Location: Animal Parasitic Diseases Laboratory2016 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:
This is the final report for the project 8042-32000-094-00D that terminates in November 2016. Substantial and impactful results were obtained over the 5 years of the project. All milestones were either Met or Substantially Met during the period of the Project plan. A class of enzymes called apyrases was studied to determine if they have the capability to 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. One such enzyme was identified in the large round worm nematode parasite, Ascaris suum (A. suum), and was cloned and expressed with activity to degrade only certain nucleotides (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). 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. Mice were immunized with the cloned apyrase using different adjuvant products and routes of immunization, and given a challenge infection that includes a novel application of several different nematode species, including A. suum, Trichuris muris (T. muris), and Heligmosomoides polygyrus bakeri (H. p. bakeri), to look concurrently for a pan-nematode protection against several parasitic worms. Immunization failed to show immune protection. To test an alternative strategy, we used a computational based approach to predict candidate proteins with immunogenic potential by screening for conserved protein families in a pan-nematode database of parasite secreted proteins (secretome). Among the top 17 candidates derived from our prioritization scheme, three were chosen for cloning, expression and purification of recombinant proteins for a vaccine trial in mice. Parasite recovery data showed a significant reduction in A. suum larvae in the lungs and H. p. bakeri adults in the intestines of mice vaccinated with the cloned parasite proteins; there was no significant reduction in the recovery of T. muris larvae. To further refine this strategy, vaccine candidates were developed from parasitic worm excretory/secretory products (ESPs or ‘secretome’) that activate host immunity. In T. suis infections, a gastrointestinal nematode of swine, there is a general trend for “resistant” pigs to harbor low numbers of parasites, and to exhibit a high IgG1 immune response and a lower IgG2 response. In contrast, “susceptible” pigs with high worm burdens have a low or equivalent IgG1 to IgG2 ratio. To examine this phenomenon, parasite secreted proteins were separated on 2 dimensional polyacrylamide gels and used to select immunodominant, putative protective antigens by differentially and immunologically screening the gels with sera pooled from susceptible animals and sera pooled from resistant animals. Following the analysis, a subset of sequences was selected in both categories for database analysis and DNA sequencing. The selected proteins were first identified by database mining, three of which have been cloned and sequenced. The cloned fragments have been modified for protein production for subsequent analysis in cellular and whole mouse assays for protective responses. Once validated, the experiments will be scaled up and tested in a larger pig immunization assay. Another approach to achieve the same goal involved monitoring nutrition levels to identify appropriate 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 (type 3) 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, 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. Work on the transcriptome of macrophages from pig lungs continued to provide bioinformatic tools to evaluate epigenetic features (nongenetic influences on gene expression) of exposure of swine to parasitic nematodes and vitamin A. Much of this information is regularly recorded in the Porcine Translational Research Database that fosters comprehensive and integrated analysis of the pig genome, and provides important tools for global analysis and data-mining of pig immune responses.
1. Developmental acquisition of the regulome (all the regulatory components within a cell) during innate immunity. Innate lymphoid cells (ILCs) play a key role in the initial host defense against early infection. ARS researchers in Beltsville, Maryland, observed that genome-wide chromatin in proximity to effector genes was selectively accessible after activation of ILCs in a stepwise manner during development of the mouse, but did not change after stimulation of these cells. 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.
2. Evidence for gene transfer between worms and either bacteria or plants may play a role in the evolution of nematode parasitism. Parasitism among nematodes has occurred in multiple, independent events. Deciphering processes that drive species diversity and adaptation are keys to understanding parasitism and advancing control strategies. ARS scientists in Beltsville, Maryland, explored the association between changes in conserved protein regions (domains) over the course of metazoan evolution, and the relationship between these changes and the ability and/or result of nematodes adapting to their environments in the hope of identifying targets for immune intervention and control. A protein, cyanase, was found in a select few parasitic nematodes, including those belonging to parasites grouped into the genera Trichinella and Trichuris, appears to have bacterial or fungal origins. The cynase gene produces a functionally active protein that is not present in any mammals which makes it a prime candidate for industry partners interested in development of anti-parasitic compounds.
3. Cholinergic muscarinic receptors contribute to intestinal homeostasis. The contribution of acetylcholine activated muscarinic receptors to intestinal homeostasis, clearance of intestinal pathogens, and modulation of immune function remains relatively undefined. Wild-type (WT) and type 3 muscarinic receptor (M3R)-deficient mice (Chrm3-/-) were infected with a parasitic worm to determine the contribution of M3R to host defenses. ARS scientists in Beltsville, Maryland, showed that worm-infected, Chrm3-/- mice expressed diminished protective immune-related proteins and delayed parasite clearance compared to WT mice. This information is important to a One Health model where observations in animals help to develop strategies that not only control parasitic infection but modulation of human intestinal physiology and health.
4. Naturally produced acidic chitinases are protective against parasitic worms. Acidic mammalian chitinase (AMCase) is known to be induced by allergens and parasitic worm infection, yet its role in immunity is unclear. ARS researchers in Beltsville, Maryland, showed that AMCase deficient (-/-) mice had a normal response to allergens or to parasitic worms in the lung, but showed a profound defect in protective immune responses in the intestines. The impaired immunity in the intestines was associated with reduced mucus production; a decrease in the expression of proteins associated with immune protection; and reduced numbers of specialized dendritic cells that regulate cellular homing to the intestine. This observation showed a distinct role for AMCase in localized protection against worm infection that is distinct from responses to soluble allergens in the lung. This is important because it shows that worms can be eliminated by a protective response without deleterious allergic responses that can accompany worm clearance.
