Progress 11/17/16 to 05/14/17
Outputs
Progress Report 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. 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. This is the final report for the bridge project 8042-32000-104-00D that terminates in November 2017. Substantial and impactful results were obtained over the five years of the project. All milestones were either Met or Substantially Met during the period of the Project plan. There were animal facilities and production issues that limited the availability of sows to test hypotheses related to in vivo epigenetic effects of parasite infection and vitamin A supplementation on piglets. Using bioinformatics and computational tools to evaluate conserved protein families in a pan-nematode database of parasite secreted proteins (secretome) a series of candidate proteins with immunogenic potential were chosen for cloning, expression and purification for vaccine trials in mice and pigs. Two dimensional polyacrylamide gel electrophoresis was used as a proteomic tool to select immune dominant and putative protective antigens from parasite secreted proteins by screening the gels with sera from parasite susceptible and resistant swine. Likewise, a parasite secretome protein array is in production for summer 2017 to expand the scope of proteomic tools used to select proteins for induction of protective immune responses. Another more empirical approach generated molecular evidence for gene transfer between worms and either bacteria or plants that play a role in the evolution of nematode parasitism and identified parasite unique targets for immune intervention and control. A cyanase protein was found in the parasitic genera Trichinella and Trichuris with apparent bacterial or fungal origins that is a functionally active protein not present in any mammals. Similarly, a class of enzymes called apyrases with activity to degrade only certain nucleotides, involved in signaling and stress responses, was identified in the large round worm nematode parasite of pigs, Ascaris suum, and was cloned and expressed. These molecules can modify the innate immune responses by removing key host cell signaling molecules i.e., nucleotides, from the surrounding medium that are released from damaged cells. This work relates to finding parasite target proteins involved in potentiating colonization and survival. By controlling the activities of these types of proteins, strategies can be devised to attenuate infection. Mice were immunized with the cloned apyrase and other selected proteins using different immune activating adjuvant products and routes of immunization, and given a challenge infection that includes several different nematode species, including A. suum, Trichuris muris, and Heligmosomoides polygyrus bakeri, to look concurrently for a pan-nematode protection against several parasitic worms. Immunization failed to show immune protection but did show that the model can be used to screen for immune responses to multiple parasites and parasite products. Testing of these cloned antigens in pigs is underway to evaluate immune responses in the host animal species. Another approach developed in the Project Plan tested nutrient effects on innate immune responses. 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 a common micronutrient deficiency and is associated with profound defects in adaptive immunity. It was found that one type of innate lymphoid cells (ILC3s) is 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 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. The dietary trace mineral selenium supported optimal clearance of parasitic worms through macrophage activation. Selenium induces a switch in macrophage activation from a pro-inflammatory M1-type cell to an anti-inflammatory M2-type cell where the production of prostaglandin J2 (PGJ2) molecules plays a key regulatory role and intestinal M2 cells contributed to decreasing adult worms and egg production. These results demonstrated that optimal expression of selenium-dependent proteins and certain prostaglandins regulate M2 activation to enhance anti-parasite responses, and how overt and functional deficiencies in dietary trace minerals can have deleterious effects on worm infection. Related studies on developmental acquisition of the regulome (all the regulatory components within a cell) during innate immunity of innate lymphoid cells (ILCs) showed a stepwise development in the mouse that did not change after stimulation of these cells. Similar work on the transcriptome of macrophages from pig lungs using bioinformatics tools to evaluate epigenetic features (non-genetic influences on gene expression) during exposure to parasitic nematodes and vitamin A showed evidence suggesting that pigs are a functionally intermediate species between rodents and humans that can better model common immune and inflammatory responses. Conservation in sequence and structure among immune genes revealed a much higher level of sequence similarity among humans and pigs then to mice. The scope of the Porcine Translational Research Database fosters comprehensive and integrated analysis of the pig genome, and provides important tools for global analysis and data-mining of pig immune responses. The database