Location: Animal Parasitic Diseases Laboratory2014 Annual Report
1a. Objectives (from AD-416):
Objective 1: Determine the immune relationship between parasites and the mucosal immune response concentrating on epigenetic targets and the innate immune system. The goal of the proposed research project is to evaluate the influence of parasitic infection during gestation and in the pre-weaning period on mucosal macrophages and to explore dietary effects that regulate mucosal immune responses in pigs. Objective 2: Evaluate the ability of nutritional supplements and pathogen-associated molecules in modulating the immune response. Macrophages and related dendritic cells at mucosal surfaces provide the first line of defense as they respond to pathogen-associated molecular pattern (PAMP) molecules that bind toll-like receptors (TLRs) and trigger innate immune responses that link them to components of acquired immunity. They also respond to danger-associated molecular pattern (DAMP) molecules that trigger responses to cell injury and inflammation. The inherent potential of molecules from the parasite to modulate immune function to secure the parasitic relationship with the host may be met by nutritional conditions that influence host immunity. This objective will begin to evaluate these features of macrophage biology as they contribute to resistance to parasitic infection and the influence of nutrients on this process.
1b. Approach (from AD-416):
The approach for Objective 1 is to determine the immune relationship between parasites and the mucosal immune response concentrating on epigenetic targets and the innate immune system. Stimulation of primary pig alveolar macrophages (AM) by all-trans retinoic acid (ATRA), parasites, or parasite-derived products in vitro will provide information on transcriptomic markers and epigenetic sites to evaluate in later in vivo-treatment studies of pigs given ATRA and infected with Ascaris suum. Exposure of sows during gestation and neonates during the first 21 days of life to ATRA or infection with A. suum will polarize pig AM and imprint epigenetic traits that influence functional activity at mucosal surfaces. The approach used for Objective 2 is to evaluate the ability of nutritional supplements and pathogen-associated molecules in modulating the immune response. The aim is to identify parasite-derived nucleotide metabolizing enzymes, and in particular apyrases, that may control local inflammatory responses by modulating ATP levels in surrounding tissues. The AM will be used as a functional readout cell for parasite products and metabolites derived from parasite enzymatic activity. ATRA acting as a supplemental nutrient in the presence of adenosine will modulate adenosine receptor signaling of primary pig AM leading to synergistic effects on macrophage function, cytokine production, and gene expression. The study is designed to determine if ATRA co-stimulation with adenosine alters pig AM function in vitro.
3. Progress Report:
The cloning and expression of Ascaris apyrase has made sufficient material available for testing its immune protective features in a mouse model of experimental ascariasis. Mice have been immunized with the cloned apyrase using different adjuvant products and routes of immunization. The challenge infection will include a novel application of several different nematode species, including A. suum, Trichuris muris, and Heligmosomoides polygyrus, to look for a pan-nematode protective potential as well as specific protection against A. suum. Work on the transcriptome of pig alveolar macrophages provides the bioinformatic tools to evaluate epigenetic features of exposure of swine to parasitic nematodes and vitamin A in utero.
1. Intestinal cells monitor nutrition levels to provide appropriate immune response. The immune system adapts to nutritional levels in the intestine to provide immunity at barrier surfaces to respond appropriately to different pathogens. Vitamin A deficiency is one of the most common micronutrient deficiencies in humans and is associated with profound defects in adaptive immunity. It was found that type 3 innate lymphoid cells (ILC3s) are severely diminished when vitamin A is deficient in the diet and this can compromise immunity to certain bacterial infections. However, vitamin A deficiency has a paradoxical effect because there is also a dramatic expansion of type 2 innate lymphoid cells (ILC2s) that produce a protein called interleukin-13 which, in turn, enhances resistance to worm infection. It was observed that ILCs are a primary sensor of dietary deficiency. The flexibility to switch ILC activity based on changes in dietary vitamin A show an adaptation of the immune system that promotes survival in the face of pathogens that invade mucosal surfaces.
2. Intestinal cells responding to worm infection alter sugar metabolism. Worm infection is associated with changes in epithelial permeability and inhibition of sodium-linked glucose absorption and that is dependent on a category of cells called M2 macrophages. It was found that the M2 cell inhibits the activity of receptor for glucose on intestinal cells that results in a “lean” phenotype. These cells do not play a role in changes in intestinal permeability and barrier function. The data provides evidence for the ability of M2 macrophages to alter sugar metabolism of neighboring cells and thus control the amount of fluid in the intestine to protect against worm infection. It also explains changes in sugar metabolism and body composition that are associated with parasitic worm infections.
