Research Project: Polyphenol-Rich Foods and Promotion of Intestinal Health

Location: Diet, Genomics and Immunology Laboratory

2021 Annual Report

Objectives
Objective 1: Determine if polyphenol rich diets prevent microbiome dysbiosis, inappropriate activation of Toll and NOD-like receptor (TLR/NOD-like receptors) and reduce the severity of colitis in pigs. [NP107, C3, PS3B] Objective 2: Compare consumption of polyphenol-rich foods combined with probiotics early in life for amelioration of systemic inflammation induced by a westernized high fat diet. [NP107, C3, PS3B, C4, PS4A] Objective 3: Establish if consumption of polyphenol-rich foods will normalize westernized high fat diet-induced microbiome dysbiosis and prevent generalized inflammation. [NP107, C3, PS3B C4, PS4A]

Approach
The overall goal of the proposed research is to understand the complex interactions between diet, gut microbiome and host responses that are critical for the prevention of diseases associated with poor diet choices. The objective is to use a human-relevant pig model to understand mechanisms of intestinal dysregulation during consumption of a westernized-diet, which is strongly associated with obesity and related metabolic diseases, and to evaluate the incorporation of dietary probiotics, fruits and vegetables as an approach to attenuate the adverse consequences of consuming a westernized-diet. To achieve this goal, we propose to study the time-dependent changes in broad host health biomarkers within the immunome, microbiome and metabolome, and the dietary interventions that modulate these biomarkers. Our central hypothesis is that a modified westernized dietary pattern that contains recommended levels of fruits and vegetables will promote a healthier host microbiome due a polyphenol-induced prebiotic effect and anti-inflammatory responses.

Progress Report
A pilot study evaluated the effect of a dietary intervention with a standard 16% protein diet containing macro and micronutrients requirements for growing pigs and supplemented with 1.25 cups of fruits (containing equal parts of green grapes, strawberries, red apples, blackberries, and blueberries) and 1.75 cups of vegetables (containing equal parts of spinach, kale, broccoli, and celery) equivalent to half the daily recommendation by Dietary Guidelines for Americans (DGA) for daily consumption of fruits and vegetables. Fecal microbiome analysis by 16S rRNA sequencing indicated diet-related changes on the relative abundance of several amplicon sequence variants (ASV) within the bacterial family and genus taxonomic levels, including Muribaculaceae, a versatile bacterial taxon concerning complex carbohydrate degradation, Rikenellaceae RC9 group, Bacteroidaceae within Bacteroidales; Ruminococcaceae UCG010, Lachnospiraceae NK3A20 group and Family XIII UCG001 within Clostridiales and genus from Erysipelotrichaceae within Erysipelotrichales consistent with a shift in host-microbiome composition after consumption of diets high in fiber, and relatively greater vegetable than animal protein sources, which are known to promote fermentation and functionality of the intestinal microbiome. The impact of dietary supplementation with Fruits and Vegetables (FV) on host transcriptome was assessed by whole blood RNA sequencing. A Principal component analysis (PCA) of whole blood transcriptome expression showed an FV-diet induced separation of the treated group relative to control after a three-week intervention. Blood derived from pigs fed FV-supplemented diet showed 111 differentially expressed genes (DEG) when compared against paired baseline sample or 37 DEG when compared among dietary treatments after three weeks (FDR<0.05). Both comparisons showed an up-regulation of genes associated with regulation of B-cell development, survival, and signaling (TNFRSF13C, FCRL5, CD79B), regulation of antigen processing (CD1A, SLA-DQA1), immunoglobulin receptor binding (IGHA1), and downregulation of genes associated with cytokine receptor activation (IL7R, IL6ST, ILR2) cell proliferation and differentiation (EP300) or apoptosis (NOTCH2) among other biological functions. A follow-up experiment with 30 pigs was done to compare the dextran sodium sulfate (DSS) induced colitis response after a four-week-dietary intervention with an FV diet containing the equivalent to a full or half daily DGA recommendation for daily consumption of fruits and vegetables. Histopathological analysis of proximal colon sections indicated reduced crypt depth mucosa in pigs fed the full FV diet relative to pigs from half FV or control diets suggesting an FV-induced amelioration of colitis response. RNA-sequencing of proximal colon mucosa indicated 69 DEG (FDR<0.1) in DSS treated pigs, including; the upregulation of genes associated with mucus production (MUC2), chloride ion homeostasis (SLC12A8), paracellular barrier (CLDN8), protection and healing of intestinal mucosa from insults (TFF2) and downregulation of genes associated with detoxification and protection against reactive oxygen species and peroxidation (GSTA1) or a T-cell/NK-cell specific serine protease necessary for lysis of bacterially-infected cells (GZMA) suggesting an impairment in intestinal function and immune response that was ameliorated or prevented in FV-DSS treated groups. We continue to work on the analysis of these data and expect to construct correlations between transcriptomic changes and fecal microbiome taxonomical and functional data once it is finalized. Continued progress has been made to compare the effect of a Westernized high fat (HF) and a -healthy heart diet with and without statin supplementation on the transcriptome of different areas of the brain. RNA-sequencing libraries of the frontal cortex, hypothalamus, hippocampus, Circle of Willis, and vessels associated with frontal cortex samples derived from 30 pigs have been synthesized. Differentially expressed gene (DEG) analysis is underway to determine the impact of diet and statin intervention on brain function. Substantial progress has been made on determining the impact of diet supplemented with edible white button mushroom (WBM) on brain transcriptome and metabolome and its potential association with changes previously found in fecal microbiome composition. Regardless of a lack of differential expression of genes(DEG) in brain transcriptome (cortex, hippocampus), targeted metabolomics analysis on brain cortex, hippocampus, and liver indicated a diet-related modulatory role of lipidome composition with a reduced concentration of phosphatidylcholine (PC), sphingolipids (SM) in the liver and increased amino acid concentration in the brain cortex. Changes in PC concentration in the hippocampus were correlated with fecal microbiota changes induced by the WBM diet suggesting a modulatory effect of WBM supplemented diet in brain metabolome with potential contribution of gut microbiota.

