Progress 10/01/15 to 09/30/16
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 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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. 08 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. 09 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.
Impacts
(N/A)
Publications
- Guo, L., Huang, Y., Chen, X., Hu-Li, J., Urban Jr, J.F., Paul, W.E. 2015. Innate immunological function of TH2 cells in vivo. Nature Immunology. 16:1051-1059.
- Vannella, K.M., Ramalingam, T.R., De Queiroz, P.R., Sciurba, J., Barron, L. , Borthwick, L., Smith, A.D., Mentink-Kane, M., White, S., Thompson, R.W., Cheever, A.W., Bock, K., Moore, I., Fitz, L.J., Urban Jr, J.F., Wynn, T.A. 2016. Acidic Chitinase Limits Allergic Inflammation and Promotes Intestinal Nematode Expulsion. Nature Immunology. 17(5):538-44 doi: 101038/ ni.3417.
- 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. CD103+ dendritic cells suppress Helminth-driven Type 2 immunity through constitutive expression of IL-12. Immunity. 213(1):35-51. doi: 10.1084/jem.20150235.jeq201612
- 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.
- 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. doi: 10.1152/ajpgi.00461.2014.
|
Progress 10/01/14 to 09/30/15
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. Immunization of mice with cloned Ascaris suum apyrase, as a model to detect immunological activity for use in pigs, failed to show immune protection against a challenge infection with A. suum infective eggs. To test an alternative strategy, we used a computational based approach to predict candidate proteins with immunogenic potential by screening orthologous protein families of a pan-nematode secretome database. 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 challenged with three parasitic worms that inhabit different regions of the intestine; A. suum, Heligmosomoides polygyrus bakeri and Trichuris muris. 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. This very promising result is now being tested in a repeat study to confirm the initial results. The work is partially supported through a reimbursable cooperative agreement with Washington University, St. Louis, MO with funding from the National Institute of Food and Agriculture (8042- 32000-094-03R). Accomplishments 01 Parasite modulation of host immunity. Ascaris suum, the large roundworm, is a zoonotic parasite of swine and closely related to a roundworm species that infects nearly 25% of the world�s population. It and related parasites have evolved methods for controlling the host immune response. Agricultural Research Service (ARS) scientists in Beltsville, Maryland, studied a class of parasite enzymes (called ATP diphosphohydrolases) and determined that they modify the innate immune responses by removing key nucleotide substrates that are released from damaged cells and that would otherwise provoke an inflammatory response. Thus, proteins secreted by the parasites degrade factors that mediate the host�s cell-to-cell signaling and stress responses. Finding parasite proteins that potentiate colonization and survival may hasten progress towards vaccines or drugs that prevent or minimize the damage caused by these infections. Vaccines are being developed for other livestock parasites using this technology because this feature of parasite inhibition of signals that activate the host are common to other parasites and hosts. 02 Resource limitation has consequences on co-infection for both hosts and parasites. Most animals are concurrently infected with multiple parasite species and live in environments with fluctuating resource availability. Resource limitation can influence host immune responses and the degree of competition between co-infecting parasites. To test how resource limitation affects immune trade-offs and co-infection outcomes, a factorial experiment using laboratory mice was conducted where mice were given a standard or low protein diet, dosed with two species of parasitic worms (alone and in combination), and then challenged with a micro-parasite. Co-infection was found to influence parasite survival and reproduction via host immunity, but the magnitude and direction of responses depended on both resources and the combination of co-infecting parasites. These findings highlight that resources and their consequence for host defenses are a key context that shapes the magnitude and direction of parasite interactions. The work, a collaborative effort with scientists from the University of Georgia, will be used to determine how changes in diet can improve the health of animals co-infected with different parasites. 