Location: Diet, Genomics and Immunology Laboratory
2022 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
Continued progress has been made on metagenome characterization of proximal colon contents derived from pigs fed a FV-supplemented diet and later exposed to a Dextran sodium sulfate (DSS) treatment to induce colitis. Shotgun metagenomic sequencing has been completed generating an average of 3.5 M reads per sample after host filtering. Taxonomic profiles at species level were compared using Bray-Curtis dissimilarity index and samples were visualized using non-metric multidimensional scaling (NMDS). Permutational multivariate Analysis of Variance (ANOVA) are being done to estimate the effects of experimental factors that may explain variation in microbial beta diversity on both taxonomic and functional profiles with pairwise comparison calculations among treatments groups. Relative to non-DSS control group, the Shannon alpha diversity index was reduced in response to DSS-challenge but was maintained in DSS-challenged groups that received the FV-supplemented diet. For functional profiling, high-quality (filtered) reads have been aligned against the SEED database via translated homology search and annotated to Subsystems, or functional levels using Super-Focus. Additional protein annotations used by HUMAnN2 such as Enzyme Commission (EC) number, COG, KO, Pfam domain and GO term assignments will also be used and further collapsed to enzyme/reaction abundances prior to metabolic pathway reconstruction to compare responses among dietary groups.
Continued progress has been made to compare the longitudinal effect of time and dietary supplementation with Fruits and Vegetables (FV) on host fecal microbiome composition by 16S rRNA (bacteria) and ITS (fungi) sequencing analysis after a three-week intervention with FV-supplemented diets containing the equivalent to a full or half daily Dietary Guidelines for Americans (DGA) consumption recommendations. Further longitudinal characterization of fecal microbiome changes in bacteria and fungi have been extended by collecting two additional times after pigs were exposed to a week-long challenge with dextran sodium sulfate (DSS) to induce colitis. FV-dietary supplementation was able to ameliorate DSS-induced changes in fecal microbiome alpha-diversity by maintaining abundance of bacterial taxa involved in carbohydrate metabolism. Further statistical analysis is being conducted to characterize potential association of dietary FV levels with changes in bacteria and fungi abundance and their predicted function.
Longitudinal gene expression analysis by RNA-sequencing of whole blood cells (WBC) derived from FV supplemented dietary intervention and collected at three different times have been completed. Differential gene expression showed a time-dependent response in WBC transcriptome after FV-intervention with an overall reduction of DSS-induced differentially gene expression in pigs fed the Full-FV supplemented diet. Further analysis to determine differentially expressed genes and associated biological pathways is currently in progress.
Processing of synchronous longitudinal fecal content samples collected at five times after FV-supplementation and DSS challenge has been finished and sent for metabolite analysis.
Progress for experimental analysis under Objective 1 have been slowed due to limited access to laboratory facilities or malfunctioning equipment compromised during pandemia. Planned research related to Objectives 2 and 3 have also been impacted due to lack of available attending veterinarian that can approve submitted animal protocol.
Accomplishments
1. Dietary consumption of Fruits and Vegetables (FV) modulates host gene expression and microbiome composition. A higher intake of FV or derived polyphenols has been associated with improved inflammatory and immune status in subjects with metabolic diseases, however research is needed to understand the mechanisms of immunomodulation. Using the pig as a pre-clinical translational model for evaluating nutritional interventions in healthy humans, ARS scientists at Beltsville, Maryland, measured changes in fecal microbiome (FM) and gene expression in whole blood cells (WBC) after a two-week controlled feeding study with a FV-supplemented diet as recommended by Dietary Guidelines for Americans (DGA). Feeding an FV-supplemented diet induced differential expression of several genes associated with an increase in B-cell development, differentiation and the regulation of cellular movement and inflammatory response, concomitant with changes in bacterial taxa abundance within Order Clostridiales.
2. White button mushroom consumption affects host metabolome composition. White bottom mushroom (WBM) dietary supplementation has prebiotic effect in WBM-treated pigs by changing the composition of gut microbiome and inflammatory responses after lipopolysaccharide stimulation. Edible mushroom derived proteins and polysaccharides have also been proposed as bioactive with neuroprotective and anti-ageing properties. ARS scientists performed a targeted metabolomic analysis on brain and liver tissues derived from pigs fed WBM-supplemented diets and associated metabolite abundance with WBM-induced changes in fecal microbiome composition. Metabolite changes were positively associated with increased abundance of bacteria responsive to WBM-dietary intervention suggesting dietary-WBM affects liver and brain metabolite composition.
3. Improved detection and annotation of tissue metabolome. Sterols, bile acids, and acylcarnitines are key players in lipid metabolism. Metabolite annotation with mass spectrometry analytics are challenging because of the presence of several isomers and stereoisomers, variability in ionization, and their relatively low concentrations in biological samples. A sensitive and simple qualitative LC-MS/MS (liquid chromatography with tandem mass spectrometry) method concurrently analyzing Bile Acid, Carnitine and Sterol Library of standards along with biological samples in the same analytical run has been tested by scientists from University of Eastern Finland, Kuopio, Finland, and collaborating ARS scientists at Beltsville, Maryland. This semi-targeted method provided retention time and mass spectrometry information for the reference library to substantiate the presence of lipid-metabolites in biological samples from humans, rodents and pigs. The simultaneous detection and annotation of sterols, bile acids, and acylcarnitines from reference standards and biological samples with high precision across species enables the automated identification of compounds with variable structure and low concentration providing a valuable tool for screening these metabolites in biological samples.
Review Publications
Babu, A.F., Koistinen, V.M., Turunen, S., Solano-Aguilar, G., Urban, Jr. J.F., Zarei, I., Hanhineva, K. 2022. Identification and distribution of sterols, bile acids, and acylcarnitines by LC-MS/MS in humans, mice, and pigs -- a qualitative analysis. Metabolites. 12(1):49. https://doi.org/10.3390/metabo12010049.
Solano-Aguilar, G.I., Lakshman, S., Jang, S., Gupta, R., Molokin, A., Schroeder, S.G., Gillevet, P.M., Urban Jr., J.F. 2021. The effects of consuming white button mushroom Agaricus bisporus on the brain and liver metabolome using a targeted metabolomic analysis. Metabolites. 11(11):779. https://doi.org/10.3390/metabo11110779.
Solano-Aguilar, G.I., Lakshman, S., Shao, J.Y., Chen, C.T., Beshah, E., Dawson, H.D., Vinyard, B.T., Schroeder, S.G., Jang, S., Molokin, A., Urban Jr., J.F. 2021. Fruit and vegetable supplemented diet modulates the pig transcriptome and microbiome after a two-week feeding intervention. Nutrients. 13:4350. https://doi.org/10.3390/nu13124350.
Maloney, J.G., Molokin, A., Solano Aguilar, G., Dubey, J.P., Santin, M. 2022. A hybrid sequencing and assembly strategy for generating culture free Giardia genomes. Current Research in Microbial Science. https://doi.org/10.1016/j.crmicr.2022.100114.