5. An intestinal receptor for the cytokine Interleukin-13 (IL-13) is critical to parasite clearance and is functionally similar to the immune protein, interleukin-4 (IL-4). Worm infection upregulates cell receptors for both IL-4 and IL-13 to promote worm clearance. ARS researchers in Beltsville, Maryland, used mice deficient in the receptor IL-13Ra1 (IL-13Ra1-/-) to examine the contribution that the IL-13 protein may have on the receptor for IL-4 and its abilities to activate immune responses to either a primary gastrointestinal nematode infection or to a secondary infection following a memory response. Results showed that 13Ra1-/- mice had impaired worm expulsion and higher worm egg production relative to wild-type (normal) mice. Also, goblet cell numbers, and production of the resistin-like molecule beta were attenuated significantly in 13Ra1-/- mice following a secondary infection. There were no apparent differences on smooth muscle function or epithelial permeability, between wild-type and 13Ra1-/- mice following a primary infection; however, these responses were absent 13Ra1-/- mice during a secondary worm infection. These results show that activation of IL-13Ra1 is critical for key aspects of immune and functional responses to worm infection and in particular, to secondary infections that are normally expelled from the intestine.
6. Specialized immune cells i.e., dendritic cells, that migrate to the gut, express a protein that facilitates worm expulsion. CD8a(+) and CD103(+) are dendritic cells (DCs) that play a central role in the development of responses to microbial pathogens. ARS researchers in Beltsville, Maryland, examined host responses to parasitic worm infection in Batf3-/- deficient mice that do not have cellular DC subsets. It was observed that several of the typical host responses to worm infection were enhanced in Batf3(-/-) mice. The key to altering the activity of these DCs was the absence of production of a cytokine called IL-12 that is normally produced after these cells are stimulated by microbial products. These findings identify a previously unrecognized role for migratory CD103(+) DCs in antagonizing worm infection and the associated cellular and antibody responses that can have deleterious effects on host tissues.
7. The dietary trace mineral selenium is critical for optimal clearance of parasitic worms. The micronutrient 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. ARS researchers in Beltsville, Maryland, tested the host response to a worm infection in mice fed diets that were selenium-adequate (0.08 parts per million - ppm) and found that intestinal M2 cells contributed to decreasing adult worms and egg production when compared with infection of mice on selenium-deficient (<0.01 ppm) diets. Further, increases in dietary selenium to supra-physiological levels (0.4 ppm) were not different from selenium adequate diets. These results demonstrate that optimal expression of selenium-dependent proteins and selenium-dependent production of 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.
8. Some human and pig inflammatory responses are more similar to each other than either is to mice. Emerging evidence suggests that pigs are a scientifically acceptable intermediate species between rodents and humans and therefore able to better model immunity and inflammation. ARS scientists in Beltsville, Maryland comparatively evaluated genes in the inflammasome (genes associated with inflammation) which involves responses to microbial products. Conservation in sequence and structure among these genes revealed a much higher level of sequence similarity among humans and pigs then to mice. This work supports using pigs to model both human immune and inflammatory responses to infection. Caution must be exercised, however, as pigs differ from humans in several fundamental pathways.
9. Expanded scope of the Porcine Translational Research Database. 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. ARS scientists in Beltsville, Maryland increased the content of this database by approximately 1.5 fold by adding more than 2,200 gene entries during this report period (33.6% of the pig genome and including 1,212 genes not found in the current publically available pig genome assembly). The database now contains a complete representation of the "Transportome" (Solute Carrier and the ATP binding Cassette Super-families), complete annotations of 356 genes that compose porcine cell surface leukocyte 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 porcine members), and mucus-related Mucin Super-families (20 porcine members). This information will be useful for modeling human disease in pigs because of the close evolutionary and functional features of the two species. The database is part of a collaborative initiative with scientists in the Beltsville Human Nutrition Research Center and has been accessed over 50,000 times by investigators from more than 30 laboratories worldwide.
5. Significant Activities that Support Special Target Populations:
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.
Everts, B., Tussiwand, R., Dreesen, L., Fairfax, K.C., Huang, S.C., Smith, A.M., Oneil, C.M., Lam, W.Y., Edelson, B.T., Urban Jr, J.F., Murphy, K.M., Pearce, E.J. 2016. Migratory CD103+ dendritic cells suppress Helminth-driven Type 2 immunity through constitutive expression of IL-12. Journal of Experimental Medicine. 213(1):35-51. doi: 10.1084/jem.20150235..
Huang, Y., Guo, L., Qiu, J., Chen, X., Hu-Li, J., Siebenlist, U., Williamson, P.R., Paul, W.E., Urban Jr, J.F., Paul, W.E. 2015. IL-25-responsive, lineage-negative, KLRG1(hi) cells are multipotential “inflammatory” type-2 innate lymphoid cells. Nature Immunology. 16(2):161-169. doi: 10.1038/ni.3078
Suo, S., Wang, X., Zarlenga, D.S., Ri-E, B., Ren, Y., Ren, X. 2015. Phage-display for identifying peptides that bind the spike protein of transmissible gastroenteritis virus and possess diagnostic potential. Journal of Parasitology. 51(1):51-56.
Cao, L., Ge, X., Gao, Y., Zarlenga, D.S., Li, X., Yin, X., Qin, Z., Liu, J., Ren, X., Li, G. 2015. Putative phage-display epitopes of the porcine epidemic diarrhea virus S1 protein and their anti-viral activity. Virus Genes. 51(2):217-224.