03 Immune antibodies and helminth (parasitic worm) products drive chemokine and inflammatory cell crosstalk to promote intestinal repair. Worm parasites can cause damage when migrating through host tissues then rapid tissue repair is needed to prevent bleeding and bacterial dissemination, particularly from the intestine. Mice lacking antibodies or activating antibody receptors had impaired intestinal tissue repair following worm infection, but supplying antibody-rich immune serum partially rescued wound healing even in infected mice lacking antibodies. Impaired healing was associated with inflammatory cells expressing messenger proteins and chemokines at reduced levels, and by smooth muscle cells within intestinal lesions. Antibodies and worms together triggered chemokine production by inflammatory cells in test tube studies via engagement of Fc receptors, and chemokine secretion by intestinal muscle cells was directly activated by worms. Blocking chemokine production during worm infection reproduced the delayed wound repair observed mice lacking endogenous antibodies and receptors. Conditioned media used to grow human inflammatory cells stimulated with worms that infect humans together with immune serum, promoted chemokine- dependent wound closure human muscle cells in the test tube. These findings suggest that worms and antibodies instruct a chemokine driven crosstalk between inflammatory cells and muscle cells in the intestine to promote intestinal repair. This work, in collaboration with colleagues in Switzerland, will be expanded to look for parasite products that can be harnessed in clinical settings of impaired wound healing. 04 Immune and inflammatory responses in pigs concurrently infected with two parasitic worms. The immune response mounted by pig to a parasitic infection can vary considerably depending on the presence or absence of other parasites. For example, whipworm elicits a strong immune response in the large intestine leading to its rapid expulsion from the pig, but also has a strong antagonistic effect on hookworms when pigs are co- infected. ARS scientists in Beltsville, Maryland, in collaboration with scientists in Denmark examined the basis of such interactions by comparing components of the immune responses mounted by pigs harboring one or a pair of infections. Whipworm was found to markedly enhance antibody responses against hookworm. Pigs infected with only whipworm exhibited a strong immune response whereas those infected only with hookworm displayed a weaker, delayed response. Interactions were found between the two worm parasites with regard to the production of several immune activating proteins or cytokines. An understanding of the interactions between these two common worm infections in pigs can help devise more efficacious vaccines to protect animal and human health. 05 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 the pig immune response. ARS scientists in Beltsville, MD increased its content approximately 1.5 fold by adding more than 1,600 gene entries since last year and by manually assembling and annotating 5,820 other transcripts of immune and metabolism related genes as well as all known epigenetic regulators from 5,428 genes (28% of the pig genome and including 931 genes not found in the current publically available pig genome assembly); 1,395 of these full-length gene sequences were submitted to Genbank (Bioproject: PRJNA80971). These sequences were also used to perform several transcriptomic and epigenetic�based studies (miRNA and mRNASeq expression profiling) including responses of pig inflammatory cells from the lung to vitamin A or to protein messenger cytokines like interleukin-4 (IL-4) or interferon-gamma (IFN-g) and the bacterial product LPS, as well as in the pig response to whipworm. We observed 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/IFN-g. 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 45,000 times by investigators from more than 30 laboratories worldwide.
Impacts
(N/A)
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.
- Dong, B., Zarlenga, D.S., Ren, X., 2014. An overview of live attenuated recombinant pseudorabies viruses for use as novel vaccines. Journal of Immunology, 2014. 1-10. Article ID 824630, DOI:10.1155/2014/824630.
- 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.
- Zarlenga, D.S., Hoberg, E.P., Rosenthal, B.M., Mattiucci, S., Nascetti, G. 2014. Anthropogenics: Human influence on global and genetic homogenization of parasite populations. Journal of Parasitology. 100(6):756-772. doi: 10. 1645/14-622.1.
|
Progress 10/01/13 to 09/30/14
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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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. 08 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.
Impacts
(N/A)
Publications
- Fayer, R., Elsasser, T.H., Gould, R., Solano Aguilar, G., Santin, M., Urban Jr, J.F. 2014. Blastocystis tropism in the pig intestine. Parasitology Research. 113:1465-1472.
- 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.