3. Specific classes of antibody and cells combine to regulate worm infection and inflammation. Both worm infection and tumor growth induce myeloid-derived suppressor cells (MDSC) that enhance the activity of other mast cells to control infection with worms. This effect is further dependent on a specific class of antibodies called IgE antibodies that develop after worm infection by different developmental pathways to increase the strength of the binding or affinity to worm products. Blocking the development of MDSCs using the FDA-approved drug gemcitabine exacerbated the worm infection but also reduces lung inflammation. The work showed that these cells act as a dual edged sword that must be carefully monitored to ensure that the worm burden from infection is not increased and inflammation is minimized to improve the health of the animal or human exposed to worm infection.
4. Ascaris apyrase as a candidate for parasite vaccination. Parasites have at their disposal many ways in which they can modulate their local environments and host immunity to make it easier for them to live within the host. Some of these proteins are parasite specific and others are evolutionarily conserved proteins that the parasite has adopted and changed for its own personal use. We ventured to study a class of enzymes in Ascaris called apyrases, to determine if they have the capability to modify the innate immune responses by removing key substrates i.e. nucleotides, from the surrounding medium that are released from damaged cells. We identified one such enzyme in the nematode A. suum that when cloned and expressed is able to degrade only GDP and UDP which are believed to be involved in signaling and stress responses. Comparative genomics allowed us to identify conserved regions within the protein and mutate these regions in the hope of identifying the active site(s). The sequences of the putative conserved regions were genetically modified and we demonstrated that such modifications also modified enzyme activity. This work relates to finding parasite target proteins involved in potentiating colonization and survival. By controlling the activities of these types of proteins, we may be able to attenuate infection. This research is important because only recently Ascaris suum has been recognized not only as a parasite of swine, but a zoonotic pathogen as well, and because it is a close relative of Ascaris lumbricoides, the sister species of A. suum, that infects nearly 25% of the world’s population.
5. Identification of novel treatments against swine viruses. Porcine parvovirus (PPV) is among the most common and important causes of infectious infertility in pigs and can result in significant economic losses to the swine industry. It is one of the few viruses that can survive in the environment for extended periods of time and is refractory to most disinfectants. Currently there are no viable treatments for the disease. Vaccination is effective, but cost and safety issues have been deterrents to routine vaccination in many regions of the world. At present, attenuated and killed viral vaccines are being used to control PPV infection; however, attenuated vaccines stand the danger of restructuring and reacquiring virulence. Furthermore, evidence has been advanced that vaccination against PPV may protect against the disease, but it does not necessarily prevent viral infection and virus shedding of heterologous strains. Diammonium glycyrrhizinate (DG), the active ingredient of Glycyrrhiza extraction, has significant anti-inflammatory effects and is active in treating human immunodeficiency virus hepatitis A virus, hepatitis B virus, coronavirus, and herpes virus. In this study, the antiviral effects of DG on PPV were analyzed in vitro. Data show that DG decreases infectivity of PPV significantly if incubated with the virus prior to contact with the cells and can reduce virus propagation by 75%. The work demonstrates a mechanism of action to reduce infection.
6. The diagnosis of swine transmissible gastroenteritis. Transmissible gastroenteritis (TGE) is a highly contagious disease of swine characterized by up to 100% mortality in suckling piglets though pigs of all ages and categories are susceptible. The virus responsible for TGE (TGEV) consists of four structural proteins: spike (S), small membrane (sM or E), membrane (M), and nucleocapsid (N) proteins. The M protein is one of the major structural proteins of all coronavirus particles. In this study, the M protein of TGEV was used as a screening mechanism to identify proteins that bind to the surface of the phage and exhibit antiviral activity and can be used to differentiate different viral pathogens of swine. Three peptides expressing TGEV-M-binding peptides were identified and characterized in more depth. One peptide exhibited greater diagnostic sensitivity than the tests currently available. Also, when another peptide was examined for antiviral activity, results showed that it was able to prevent TGEV infection in vitro if the virus was first pretreated with the peptide. These results demonstrated that the TGEV peptides can be utilized for virus-specific diagnostics and anti-virals to better diagnose and treat infected animals.
