Location: Diet, Genomics and Immunology Laboratory
2017 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
Previously we demonstrated that the most bioactive vitamin A metabolite, all-trans retinoic acid (ATRA) potentiated the mRNA expression of several IL-4 induced chemokines (chemokine (C-C motif) ligand 11 ((CCL11), CCL17, CCL22 and CCL26) associated with alternative activation (M2a) in porcine macrophages in vitro. These responses are necessary to maintain immune homeostasis and limit inflammation. We extended these findings to human macrophages. In human THP-1 cells, IL-4 exposure led to an increase in the mRNA for the M2a macrophage-associated chemokines, CCL11, CCL13, CCL17, CCL18, CCL22 and CCL26, as well as the M2a-associated surface markers, CD206, CD209 and CD274 and the M2a associated enzyme TGM2. ATRA synergistically increased IL-4–induced CCL2, CCL13, CCL18, CCL22, CCL26 and TGM2 mRNA and CCL13, CCL18 and CCL26 protein levels, while having no effect or slightly down-regulating MRC1, CD209 and CD274 mRNA expression. The effects of ATRA on mRNA levels were greater at 24 compared to 48 hrs; however, greater differences in protein levels were observed at 48 hrs for CCL13, CCL18 and CCL26. In contrast to porcine monocytes and macrophages, ATRA decreased IL-4 induced CCL17 mRNA and protein expression in THP-1 cells. Thus, in porcine monocytes and macrophages and in human macrophages, ATRA selectively increased signaling in response to IL-4. Ongoing experiments include the effects of ATRA, vitamin D and/or IL-4 on nuclear factor-kappaB signaling, inflammasome activation and TGM2 activity in pig macrophages and human THP-1 cells. We are also assisting the Food Components and Health Laboratory (FCHL) in the analysis of whole blood transcriptomic data from humans fed beta carotene or lycopene.
We generated a mouse strain with KO of selenoproteins in macrophages. Much to our surprise, these mice did not exhibit a phenotype in either of our infectious disease models (Cr, Hpb), especially considering the importance of macrophages in the clearance of a Hpb infection. However, preliminary results suggest that KO of selenoproteins in BMDM may affect inflammasome activation and this will be pursued further. Mice with selenoprotein expression KO in T cells are being generated by crossing floxed TRSP mice with Lck-cre mice. We expect to see a phenotype as alterations in the immune responses in non-infectious disease models have been observed. Our studies examining the effect of VA deficiency on the fecal microbiome are progressing and significant changes have been observed with distinct groupings for VA adequate and deficient mice in a principle component analysis of the data. We have observed enlarged goblet cells in VA deficient mice that are strongly positive for staining with Periodic Acid-Schiff. We are currently determining how mucus production is altered by VA deficiency. We have identified unique changes to mucins in goblet cells and the intestinal mucus layer by staining with lectins that have specificity for different sugar moieties and plan on examining the expression of genes involved in mucin production and export.
In collaboration with ARS scientists from the Environmental Microbial and Food Safety (EMFSL) and the Invasive Insect Biocontrol and Behavior Laboratory (IIBBL), we have tested the effect of pomegranate peel extracts on infectious colitis induced by Cr. The results demonstrate that the pomegranate peel extract (POM) reduces colonic pathology and development of a systemic infection by Cr in vivo. POM treatment completely prevents infection induced mortality and reduces damage to the colonic mucosa, thus preventing translocation of bacteria to the systemic compartment that is characteristic of untreated mice. Interestingly, POM treatment does not alter peak Cr load, clearance, or the cytokine response to infection indicating that the effect is on barrier function. One gene, Ang4, which is important for the control of enteric bacteria and barrier function, is increased in POM treated mice.
Several other collaborations have occurred over the last year. Our expertise in the use of Cr has resulted in requests for collaboration. The Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia asked for our assistance in characterizing a mouse strain with a conditional knock out of A disintegrin and metalloproteinase (ADAM) 10, a member of a transmembrane zinc-dependent protease family that mediates ectodomain shedding and regulated intramembrane proteolysis, in dendritic cells. Given its important role in cell-cell and cell-matrix interactions, ADAM10 has been studied in many physiological and disease processes including neuron homeostasis and development, Alzheimer’s disease, and allergy. We tested the KO mouse strain in our Cr model and found that ADAM10 was important for preventing systemic dissemination of bacteria and mortality. A manuscript containing these and other results has been submitted for publication. A second collaboration with the University of Maryland-Baltimore Medical School also utilized our expertise using the Cr model. In this study bi-directional communication between the gut and the brain were demonstrated. Mice with moderate-level traumatic brain injury (TBI) had colonic paracellular flux and glial fribrillary acid protein expression after Cr infection that was significantly increased compared to shams. Importantly, brain lesions were significantly larger and neuroinflammation was increased in TBI-injured mice infected with Cr compared to CCI alone. These results demonstrate bi-directional communication between the gut and the brain and have implications for treatment of individuals suffering from TBI.