|
Progress 10/01/12 to 09/30/13
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. Ascaris suum is a widespread and prevalent parasite of pigs, and is closely related to Ascaris lumbricoides, which infects nearly 1/3 of the global human population. These parasites are highly capable of adapting to, and modifying, their host environments. Their ability to persist in pigs and people depends, in part, on being able to degrade excreted products that would otherwise provoke strong innate and acquired immune responses. Nucleotide metabolizing enzymes comprise one class of molecules that enables such parasites to blunt host immunity. These enzymes are generally secreted into the surrounding space and, by degrading interstitial nucleotides, prevent initiation of the innate immune response. ARS researchers at the Beltsville Agricultural Research Center and the Beltsville Human Nutrition Center cloned and expressed the gene encoding this enzyme and showed it to be biologically functional. The protein specifically degrades those nucleotides that would otherwise initiate stress responses. The team identified the active site within the enzyme by demonstrating that changes to it abrogated enzymatic function. Establishing the biological function and mechanistic basis for this enzyme�s action lays the groundwork for vaccines that might substantially limit the damage incurred by these prevalent and damaging parasites to veterinary and human health. Accomplishments 01 Modeling the spread of viral and bacterial pathogens. Modeling is an important way to anticipate how diseases spread and to intervene early to curtail or eliminate dissemination. Insufficient epidemiological data on animal pathogens makes testing new algorithms on this broad group of organisms problematic. As such, developing systems for modeling animal pathogens can often be tested on human diseases then adapted to livestock when sufficient data comes available. Herein, we developed an algorithm (D-R model) and tested it using data related to the spread of ceftazidime-resistant Escherichia coli. The availability of extensive human data provided support that such a model can be adapted to swine viral diseases. Results showed that the D-R model, which was originally created to define trends in the transmission of swine viral diseases, could be successfully modified to evaluate trends in the appearance of ceftazidime-resistant E. coli. Predictions based on limited data successfully mirrored actual increases in drug resistance through 2005, a decrease between 2005 and 2008, and a dramatic inflection point and abrupt increase beginning in 2008. This is consistent with a resistance profile where changes in drug intervention temporarily delayed the upward trend in the appearance of the resistant phenotype; however, resistance quickly resumed its upward momentum in 2008. The same model was successfully tested in evaluating the incidence and death rates caused by tuberculosis in the U.S. and in Germany. The results exceeded the current algorithms used by the World Health Organization to monitor common human diseases. The success of this algorithm suggests it can be applied to a wide range of viral and bacterial infections and can be adapted to animal infections. Its strength lies in the predictive capabilities using minimal amounts of high quality data and its ability to �self-correct� amidst data anomalies. Current algorithms falter under both these nature conditions. 02 Description of a new class of anti-parasite drugs against pig roundworms. Ascaris suum is an intestinal parasitic roundworm of pigs that is very closely related to Ascaris lumbricoides, a major intestinal parasitic roundworm of humans that infects more than one billion people worldwide. Because of reduced efficacy and the threat of resistance to the current small set of approved drugs to treat Ascaris infections, new treatments are needed. ARS scientists at the Beltsville Agricultural Research Center and the Beltsville Human Nutrition Center in collaboration with scientists at the Division of Biological Sciences, University of California, San Diego tested the effectiveness of Cry5B, a roundworm-killing protein made by the natural soil bacterium Bacillus thuringiensis which represents a promising new class of anti-worm proteins (anthelmintics). They demonstrated, for the first time, that this drug binds receptors on A. suum and kills cultured larval and adult worms. Moreover, oral administration of the drug to infected pigs resulted in near elimination of the infection from the intestine. This novel approach holds excellent potential to protect swine and human health. 03 Culture-based testing of anti-parasite drugs against economically important worm species. Parasitic worms infect humans and livestock worldwide with negative effects on health and productivity. Little research has been conducted to develop new anti-worm drugs (athelmintics) owing to low perceived returns on such investment; nevertheless, diminishing efficacy of available drugs is cause for concern among livestock producers and human health professionals. Testing new drugs in livestock or humans can be prohibitively expensive. Reliable, low-cost systems for preliminarily screening new drugs would therefore be of tremendous value. ARS Researchers working at the Beltsville Agricultural Research Center and the Beltsville Human Nutrition Center in collaboration with scientists at the Division of Biological Sciences, University of California, San Diego demonstrated the feasibility of this approach by testing a number of established anti-parasitic drugs in culture, and then in rodents. This approach holds promise for evaluating many new compounds. 04 Comparing the human and pig genome to model disease. The domestic pig is known as an excellent model for human immunology and the two species are infected by many similar pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the gene complex that regulates the immune system, known as the pig immunome, is not well-characterized. The complete sequencing of the pig genome has provided the opportunity to annotate and categorize the pig immunome, and compare and contrast pig and human immune systems. This extensive annotation dramatically extends the genome-based knowledge of a major portion of the porcine immunome. The functional approach of using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Analysis of this core immunome cluster confirmed rapid evolutionary change in this set of genes, and the importance of the pig�s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence 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.
