamics from each fermentation will be measured using 16S rRNA sequencing of microbial DNA, which was collected daily. The 16S rRNA amplicons have so far been sequenced from two subject stool fermentations. The final fermentation will be repeated this summer. Short chain fatty acides (SCFAs) and 16SrRNA amplicons will be analyzed for all remaining fermentations over the course of FY24. In support of Sub-objectives 1D, 2D and 3C, a new study coordinator was hired, and we began enrolling participants in this clinical trial in January 2023. Over 80 participants have been screened, nine participants enrolled, and five have completed the 66-day trial. The primary reason for disqualification is due to participant availability for the full trial, but other reasons include a high inulin score, high body mass index, unwillingness to take the vaccine, enrollment in a different study, and inadequate transportation. In support of Sub-objective 2A, ARS scientists completed fecal water experiments with Caco-2 cells using samples from 112 participants in the USDA Nutritional Phenotyping Study (n=211 to-date). SCFAs were measured from all fecal and plasma samples available from the USDA Nutritional Phenotyping Study. Statistical analyses of fecal and plasma SCFAs in the context of diet are on-going. In support of Sub-objective 2B, all shotgun metagenomes were mapped to microbial genes in the Carbohydrate-Active Enyzme (CAZyme) Database and to other databases (KEGG, MetaCyc, etc.) A new metric, Muc2Plant, was defined as the ratio of mucin-unique CAZymes to plant unique CAZymes. Muc2Plant was positively associated with gastrointestinal (GI) inflammation, in support of the hypothesis that mucin degradation promotes or co-occurs with GI inflammation. However, dietary fiber intake was not associated with Muc2Plant. A draft manuscript has been prepared. A higher diversity of non-glucose monosaccharides in the diet was associated with lower GI inflammation. In support of Sub-objective 3A, data from the USDA Nutritional Phenotyping Study was used to examine the association of intestinal microbiota at the family and genus levels (using 16S rRNA gene sequences) with 17 immune factors made up of a total of 79 individual markers of systemic immune activation. Results showed the families S24-7, Rikenellaceae, Pseudomonoadaceae and one uncharacterized family (and genera within these families) were associated with four different immune factors describing systemic inflammation, T lymphocyte activation, monocyte activation and matrix metalloproteinase activity, indicating that intestinal bacteria may directly affect systemic immunity in healthy adults. A manuscript has been submitted for publication. Additionally, as a follow-up to the USDA Nutritional Phenotyping Study, the WHNRC postprandial monocyte study aims to delineate the function of postprandial non-classical monocytes following consumption of a mixed macronutrient challenge meal. In addition, the study aims to evaluate whether the type of dietary fat in the challenge meal contributes to changes in postprandial monocytes. We currently have an IRB-approved protocol, a study coordinator, and have nearly completed optimizing our experimental protocols. In a subordinate project, as part of the ARS Dairy Grand Challenge, relationships that were identified in fiscal year (FY) 2022 among milk components – lactose, oligosaccharides and fatty acids- and microbial taxa in raw milk were published in a peer reviewed manuscript. The hypothesis that milk fatty acids may have an antibiotic effect on opportunistic pathogens within raw milk was presented in the manuscript. Additionally, in a second experiment, the U.S. Dairy Forage Research Center in Madison, Wisconsin, provided both Holstein and Jersey cows with diets containing two levels of dietary fiber (forage). Bacterial DNA was extracted from the raw milk collected from these cows. In future FYs the microbial community of raw milk will be compared between Holstein and Jersey cows, as well as the relative effect of diet on the milk microbial community in each breed. In a subordinate project with the California Dairy Research Foundation on dairy consumption, lactase persistence genotypes, and gut microbiome (2032-51530-026-023T), the objective was to determine how lactase persistence genotypes and dairy consumption interact to impact the gut microbiome and fecal SCFAs. Increased abundance of families Lactobacillaceae and Lachnospriaceae was found in the gut microbiomes of lactase non-persistent (LNP) individuals consuming over 12.4 g lactose/day (manuscript submitted). Independent of lactose consumption, fecal samples from LNP individuals contained more SCFAs than fecal samples from lactase persistent individuals. Progress on this project supports Objectives 1 and 2 to understand the relationship between diet, microbiome, and GI health. In a subordinate project on honey (2032-51530-026-09T) with the National Honey Board, the primary aim is to assess the effects of minor components of honey on the composition and function of the small intestine microbial community. In vitro gastric digestion of honey or sugar control together with Enterotoxigenic Escherichia coli (ETEC) showed that honey inhibited ETEC growth under gastric conditions relative to sugar control. Fermentation of the gastric digests with a small intestine microbial community showed that honey does not appear to inhibit ETEC growth outside of the gastric environment. Analysis of bacterial DNA collected during fermentations is under way and a manuscript describing these results has been drafted. In a subordinate project on gut microbiomes of 1000 mother-infant dyads (2032-51530-026-10T) with the Bill & Melinda Gates Foundation, ARS researchers in Davis, California, extracted DNA from approximately 3000 fecal samples. Analysis of infant gut microbiomes in the context of milk cells and infant morbidity is ongoing. Progress on this project supports Objective 1 to understand the relationship between diet and microbial function. In a subordinate project on glycans as food biomarkers (0000065329), ARS researchers in Davis, California, collaborated with colleagues at University of California, Davis (UCD) to prepare a second version of the Davis Food Glycopedia (DFG) that provides more detailed glycan content of 250 high-priority foods. ARS researchers are preparing a food image database to accompany the DFG and collaborating with UCD colleagues on the interpretation of fecal fermentations with foods of known glycan content and on the design of a clinical trial in toddlers. Progress on this project supports Objective 1 to understand the relationship between diet and microbial function. In a subordinate project on the use of Artificial Intelligence (AI) in nutrition (2032-51530-026-18R), ARS scientists in Davis, California, together with UCD collaborators, completed the quality control and release of the first benchmark data set of food photo diaries with companion food records for the application of AI algorithms to assess dietary intake. Publicly available algorithms were evaluated using the benchmark and found to perform poorly on single-ingredient foods and beverages (manuscript submitted). In collaboration with colleagues at UCD, ARS scientists are now working to fill this domain gap. Progress on this project supports Objectives 1, 2, and 3, as all rely on accurate dietary intake assessment.

