Location: Diet, Genomics and Immunology Laboratory
2022 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
Continued progress was made on Objective 1. Experiments were conducted using a Total Western Diet (TWD) based on National Health and Nutrition Examination Survey (NHANES) data that mimics the protein, fat, sugar, vitamin and mineral composition of a typical American diet. The effect of an addition of a type 2 resistant starch, raw potato starch (RPS, 2, 5, 10% of diet), to the TWD on morphological, microbiome and gene expression changes to the colon and cecum were evaluated. These studies resulted in a publication (listed below). A principal component analysis (PCA) of the cecal 16S rRNA sequencing data showed 4 discreet groupings based on the dose of RPS in the diet. Alpha-diversity analysis indicated that increased consumption of RPS was associated with a decrease in Alpha-diversity with the relative abundance of OTUs increasing or falling with different RPS doses. This was primarily driven by a large increase in genus Lachnospiraceae NK4A136 group that went from 7% in mice fed the basal diet to 50% in mice fed the 10% RPS diet in a dose dependent manner. Linear discriminant analysis effect size (LEfSe) analysis identified differentially abundant genera as biomarkers of RPS consumption.
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.
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. Additional studies indicate this effect is restricted to the 10% RPS level. However, more subtle effects on initial colonization of the host were noted. Initial colonization of the host by Cr occurs in the cecal patch and then spreads to the distal colon where significant Cr fecal titers can be detected by day 4 in productively infected mice. We noted that 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.
To investigate possible mechanisms by which RPS is altering Cr infection, 16S sequencing of the microbiome and gene expression analyses on cecal and colon tissue were performed. As seen before, increasing dietary RPS decreased microbiome diversity, but this was partially ablated by Cr infection. This was due in part to a reduction in the large increase in the genus Lachnospiraceae NK4A136 group that is observed in uninfected mice. A PCA analysis at the genus level showed distinct groupings based on diet and infection status indicating a unique microbial signature for each group. Analysis of deep sequencing gene expression data obtained by RNAseq analysis of colon and cecal tissue are nearing completion and will provide insight into unique gene expression changes associated with diet and infection. Results from these studies are being prepared for publication this year.
Work on Objective 1 also progressed with the initiation of two new sets of studies. We completed a time course experiment examining the effect of increasing the length of time on the RPS diets from 3 to 7 weeks with fecal samples collected weekly for microbiome analysis and collected cecal samples at week 7. We also collected cecal contents at 3 weeks post-RPS feeding to investigate the effect of feeding RPS on bacterial gene expression to complement the previously obtained 16S data. We also conducted new studies using a type 4 resistant starch. We have completed the initial phase of two studies looking at Versafibe 1490, a chemically modified type 4 resistant starch to compare to our results obtained with the RPS. Usage of this type of starch is increasing rapidly in the food supply. Samples obtained from the first study are being analyzed for changes to the microbiome and gene expression in uninfected mice and the second study in mice challenged with Cr to see if a RS4 starch also produces a more acute infection as was observed with the type 2 RS, RPS.
We continued examining the potential beneficial or deleterious effects of butyrate (at levels at or below the cecal contents of our RPS-treated mice), on the response of pig and human intestinal epithelial cells to E. coli-derived ligands; outer membrane vesicles (OMVs), Pam3CysSerLys4 (Pam3CSK4, a TLR1/2 ligand), and ultrapure lipopolysaccharide (LPS), a TLR4 ligand. We discovered that 1 mM butryrate increased IL-8 production in OMV-treated human HT-29 cells; however, butyrate from 5-100 mM decreased IL-8 production in a dose-dependent fashion. Using HT-29 cells engineered to express a luminescent protein when the arylhydrocarbon receptor (AHR) promoter is activated, we found that butryrate increased the expression AHR in a dose dependent response from 0.1 to 12.5 mM and inhibited it in a dose-dependent response from 25 to 100 mM. Butyrate also enhanced AHR activation by an aryl hydrocarbon ligand derived from glucobrassicin (found in CV), ITE (2-(1' H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester), and the tryptophan derivative FICZ (6-Formylindolo[3,2-b]carbazole). We discovered that ITE and FICZ increased the production of the proinflammatory cytokine IL-8, in response to OMV and LPS; however, when cells were pretreated with 12.5 mM butyrate, this effect was blunted. Thus, our in vitro work provides potential mechanistic explanations for the changes to the microbiome and gene expression from our in vivo studies. In addition, it illustrated potential complex relationships between RS and CV at the molecular level.
