Location: Diet, Genomics and Immunology Laboratory
2024 Annual Report
Objectives
Objective 1. Study the effect of resistant starch on the function of innate lymphoid cells, regulatory T cells, and regulatory macrophages in mucosal immunity and resistance to gastrointestinal infection. [NP107, C3, PS3B]
Objective 2. Examine the effect of cruciferous vegetables on the function of innate lymphoid cells, regulatory T cells, and regulatory macrophages in mucosal immunity and resistance to gastrointestinal infection. [NP107, C3, PS3B]
Objective 3. Define the effect of combining resistant starches with cruciferous vegetables on the function of innate lymphoid cells, regulatory T cells, and regulatory macrophages in mucosal immunity and resistance to gastrointestinal infection. [NP107, C3, PS3B]
Approach
The mucosal immune system is the first line of defense against a wide variety of bacterial, viral, and parasitic pathogens and must also regulate intestinal homeostasis. There is substantial cross-talk between the host immune system and the microbiome that modulates development of mucosal immunity and maintenance of intestinal homeostasis. Diet can affect the microbiome and, therefore, gut mucosal immunity and intestinal homeostasis. The composition of the microbiome can be altered by consumption of resistant starches (RS) or cruciferous vegetables (CV); but how this translates to changes in gut mucosal immunity and resistance to disease is largely unexplored. The goal of this project is to define how RS and CV affect the interaction between the gut microbiome and immune cells. This will be accomplished using rodent and porcine models to study the effect of feeding type 2 or 3 RS, or CV on activation of innate lymphoid cells (ILCs), as well as the activity/polarization of tissue macrophages (M's), and induction of T regulatory (Treg) cells at homeostasis and after challenge by enteric pathogens. This work will lead to the development of new biomarkers of immune status responsive to changes in nutrition, the microbiome, and identify nutrient-immune interactions potentially beneficial to human health. The studies will use a complementary approach to take advantage of the strengths of each animal model system. Mice will be used as a lower cost, high-throughput screening tool to evaluate the effect of RS and CV rich in dietary aryl hydrocarbon receptor (AhR) ligands on the microbiome and gut immune parameters. The results from these studies will be distilled into candidate foods to test mechanism-based effects in a pig model that are likely to yield data highly relevant to humans. The proposed mouse models in this project plan will provide flexibility to evaluate several classes of dietary RS and CV at various concentrations and combinations to evaluate mucosal responses to both bacterial and parasitic worm infections. Changes in mucosal cell populations of ILCs, Tregs and regulatory Mfs and their functional expression in explanted cells in vitro will provide a context for a diet-dependent mechanism in disease resistance. The effects RS and CV on the microbiome will be evaluated and correlated with changes to mucosal immunity. Subsequent studies in pigs using diet combinations optimized in mice and the use of a more human-like food matrix in pig feeding studies will inform recommendations for dietary RS and CV compositions predictive of improved intestinal health in humans. This will include challenge studies using infections in pigs caused by zoonotic E. coli and Trichuris suis (Ts) that are comparable to E. coli and whipworm infections in mice and humans. We have previously reported on the changes in metabolome and microbiome of Ts infected pigs affording us the opportunity to test the effects of dietary interventions on important diseases affecting humans.
Progress Report
This is the final report for project 8040-53000-021-000D entitled “Effect of Resistant Starch and Cruciferous Vegetables on Mucosal Immunity and Disease Resistance” which has been replaced by a new project 8040-10700-005-000D. For additional information, see the new project report. Work was completed on the three objectives which fall under National Program 107, and directly addresses Statement 3B: Identify Roles of Food, Food Components and Physical Activity in Promoting Health and Preventing Disease. This work resulted in 26 publications. Data and technologies developed during the current reporting period laid the foundation for studies proposed in the new project plan.
