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

Location: Immunity and Disease Prevention Research

2020 Annual Report

1a. Objectives (from AD-416):
Our long-term objective is to determine how dietary fiber contributes to immunological health. 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 approaches to examine our central hypothesis: a cross-sectional Phenotyping Study, a Longitudinal Study, and a Fiber Intervention Study. Sub-objectives will be addressed by each study as follows: 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.

1b. Approach (from AD-416):
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.

3. Progress Report:
In support of Sub-objective 1A, sequencing of stool samples from the USDA Nutritional Phenotyping study for 16S rRNA (n=365) and shotgun metagenomes (n=288) is now complete. Pre-processing of both data sets is also now complete. Analysis of the 16S rRNA microbiome in relation to diet is currently in-progress. The dietary data has been converted into a hierarchical food tree for analysis of food-microbiome relationships. Analysis of the metagenomes for carbohydrate degrading enzymes is in-progress. In support of Sub-objective 1B, fermentation studies will be conducted using participant stool. A DASBox fermentation system has been installed and standard operating procedures for maintenance and set-up have been developed. Additionally, pilot fermentations have been run using individual strains of bacteria typically found in the gut to develop standard operating procedures for running the instrument and performing sampling. These drafts will be finalized with pilot fermentations of complex gut microbial communities when we are able to return to the lab. In support of Sub-objective 2A, several fecal and plasma markers have been tested using the samples from the phenotyping study. An analysis of stool consistency and stool weight in relationship to diet, physical activity, and stress is under way. Measurements of fecal calprotectin, neopterin, and myeloperoxidase (MPO) are complete, as is plasma lipopolysaccharide-binding protein (LBP). Measurements of fecal pH are complete. Measurements of fecal sIgA and beta-defensin 2 as well as fecal Short Chain Fatty Acids (SCFAs) will resume when the lab is re-opened post-COVID-19. In addition, a method for the quantification of host-derived transcripts from exfoliated colonocytes in participant stool has been optimized. In support of Sub-objective 3A, cytokines have been measured in plasma and immune activation is being measured by flow cytometry. Data is now available in a Research Electronic Data Capture (REDCap) database. A statistical analysis is in-progress. In a subordinate project on milk, health, and genetics with University of California, Davis, (61752), the objective is to determine how lactase persistence genotypes and dairy consumption interact to impact human health. A method was developed and published to estimate lactose consumption from 24-hour food recalls using machine learning and database matching. Lactose intake was estimated in the USDA Nutritional Phenotyping cohort using these methods. Analysis of the microbiome in the context of lactose intake and lactose tolerance genotype is in progress. Progress on this project supports Sub-objective 1A to understand the relationship between diet and microbial function. In a subordinate project on honey with the National Honey Board (2032-51530-026-09T), the primary aim is to assess the effects of minor components of honey on the composition and function of the small intestine microbial community. We completed a proof of concept in vitro digestion and fermentation study of honey with a microbial community from human stool and compared the results to those of a simple sugar control (38% fructose and 31% glucose). Gut microbial fermentation of 1% digested honey resulted in reduced pH relative to 1% digested sugar control, indicating that acid production (fermentation) is likely stimulated by the complex sugars present in honey. Development of a mock community resembling a typical small intestine microbial community, in addition to collection of actual small intestine communities from healthy humans undergoing colonoscopy, will resume when we are able to access our laboratory. Progress on this project supports Sub-objective 1B, to understand the relationship between diet and microbial function using an in vitro system. In a subordinate project on diet and antimicrobial resistance genes with University of California (2032-51530-026-07S), the sequencing reads from shotgun metagenomes of participant stool (n=288) have been mapped to antimicrobial resistance genes. Taxonomic assignments are underway. Progress on this project supports Sub-objective 1A, to understand the relationship between diet and microbial function.

4. Accomplishments
1. Building bridges between unconnected databases for human nutrition. The food composition database underlying a publicly available dietary assessment tool, Automated Self-Administered 24-Hour (ASA24), includes 65 nutrients; however, an independent and unconnected database could provide estimates for nearly 100 additional nutrients, if it were linked. ARS scientists in Davis, California, and collaborators at the University of California, Davis, used machine learning and database mapping methods to estimate dietary intake of one such nutrient, lactose, that existed only in the unconnected database. Food items between the two databases were acceptably matched based on their nutrient profiles and text descriptions. Compared to manual mapping of food items by experts, this method reduced the time required for estimation of lactose by 99.7%. These results suggest that unconnected food composition databases may be linked using computer-assisted methods, expanding assessment of up to 100 additional nutrients for researchers assessing nutrients not available in the output of ASA24. This increased capacity benefits the nutrition research community by expanding the tools they have available for their studies, and will also benefit nutrition professionals, and eventually consumers, by improving dietary guidance for Americans.