Accomplishments
1. Diverse dietary monosaccharides associated with a healthier gut. The human gut microbiome influences health, but what aspects of diet support a healthy microbiome is not understood. ARS scientists in Davis, California, mapped dietary data of a human cohort to the Davis Food Glycopedia to determine the level of 10 different sugar monomers, or monosaccharides, in the diets of healthy U.S. adults. A higher diversity of monosaccharides in the diet was associated with greater diversity in the gut microbiome and lower gastrointestinal inflammation. Specific food sources were enriched for individual monosaccharides which were associated with particular gut microbes. These results suggest that diets could be tailored to include foods that are rich sources of specific monosaccharides to support the growth of specific gut microbes. Such an approach could provide specific health benefits to individual Americans, which is a focus of the new USDA initiative, Agricultural Science Center of Excellence for Nutrition and Diet for Better Health (ASCEND for Better Health).

2. Consumption of a high-fat meal alters circulating monocytes. Monocytes are a heterogenous population of circulating immune cells contributing to vascular integrity and immune defense as well as to the initiation and progression of atherosclerosis. To better understand how circulating monocyte subsets respond to a dietary challenge, ARS researchers in Davis, California, examined blood from healthy human subjects before and after consumption of a fat-containing meal. Consumption of the meal induced changes in monocyte frequencies and subsets independently of subject age. In relation to fasting conditions, the circulating monocyte pool after the meal was significantly larger and was comprised of fewer “classical” monocytes which are important for replenishing immune cells in tissues, but more “patrolling” monocytes which are important for maintaining vascular integrity. These findings suggest that increased “patrolling” monocytes in blood after consuming a meal may provide enhanced surveillance of vascular tissue. This very new observation raises the question of whether different types of fats in the meal may help in reducing vascular inflammation and the initiation and progression of atherosclerosis.

3. Increased forages in cow diets may reduce opportunistic pathogen load in milk. There is a great need to help reduce antibiotic exposure through consumption of animal-based foods. Understanding alternate dairy management practices that can help reduce the risk of mastitis pathogens in the bovine udder will facilitate this goal. ARS researchers in Davis, California, conducted sequence-based analysis of bovine milk microbiota in cows fed different fiber types/amounts. Increased fiber from forages was associated with decreased Gammaproteobacteria, a class containing opportunistic pathogens. Fatty acids within the milk were negatively correlated with specific opportunistic pathogens to a greater degree than milk oligosaccharides (complex sugars). ARS researchers propose that dietary fiber from forages and the resulting changes in milk fatty acids might contribute to reduced pathogen load in cow udders, which could improve the quality and quantity of milk produced and decrease the need for antibiotic treatment in dairy cattle.

Review Publications
Calder, P.C., Ortega, E.F., Meydani, S.N., Adkins, Y.C., Stephensen, C.B., Thompson, B., Zwickey, H. 2022. Nutrition, immunosenescence and infectious disease: An overview of the scientific evidence on micronutrients and on modulation of the gut microbiota. Advances in Nutrition. 13(5):S1-S26. https://doi.org/10.1093/advances/nmac052.
Perez-Plazola, M., Diaz, J., Stewart, C., Arnold, C.D., Caswell, B.L., Lutter, C., Werner, E., Maleta, K., Turner, J., Prathibha, P., Liu, X., Gyimah, E., Iannotti, L.L. 2023. Plasma mineral status after a six-month intervention providing one egg per day to young Malawian children: A randomized controlled trial. Scientific Reports. 13. Article 6698. https://doi.org/10.1038%2Fs41598-023-33114-1.