contains a complete representation of the "Transportome" (Solute Carrier and the ATP binding Cassette Super- families), complete annotations of 356 genes that compose pig cell surface markers that conform to the human markers, and complete representation of the Tetraspanin membrane proteins (32 porcine members) and Protocadherin beta cell adhesion proteins (12 pig members), and mucus- related Mucin Super-families (20 porcine members). The database is part of a collaborative enterprise with scientists in the Beltsville Human Nutrition Research Center and the Beltsville Agricultural Research Center and has been accessed over 50,000 times by investigators from more than 30 laboratories worldwide. This information is important in the design of immunization strategies that are needed to induce activation of immune compartments to provide more effective vaccines for integrated control of parasitism through immunization in both pigs and humans. Accomplishments 01 Innate lymphoid cells (ILC) sense nutrients in the gut. The identification of immune cells important in the early activation of immunity and inflammatory can be used to control both bacterial and parasitic worm infection in the gut; however, it is not known if ILCs respond only to infection or to changes in nutrients in the gut, and if these changes can direct a more appropriate response that reduces infection while minimizing inflammation. ARS scientists in Beltsville, Maryland, and colleagues at the National Institutes of Health, Bethesda, Maryland, showed that ILC2 switch their metabolism to utilize fatty acids (FA) that are increased during a worm infection. Further, the ILC2 use FA metabolisms to preferentially produce anti-parasitic immune proteins when vitamin A is limited; a condition often caused by worm infection. These results demonstrate that ILC2 use FAs to maintain their protective function during worm infection even in the face of diets where vitamin A is deficient. This could represent a host adaptation to maintain intestinal barrier immunity under dietary restriction. The work is important to those interested in the effects of diet on immunity and inflammation. 02 Innate lymphoid cells (ILC) subsets protect against worm infection and initiate repair of damaged tissues. A class of lymphoid cells called ILCs are first responders to bacterial and parasitic infections in animals and humans and they play key roles in host defense, gut barrier integrity, and homeostasis. The ILCs functionally mimic the better- defined class of adaptive lymphoid cells that provide immune memory against infection. ARS scientists in Beltsville, Maryland, and colleagues at the National Institutes of Health, Bethesda, Maryland, showed that ILC2 acquire functional activity in a stepwise manner during their development in mouse bone marrow and change little after external activation; unlike adaptive immune memory cells. There is extensive sharing of the regulatory circuits that control both ILC and adaptive immune cells. The discovery of specialized ILC subsets and the understanding of the molecular relationship between these diverse cell populations represent a major advance into how the immune system copes with infection, inflammation, tissue repair, and metabolic homeostasis; further, deciphering the precise nature of these circuits will help to better utilize environmental cues, such as changes in diet, to better control inflammation and responses to different types of infection. 03 Immune mechanisms against worm infection may also prevent colitis. Mice infected with worm parasites are also protected against intestinal colitis by the induction of regulatory cells that limit inflammation. ARS scientists in Beltsville, Maryland, and colleagues at the Tufts Medical Center in Boston, Massachusetts, showed that there is decreased expression of an intracellular signaling molecule called spleen tyrosine kinase (Syk) in intestinal regulatory cells in worm-infected mice and it was further shown that when these cells have a genetic deletion of Syk that colitis is reduced in mice. These regulatory cells normally sense pathogenic bacteria that damaged the gut barrier through the expression of special sugar binding receptors that often signal through the Syk pathway. The worm, and isolated worm products, reduce expression of these receptors and, in turn, reduce a strong inflammatory response. These results showed that inhibition of Syk induced regulatory cells that limit colitis. Understanding the molecular interactions that activate these regulatory cells, including changes in diet, will help reduce inflammation from both infectious and metabolic disease. 04 Novel screening system developed to identify anti-parasite molecules. A microfluidic device (�chip�) was developed to record the rhythmic contraction of the pharynx of parasitic worms that control worm feeding. These electropharyngeograms (EPGs) record responses from multiple worms per chip and can be used to evaluate novel drugs that target worm feeding to damage worm development. ARS scientists in Beltsville, Maryland, and colleagues at the University of California � San Francisco, George Washington University, and the University of Oregon validated microfluidic technologies using larval stages of parasitic human hookworms and the pig large round worm Ascaris suum against known anti-parasite drugs. Novel drugs are not being tested with this device. This showed that the microfluidic EPG platform provides a new tool for screening candidate drugs that can be further tested to eliminate parasitic worms from animals and humans. 