Impacts
(N/A)
Publications
- Ding, F., Zarlenga, D.S., Ren, Y., Li, G., Luan, J., Ren, X. 2011. Use of the D-R model to define trends in the emergence of Ceftazidime-resistant Escherichia coli in China. PLoS One. 6(12):e27295.
- Ren, Y., Ding, F., Zarlenga, D.S., Ren, X. 2012. Incidence rates and deaths of tuberculosis in HIV-negative patients in the United States and Germany as analyzed by new predictive model for infection. PLoS One. 7(10) :e42055.
- Yang, Y., Qina, W., Zarlenga, D.S., Cao, L., Tian, G. 2013. TsDAF-21/Hsp90 is expressed in all examined stages of Trichinella spiralis. Veterinary Parasitology. 194(2-4):171-174.
- Urban, Jr., J.F., Hu, Y., Miller, M.M., Scheib, U., Yiu, Y.Y., Aroian, R.V. 2013. Bacillus thuringiensis-derived Cry5B has potent anthelmintic activity against Ascaris suum. PLOS Neglected Tropical Diseases. 7(6) :e2263.
- Blum, A.M., Hang, L., Setiawan, T., Urban Jr, J.F., Stoyanoff, K., Leung, J., Weinstock, J.V. 2012. Heligmosomoides induces tolerogenic dendritic cells that block colitis and prevent antigen-specific gut Tcell responses. Journal of Immunology. Sept 1:189(5):2512-2520.
- Smith, A.D., Cheung, L., Shea-Donohue, T., Urban Jr, J.F. 2013. Selenium status alters the immune response and expulsion of adult Heligmosomodies bakeri in mice. Infection and Immunity. 81(7):2546-2553.
- Yang, Z., Grinchuk, V., Smith, A.D., Qin, B., Bohl, J.A., Sun, R., Notari, L., Zhang, Z., Sesaki, H., Urban Jr, J.F., Shea-Donahue, T., Zhao, A. 2013. Parasitic nematode-induced modulation of body weight and associated metabolic dysfunction in mouse models of obesity. Infection and Immunity. 81(6):1905-1914.
- Zhao, A., Yang, Z., Sun, R., Grinchuk, V., Netzel-Arnett, S., Anglin, I.E., Driesbaugh, K.H., Notari, L., Bohl, J.A., Madden, K.B., Urban Jr, J.F., Antalis, T.M., Shea-Donahue 2013. SerpinB2 is critical to Th2 immunity against enteric nematode infection. Journal of Immunology. 190(11):5779-87.
- Yang, Z., Grinchuk, V., Urban Jr, J.F., Bohl, J., Sun, R., Notari, L., Yan, S., Ramalingam, T., Keegan, A.D., Wynn, T.A., Shea-Donohue, T., Zhao, A. 2013. Macrophages as IL-25/IL-33-responsive cells play an important role in the induction of type 2 immunity. PLoS One. 8(3):e59441,1-11.
