Research Project: Effect of Resistant Starch and Cruciferous Vegetables on Mucosal Immunity and Disease Resistance

Location: Diet, Genomics and Immunology Laboratory

2020 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
Substantial progress was made on Objective 1. Experiments were conducted using a Total Western Diet (TWD) 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 short-chain fatty acid (SCFA) production, morphological changes to the colon and changes to the microbiome were evaluated. We saw a dose-dependent increase in butyric acid and dose-dependent decreases in isobutyric, isovaleric and valeric acid suggesting an increase in saccharolytic fermentation and a decrease in proteolytic fermentation (branched chain SCFAs). The principle component analysis of the 16S rRNA sequencing data clearly showed 4 discreet groupings based on the dose of RPS in the diet (0-10%). Additional analyses are underway to correlate changes in bacterial species with RPS dose, SCFA levels and specific bacterial metabolic pathways. Feeding mice the TWD/10% RPS diet led to an increase in the number and size of goblet cells in the colon, suggesting alteration of mucus synthesis and/or secretion pathways. Contrary to our hypothesis that RPS should improve the outcome of Citrobacter rodentium (Cr)-induced colitis, the 10% RPS diet led to an increased colonization of the colon, enlarged spleens, and colon pathology. Additional studies carrying over from the previous project plan on the effect of all trans-retinoic acid (ATRA) treatment on IL4-induced Th2 response in macrophage and epithelial cells were completed. Following up on previously published studies, we tested the effect of ATRA on IL-4 responses in two porcine intestinal epithelial cell lines, IPEC1 and IPEC-J2, and observed that ATRA increased mRNA for the IL-4 receptor alpha chain. ATRA also increased IL-4 induced phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mRNA expression of the chloride channel, calcium activated, family member 1 (CLCA1), important for mucus formation, and chemokine (C-C motif) ligand 26 (CCL26), a potent eosinophil chemoattractant. We extended these findings to human macrophage THP-1 cells and showed that ATRA synergistically increased IL-4–induced CCL2, CCL13, and CCL26 mRNA and protein levels. Transglutaminase 2 mRNA, protein, and enzyme activity were synergistically induced in THP-1 cells pretreated with ATRA and then treated with IL-4, thus, ATRA increased signaling in response to IL-4 in porcine epithelial cells and porcine and human macrophages. Given the prevalence of allergic and parasitic diseases worldwide and the close similarities in the porcine and human immune responses, these findings have important implications for the nutritional regulation of allergic inflammation at mucosal surfaces. In collaboration with our DGIL Research Leader we examined the effect of feeding indole 3-carbinol, a bioactive component in cruciferous vegetables, on Cr-induced colitis (Objective 2). The consumption of I3C had no effect on the colonization of the colon by Cr in a susceptible strain of mice, C3H/HeNCr, and this contrasted with the result obtained with a resistant strain of mice, C57Bl/6. However, I3C treatment led to a significant reduction in the production of pro-inflammatory cytokines in the colon including IL17A, IL6, IL1ß, TNF-a, IFN-g and IL17, TNF-a, IL12p70 and G-CSF in serum. In contrast, the production of IL-22, which is associated with protection, was not decreased and IL22 expression is modulated by signaling through the aryl hydrocarbon receptor, a target for I3C. Systemic inflammation in mice fed the control diet was attenuated by I3C treatment. In addition, serum antibody production was reduced in I3C-treated infected mice. Together, these results suggest that I3C, and by extension, cruciferous vegetables, can attenuate the inflammatory response induced by Cr infection. In collaboration with the Animal Parasitic Diseases Laboratory, we investigated the effect of krill oil (KO) on Cr and parasitic infections. KO is a natural product rich in PUFA and the potent antioxidant astaxanthin and may help in the resolution of inflammation and promote mucosal healing. KO significantly decreased LPS-induced IL1ß and TNFa expression in human macrophages in vitro in a dose-dependent manner by regulating a broad spectrum of signaling pathways, such as NF-KB and NOD-like receptor signaling. KO displayed a synergistic effect with COX2 and IKK2 inhibitors in attenuating inflammation by promoting M2 polarization and enhancing macrophage-mediated intracellular bacterial killing. Feeding mice KO mitigated intestinal mucosal damage induced by helminth infection and partially restored helminth-induced microbial dysbiosis. Several microbial signatures with strong predictive power for the status of both infection and supplementation were identified. Moreover, the pro-resolving properties of KO was validated using a Th1/Th17-inducing Cr-induced colitis model. Further, microbial signatures with high prediction accuracy for colitis related pathophysiological traits were identified in mice. The findings from this study provided a mechanistic basis for optimizing microbiome-based alternative therapeutics in the management of IBD. Key T cell regulatory molecule controls anti-parasite immunity. Worm infections in livestock are most economically harmful when they are persistent and compromise the health and well-being of animals to reduce productivity. Parasitic worm infections can activate host signaling molecules that regulate the expulsion of worms from the host species. ARS researchers at Beltsville, Maryland, along with colleagues from Washington University School of Medicine in St. Louis, Missouri, used genetically modified rodent models to show that the infecting parasitic worms activate a regulatory factor called Bhlhe40 within T helper 2 lymphocytes that is critical to control repeated parasite infections. These observations point to a need to demonstrate the role of similar factors in livestock and humans since they can be used as a target to better control parasitic infections that increase the cost of animal production and the quality of food available to the US public. This information is important to those interested in immune regulation of the responses to parasitic infection because the molecular tools available for use in rodents cannot generally be used in livestock and humans but identifying these regulatory molecules can be applied to improve the health and productivity of both livestock and humans.

