Location: Diet, Genomics and Immunology Laboratory
2023 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
Work on Objective 1 progressed with the completion and publication of data describing the effects of a type 2 RS, raw potato starch (RPS) on a Citrobacter rodentium (Cr) infection. In addition to the published experiments highlighted in the Accomplishment section, we completed time course experiments examining the effects of increasing the length of time on the RPS diets from 3 to 7 weeks. The data showed that the effect of RPS on the fecal microbiome was stable after 3 weeks.
We also conducted several new studies using a type 4 resistant starch, Versafibe 1490 (VF), a chemically modified RPS used in food products to increase the fiber content. Compared to RPS, VF is primarily an insoluble fiber. As seen with RPS, feeding VF decreases the microbiome’s alpha-diversity. However, VF did not cause a large increase the relative abundance of the genus Lachnnospireceae nor a reproducible decrease in fecal pH, as was observed in mice fed RPS. Changes to the microbiome were largely associated with the 10% VF fed mice and this correlated with an increase in the fermentation product butyrate (BUT). Gene expression in the cecum and distal colon was analyzed by RNASeq and very few changes were observed compared to our previous results with RPS. This suggest that VF is metabolized very differently in the cecum and colon compared to RPS. A manuscript describing these results will be submitted this year. 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 in mice. Additionally, analyses are being conducted to determine if there are specific changes to the microbiome or gene expression that can be corrected with increased susceptibility to Cr infection. We have also initiated two studies looking at the effect of broccoli consumption on the microbiome, metabolome and gene expression in the liver, cecum, small and large intestine. These studies are in progress and will be completed in the early fall and will provide necessary information on the broccoli level to use in RPS/CV studies proposed in Objective 3.
We continued examining the potential inhibitory effects of butyrate (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. Using RNASeq, we demonstrated that BUT levels below the highest cecal contents of our RPS-treated mice, reduced the mRNA expression of resistin like beta (Retnlb) involved in antibacterial responses, the inflammasome component, NLR Family Pyrin Domain Containing 3 (NLRP3) and the central Nuclear Factor Kappa B (NF-kB) component, NFKB1. NLRP3 and NFKB1 are important in control of Cr infections. BUT decreased the inflammatory responses to OMV and LPS, including inhibition of Interleukin -8, C-X-C motif chemokine ligand 5, C-C motif chemokine ligand 20 and Elafin RNA/protein induction. All of these proteins are controlled by NF-kB and are important for in vitro or in vivo responses models of colitis. Thus, our in vitro work provides potential mechanistic explanations for the changes to the microbiome and gene expression from our in vivo studies.
We continued our attempts to improve the assembly, annotation, and analysis of the porcine genome. To date, we have comparatively examined > 14,000 genes in the 3 latest builds of the genome (National Center for Biotechnical Information (NCBI) build 11.1, Ensemble build 11.1 and Machine Readable Cataloging build 1.0). Our analysis reveals that the percentage of correctly 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. A manuscript describing these results will be submitted this year. We have also started work on the annotation of the Ossabaw genome.
Accomplishments
1. Feeding raw potato starch alters the microbiome, colon and cecal gene expression, and resistance to a gastrointestinal infection in mice. Resistant starches (RS) are found in foods and are digested in the cecum and colon to produce compounds that may contribute to human gut health; however, most experimental animal work to date has used diets that do not approximate what humans eat. To address this problem, mice were fed a Western-style diet (WD) for 6 weeks and then supplemented the WD with 0, 2, 5, or 10% of resistant potato starch (RPS) for an additional three weeks, and then infected with Citrobacter rodentium (Cr), a mouse pathogen that mimics many aspects of pathogenic E. coli infections in humans. ARS scientists in Beltsville, Maryland, found that mice fed 10% RPS, had greater infection by Cr, levels of fecal Cr excretion, colonization of colonic tissue and colonic pathology. Both diet and infection altered the fecal and cecal microbiome composition with increased levels of RPS resulting in decreased a-diversity that was partially reversed by Cr infection. RNASeq analysis identified several mechanistic pathways that could be associated with the increased colonization of Cr-infected mice fed 10% RPS including a decrease in enrichment for genes associated with T cells, B cells, genes associated with the synthesis of DHA-derived SPMs and VA metabolism/retinoic acid signaling. Potatoes are widely consumed in the US and these results suggest that high-level consumption of RPS, in the context of a typical American diet, may alter susceptibility to gastrointestinal bacterial infections, and warrants further investigation.
Review Publications
Liu, F., Smith, A.D., Wang, T.T., Pham, Q., Yang, H., Li, R.W. 2023. Multi-omics analysis detected multiple pathways by which pomegranate punicalagin exerts its biological effects in modulating host–microbiota interactions in murine colitis models. Food & Function. 14:3824. https://doi.org/10.1039/D3FO00286A.
Liu, F., Smith, A.D., Wang, T.T., Pham, Q., Yang, H., Li, R.W. 2023. Ellagitannin punicalagin disrupts the pathways related to bacterial growth and affects multiple pattern recognition receptor signaling by acting as a selective histone deacetylase inhibitor. Journal of Agricultural and Food Chemistry. 71(12):5016-5026. https://doi.org/10.1021/acs.jafc.2c08738.
Vonderohe, C., Guthrie, G., Stoll, B., Melendez Hebib, V., Dawson, H.D., Burrin, D.G. 2022. Increased circulating cortisol after vaginal birth is associated with increased FGF19 secretion in neonatal pigs. Endocrinology. 164(1). https://doi.org/10.1210/endocr/bqac188.
Cortesa, L.M., Brodsky, D., Chen, C.T., Pridgen, T., Odle, J., Snyder, D., Cruse, G., Putikova, A., Masuda, M.Y., Doyle, A.D., Wright, B.L., Dawson, H.D., Blikslager, A., Dellon, E.S., Laster, S.M., Kaser, T. 2022. Immunologic and pathologic characterization of a novel swine biomedical research model for eosinophilic esophagitis. Frontiers in Allergy. 3:2022. https://doi.org//10.3389/falgy.2022.1029184.
Smith, A.D., Chen, C.T., Cheung, L., Dawson, H.D. 2023. Raw potato starch alters the microbiome, colon and cecal gene expression, and resistance to citrobacter rodentium infection in mice fed a western diet. Frontiers in Nutrition. 9:2022. https://doi.org//10.3389/fnut.2022.1057318.
