Location: Immunity and Disease Prevention Research
2019 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
This report documents progress for Project 2032-51530-026-00D, which began in January 2019 and continues research from expired projects 2032-53000-001-00D, "Assessing the Impact of Diet on Inflammation in Healthy and Obese Adults in a Cross-Sectional Phenotyping Study and a Longitudinal Intervention Trial" and 2032-51530-024-00D, "Metabolism and Health Effects of Phospholipid and Triglyceride Forms of Omega-3 Polyunsaturated Fatty Acids."
In support of Sub-objective 1A, stool sample collection from the phenotyping study participants is complete. To determine microbial taxa and function, 16S rRNA sequencing is being conducted on all available samples and shotgun metagenomics sequencing on a subset. Bacterial DNA extraction from the fecal samples for 16S rRNA sequencing will be complete by the end of the fiscal year. Based on DNA yield and quality, as well as quality of the associated dietary data, 192 samples have been selected for shotgun metagenomics sequencing. Pre-processing of sequences from the first 96 samples suggest that library preparation and size-selection methods have been successful.
To determine microbial function and its relation to diet, fermentation studies will be conducted in support of Sub-objective 1B using participant stool. The pH of fermentation media may influence the carbohydrate fermentation capacity of whole microbial communities. Therefore, a set of experiments is being conducted to optimize fermentation conditions to reflect the physiological environment under which these fermentations would naturally occur.
To determine the relationship between dietary intake and gastrointestinal health, several fecal and plasma markers are being tested using the samples from the phenotyping study related to Sub-objective 2A. It is anticipated that measurements of fecal calprotectin will be complete by the end of the fiscal year. In addition, a method for the quantification of host-derived transcripts from exfoliated colonocytes in participant stool has been optimized.
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 in the phenotyping study, cytokines are being measured in plasma and immune activation is being measured by flow cytometry. It is anticipated that all data will be available by end of FY19. A statistical analysis plan is being prepared. This work is in support of Sub-objective 3A.
In a subordinate project on milk, health, and genetics (0000061752) with University of California, Davis, the objective is to determine how lactase persistence genotypes and dairy consumption interact to impact human health. Genotype and ethnicity influenced total dairy intake as well as intake of specific dairy product types (fluid milk, cheese, yogurt) and alternative milks. A manuscript on that topic has been accepted. A method was developed to estimate lactose consumption from 24-hour food recalls using machine learning and database matching. A manuscript on the topic of lactose estimation from 24-hour food recall data is in preparation.
Accomplishments
1. Diet, gut bacteria, and chronic diarrhea. Over 1.2 million people in the U.S. suffer from inflammatory bowel disease, a form of which also spontaneously occurs in monkeys. Gut bacteria aid nutrient absorption, while nutrients influence which gut bacteria live and die. ARS researchers in Davis, California, in collaboration with University of California, Davis discovered that even though both healthy and diseased animals consumed the same foods, the "diet" of their gut microbes was not the same. Gut microbes in diseased animals consumed more of the protective layer produced by intestinal cells. The microbes also participated in cross-feeding relationships where one microbe degraded part of the protective layer, leaving another part for another microbe. These discoveries will enable new strategies to prevent chronic gastrointestinal diseases, such as ulcerative colitis, in humans.
2. Vitamin A supplementation of newborns at risk of vitamin A deficiency does not improve thymic function. Vitamin A (VA) deficiency impairs immune function and increases the risk of death from common childhood infections, thus VA supplementation is recommended for children above six months of age who are at risk of deficiency. Because VA might also help younger infants, ARS scientists from Davis, California, worked with colleagues in Bangladesh to conduct a randomized, placebo-controlled trial to determine if VA supplementation at birth improved functioning of the thymus, an organ of the immune system responsible for development of T lymphocytes, which play a key role in protection against infectious diseases. Contrary to expectations, VA supplementation decreased thymus size and its output of T lymphocytes, but the decrease was transient and did not affect T lymphocyte levels in blood. One possible reason for this unexpected finding is that the VA deficiency among study mothers was lower than expected, suggesting that the infants may have been less likely to benefit than in other settings with a higher risk of VA deficiency. In summary, these findings do not support the use of VA supplementation at birth to improve immune function and suggest caution in the use of settings where the risk of deficiency is low among women of childbearing age. In summary, these findings do not support the use of VA supplementation at birth to improve immune function and suggest caution when the risk of deficiency is low.
Review Publications
Westreich, S.T., Ardeshir, A., Alkan, Z., Kable, M.E., Korf, I., Lemay, D.G. 2019. Fecal metatranscriptomics of macaques with idiopathic chronic diarrhea reveals altered mucin degradation and fucose utilization. BMC Microbiome. 7:41. https://doi.org/10.1186/s40168-019-0664-z.
Huda, N., Ahmad, S.M., Alam, M.J., Khanam, A., Kalanetra, K.M., Taft, D.H., Raqib, R., Underwood, M.A., Mills, D.A., Stephensen, C.B. 2019. Bifidobacterium abundance in early infancy and vaccine response at 2 years of age. Pediatrics. 143(2):e20181489.