|
Progress 10/01/11 to 09/30/12
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. Alveolar macrophages (AM) normally make up greater than 90% of the cells in the airspaces of the lung and are the first line of defense against airborne particles and pathogens that contribute to lung health and disease. Information on the biology and function of these cells comes largely from studies in mice with little comparable information available for pig and human. To address this, we studied basal gene expression in porcine AM by Illumina-based next generation sequencing technology. On average, 25 million 78-base pair reads per sample were obtained. These were assembled into contigs by the RNA-Seq module of the CLC Genomics Workbench using four different pig sequence databases as the scaffolds; Ensembl version 9, University of California Santa Cruz (UCSC) redundant RNA database, National Center for Biotechnology Information's RefSeq database, and the Harvard pig assembled expressed sequence tag EST database. To address expression of sequences that may be missing from the analysis due to the unfinished pig genome, human and bovine genomes also were used as scaffolds. To expand the scale of gene expression in AM, primary explanted porcine AMs were plated and cultured for 42 hours with inducing molecules that included pig cytokines and derivatives of vitamin A followed by RNA isolation from the cells. The cDNA was then synthesized via reverse transcription to measure gene expression through large scale real-time polymerase chain reaction (PCR). The levels of gene expression were calculated after normalization via the Reads Per Kilobase of exon model per Million mapped reads. Canonical pathway analysis of highly expressed transcripts was done with Ingenuity Pathway Analysis. Information on genes and annotations as well as real-time PCR assay sequences can be found in our online Porcine Immunology and Nutrition (PIN) database. A total of 130 new full or partial length porcine genes were cloned from this project including many key immune regulatory molecules and splice variants. Real time PCR results confirm the deep sequencing data from our bioinformatic pipeline with a correlation of 0.999. Correlations of gene expression among animal samples generally range from 0.928-0.989 including both biological and technical duplicates. Results from pathways and bio-function analysis were consistent with the biological roles of AM defined in the mouse. This data is a proof of principle for transcriptomic evaluation of AM in the pig and is the basis for completing the objectives of the project plan. Ascaris suum apyrase was successfully cloned and an enzymatically functional molecule was produced. This parasite-derived product can now be used as a test substance on pig AM. Accomplishments 01 Worm burden disrupts pig intestinal microbiota and improves function. Pigs infected with Trichuris suis (whipworm) had major changes in the composition of bacterial populations in the intestine. Some pigs expres resistance to infection and expel the worms while others are susceptible and worms persist. There are significant differences in the bacterial populations that are associated with the worm burden. In particular, Campylobacter bacteria, a group that can cause disease in pigs and human were significantly reduced in whipworm resistant pigs but increased in susceptible pigs. The host response in the intestine of worm resistant pigs was also enriched for protective gene expression that cleared the worm infection and improved tissue healing and function. These findings should facilitate development of strategies for parasitic control in pig and humans and could optimize the successful application of helminth (wo therapy to reduce inflammation during autoimmune diseases in humans. T application of helminth therapy is currently part of a multi-center National Institutes of Health and European Union trail of safety and efficacy. 02 Methods to neutralize porcine transmissible gastroenteritis virus (TGEV) The spike (S) protein is a key structural protein of TGEV as well as oth coronaviruses. This protein is membrane bound, is located in the viral envelope, and assists in the binding of viral particles to cell receptor that facilitate invasion. It is an important immune target for the host in neutralizing infective virus. Four antigenic sites, A, B, C and D, have been defined in the S protein. Of these, the region encoding antigenic sites A and to a lesser extent D, herein defined as S-AD, are most critical in eliciting host neutralizing antibodies. We amplified, cloned, and expressed the S-AD fragment from the porcine TGEV in a prokaryotic expression vector and rabbit polyclonal antiserum was generated using the recombinant S-AD (rS-AD) protein. Results clearly indicated that polyclonal serum recognized TGEV and reduced cell infectivity by 100%. Furthermore, the truncated rS-AD peptide itself wa able to bind to the surface of cells in a competitive manner and completely inhibit viral infection. These results indicate that this construct and this approach can be used to attenuate TGEV infections in swine and may be applicable to other coronaviruses. 