7. Identifying novel vaccines against Porcine Epidemic Diarrhea Virus (PEDV). The PEDV causes porcine epidemic diarrhea (PED) which in turn can result in very high mortality in newborn piglets. The disease was first reported in England in 1971 and has since been reported worldwide. In the USA, it is fast becoming one of the most important pathogens of swine. Although conventional inactivated and attenuated vaccines are used in some areas, vaccines of this nature have drawbacks such as recovery of virulence, spread of viruses, high cost, and poor protection efficacy. Therefore, development of novel and effective methods are necessary for the control of PED. In this study, phage libraries capable of expressing random peptides on their surface were bound to a key virus surface protein in order to find phages producing peptides capable of binding to the virus and inhibiting infection. Three peptides/phage were identified, the most important of which (peptide H) was shown to be highly effective in reducing infection of swine cells by PEDV. The studies which followed corroborated our hypothesis that peptide H functions in part by interacting with the ability of PEDV to bind to the cell surface. Future studies will focus on identifying the specific site of interaction of peptide H and whether or not such a peptide can be used to eliminate or reduce PEDV infections through an effective viral gene vaccines.
8. Expanded scope of the Porcine Translational Research Database. The content of the Beltsville Human Nutrition Research Center's Porcine Translational Research Database approximately 1.5 fold. On June 26, 2013 the data base contained 6,676 porcine gene entries (more than 1,600 from last FY). Several transcriptomic and epigenetic –based (Including miRNA and mRNASeq expression profiling) studies on the responses of porcine alveolar macrophages to vitamin A were conducted. Manually assembled and annotated 3,835 high-interest (all known immune and metabolism related genes) transcripts from 2,285 genes (17% of genome), including 700 genes not found in the current genome assembly. These comprehensive and integrated analyses increase the value of the porcine genome sequences and provide important tools for global analyses and data-mining of the porcine immune response. This information will be useful for modeling human disease in pigs because of the close evolutionary and functional features of the two species.Hu, Y., Ellis, B.L., Yiu, Y.Y., Miller, M.M., Urban Jr., J.F., Shi, L.Z., Aroian, R.V. 2013. Comprehensive comparison of anthelmintic classes on diverse nematodes. PLoS One. DOI: 10.1371/journal.pone.0070702.
Yang, Z., Sun, R., Grinchuk, V., Blanco, J.A., Notari, L., Bohl, J.A., Mclean, L.P., Ramalingam, J.R., Wynn, T.A., Urban Jr, J.F., Vogel, S.N., Shea-Donohue, T., Zhao, A. 2013. IL-33-induced alterations in murine intestinal function and cytokine responses are MyD88, STAT6, and IL-13-dependent. American Journal of Physiology - Gastrointestinal and Liver Physiology. 304(4):G381-389.
Meng, F., Suo, S., Zarlenga, D.S., Cong, Y., Ma, X., Zhao, Q., Ren, X. 2014. Phage displayed peptide recognizing porcine aminopeptidase N is a potent small molecule inhibitor of PEDV entry. Virology. 456-457:20-27.
Zou, H., Ren, Y., Tao, Y., Zarlenga, D.S., Ren, X. 2014. Antiviral effect of diammonium glycyrrhizinate on cell infection by porcine parvovirus. Current Microbiology. 69(1):82-87.
Zou, H., Zarlenga, D.S., Sestak, K., Suo, S., Ren, X. 2013. Transmissible gastroenteritis virus; identification of M protein-binding peptide ligands with antiviral and diagnostic potential. Antiviral Research. 99:383-390.
Cong, Y., Zarlenga, D.S., Richt, J., Wang, X., Wang, Y., Wang, J., Ren, Y., Li, G., Ren, X. 2013. Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus. Virus Genes. 47(1):66-74.
Morales, J.K., Saleem, S.J., Martin, R.K., Barnstein, B.O., Graham, L., Bear, H.D., Urban Jr, J.F., Conrad, D.H., Ryan, J.J. 2014. Myeloid derived suppressor cells enhance IgE-mediated mast cell responses. Journal of Leukocyte Biology. 95(4):643-650 DOI: 10.1189/jlb.0913510.
Hang, L., Blum, A.M., Setiawan, T., Urban Jr, J.F., Stoyanoff, K., Weinstock, J.V. 2013. Heligmosomoides polygyrus bakeri infection activates colonic FoxP3+ T cells enhancing their capacity to prevent colitis. Journal of Immunology. 191(4):1927-34.
Notari, L., Riera, D.C., Sun, R., Bohl, J.A., Mcclean, L.P., Madden, K., Vanrooijen, N., Vanuytsel, T., Urban Jr, J.F., Zhao, A., Shea-Donohue, T. 2014. Role of macrophages in the altered epithelial function during a type 2 immune response induced by enteric nematode infection. PLoS One. 23:9(1)e84763.