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

Location: Immunity and Disease Prevention Research

2021 Annual Report

Objectives
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.

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, analysis of the marker gene sequences 16S rRNA (n=365) and shotgun metagenomes (n=290) is now complete with respect to taxonomic designations. Dietary data has been converted into a hierarchical food tree with leaf-node annotations for calories, fiber, protein, carbohydrates, and fat. Diet-microbiome relationships with respect to taxonomies identified via 16S rRNA analysis are complete, and a manuscript is in preparation. The shotgun metagenomes have been mapped to functional databases to estimate abundances of microbial genes. Analysis of shotgun metagenomes with respect to diet is underway. To determine microbial function and its relation to diet, fermentation studies will be conducted in support of Sub-objective 1B using participant stool. A set of control fermentations have been run using whole stool communities for 10 days each to determine the duration of time needed to establish a stable and representative gut microbial community using the DASBox fermentation system. Bacterial DNA has been extracted from samples taken daily during fermentation. 16S rRNA amplicon sequences will be analyzed in the coming months to understand the number of fermentation days needed to establish microbial community stability. An Institutional Review Board (IRB) protocol has been approved for the clinical trial for Sub-objective 1C. However, we have not been able to begin the clinical trial due to COVID-19 restrictions. To determine the relationship between dietary intake and gastrointestinal health, several fecal and plasma markers have been tested using the samples from the phenotyping study. This work supports Sub-objective 2A. Measurements of the following gastrointestinal (GI) markers are complete: fecal calprotectin, fecal neopterin, fecal myeloperoxidase, fecal sIgA, fecal beta-defensin-2, plasma lipopolysaccharide-binding protein, and fecal pH. Measurements of fecal Short Chain Fatty Acids (SCFAs) and quantification of host-derived transcripts from exfoliated colonocytes have been delayed due to COVID-19 restrictions. Analysis of the relationships between diet and GI markers is in progress. An analysis of stool consistency in relation to diet, physical activity, and stress was completed, and the publication was accepted. Sub-objective 2B relates to determining whether dietary fiber intake and gut microbiome functional capacity are correlated with markers of gut health. Total gut microbiome carbohydrate enzyme capacity was analyzed in relationship to dietary fiber diversity. All shotgun metagenomes have been mapped to microbial genes, and a method to aggregate these genes at the level of enzymatic pathways was identified. Higher resolution analyses (e.g., specific carbohydrate-active enzyme families and pathways) are underway. An IRB protocol has been approved for the clinical trial for Sub-objective 2C. However, we have not been able to begin the clinical trial due to COVID-19 restrictions. We have not been able to begin the clinical trials in Sub-objective 1D and Sub-objective 2D due to COVID-19 restrictions. To 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, cytokines have been measured in plasma and immune activation is being measured by flow cytometry. This work in the phenotyping study is in support of Sub-objective 3A. Data is available in a RedCap database. A statistical analysis examining the association of the Healthy Eating Index (HEI) with the 90 immune variables in 360 Phenotyping Study volunteers has been completed and a manuscript is in preparation. In brief, a higher HEI score, indicating a healthier diet, is associated with lower levels for several markers of systemic inflammation, including plasma C-reactive protein (CRP) and leukocyte counts, including neutrophil and lymphocyte concentrations. An IRB protocol has been approved for the clinical trial in support of Sub-objective 3B. However, we have not been able to begin the clinical trial due to COVID-19 restrictions. We are not able to begin the clinical trial for Sub-objective 3C due to COVID-19 restrictions on building occupancy and conducting human studies. In addition, a pilot study to prepare for this trial was terminated early due to the pandemic. Although the pilot study ended early with a smaller number of volunteers than planned, we were able to confirm that we can successfully measure the primary vaccine endpoint for the study. This primary endpoint is the measurement of plasma cells secreting vaccine-specific antibody in peripheral blood one week after vaccination. We also confirmed that we can measure antibody responses to two different vaccine antigens, bacterial lipopolysaccharide (LPS) and bacterial outer membrane protein. In addition, we are currently determining if T-cell responses are detectable in these volunteers. Measurement of T-cells would be a secondary study endpoint. In a subordinate project, ARS scientists in Davis, California, in collaboration with the researchers at the U.S. Dairy Forage Research Center in Madison, Wisconsin, are working on the ARS Dairy Grand Challenge project to determine if feeding lactating cows a more sustainable, high fiber/low starch (HFLS) diet, is associated with changes in raw milk microbial community composition and somatic cell count. Raw milk samples were collected from cows fed HFLS and low fiber/high starch diets in a crossover design. The microbiota alterations detected in raw milk during consumption of the HFLS diet suggest that microbes involved in fiber digestion were selectively enriched and may be associated with reduced udder inflammation. This work expands the research efforts to understand the potential for dietary fiber to influence health in support of Objective 1. In a subordinate project on dairy consumption (2032-51530-026-04T), the objective is to determine how lactase persistence genotypes and dairy consumption interact to impact human health. Lactose intake was estimated for all participants in the USDA Nutritional Phenotyping cohort using the methods we developed for this purpose. Analysis of the microbiome in the context of lactose intake and lactose tolerance genotype is complete and a manuscript has been prepared. Analysis of data from this cohort also demonstrates that dairy consumption does not cause GI inflammation, as there is no association between dairy intake of any type and fecal or plasma markers of GI inflammation. Progress on this project supports Objective 1 to understand the relationship between diet and microbial function and Objective 2 to understand the relationship between diet and GI health. In a subordinate project on honey (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. Progress was halted due to limited access to the lab during the COVID-19 pandemic. However, we recently resumed development of a mock community resembling a typical small intestine microbial community, confirming growth conditions needed to culture small intestine microbes. Progress on this project supports Objective 1 to understand the relationship between diet and microbial function. In a subordinate project on diet and antimicrobial resistance genes (2032-51530-026-07S), the sequencing reads from shotgun metagenomes of participant stool (n=288) have been mapped to taxonomic assignments at both the whole community level and to assemblies of contigs to identify the taxonomic source of antimicrobial resistance genes (ARGs). We have analyzed ARGs in relationship to diet and a manuscript is in progress. 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), the collaborating lab at University of California, Davis, has completed the monosaccharide compositions of more than 500 foods. ARS scientists identified additional foods commonly consumed by adults using the USDA Nutritional Phenotyping Study, What We Eat in America, and Food and Nutrient Database for Dietary Studies ingredient databases to expand the monosaccharide composition library. 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 (A.I.) in nutrition (2032-51530-026-18R), ARS scientists together with collaborators from University of California, Davis, have received Institutional Review Board (IRB) approval for a clinical trial in which participants collect food photo diaries with companion food records to create a reference data set for the application of A.I. algorithms to assess dietary intake, particularly for food glycans. ARS scientists have also tested near infrared technology and machine learning to distinguish a pair of visually similar foods. Progress on this project supports Objective 1 to understand the relationship between diet and microbial function. In a subordinate project on walnuts (0000065365), the primary aim is to use in vitro digestion and fermentation to understand which bacterial genes within the gut microbiome respond to consumption of walnuts. This will facilitate understanding how bacterial genes might influence health effects of walnut consumption. Because no in vitro laboratory methods were deemed sufficient to replicate biological mastication of walnuts, we began a human study to collect chewed walnuts from 10 subjects for use in the in vitro digestion and fermentation. Samples have been collected from four subjects. Six additional subjects have been recruited and are undergoing screening. Progress on this project supports Objective 1.