03 Transmission models for viral and bacterial pathogens. Modeling can be important method to predict the spread of disease. However, the dearth epidemiological data on animal pathogens makes testing new algorithms difficult. Often it is possible to develop and adapt systems for modeli human diseases to those that affect livestock animals. Herein we developed an algorithm (D-R model) and tested it using data related to t spread of ceftazidime-resistant Escherichia coli. The availability of extensive human data provided support to model swine viral diseases. Results showed that the D-R model, which was originally created to defin trends in the transmission of swine viral diseases, can be adapted to evaluating trends in the appearance of ceftazidime-resistant E. coli. Using only a limited amount of data to initiate the study, our predictio closely mirrored the changes in drug resistance rates which showed a steady increase through 2005, a decrease between 2005 and 2008, and a dramatic inflection point and abrupt increase beginning in 2008. These changes were predicted using the D-R model. The success of this algorit suggests its applicability to a wide range of animal viral and bacterial infections with predictive capabilities using minimal amounts of high quality data and its ability to "self-correct" amidst data anomalies. Current algorithms falter under both these naturally-occurring condition 04 Cytokine DNA as an adjuvant for vaccinating against swine viral diseases Direct injection of plasmid DNA has been evaluated as a candidate strate for vaccination. A great number of studies have shown that eukaryotic expression plasmids can induce protective immune responses. The porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemi diarrhea, a highly contagious enteric disease of swine. The Spike (S) protein is one of the main structural proteins of PEDV that is capable o inducing neutralizing antibodies in the host. We generated three distin plasmid DNA constructs in a eukaryotic expression vector; one encoding t full-length S protein, the second encoding the N-terminal fragment (S1) which contains potent antigenic sites, and the third expressing the porcine interleukin-18 (pIL-18). Using a mouse model, the host immune responses triggered by the full-length and truncated S proteins in the presence of pIL-18 was used to determine if IL-18 offered adjuvant activity. Results presented here showed that the full-length S gene unilaterally induced a better immune response than the truncated form of the protein. This study will help develop better ways to enhance host protection against common swine viruses and facilitate the development o efficacious viral gene vaccines. 05 Secreted parasite enzymes capable of modulating host immunity. Parasite are inherently capable of adapting to change and new environments in the host. Fundamental to successful parasitism is modulation of the immune system by parasite-derived products that facilitate parasite development Parasite co-evolution with the host and its ability to flourish amidst strong innate and acquired immune responses, have played key roles in th adaptive process. Nucleotide metabolizing enzymes are a class of molecules that parasites use to control local environments. These enzym are generally secreted into the surrounding space to degrade interstitia nucleotides and prevent initiation of the innate immune response. We identified one such enzyme in the large parasitic round worm Ascaris suu It has been cloned and a biologically functional protein has been expressed. The protein is capable of degrading only guanine diphosphate and uridine diphosphate nucleotides which are believed to be involved in signaling and stress responses. The role of this enzyme in facilitating parasite survival is under investigation. This could provide a strategy to vaccinate animals and humans because A. suum widely infects swine and related species, A. lumbricoides, infects nearly 1 of 3 of all humans worldwide.
Impacts
(N/A)
Publications
- Butler, J.E., Sun, X., Wertz, N., Lager, K.M., Chaloner, L., Urban Jr., J., Francis, D.L., Nara, P.L., Tobin, G.J. 2011. Antibody repertoire development in fetal and neonatal piglets XXI. Usage of most VH genes remains constant during fetal and postnatal development. Molecular Immunology. 49(3):483-494.
- Vlaminck, J., Martinez-Valladares, M., Tilleman, K., Deforce, D., Dewilde, S., Moens, L., Urban Jr, J.F., Claerebout, E., Vercruysse, J., Geldhof, P. 2011. Immunizing pigs with Ascaris suum hemoglobin increases the inflammatory response in the liver but fails to induce a protective immunity. Parasite Immunology. 33:250-254.
- Flores-Mendoza, L., Sotelo-Mundo, R.R., Dawson, H.D., Mwangi, W., Hernandez, J. 2010. Characterization of porcine CD205. Developmental and Comparative Immunology. 34(7):715-721.
- Kvist, P.H., Iburg, T., Dawson, H.D., Jensen, H.E. 2010. Effect of subcutaneous glucose sensor implantation on skin mRNA expression in pigs. Diabetes Technology & Theraputics. 10:791-799.
- Meng, F., Zhao, Z., Li, G., Suo, S., Shi, N., Yin, J., Zarlenga, D.S., Ren, X. 2011. Bacterial expression of antigenic sites A and D in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection. Virus Genes. 43:335-341.
- Suo, S., Li, X., Li, P., Li, G., Ren, Y., Zarlenga, D.S., Ren, X. 2012. Immune responses induced by DNA vaccines bearing Spike gene of PEDV combined with porcine IL-18. Virus Research. DOI: 10.1016/j.virusres.2012. 05.007.
- Shea-Donohue, T., Notari, L., Stiltz, J., Sun, R., Madden, K.B., Urban Jr, J.F., Zhao, A. 2010. Role of enteric nerves in immune-mediated changes in protease activated receptor 2 effects on gut function. Neurogastroenterology & Motility. 10:1138-e291.
- Santiago, H.C., Leevan, E., Bennuru, S., Ribeiro-Gomes, F., Mueller, E., Wilson, M., Wynn, T., Garboczi, D., Urban Jr, J.F., Nutman, T.B. 2012. Molecular mimicry between cockroach and helminth glutathione S- transferases promotes cross-reactivity and cross-sensitization. Journal of Allergy Clinical Immunology. 130:248-256.
|
|