Work on Objective 1 was completed. In total, we conducted studies with 3 different resistant starches, a raw potato starch (RPS, RS2), a type 4 resistant starch (RS4), Versafibe 1490 (VF), made by chemically modifying RPS, and a RS4 derived from tapioca starch. Unique to our RS studies was the use of a basal rodent diet based on the National Health and Nutrition Examination Survey (NHANES) data that allows us to examine the effects of RS consumption in a diet that mimics the macro- and micro-nutrient content of an American diet. Thus, the data generated is more applicable to the human situation. Several common themes emerged from the studies of RPS and Versafibe 1490 (VF) with additional data pending final analysis with the tapioca RS4 (RTS). Addition of resistant starches causes dose-dependent changes in alpha- and beta-diversity. RS addition decreased alpha-diversity and increased beta-diversity but the effects were more pronounced in RPS than VF fed mice. This may result from the fact that compared to RPS, VF is primarily an insoluble fiber, and metabolized differently by the host microbiome. The decrease in alpha-diversity is notable as having a highly diverse microbiome is thought to be desirable. Our data contributes to a growing body of evidence that consumption of a particular RS can select for specific microbiota that benefit from the resistant starch and increase in abundance at the expense of other bacterial strains resulting in decreased diversity. For example, RPS but not VF led to a major increase in the genus Lachnospireceae NK4A136 group indicating preferential use of RPS by this genus. RS starch consumption led to changes in short chain fatty acids (SCFAs) with a dose dependent increase in butyrate (BUT) and decreased levels of branched SCFA indicating a shift from proteolytic to carbohydrate fermentation. One notable difference between RPS, RTS and VF is their effect on fecal pH with RPS and RTS causing a decrease in fecal pH with increasing levels of RS while VF did not appreciably affect fecal pH, suggesting that starch structure affects the microbiome metabolites produced that affect fecal pH.
Gene expression was analyzed using RNASeq. Cecum gene expression showed a RPS dose-dependent segregation into 4 groups analogous to the results obtained from 16S sequencing. The group separation decreased in the proximal colon and to an even greater extent in the distal colon. In each tissue, the greatest number of gene expression changes occurred in mice fed the 10% RPS diet with most changes in gene expression unique to each tissue but there were common pathways altered by RPS consumption in the three tissues. These included changes in genes involved in antibacterial and antiparasitic responses, multiple genes involved carbohydrate, lipid, minerals, and vitamin metabolism and multiple IFN induced genes. These data indicate that RPS has wide-ranging effects on genes involved in immunity and metabolism. Compared to RPS, VF induced fewer gene expression changes, mostly observed in mice fed a 10% VF diet and suggests that VF is metabolized very differently in the cecum and colon compared to RPS.
Work on Objective 2 was completed. Contrary to our hypothesis that RPS should improve the outcome of Citrobacter rodentium (Cr)-induced colitis, we found that mice fed the 10% RPS diet led to an increased colonization of the colon, enlarged spleens, increased colonic hyperplasia, and increased colon pathology. In addition, initial colonization and Cr fecal excretion was greater in RPS fed mice than in mice fed the basal TWD had slightly lower titers on day 4 and that a greater percentage of mice failed to become productively infected by day 4. In comparison, all mice receiving RPS had robust fecal titers by day 4 that increased with increasing RPS dose suggesting that RPS enhances the ability of Cr to establish a productive infection. Even though we did not observe the same level of changes in the microbiome and gene expression in VF-fed mice, we still observed an increase in susceptibility to Cr infection. Other researchers have recently reported similar results when comparing mice fed a low vs a high fiber diet. The commonality is that both fiber and RS can be fermented by the microbiome and create an environment conducive to increased colonization by Cr. In total, these results suggest that diets high in fiber or RS may increase our susceptibility to certain food-borne pathogens, an important factor when considering the risk of acquiring food-borne bacterial infections.