Accomplishments
1. Stool consistency is related to diet and stress. ARS researchers in Davis, California, sought to understand how diet, physical activity, and stress influence whether stool produced by healthy people is normal, hard, or soft by examining dietary records, blood tests, physical activity data, and stool samples. Dietary factors which influenced stool form included moisture, saturated fat, dairy, sodium, vegetables, fruit, and alcohol. Stool was less likely to be normal with a high physiological stress load, particularly influenced by the stress hormones norepinephrine and cortisol. These results suggest that manipulation of diet and/or stress could help improve stool consistency, even in healthy adults.

2. Dietary fiber-enriched wheat promotes health. Growing evidence indicates a connection between the gut microbiome and development of obesity. Consumption of dietary fiber can shift the microbiome composition and increase the production of Short Chain Fatty Acids (SCFA) in the gut, which may reduce the inflammation associated with negative health impacts of obesity. ARS researchers in Davis, California, administered baked rolls made with regular wheat or wheat enriched for the dietary fiber, resistant starch type 2 (RS2), to healthy adults for one week each. Consumption of the RS2-enriched rolls increased the abundance of bacterial taxa known to produce the SCFA butyrate, increased bacterial fermentation and reduced postprandial glycemia relative to the regular wheat rolls. This research indicates that replacement of dietary fiber enriched wheat for regular wheat in commonly consumed products in the American diet could help alter the gut microbiome and reduce glycemia to decrease the risk of developing type II diabetes.

