Location: Diet, Genomics and Immunology Laboratory
2015 Annual Report
Objectives
Objective 1: Determine whether bioactive food components, such as vitamins A and D or lycopene, acting via vitamin A and D-receptor-mediated pathways and nuclear factor-kappaB signaling, exhibit dose-dependent inhibitory effects on macrophage-mediated remodeling of adipose tissue toward a pro-inflammatory phenotype in response to high fat diets in a swine model. [NP 107 Component 3, Problem Statement 3B].
Objective 2: Determine whether bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Compare responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling. [NP 107 Component 3, Problem Statement 3B]
Approach
For Objective 1, in vitro and in vivo porcine models will be used to test the hypothesis that vitamin A or vitamin D or metabolites of dietary compounds that signal through retinoic acid receptor signaling pathways, such as lycopene, will promote an anti-inflammatory phenotype of adipose tissue macrophages and inhibit pro-inflammatory responses of adipose tissue macrophages to inflammatory ligands via inhibition of NF-kappaB signaling and epigenetic regulation of macrophage polarization.
For Objective 2, a mouse model will be used to test the hypothesis that bioactives from food, including selenium, vitamin A, and thiol compounds, alter the immune response to model infectious organisms in mice through epigenetic changes, redox-sensitive signaling pathways, and tissue remodeling by controlling cellular thiol levels, redox tone, and/or mitochondrial function. Responses of wild-type and genetically engineered mice with altered expression of one or more selenoproteins or proteins important for vitamin A or redox function, will be used to identify specific proteins or pathways important for the effect of the nutrients under study on immune function and tissue remodeling.
Progress Report
To use the pig as a translational species to determine the most fruitful nutritional interventions in the human inflammatory pathway it is necessary to understand mechanisms involved in the inflammatory process in multiple species. To this end, we conducted several in silico and in vitro analyses to determine whether central pathways involved in inflammation and alternatively activated macrophage development are conserved among the three species (mouse, pig and human). We determined shared functional domains and regulatory family members of nine related pathways involved in inflammation, the so called inflammasomes. Several key members of these pathways were sequenced in our laboratory. Based upon our analysis we determined that several pig inflammasome pathways are intermediate in similarity between the mouse and the human; however; some of them are not conserved in pigs or mice. A preliminary analysis of whole genome expression profiles of macrophages indicates that the lipopolysaccharide (LPS, a proinflammatory mediator) -induced transcriptome appears to be more conserved among humans and pigs while the LPS-induced microRNAome and targets appears similar in all 3 species. However, the pig and human IL-4-induced, alternatively activated macrophage, transcriptomes are dramatically more similar. Several novel observations were also noted including selective, high-level induction of the epigenetic regulator histone deacetylase 9 (HDAC9) and the histone demethylase, KDM6B by IL-4 and numerous histone demethylases and methyltransferases by LPS. These analyses strengthen the notion that swine are a scientifically and ethically acceptable intermediate species (rodent-human) for conducting inflammation-related research relevant to humans.
The central hypothesis being tested in objective 1 is that vitamin A (VA), either alone or in concert with cytokines like IL-4 or nutrients like vitamin D (VD), will foster the development of alternatively-activated macrophages. We determined that the bioactive vitamin A metabolite, all-trans retinoic acid (ATRA) increases vitamin D3 receptor (VDR) mRNA and protein level in macrophages and epithelial cells. We found that vitamin D (VD) significantly regulated a total of 464 genes out of 5,000 compared. Of these, 256 genes were significantly regulated only by the combination of VA and VD (160 down-regulated and 96 up-regulated). ATRA- regulated 32 VD3-regulated genes whose expression was potentiated (>50%) in the same direction by ATRA, but not significantly affected by ATRA alone. Several of these genes are associated with alternatively-activated macrophages (MRC1, IL1RN). Fifteen genes were additively and three genes were synergistically affected by the combination of VA and VD. Several of these genes (PLXNA1, TIMP1) are also associated with alternatively activated macrophages. It is clear from these data that VA increases VD’s effect on the macrophage transcriptome as 2/3 of the VD regulated genes (305/464) were additively or synergistically induced or repressed by the combination of VA and VD.