Accomplishments
1. Dietary composition modulates lipid metabolism and inflammatory response in small intestine. Cardiovascular disease (CVD) is the leading cause of death in the U.S. Dietary composition and statin therapy are known modulators of coronary artery disease (CAD), a type of CVD characterized by the development of cholesterol plaques in coronary arteries exacerbated by inflammation and dyslipidemia. ARS scientists from Beltsville, Maryland, and Boston Human Nutrition Research Center, Boston, Massachusetts, have shown in a porcine translational model for humans that a Westernized type diet influenced the differential expression of lipid metabolism genes (SCD, FADS1, SQLE) relative to a heart-healthy diet (HHD) in the jejunum of pigs with associations with atherosclerotic lesion severity and systemic cholesterol concentrations. In addition, the westernized diet was also associated with a higher interferon signaling and inflammation response together with systemic inflammatory markers relative to HHD. This work is important as it addresses the differential relationships between dietary patterns, statin therapy, and their impact on intestinal health.

2. Dietary pattern and atorvastatin interaction modify large intestine transcriptome. Dietary quality improvement and atorvastatin therapy are widely used for coronary artery disease (CAD) management. However, the impact of these interventions in the colon and subsequent association with disease progression are not known. ARS scientists from Beltsville, Maryland, and Boston Human Nutrition Research Center, Boston, Massachusetts, have shown that a westernized type diet (WD) upregulated the LXR/RXR activation pathway relative to a Heart-healthy diet (HHD), and the addition of atorvastatin induced a colitis-like gene expression phenotype in WD group while atorvastatin lowered inflammatory status in HHD fed pigs. These data indicated that diet quality modulates the response to atorvastatin therapy in the colon, having implications for the maintenance of a healthy intestine.

Review Publications
Shumao, Y., Nirupa, M., Lamon-Fava, S., Solano Aguilar, G., Turner, J.R., Walker, M.E., Zhi, C., Lakshman, S., Chen, C.T., Dawson, H.D., Urban Jr, J.F., Lichtenstein, A.H. 2021. Colon transcriptome is modified by dietary pattern/Atorvastatin interaction in the Ossabaw pig. Journal of Nutritional Biochemistry. 90:108577. https://doi.org/10.1016/j.jnutbio.2020.108570.
Ye, S., Matthan, N., Lamon-Fava, S., Solano Aguilar, G., Turner, J., Walker, M.E., Chai, Z., Lakshman, S., Urban Jr, J.F., Lichtenstein, A.H. 2021. Western and heart healthy dietary patterns differentially affect the expression of genes associated with lipid metabolism, interferon signaling and inflammation in the jejunum of Ossabaw pigs. Journal of Nutritional Biochemistry. 90:108577. https://doi.org/doi: 10.1016/j.jnutbio.2020.108577.