2. Daily zinc supplementation affects immune function in Laotian children at risk of deficiency. Zinc deficiency impairs immune function, thus increasing the risk of infectious disease, and is common among children in South-East Asia. However, the best approach to providing zinc supplements (daily by itself, daily with other micronutrients, or intermittently by itself when children are treated for episodes of diarrhea) is not clear. ARS scientists at Davis, California, worked with collaborators from the University of California, Davis, the Khon Kaen University in Thailand, and the Lao Tropical and Public Health Institute in the Lao People's Democratic Republic, to determine which method of supplementation affected immune function by conducting a randomized, placebo-controlled community intervention trial among 512 rural Laotian children. Children six to 23 months of age received one of four treatment regimens for 9 months. The results of the study showed that only the daily preventive zinc tablets produced significant changes in any of the immune endpoints relative to the placebo group, decreasing lymphocyte and eosinophil concentrations in blood, which suggests that this supplement may have improved an underlying infection, thus decreasing immune activation involving these cell types. This result provides important evidence for public health experts in Laos and internationally showing that daily zinc supplementation by itself (at 7 milligrams elemental zinc per day) affects immune function compared to other modes of supplementation currently being evaluated, a result that will be helpful in making decisions on which type of supplement to use in programs to prevent zinc deficiency.

5. Record of Any Impact of Maximized Teleworking Requirement:
The maximum teleworking requirement and closure of our facility at the Western Human Nutrition Research Center for over four months has had a negative impact that resulted in (a) the pause and delay of all human trials (Pilot, Intervention, Longitudinal), and (b) the pause and delay of all bench lab work.

Review Publications
Westreich, S.T., Salcedo, J., Durbin-Johnson, B., Smilowitz, J.T., Korf, I., Mills, D.A., Daniela, B., Lemay, D.G. 2020. Fecal metatranscriptomics and glycomics suggests that bovine milk oligosaccharides are fully utilized by healthy adults. Journal of Nutritional Biochemistry. 79.
Treiber, M.L., Taft, D.H., Korf, I., Mills, D.A., Lemay, D.G. 2020. Pre- and post-sequencing recommendations for functional annotation of human fecal metagenomes. BMC Bioinformatics. 21(74).
Chin, E.L., Simmons, G., Bouzid, Y.Y., Kan, A., Burnett, D.J., Tagkopoulos, L., Lemay, D.G. 2019. Nutrient estimation from 24-hour food recalls using machine learning and database mapping: a case study with lactose. Nutrients. 11(12):3045.
Wessells, R.K., Hinnouho, G., Barffour, M., Arnold, C., Kounnavong, S., Kewcharoenwong, C., Lertmemongkolchai, G., Schuster, G., Stephensen, C.B., Hess, S. 2020. Impact of daily preventive zinc or therapeutic zinc supplementation for diarrhea on plasma biomarkers of environmental enteric dysfunction among rural laotian children: a randomized controlled trial. American Journal of Tropical Medicine and Hygiene. 102(2):415-426.
Kewcharoenwong, C., Schuster, G.U., Wessells, R.K., Hinnouho, G., Barffour, M.A., Kounnavong, S., Brown, K.H., Hess, S.Y., Samer, W., Tussakhon, I., Lertmemongkolchai, G., Peerson, J.M., Stephensen, C.B. 2020. Daily preventive zinc supplementation decreases lymphocyte and eosinophil concentrations in rural laotian children from communities with a high prevalence of zinc deficiency: results of a randomized controlled trial. Journal of Nutrition. 150(8):2204-2213.
Liu, J., Zhu, Y., Jay-Russell, M., Lemay, D.G., Mills, D.A. 2020. Reservoirs of antimicrobial resistance genes in retail raw milk. Microbiome. 8(99).
Peters, D.C., Rivers, A.R., Hatfield, J.L., Lemay, D.G., Liu, S.Y., Basso, B. 2020. Harnessing AI to transform agriculture and inform agricultural research. IEEE IT Professional. 22(3):16-21.