We used gene expression studies to identify candidate selenoprotein genes affected by H. polygyrus bakeri (Hpb) infection in both selenium-adequate and -deficient mice, and associated with the rebound in immunity upon re-feeding an adequate diet. To gain further insights into which selenoproteins are critical for immune function we have obtained nine Sel KO mouse strains for analysis, including two conditional KOs of TR1 and GPX4 (normally embryonically lethal) and are currently breeding them to obtain sufficient numbers for testing. Preliminary results suggest that deletion of the peroxidase-like domain of selenoprotein P increases the pathology of a C. rodentium (Cr) infection. Conditional KO of TR1 and GPX4 in T-cells, macrophages and gut epithelial cells will be generated for testing with our two model systems (Hpb, Cr). Additional experiments testing thiol/antioxidant compounds for their ability to substitute for selenium, including N-acetyl cysteine, 2-ME, lipoic acid, CoQ10 and its reduced form, and vitamin E in the Cr and/or Hpb models have not yielded any antioxidants that can substitute for the role of selenium. We demonstrated that Hpb cyst formation is a STAT6 dependent process and knock-out of IL-13 produces a cyst phenotype similar to that seen in selenium deficient mice where IL-13 expression is decreased suggesting a role for IL-13 in cyst formation and are currently looking for effects of selenium on collagen deposition in these cysts. We have completed studies on the effect of VA deficiency on immunity to Cr infection and demonstrate that VA deficiency increases the colonization of the colon, increased colonic pathology that was associated with disruption of barrier function, and produces mice with enlarged goblet cells that stain heavily for mucins suggesting altered goblet cell function in VA deficient mice.
A collaborative study with another ARS scientist from the Environmental Microbial and Food Safety Laboratory (EMFSL) was undertaken to compare gene expression in Cr grown under three different nutrient conditions and examine changes in bacterial gene expression. Major nutrient-dependent changes in bacterial gene expression were observed indicating that we will be able to determine dietary-induced bacterial metabolomic changes in addition to compositional changes to the microbiome. In collaboration with ARS scientists from Environmental Microbial and Food Safety (EMFSL) and the Invasive Insect Biocontrol and Behavior Laboratory (IIBBL), we tested the effect of pomegranate peel extracts on pathogenic bacteria, both in vitro and in vivo. The results suggest that the pomegranate peel extract alters swarming properties of Salmonella in vitro and reduced the pathogenicity of Cr infections in vivo. As a result of these studies a new project has been developed in EMFSL that includes collaboration with the Diet, Genomic, and Immunology Laboratory (DGIL).
Accomplishments
1. Muscarinic receptors that bind acetylcholine improve resistance to bacterial pathogens in the intestine. Citrobacter rodentium is a bacterial infection of mice that mimics bacterial induced gastrointestinal disease in humans. The type 3 muscarinic receptors (M3R) is known to regulate mucosal homeostasis within the colon and mice deficient in M3R were more susceptible to Cr infection. ARS scientists in the Beltsville Human Nutrition Research Center, Beltsville, Maryland, found most of the systemic immune response in the M3R deficient mice was intact, but mucus-producing cells in the colon were reduced, making it easier for the bacteria to bind to the intestine and begin the disease process. They also found that M3R contributed to the development of inflammatory macrophage cells that play a role in eliminating bacterial infection. This study showed that M3R is important in host defense against Cr infection through effects on goblet cell mucus production and in the modulation of macrophage function. These results demonstrate that M3R plays a previously unrecognized role in gut mucosal immunity and suggests that engagement of these receptors by activating compounds in the diet could enhance resistance to certain bacterial infections.
2. Diets low in protein can influence host immune responses and the degree of competition between co-infecting parasites. Most humans are concurrently infected with multiple parasite species and live in environments transmission in co-infected hosts under these conditions. To test how diets low in protein affect immunity and co-infection outcomes, ARS scientists in the Beltsville Human Nutrition Research Center, Beltsville, Maryland, gave mice a standard or low protein diet, dosed with two species of worm parasites (alone and in combination), and then challenged with a bacterial pathogen. They found that co-infection influenced parasite survival and reproduction via the type of host immunity that was affected by the diet. In pigs infected with whipworm and nodular worm and each worm parasite induced unique immune response types with both positive and negative effects on survival of each of the parasites. This information can be used by livestock producers to develop low cost diets that are appropriate to control worm infection when conventional drug treatments cannot be used because of organic farming restrictions with fluctuating dietary resources, yet little is known about individual health and pathogen.
3. Cell “cross-talk” promotes resistance to infection and tissue repair. Worm parasites can cause considerable damage when migrating through host tissues, which requires rapid tissue repair important to prevent bleeding and release of intestinal bacteria throughout the body. Studies showed that mice lacking the ability to make antibodies or one of the general cellular receptors for these antibodies had impaired intestinal repair after invasion of the tissue by a parasitic worm. The impairment was also associated with suppressed production of a protein messenger chemokine released by muscle cells in the intestine and inflammatory macrophage cells, indicating that inflammatory cells and muscle cells communicate to promote intestinal repair. This “cross-talk” feature could be harnessed in clinical settings of impaired wound healing. Other studies in mice demonstrated cell “cross-talk” between inflammatory neutrophil cells in the lungs and macrophages promoted the killing of parasitic worm larvae migrating through the lungs. In addition, cells of the innate immune system called innate lymphoid cells-2 or ILC2 were activated by specific protein cytokine messenger molecules to facilitate expulsion of adult worms from the intestine, while ILC3 cells that are generated by a different set of cytokines can control fungal infection. These studies provide insight into some of the protein messenger molecules and related receptors for these protein that provide communication signals between cells to improve resistance to infection and promote tissue repair. How diet can affect cellular communication to improve health is under study.
