Location: Immunity and Disease Prevention Research
2022 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.
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 16S rRNA (n=365) and shotgun metagenomes (n=290) is now complete. A manuscript has been published on diet-microbiome relationships, using tree-based taxonomic metrics for both diet and microbiome. Further analyses of diet and microbial gene content using the shotgun metagenomes is underway.
To determine microbial function and its relation to diet, fermentation studies are being conducted in support of Sub-objective 1B using participant stool. The daily dynamics of three microbial communities continuously cultured from participant stool over a 10-day period were measured by 16S rRNA marker gene sequencing. Sequence data showed that four days of continuous culture is needed to establish a stable community using the DASBox fermentation system. Two stools from subjects who habitually consumed at least 14g/1000kcal (adequate) or less than 8g/1000kcal (low) dietary fiber were cultured for four days to establish stable communities and subsequently used to ferment mucin, arabinoxylan, pectin and resistant starch type 3. The latter was generated from whole potatoes and assessed for quality on site. Additional fermentations are in progress.
An Institutional Review Board (IRB) protocol has been approved for the clinical trial related to Sub-objective 1D. However, we have not been able to begin the clinical trial due to COVID-19 restrictions and supply chain delays. See Sub-objective 3C, which relies on the same clinical trial, for further information.
To determine the relationship between dietary intake and gastrointestinal health, in support of Sub-objective 2A, ARS scientists measured short chain fatty acids (SCFA) in fecal samples from the USDA Nutritional Phenotyping Study. In a subset of the fecal samples that had been RNA-preserved, ARS scientists quantified host-derived transcripts from exfoliated colonocytes. A manuscript on the relationship between diet and gastrointestinal (GI) markers of GI health is in-progress.
In support of Sub-objective 2B, all shotgun metagenomes have been mapped to microbial genes in the Carbohydrate-Active Enyzme (CAZy) Database and to other databases (KEGG, MetaCyc, etc.). A manuscript on the relationship between fiber consumption and these microbial genes is underway.
In Sub-objective 2D, an IRB protocol has been approved for the clinical trial. However, we have not been able to begin the clinical trial due to COVID-19 restrictions and supply chain delays. See Sub-Objective 3C, which relies on the same clinical trial, for further information.
A statistical analysis was performed in support of Sub-objective 3A to determine if the plasma concentration of metabolites of the essential amino acid tryptophan produced from intestinal bacteria (indole, indole acetic acid [IAA], indole propionic acid [IPA]) were associated with 88 systemic immune markers in healthy adults from the Nutritional Phenotyping Study. These indole metabolites act on immune function by transcriptional regulation via the aryl hydrocarbon (AhR) nuclear receptor in both intestinal and systemic immune tissue. Regression analysis showed that after adjustment for multiple comparisons, IAA and indole were positively associated with Natural Killer (NK) T-cell levels, suggesting that these bacterial metabolites may enhance protection against viruses and intracellular bacteria, a key function of this cell type. A manuscript reporting these results has been submitted for publication. In addition, a goal of our project is to determine how intestinal bacteria themselves are associated with systemic immune function. To address this goal, a statistical analysis has begun examining the association of intestinal bacteria at the family and genus levels with this same set of immune markers and will result in the preparation of a manuscript for submission later this year.
Work resumed this year under Sub-objective 3C to initiate the clinical trial. In brief, using data from our pilot study completed in March of 2019, we modified the study protocol to shorten the duration of the trial and simplify the study visits for study volunteers. In addition, work completed this year showed us that addition of a second endpoint to this study, examining the T-cell response to oral typhoid fever vaccination, was not feasible, as the vaccine antigens that are useful in measuring our primary endpoint, the antibody response, were not sufficiently specific for the vaccine strain of typhoid fever in the T-cell assays that we evaluated. Thus T-cell analysis was dropped from the study. Enrollment of volunteers into the study should begin later this year but has been hampered by many issues. First, our Center was restricted to 25% occupancy through January 2022 making conduct of this study impossible. In addition, we had problems securing key materials for the study. For example, the manufacturer stopped production of the typhoid fever vaccine to be used in the study early in the pandemic and it only became available again in May 2022. In addition, we have not yet been able to secure the dietary fiber products (food-grade inulin, and the control material, food-grade maltodextrin) but orders have been placed and delivery is expected soon. Presuming no other impediments emerge, the trial should begin later this year.