We continued our attempts to improve the assembly, annotation, and analysis of the porcine genome. To date, we have comparatively examined 8,100 genes in the 3 latest builds of the genome. Our analysis reveals that the percentage of error-free assembled and annotated genes in National Center for Biotechnical Information (NCBI) build 11.1, Ensembl builds 11.1 and MARC build 1.0 are 70.8, 66.9, and 63.5%, respectively. A comprehensive examination of these errors revealed 10 systematic sources of errors; an indel that is present every 12,264 bp in Ensembl build 11.1, intronless or single exon coding region genes being assigned additional exons, endogenous retroviral sequences being annotated as protein coding genes, selenoprotein genes being assigned a premature stop codon, failure to annotate TCR variable regions, merging of most of the immunoglobulin heavy and light chain constant regions, failure to correctly assemble protocadherin genes, merging of multiple genes into 1 locus, and failure to annotate pseudogenes. Correcting these errors 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 internationally regardless of the genome build source. We are working with Ensembl build 11.1 curators to implement these findings.
Accomplishments
1. Feeding resistant potato starch alters the microbiome and gene expression in mice fed a Western diet. Resistant starches (RS) are found in foods and are digested in the cecum and colon. RS consumption can lead to changes in the microbiome and production of compounds called short-chain fatty acids (SCFA) that may contribute to gut health. However, many RS studies have been done with diets that do not resemble a typical American diet and may not reflect changes that occur in people eating an American-style Western diet. To address this issue, we fed mice a Total Western Diet (TWD), based on NHANES data obtained by surveying what kinds of food people eat, that mimics an American diet, for six weeks, and then supplemented the diet with 0, 2, 5, or 10% of resistant potato starch (RPS) for an additional three weeks. We analyzed cecum samples for the levels of SCFA, changes to the microbiome in the cecum and differences in gene expression in the cecum and colon of mice fed the different diets. Feeding the mice RPS had profound effects on SCFA levels, decreased the diversity of themicrobiome and significantly altered gene expression in the cecum and colon with the greatest effects seen in mice fed the 10% RPS diet. Of note, feeding RPS induced gene expression changes unique to the cecum and colon that are important for immune responses to bacteria, parasites, and viruses. These results demonstrate that consumption of resistant starches may significantly alter the microbiome and gene expression in ways that can impact our health and resistance to disease and warrants further investigation.
Review Publications
Smith, A.D., Fan, A.X., Qin, B., Desai, N., Zhao, A., Shea-Donohue, T. 2021. IL-25 treatment improves metabolic syndrome in high-fat diet and genetic models of obesity. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy. 14:4875-4887. https://doi.org/10.2147/DMSO.S335761.
Smith, A.D., Chen, C.T., Cheung, L., Ward, R., Hintze, K., Dawson, H.D. 2022. Resistant potato starch alters the cecal microbiome and gene expression in mice fed a Western diet based on NHANES data. Frontiers in Nutrition. 9. Article 782667. https://doi.org/10.3389/fnut.2022.782667.
Spencer, T.E., Wells, K.D., Lee, K., Telugu, B.P., Hansen, P.J., Bartol, F.F., Blomberg, L., Schook, L.B., Dawson, H.D., Lunney, J.K., Driver, J.P., Davis, T.A., Donovan, S.M., Dilger, R.N., Saif, L.J., Moeser, A., McGill, J.L., Smith, G., Ireland, J.J. 2022. Future of biomedical, agricultural and biological systems research using domesticated animals. Biology of Reproduction. 106(4):629-638. https://doi.org/10.1093/biolre/ioac019.
Vonderohe, C., Guthrie, G., Stoll, B., Chacko, S., Dawson, H.D., Burrin, D.G. 2021. Tissue-specific mechanisms of bile acid homeostasis and activation of FXR-FGF19 signaling in preterm and term neonatal pigs. American Journal of Physiology. https://doi.org/10.1152/ajpgi.00274.2021.