4. Construction of a porcine non-redundant transcriptome and analysis pipeline. Because the porcine genome is in its first draft, there are a significant number of problems that impede its use in determining whole genome analysis of gene expression, including gene fragmentation, duplicate sequencing of genes and failure to sequence certain areas of the genome. To address this ARS scientists in the Beltsville Human Nutrition Research Center, Beltsville, Maryland, constructed a non-redundant transcriptome containing 6,545 sequences that include the vast majority of genes that have been deemed biologically important enough to study, including the vast majority of genes that are related to epigenetics, nutrition, metabolism, immunity and inflammation (approximately 32% of the pig genome). These sequences include 1,347 fragmented genes, 970 duplicated genes and 1,042 genes missing from the genome and 1,395 full-length gene sequences that have been submitted to Genbank (Bioproject: PRJNA80971). These sequences and pipeline were used to perform several transcriptomic and epigenetic–based studies (miRNA and mRNASeq expression profiling) including responses of porcine alveolar macrophages to vitamin A and vitamin D or IL-4 or LPS/IFN-g and in vivo intestinal immune responses to T. suis. Since its introduction this year, this non-redundant transcriptome and analysis pipeline has also been used by 3 other labs within ARS, an external MTA has been executed to transfer this technology to the Arkansas Children’s Hospital Research Institute and commercial and international (Denmark, Belgium) interest has been expressed in this technology. These comprehensive and integrated analysis tools will be useful for global analysis and data-mining of the porcine immune and inflammatory responses as well as modeling human disease in pigs because of the close evolutionary and functional features of the two species.
Review Publications
Andreasen, A., Petersen, H., Kringel, H., Iburg, T., Skovgaard, K., Dawson, H.D., Urban Jr, J.F., Thamsborg, S. 2015. Immune and inflammatory responses in pigs infected with Trichuris suis and Oesophagostomum dentatum. Veterinary Parasitology. 207(3-4):249-258.
Budischak, S.A., Sakamoto, K., Megow, L.C., Cummings, K.R., Urban Jr, J.F., Ezenwa, V. 2015. Resource limitation alters the consequences of co-infection for both hosts and parasites. International Journal for Parasitology. 45(7):455-463.
Von Bieren, J.E., Volpe, B., Sutherland, D.B., Burgi, J., Verbeek, S., Marsland, B.J., Urban Jr, J.F., Harris, N.L. 2015. Immune antibodies and helminth products promote CXCR2-dependent repair of parasite-induced injury. PLoS Pathogens. 11(3):e1004778.
Mahadwar, G., Chauhan, K.R., Bhagavathy, G., Murphy, C.F., Smith, A.D., Bhagwat, A.A. 2015. Swarm motility of Salmonella enterica serovar Typhimurium is inhibited by compounds from fruit peel extracts. Letters in Applied Microbiology. 60:334-340.
Wang, A., Smith, A.D., Li, Y., Urban Jr, J.F., Thirumalai, R.R., Wynn, T., Lu, N., Shea-Donohue, T., Yang, Z., Zhao, A. 2014. Genetic deletion of IL-25 (IL-17E) confers resistance to dextran sulfate sodium-induced colitis in mice. Cell & Bioscience. DOI: 10.1186/2045-3701-4-72.
Merenstein, D.J., Tan, T.P., Molokin, A., Smith, K.H., Roberts, R.F., Shara, N.M., Mete, M., Sanders, M.E., Solano Aguilar, G. 2015. Safety of Bifidobacterium animalis subsp. lactis (B. lactis) strain BB-12-supplemented yogurt in healthy adults on antibiotics: A phase I safety study. Gut Microbes. 6(1):66-77. DOI: 10.1080/19490976.2015.
Hibberd, P.L., Kleimola, L., Florina, A.M., Botelho, C., Haverkamp, M., Andreyeva, I., Poutsiaka, D., Fraser, C., Solano Aguilar, G., Syndman, D.R. 2014. No evidence of harms of probiotic Lactobacillus rhamnosus GG ATCC 53103 in healthy elderly-a Phase I Open Label Study to assess safety, tolerability and cytokine responses. PLoS One. 1:9(12):e113456. DOI: 10.1371/journal.pone.0113456.
Mclean, L.P., Smith, A.D., Cheung, L., Sun, R., Grinchuk, V., Vanuytsel, T., Desai, N., Urban Jr, J.F., Zhao, A., Raufman, J.P., Shea-Donohue, T. 2015. Type 3 Muscarinic Receptors Contribute to Clearance of Citrobacter rodentium. Inflammatory Bowel Diseases. DOI: 10.1097/MIB.0000000000000408.
Stephensen, C.B., Dawson, H.D., Claycombe, K.J. 2015. Impact of nutrition on immune function and the inflammatory response. Journal of Nutrition. 145(5):1039S-1108S. DOI: 10.3945/jn.114.194571.