Accomplishments
1. Krill oil reduces inflammation. The anti-inflammatory properties of Omega-3 fatty acids may help with the prevention and management of inflammatory bowel diseases and promote mucosal healing. Krill oil is a natural product rich in Omega-3 and is potent with the antioxidant astaxanthin. ARS researchers at Beltsville, Maryland, and the Ocean University of China, Qingdao, China, showed that Krill oil reduced inflammation by decreasing the levels of colitis-inducing bacteria that cause inflammation in mice and intestinal mucosal damage induced by a gastrointestinal parasite infection. The findings from this study show that Krill oil may help with inflammatory bowel syndrome.

2. Analysis of the host transcriptome and metabolome identified novel metabolic pathways associated with parasite-infected pigs. Prevention of the pig whipworm Trichuris suis is important in swine production because of its negative effects on pig performance and Trichuris trichuria, is a whipworm that infects about ¼ of the world’s human population. ARS researchers at Beltsville, Maryland, and the University of Maryland School of Medicine demonstrated that T. suis infection of pigs led to the differential expression of about 1,200 genes in the proximal colon and was nearly 90% lower in pigs that had expelled worms. Genes that were differentially expressed include genes involved in de novo cholesterol synthesis, fructose and glucose metabolism, basic amino acid metabolism, and bile acid transport. Metabolic analysis indicated changes in fatty acid profiles, antioxidant capacity, sugars, Krebs cycle intermediates, microbe-derived metabolites and altered metabolite transport in infected animals. The results support a model to test novel diets that favorably alter the microbiome and improve host intestinal health in both pigs and humans exposed to Trichuris.

3. Proteins in the intestine control the response to worms. Mucosal tissues that line the intestines and lungs have nerves that can help support immune cells in responding early to parasitic infection. The mucosal neurons produce a messenger protein that reacts to parasitic infection and promotes allergic responses in animals and humans. ARS researchers at Beltsville, Maryland, and colleagues at the NIH in Bethesda, Maryland, showed that a class of molecules that act as alarming messengers help the intestine to respond to parasitic infection. This knowledge is important for livestock producers who need new methods to eliminate parasitic infections that compromise the health of animal health and decrease productivity and clinicians who require therapeutic tools to control allergic disease.

Review Publications
Liu, F., Xie, Y., Zajaz, A.M., Hu, Y., Aroian, R.V., Urban Jr, J.F., Li, R.W. 2020. Gut microbial signatures with predictive power for moxidectin treatment outcomes in. Journal of Parasitology. 242(2020):108607. https://doi.org/10.1016/j.vetmic.2020.108607.
Jarjour, N.N., Bradstreet, T.R., Schwarzkopf, E.A., Cook, M.E., Lai, C., Huang, S., Taneja, R., Stappenbeck, T.S., Urban Jr, J.F., Edelson, B.T. 2020. BBhlhe40 promotes TH2 cell-mediated anti-helminth immunity and reveals cooperative Csf2rb family cytokines. Journal of Immunology. 204(4):923-932. https://doi.org/10.4049/jimmunol.1900978.
Liu, F., Smith, A.D., Solano Aguilar, G., Wang, T.T., Pham, Q., Tang, Q., Urban Jr, J.F., Xue, C., Li, R.W. 2020. Mechanistic insights into the attenuation of intestinal inflammation and modulation of the gut microbiome by krill oil using in vitro and in vivo models. Microbiome. 8:83. https://doi.org/10.1186/s40168-020-00843-8.
Jasmer, D.P., Rosa, B.A., Tyagi, R., Bulman, C.A., Beernsten, B., Urban Jr, J.F., Sakanari, J., Mitreva, M. 2020. An integrated computational and evidence-based approach identifies small molecule inhibitors detrimental to intestinal cells and tissue of parasitic nematodes. PLOS Neglected Tropical Diseases. 14(5):e0007942. https://doi.org/10.1371/journal.pntd.0007942.
Smith, A.D., George, N.S., Cheung, L., Bhagavathy, G.V., Luthria, D.L., John, K.M., Bhagwat, A.A. 2020. Pomegranate peel extract reduced the pathogenicity of Citrobacter rodentium-infections in mice. Nutrition Research. 73:27-37. https://doi.org/doi:10.1016/j.nutres.2019.11.001.
Dawson, H.D., Sang, Y., Lunney, J.K. 2020. Porcine cytokines, chemokines and growth factors: 2019 Update. Research in Veterinary Science. 131:266-300. https://doi.org/10.1016/j.rvsc.2020.04.022.
Nagashima, H., Mahlakoiv, T., Shih, H., Davis, F.P., Meylan, F., Huang, Y., Harrison, O., Yao, C., Mikami, Y., Urban Jr, J.F., Caron, K., Belkaid, Y., Kanno, Y., Artis, D.C., O'Shea, J.J. 2019. Neuropeptide CGRP limits group 2 innate lymphoid cell responses and constrains type 2 inflammation. Immunity. 51(4):682-695e6. https://doi.org/10.1016/j.immuni.2019.06.009.
Chen, C.T., Smith, A.D., Cheung, L., Pham, Q., Urban Jr, J.F., Dawson, H.D. 2020. Potentiation of IL-4 signaling by retinoic acid in intestinal epithelial cells and macrophages - mechanisms and targets. Frontiers in Immunology. 10.3389. https://doi.org/10.3389/fimmu.2020.00605.
Dawson, H.D., Chen, C.T., Li, R.W., Bell, N., Shea-Donohue, T., Kringel, H., Beshah, E., Hill, D.E., Urban Jr, J.F. 2020. Molecular and metabolomic changes in the proximal colon of pigs infected with Trichuris suis. Scientific Reports. 10, 12853. https://doi.org/10.1038/s41598-020-69462-5.