2012 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.
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.
Methods to neutralize porcine transmissible gastroenteritis virus (TGEV). The spike (S) protein is a key structural protein of TGEV as well as other coronaviruses. This protein is membrane bound, is located in the viral envelope, and assists in the binding of viral particles to cell receptors 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 was 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.
Transmission models for viral and bacterial pathogens. Modeling can be an important method to predict the spread of disease. However, the dearth of epidemiological data on animal pathogens makes testing new algorithms difficult. Often it is possible to develop and adapt systems for modeling human diseases to those that affect livestock animals. 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 to model swine viral diseases. Results showed that the D-R model, which was originally created to define 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 predictions 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 algorithm 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 conditions.
Cytokine DNA as an adjuvant for vaccinating against swine viral diseases. Direct injection of plasmid DNA has been evaluated as a candidate strategy 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 epidemic diarrhea, a highly contagious enteric disease of swine. The Spike (S) protein is one of the main structural proteins of PEDV that is capable of inducing neutralizing antibodies in the host. We generated three distinct plasmid DNA constructs in a eukaryotic expression vector; one encoding the 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 of efficacious viral gene vaccines.
Secreted parasite enzymes capable of modulating host immunity. Parasites 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 this adaptive process. Nucleotide metabolizing enzymes are a class of molecules that parasites use to control local environments. These enzymes are generally secreted into the surrounding space to degrade interstitial nucleotides and prevent initiation of the innate immune response. We identified one such enzyme in the large parasitic round worm Ascaris suum. 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 a related species, A. lumbricoides, infects nearly 1 of 3 of all humans worldwide.
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 express 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 humans, 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 pigs and humans and could optimize the successful application of helminth (worm) therapy to reduce inflammation during autoimmune diseases in humans. The application of helminth therapy is currently part of a multi-center National Institutes of Health and European Union trail of safety and efficacy.
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.
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.