3. Vitamin A (VA) supplementation improves immune function in infants. Vitamin A (VA) protects against respiratory and intestinal infections, but the mechanism is not fully known. In animals, VA increases the expression of a protein, chemokine receptor 9 (CCR9), that allows immune cells to migrate to mucosal immune sites where they protect against pathogenic microorganisms. However, this has not been shown in humans. ARS Researchers in Davis, California, working with colleagues at the International Centre for Diarrhoeal Disease Research (icddr,b), Bangladesh, conducted a randomized, controlled trial of VA supplementation in 306 Bangladeshi newborns and found that VA increased CCR9 expression by T regulatory (Treg) cells in early infancy relative to the placebo treatment. Since Treg cells play a central role in regulating immunity at mucosal surfaces, these results suggest that increased CCR9 expression by Treg cells may be one mechanism by which VA supplementation during infancy decreases the risk of death from common childhood infections in populations at risk of deficiency.

Review Publications
Stephensen, C.B. 2020. Primer on immune response and interface with malnutrition. In: Humphries D.L., Scott M.E., Vermund S.H., editors. Nutrition and Infectious Diseases. Nutrition and Health. Cham, Switzerland: Humana. p. 83-110. https://doi.org/10.1007/978-3-030-56913-6_3.
Stephensen, C.B., Liets, G. 2021. Vitamin A in resistance to and recovery from infection: relevance to SARS-CoV2. British Journal of Nutrition. 1-10. https://doi.org/10.1017/S0007114521000246.
Lemay, D.G., Baldiviez, L.M., Chin, E.L., Spearman, S., Cervantes, E., Woodhouse, L.R., Keim, N.L., Stephensen, C.B., Laugero, K.D. 2021. Technician-scored stool consistency spans the full range of the Bristol scale in a healthy US population and differs by diet and chronic stress load. Journal of Nutrition. 151(6):1443-1452. https://doi.org/10.1093/jn/nxab019.
Hughes, R.L., Horn, W.F., Finnegan, P., Newman, J.W., Marco, M.L., Keim, N.L., Kable, M.E. 2021. Resistant starch type 2 from wheat reduces postprandial glycemic response with concurrent alterations in gut microbiota composition. Nutrients. 13(2):645. https://doi.org/10.3390/nu13020645.
Joslin, S.E., Durbin-Johnson, B.P., Britton, M.T., Settles, M.L., Korf, I., Lemay, D.G. 2020. Association of the lactase persistence haplotype block with disease risk in populations of European descent. Frontiers in Genetics. 11. Article: 558762. https://doi.org/10.3389/fgene.2020.558762.
Ehrlich, A.M., Pacheco, A.R., Henrick, B.M., Taft, D., Xe, G., Huda, N.M., Mishchuk, D., Goodson, M., Slupsky, C., Barile, D., Lebrilla, C.B., Stephensen, C.B., Mills, D.A., Raybould, H.E. 2020. Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells. Gut Microbes. 20:357. https://doi.org/10.1186/s12866-020-02023-y.
Xue, Z., Brooks, J.T., Quart, Z., Stevens, E.T., Kable, M.E., Heidenriech, J., McLeod, J., Marco, M.L. 2021. Microbiota assessments for the identification and confirmation of slit defect-causing bacteria in milk and cheddar cheese. mSystems. 6(1). Article e01114-20. https://doi.org/10.1128/mSystems.01114-20.
Kable, M.E., Riazati, N., Kirschke, C.P., Zhao, J., Tepaamorndech, S., Huang, L. 2020. The Znt7-null mutation has sex dependent effects on the gut microbiota and goblet cell population in the mouse colon. PLoS ONE. 15(9). Article e0239681. https://doi.org/10.1371/journal.pone.0239681.
Ahmad, S.M., Hudaa, N.M., Raqib, R., Qadri, F., Peerson, J., Tanumihardjo, S., Stephensen, C.B. 2020. High-dose vitamin A supplementation at birth increases the percentage of CCR9+ treg cells in infants with lower birthweight in early infancy and decreases plasma sCD14 and prevalence of vitamin A deficiency at two years. Journal of Nutrition. 150(11):3005-3012. https://doi.org/10.1093/jn/nxaa260.
Parr, C.J, Lemay, D.G., Owen, C.L., Woodward Greene, M.J., Sun, J. 2021. Multimodal AI to improve agriculture. IEEE IT Professional. 23(3):53-57. https://doi.org/10.1109/MITP.2020.2986122.