In a subordinate project, as part of the ARS Dairy Grand Challenge, relationships among milk components – lactose, oligosaccharides (MO) and fatty acids (MFA) – milk microbiota, and somatic cell count (SCC), were examined in lactating dairy cows. ARS scientists in Davis, California, identified relationships between SCC, lactose and 6’-sialyllactose, which suggest a potential role of mammary inflammation in milk composition. Additionally, positive correlations between neutral fucosylated MO and mastitis-associated genera, and negative relationships between MO and beneficial genera, were identified. Datasets were generated by researchers at University of California, Davis. The U.S. Dairy Forage Research Center in Madison, Wisconsin, and the Grand Forks Human Nutrition Research Center in Grand Forks, North Dakota, were used for this work.
In a subordinate project on dairy consumption (2032-51530-026-04T), the objective was to determine how lactase persistence genotypes and dairy consumption interact to impact human health. Dairy consumption was estimated for participants in the USDA Nutritional Phenotyping Cohort. Analysis of data from this cohort also demonstrates that total dairy, cheese, fluid milk, and/or yogurt is not associated with GI inflammation and may be associated with some markers of GI protection. Consumers of fluid milk had higher levels of beta-defensin2 than non-consumers. There was a trend for consumers of yogurt to have higher secretory IgA than non-consumers. Progress on this project supports 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. Small intestine microbial communities were collected from six healthy subjects by intubating the small intestine to aspirate ileal fluid during routine colonoscopy. In vitro gastric digestion of honey and sugar control have been performed with the food-borne pathogen, enterotoxigenic E. coli, in preparation to ferment the gastric output with small intestine microbial communities. 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 (58-2032-9-03900), ARS researchers in Davis, California, together with scientists at the University of California, Davis, analyzed antimicrobial resistance genes in the fecal samples of participants of the USDA Nutritional Phenotyping Cohort. ARS researchers found that diverse diets higher in fiber and lower in animal protein were associated with lower antimicrobial resistance. A manuscript has been published. 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 (65329), ARS researchers in Davis, California, collaborated with colleagues at University of California, Davis, to release the first Davis Food Glycopedia that details the glycan content of foods. This first version lists the absolute abundances of 14 different monosaccharides in more than 800 foods. ARS scientists in Davis, California, mapped dietary data from the USDA Nutritional Phenotyping Study to foods in the Glycopedia to determine the monosaccharide composition of diets and their relationship to the gut microbiome and gastrointestinal health. 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 collaborators from University of California, Davis, conducted a clinical trial in which participants collected food photo diaries with companion food records to create the first benchmark dataset for the application of AI algorithms to assess dietary intake. The trial was completed by 95 participants and the benchmark database is now under construction. Progress on this project supports Objectives 1, 2, and 3, as all rely on accurate dietary intake assessment.
Accomplishments
1. Antimicrobial resistance is lower with diverse, high-fiber diets. Antibiotic resistance is expected to be a major cause of death worldwide in the coming decades. ARS researchers in Davis, California, sought to understand how diet is related to the antimicrobial resistance of microbes in healthy adults. Bacterial DNA from fecal samples was examined in relation to the diet of healthy men and women who were normal weight, overweight, or moderately obese. The researchers found that the intestinal bacteria of individuals who consumed diverse diets that were high in fiber had lower levels of antibiotic resistance. than individuals with less diverse diets. These results suggest that dietary modification towards a more diverse, fiber-rich diet may reduce the individual and population-scale burden of bacterial infections resistant to antibiotics.
2. More diverse gut microbiomes co-occur with diverse sources of carbohydrates from food. A high-fiber diet has strong associations with better health, likely because of fermentation products produced by the bacteria in our colon on our own physiology. However, many types of fiber exist that interact uniquely with gut bacteria and current food databases do not adequately describe these various dietary fiber types. ARS researchers in Davis, California, addressed this limitation by assembling self-reported fiber-containing foods from 343 healthy U.S. adults into a tree structure describing their relatedness to capture the diversity of fiber types found in the diet. The variety of fiber-containing foods consumed was strongly associated with the diversity of the gut bacterial community. These results suggest that current dietary guidance that recommends higher fiber intake, but does not address fiber types, should also consider fiber diversity as an approach to improving health for Americans.
3. Machine learning identifies fecal pH as a predictor of bone health. Bone mineral content (BMC) and bone mineral density (BMD) are related to bone health and risk of osteoporosis. While it is known that certain factors, such as calcium intake, affect bone health, previous studies have been limited to analysis of variables of interest or to a specific subset of the population. ARS researchers in Davis, California, used machine learning models, which can find complex patterns in data, to predict BMC and BMD in a healthy men and women. Low stool pH, which is an indicator of more fermentation of dietary fiber in the colon, was predictive of higher BMC/BMD. This discovery suggests that dietary modifications that increase fermentable fiber and lower fecal pH, may also improve bone health in a general population.