Work on Objective 3 was substantially completed with only the final data analysis to complete prior to submission for publication. The effect of adding broccoli to the TWD on the microbiome, gene expression and resistance to colitis was investigated. Addition of broccoli at 0.5, 1.0 and 2.5% to the diet resulted in dose dependent changes in the microbiome and gene expression. In cecum, there was a significant increase in the number of genes associated with activation of the AhR pathway (including the signature gene Cyp1a1) in animals fed the 2.5% broccoli diet. In contrast to our RS studies, broccoli caused a dose dependent increase in alpha-diversity and the beta-diversity analysis showed that the mice fed the 2.5% broccoli diet had a significantly different microbiome compared to the non-broccoli group with lower broccoli doses showing a more muted effect. A metagenomic analysis revealed that 13 bacterial species, including 3 closely related to species (Alistipes, Acetatifactor, and Prevotella) that have been linked to vegetable intake in rodents and humans. had their abundance increased in all groups fed broccoli compared to the basal diet in a dose dependent fashion. These were dominated by members of the Lachnospiraceae and Muribaculaceae families. In addition, 7 bacterial species had increased abundance unique to the 2.5% broccoli group, 5 of which belonged to the Lachnospiraceae family, including Lachnospiraceae bacterium MD335 which exhibited a dose dependent response. Analysis of bacterial RNA transcripts from Lachnospiraceae bacterium MD335 indicate an increase in genes associated with BUT production as well as an increase in genes associated with, cell motility, carbohydrate utilization and storage and biofilm formation. In contrast, genes involved in vitamin B12 production were downregulated.
The metagenome and bacterial transcriptomics are continuing to be mined for additional relationships. There were no bacterial species whose abundance was uniformly decreased by feeding broccoli, but different members of Lachnospiraceae family had their abundance uniquely decreased at each broccoli dose. These results indicate that members of Lachnospiraceae family were particularly affected by feeding broccoli. In addition, one bacterium, Faecalibacterium rodentium, exhibited decreased abundance in the 2.5% group. Addition of broccoli to the diet did not markedly decrease colonization by C. rodentium, an E. coli like mouse pathogen, but there was trend toward lower colonization levels in the 1.0 and 2.5% broccoli dietary groups as well as decreased colon/body weight ratios that may be indicative of less pathology. Gene expression, microbiome and histological assessments of the broccoli studies are being completed.
We continued examining the potential inhibitory effects of BUT (at levels at or below the cecal contents of our RPS-treated mice), on the response of human colon epithelial cells to E. coli-derived ligands; outer membrane vesicles (OMVs) and ultrapure lipopolysaccharide (LPS), a TLR4 ligand. In general, we found that BUT inhibited OMV and LPS-induced cytokines and chemokines. The mRNA, protein and transcriptional activator of the master regulator of these responses, nuclear-factor kappa B, were all inhibited by BUT. In contrast, we observed an increase in AHR and CYP1A1 mRNA expression by BUT, and a synergistic increase in AHR transcriptional activation in response to AHR ligands by BUT. We also observed a synergistic increase AHR ligand-induced activation of CYP1A1 enzyme by butyrate. Thus, our in vitro work continues to provide potential mechanistic explanations for the changes to the microbiome and gene expression from our in vivo studies.
We continued our improvements to the assembly, annotation, and analysis of the porcine genome. To date, we have comparatively examined 14,826 loci in the 3 latest builds of the genome (National Center for Biotechnical Information (NCBI) build 11.1, Ensembl build 11.1 and MARC build 1.0). Our analysis reveals that the percentage of incorrectly assembled and annotated genes in these builds are high, and we are working with database curators to correct some of these errors. When completed, this will lead to dramatic improvements in the assembly and annotation of the porcine genome, increase the consistency of published data and facilitate the exchange of data regardless of the genome build source.
Accomplishments
Review Publications
Smith, A.D., Chen, C.T., Cheung, L., Ward, R., Jones, B., Pletsch, E.A., Dawson, H.D. 2024. A type 4 resistant potato starch alters the cecal microbiome and gene expression in mice fed a Western diet based on NHANES data. Nutrients. 15:3141-3157. https://doi.org/10.1039/d3fo04512a.
Liu, F., Smith, A.D., Wang, T.T., Pham, Q., Hou, P., Cheung, L., Yang, H., Li, R.W. 2024. Phospholipid-rich krill oil promotes intestinal health by strengthening beneficial gut microbial interactions in an infectious colitis model. Food & Function. 15(5):2604-2615. https://doi.org/10.1039/D3FO04980A.