05 A hookworm species found in the giant panda may also infect humans. There is potential for the transmission to humans of parasitic worms from animals facilitated by rapid urbanization and increased human- wildlife interactions. Ancylostoma ailuropodae is proposed as a new hookworm species identified from the giant panda. Microscopic structural and molecular markers were used by ARS scientists in Beltsville, Maryland, and colleagues at the Sichuan Agricultural University in China to indicate that A. ailuropodae from giant pandas is related to other hookworms that can infect humans and pose a possible threat to human health. This information provides a better understanding of the taxonomy, diagnostics and evolutionary biology of the hookworms, and cautions against unsupervised contact with pandas by human because of the risk of subsequent parasitic worm infection. 06 Better DNA sequence data improved analysis and further applications. Improving our ability to generate quality sequence data is critical to evaluating similarities and differences among parasites of veterinary and medical significance; further, parasites of the genus Trichinella, that can infect humans through unprocessed meats, provide a model system to test hypotheses for improving sequencing efforts. To this end, ARS scientists in Beltsville, Maryland, showed that a new and upcoming sequencing platform enabled 10- fold improvements in the genomic sequence and assembly of this model parasite. Also, ARS scientists in Beltsville, Maryland, and colleagues at Washington University, St. Louis, Missouri, used a computational genomics based approach to predict candidate proteins that can induce immunity against Trichinella, and other worms by screening databases for conserved parasite secreted proteins (secretome). Five candidate genes/proteins from the swine parasites Ascaris and Trichuris were ultimately selected from our prioritization scheme for expression and purification of recombinant proteins for a vaccine trials. Large scale production of these proteins is currently underway for use in mouse and pig vaccine trials scheduled later in 2017 and if successful, it is anticipated that this multivalent, pan-nematode cocktail of recombinant parasite proteins will induce immunity against a broad range of parasitic worm infections in animals and man. 07 ARS-developed diagnostic tests find application in the clinical diagnosis of human parasitic disease. Assays developed for veterinary purposes on rare occasion will find application in the study and treatment of human diseases, in particular worm parasites of animals that also can infect humans. A molecular test to differentiate species of Trichinella, a worm found in infected animal tissues that can infect humans after eating unprocessed meats, was used by ARS scientists in Beltsville, Maryland, and colleagues at the Northern Ontario School of Medicine, and University of Toronto, Ontario, Canada, for clinical diagnosis of a human infection derived from wild game. Along with other clinical tests, this assay unequivocally defined the infection in meats as derived from the freeze resistant strains of Trichinella which is prevalent in regions of North America. The identification and diagnosis also enabled tracing the infection back to the tissue of origin thereby preventing further infections from the consumption of the frozen contaminated meat. The impact of ARS research in this area supports and clearly transcends applications in veterinary sciences.
Impacts
(N/A)
Publications
- Thompson, P., Zarlenga, D.S., Liu, M., Rosenthal, B.M. 2017. Long-read sequencing improves assembly of Trichinella genomes 10-fold, revealing substantial synteny between lineages diverged over seven million years. Parasitology. 6:1-14 doi:10.1017/S0031182017000348.
- Dalcin, D., Zarlenga, D.S., Larter, N.C., Boucher, D.A., Merrifield, S., Lau, R., Ralevski, F., Cheema, K., Schwartz, K.L., Hoberg, E.P., Boggild, A.L. 2017. Trichinella nativa outbreak with rare thrombotic complications associated with meat from a black bear hunted in Northern Ontario. Clinical Infectious Diseases. 64:1367-1373. doi: 10.1093/cid/cix165.
- 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.
- Shih, H., Sciume, G., Mikami, Y., Guo, L., Sun, H., Brooks, S.R., Urban Jr, J.F., Davis, F.P., Kanno, Y., O'Shea, J.J. 2016. Developmental acquisition of regulomes underlies innate lymphoid cell functionality. Cell. 165:1120-1133. doi: 10.1016/j.cell.2016.04.029.
- 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.
- 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.
- Xie, Y., Hoberg, E.P., Urban Jr, J.F., Yanga, G. 2017. Ancylostoma ailuropodae sp. n. (Nematoda: Ancylostomatidae), a new hookworm parasite isolated from wild giant pandas in Southwest China. Parasites & Vectors. 10:227-296. doi: 10.1186/s13071-017-2209-2212.
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