Review Publications
Chin, E.L., Van Loan, M., Spearman, S., Bonnel, E.L., Laugero, K.D., Stephensen, C.B., Lemay, D.G. 2021. Machine learning identifies stool pH as predictor of bone mineral density in healthy multiethnic US adults. Journal of Nutrition. 151(11):3379-3390. https://doi.org/10.1093/jn/nxab266.
Dimitratos, S., Hercules, M., Stephensen, C.B., Cervantes, E., Laugero, K.D. 2021. Association between physiological stress load and diet quality patterns differs between male and female adults. Physiology and Behavior. 240. Article 113538. https://doi.org/10.1016/j.physbeh.2021.113538.
Artegoitia Etchev, V.M., Krishnan, S., Bonnel, E.L., Stephensen, C.B., Keim, N.L., Newman, J.W. 2021. Healthy eating index patterns in adults by sex and age predict cardiometabolic risk factors in a cross-sectional study. Biomed Central (BMC) Nutrition. 7. Article 30. https://doi.org/10.1186/s40795-021-00432-4.
Kable, M.E., Chin, E.L., Storms, D.H., Lemay, D.G., Stephensen, C.B. 2021. Tree-based analysis of dietary diversity captures associations between fiber intake and gut microbiota composition in a healthy U.S. adult cohort. Journal of Nutrition. 152(3):779-788. https://doi.org/10.1093/jn/nxab430.
James, K.L., Gertz, E.R., Cervantes, E., Bonnel, E., Stephensen, C.B., Kable, M.E., Bennett, B.J. 2022. Diet, fecal microbiome, and trimethylamine N-oxide in a cohort of metabolically healthy United States adults. Nutrients. 14(7). Article 1376. https://doi.org/10.3390/nu14071376.
Oliver, A., Xue, Z., Villanueva, Y.T., Durbin-Johnson, B., Alkan, Z., Taft, D.H., Liu, J., Korf, I., Laugero, K.D., Stephensen, C.B., Mills, D.A., Kable, M.E., Lemay, D.G. 2022. Association of diet and antimicrobial resistance in healthy U.S. adults. mBio. 13(3). Article e00101-22. https://doi.org/10.1128/mbio.00101-22.
Coates, L.C., Storms, D.H., Finley, J.W., Fukagawa, N.K., Lemay, D.G., Kalscheur, K., Kable, M.E. 2022. A low starch and high fiber diet intervention impacts the microbial community of raw bovine milk. Current Developments in Nutrition. 6(6). Article nzac086. https://doi.org/10.1093/cdn/nzac086.
Trott, J.F., Schennink, A., Horigan, K.C., Lemay, D.G., Cohen, J.R., Famula, T.R., Dragon, J.A., Hovey, R.C. 2021. Unique transcriptomic changes underlie hormonal interactions during mammary histomorphogenesis in female pigs. Endocrinology. 163(3). Article bqab256. https://doi.org/10.1210/endocr/bqab256.
Castillo, J.J., Couture, G., Bacalzo, N.P., Chen, Y., Chin, E.L., Blecksmith, S.E., Bouzid, Y.Y., Vainberg, Y., Masarweh, C., Zhou, Q., Smilowitz, J.T., German, J.B., Mills, D.A., Lemay, D.G., Lebrilla, C.B. 2022. The development of the Davis Food Glycopedia - A glycan encyclopedia of food. Nutrients. 14(8). Article 1639. https://doi.org/10.3390/nu14081639.
Tagkopoulos, I., Brown, S.F., Liu, X., Zhao, Q., Zohdi, T., Earles, J.M., Nitin, N., Runcie, D.E., Lemay, D.G., Smith, A.D., Ronald, P.C., Feng, H., Youtsey, G.D. 2022. Special report: AI Institute for next generation food systems (AIFS). Computers and Electronics in Agriculture. 196. Article 106819. https://doi.org/10.1016/j.compag.2022.106819.
Norris, S., Frongillo, E., Black, M., Dong, Y., Fall, C., Lampl, M., Liese, A., Naguib, M., Prentice, A., Rochat, T., Stephensen, C.B., Tinago, C.B., Ward, K., Wrottesley, S.V., Patton, G.C. 2021. Nutrition in adolescent growth and development. Lancet. 399(10320):172-184. https://doi.org/10.1016/S0140-6736(21)01590-7.
Newman, J.W., Krishnan, S., Borkowski, K., Adams, S.H., Stephensen, C.B., Keim, N.L. 2022. Assessing insulin sensitivity and postprandial triglyceridemic response phenotypes with a mixed macronutrient tolerance test. Frontiers in Nutrition. 9. Article 877696. https://doi.org/10.3389/fnut.2022.877696.