Accomplishments
1. Protective immunity to nematode infections. ARS scientists at the Beltsville Human Nutrition Center, Beltsville Maryland, and colleagues at the University of Maryland, Baltimore Medical School demonstrated that proteins in mice genes (IL-25 and the receptor subunit IL-17RB) are increased during the primary and secondary infection of mice worm (Heligmosomoides polygyrus bakeri) (Hpb). Mice without the IL-25 gene had a reduced response and response against secondary infection of Hpb was severely impaired. Treating infected mice with the IL-25 protein restored protection against Hpb. This study demonstrates that IL-25 will be useful to individuals who are developing therapies for Hpb.
2. Impact of bidirectional brain/gut interactions on both gastrointestinal and brain health in mice. ARS scientists at the Beltsville Human Nutrition Center, Beltsville, Maryland, and colleagues at the University of Maryland, Baltimore Medical School examined changes in mucosal barrier properties and enteric glial cell response in the gut after experimental Traumatic brain injury (TBI) in mice, as well as effects of the enteric pathogen Citrobacter rodentium (Cr) on both gut and brain after injury. Moderate-level TBI induced changes in colon morphology, including increased mucosal depth and smooth muscle thickening, increased paracellular permeability and decreased and protein expression. Cr infection in chronically-injured mice worsened the brain lesion injury and increased inflammation. These results demonstrate bidirectional brain/gut interactions have repercussions for individuals suffering from TBI, but also indicates that alterations to the gut from disease, diet, or the microbiome may impact recovery from other traumatic brain events including stroke. This research is of value to those interested in how diet and inflammation may affect brain/gut interactions.
Review Publications
Bhagwat, A.A., Young, L., Smith, A.D., Bhagwat, M. 2017. Transcriptomic analysis of swarm motility phenotype of Salmonella enterica serovar Typhimurium mutant defective in periplasmic glucan synthesis. Current Microbiology. doi:10.1007/s00284-017-1267-1.
Shea-Donohue, T., Qin, B., Smith, A.D. 2017. Parasites, nutrition, immune responses, and biology of metabolic tissues. Parasite Immunology. 39(5). doi: 10.1111/pim.12422
Ma, E.L., Smith, A.D., Desai, N., Cheung, L., Hanscom, M., Stoica, B.A., Loane, D.J., Shea-Donohue, T., Faden, A.I. 2017. Bidirectional brain-gut interactions and chronic pathological changes after traumatic brain injury in mice. Brain Behavior and Immunity. pii: S0889-1591(17)30207-6. doi: 10.1016/j.bbi.2017.06.018.
Pei, C., Zhao, C., Wang, A.J., Fan, A.X., Grinchuk, V., Smith, A.D., Sun, R., Xie, Y., Lu, N., Urban Jr, J.F., Shea-Donohue, T., Zhao, A., Yang, Z. 2016. Critical role for Interleukin-25 in host protective Th2 memory response against Heligmosomoides polygyrus bakeri. Infection and Immunity. 18:84(12):3328-3337.
Wilhelm, C., Harrison, O., Schmitt, V., Pelletier, M., Spencer, S., Urban Jr, J.F., Ploch, M., Ramalingam, T., Siegel, R., Belkaid, Y. 2016. Critical role of fatty acid metabolism in ILC2 mediated barrier protection during malnutrition and helminth infection. Journal of Experimental Medicine. 213(8):1409-1418. doi: 10.1084/jem.20151448.
Mclean, L.P., Smith, A.D., Cheung, L., Urban Jr, J.F., Sun, R., Grinchuk, V., Dasai, Zhao, A., Raufman, J.P., Shea-Donohue, T. 2016. Type 3 muscarinic receptors contribute to intestinal mucosal homeostasis and clearance of nippostrongylus brasiliensis through induction of Th2 cytokines. American Journal of Physiology - Gastrointestinal and Liver Physiology. 311(1):G130-141. doi: 10.1152/ajpgi.00461.2014.
Weeks, J.C., Roberts, W.M., Robinson, K.J., Keaney, M.I., Vermeire, J.J., Urban Jr, J.F., Lockery, S.R., Hawdon, J.M. 2016. Microfluidic platform for electrophysiological recordings from host-stage hookworm Ascaris suum larvae: a new tool for anthelmenthic research. International Journal for Parasitology: Drug and Drug Resistance. 6:314-328. doi: 10.1016/j.ijpddr.2016.08.001.
Dawson, H.D., Chen, C.T., Gaynor, B., Shao, J.Y., Urban Jr, J.F. 2017. The porcine translational research database: A manually curated, genomics and proteomics-based research resource. Biomed Central (BMC) Genomics. doi: 10.1186/s12864-017-4009-7.
Hang, L., Blum, A.M., Kumar, S., Urban Jr, J.F., Mitreva, M., Geary, T.G., Jardim, A., Stevenson, M.M., Lowell, C.A., Weinstock, J.V. 2016. Downregulation of the Syk signaling pathway in intestinal dendritic cells is sufficient to induce dendritic cells that inhibit colitis. Journal of Immunology. 197(7):2948-2957. doi: 10.4049/jimmunol.1600063.