Werner, E., Haskell, M.J., Arnold, C.D., Caswell, B.L., Iannotti, L.L., Lutter, C.K., Maleta, K.M., Stewart, C.P. 2023. The effects of one egg per day on vitamin A status among young Malawian children: A secondary analysis of a randomized controlled trial. Current Developments in Nutrition. 7(3). Article 100053. https://doi.org/10.1016/j.cdnut.2023.100053.
Coates, L.C., Durham, S., Storms, D.H., Magnuson, A.D., Van Hekken, D.L., Plumier, B.M., Finley, J.W., Fukagawa, N.K., Tomasula, M.M., Lemay, D.G., Picklo, M., Barile, D., Kalscheur, K., Kable, M.E. 2023. Associations among milk microbiota, milk fatty acids, milk glycans, and inflammation from lactating Holstein cows. Microbiology Spectrum. 11(3). Article e04020-22. https://doi.org/10.1128/spectrum.04020-22.
Snodgrass, R.G., Jiang, X., Stephensen, C.B. 2022. Monocyte subsets display age-dependent alterations at fasting and undergo non-age-dependent changes following consumption of a meal. Immunity & Ageing. 19. Article 14. https://doi.org/10.1186/s12979-022-00297-6.
Yang, Z., Wang, Y.E., Kirschke-Schneide, C.P., Stephensen, C.B., Newman, J.W., Keim, N.L., Cai, Y., Huang, L. 2023. Effects of a genetic variant rs13266634 in the zinc transporter 8 gene (SLC30A8) on insulin and lipid levels before and after a high-fat mixed macronutrient tolerance test in U.S. adults. Journal of Trace Elements in Medicine and Biology. 77. Article 127142. https://doi.org/10.1016/j.jtemb.2023.127142.
Riazati, N., Kable, M.E., Newman, J.W., Adkins, Y.C., Freytag, T.L., Jiang, X., Stephensen, C.B. 2022. Associations of microbial and indoleamine-2,3-dioxygenase-derived tryptophan metabolites with immune activation in healthy adults. Frontiers in Immunology. 13. Article 917966. https://doi.org/10.3389/fimmu.2022.917966.
James, K.L., Gertz, E.R., Kirschke-Schneide, C.P., Allayee, H., Huang, L., Kable, M.E., Newman, J.W., Stephensen, C.B., Bennett, B.J. 2023. Trimethylamine N-Oxide response to a mixed macronutrient tolerance test in a cohort of United States adults. International Journal of Molecular Sciences. 24(3). Article 2074. https://doi.org/10.3390/ijms24032074.
Larke, J.A., Bacalzo, N., Castillo, J.J., Couture, G., Chen, Y., Xue, Z., Alkan, Z., Kable, M.E., Lebrilla, C.B., Stephensen, C.B., Lemay, D.G. 2022. Dietary intake of monosaccharides from foods is associated with characteristics of the gut microbiota and gastrointestinal inflammation in healthy US adults. Journal of Nutrition. 153(1):106-119. https://doi.org/10.1016/j.tjnut.2022.12.008.
Werner, E., Arnold, C.D., Caswell, B.L., Iannotti, L.L., Lutter, C.K., Maleta, K.M., Stewart, C.P. 2022. The effects of 1 egg per day on iron and anemia status among young Malawian children: A secondary analysis of a randomized controlled trial. Current Developments in Nutrition. 6(6). Article nzac094. https://doi.org/10.1093/cdn/nzac094.
Kewcharoenwong, C., Sein, M., Nithichanon, A., Khongmee, A., Wessels, R.K., Hinnouho, G., Barffour, M.A., Kounnavong, S., Hess, S.Y., Stephensen, C.B., Letmemongkolchai, G. 2022. Daily preventive zinc supplementation increases the antibody response against pathogenic Escherichia coli in children with zinc insufficiency: A randomized controlled trial. Scientific Reports. 12. Article 16084. https://doi.org/10.1038/s41598-022-20445-8.
Bragg, M., Prado, E., Caswell, B.L., Arnold, C., George, M., Oakes, L., Beckner, A., DeBolt, M., Bennett, B.J., Maleta, K., Stewart, C. 2022. The association between plasma choline, growth and neurodevelopment among Malawian children aged 6-15 months enrolled in an egg intervention trial. Maternal and Child Nutrition. 19(2). Article e13471. https://doi.org/10.1111/mcn.13471.
Snodgrass, R.G., Jiang, X., Stephensen, C.B., Laugero, K.D. 2023. Cumulative physiological stress is associated with age-related changes to peripheral T lymphocyte subsets in healthy humans. Immunity & Ageing. 20. Article 29. https://doi.org/10.1186/s12979-023-00357-5.
Durham, S.D., Wei, Z., Lemay, D.G., Lange, M., Barile, D. 2023. Creation of a milk oligosaccharide database, MilkOligoDB, reveals common structural motifs and extensive diversity across mammals. Scientific Reports. 13. Article 10345. https://doi.org/10.1038/s41598-023-36866-y.