Title:Immunomodulation of pathogen-host interactions
In this thesis we have reviewed the scientific literature related to the subject matters of our work. We have briefly summarized the literature of the innate immune response focusing on the research related to pathogen recognition. These include the Toll-like receptors (TLR) and other pathogen recognition receptors (PRR). We have also reviewed the literature of dendritic cells (DC) with the focus on the pathogen recognition receptor, dendritic cell specific ICAM grabbing non-integrin (DC-SIGN) and the roles of dendritic cell TLRs in induction of Th1 helper response. In addition, we reviewed briefly the literature of the particle based carrier systems as well as the interaction of mycoplasmas with TLRs and M. gallisepticum (MG) infection. In Experiment 1, we described the removal of a TLR-4 agonist molecule, endotoxin, from different solutions using affinity technology. The endotoxin concentrations were measured using the Limulus Amoebocyte Lysate (LAL) assay. We have demonstrated endotoxin binding from water, Pseudomonas supernatant, and sa(lt solutions by spiking the samples with known amounts of endotoxin. We have tested the reusability of the affinity resin by cleaning it with NaOH as well as hot water sanitization. Leachables from the affinity resin that could potentially contaminate the solutions were also tested for. We have also investigated if the resin changed the composition of the salt solution. In Experiment 2, we have described the removal of TLR agonist molecules, such endotoxin (TLR-4), peptidoglycan (TLR-2/Nod2), lipopeptide (TLR-2) and bacterial DNA (TLR-9) from blood and plasma using affinity technology. We have tested the efficacy of removal by spiking the anti-coagulated blood and plasma with known amount of TLR agonist and measured their capture under dynamic conditions. We have tested several affinity resins, and the result of the most efficient one is presented here. The removal of endotoxin was tested with the LAL assay, while we have used monocyte activation assay (TNF-α ELISA) for testing the removal of the other TLR agonist molecules. Tissue Factor (TF) assay was used to determine the effect of the removal of TLR agonists on the coagulation part of the innate immune system. We have tested that the affinity resin, while removing the TLR agonist molecules does not have a negative effect on the blood by assaying the parameters of the coagulation, complement activation, hemolysis and cell count. In Experiment 3, we have described mycoplasma capture from solutions, such as serum used in cell culture, by affinity technology. We have used serologically and biochemically different mycoplasmas and affinity resins that are specific for different lipid and carbohydrate moieties on the mycoplasma membranes. In Experiment 4, we have described synergistic effect of TLR agonist molecules, such as peptidoglycan (TLR-2/Nod2) and bacterial DNA (TLR-9) on the stimulation of the innate immune response. Monocyte culture-based activation assay for tumor necrosis factor-αTNF-α and Tissue Factor levels (ELISA) were used to demonstrate their effect. We have also determined the effective concentrations of these TLR agonists that synergistically induce TNF-α and Tissue Factor production. In Experiment 5, we have described the preparation of pathogen mimicking microparticles, which included the preparation of an immunoaffinity column using purified polyclonal antibodies from a M. gallisepticum-positive sera, the immunoaffinity purification of M. gallisepticum antigens and the biochemical modification of the antigens (Endoglycosidase H (EndoH) digestion, Concanavalin A (ConA) adsorption, periodate oxidization and deacylation). In addition we have described the immobilization of antigens and PRR agonists, such as TLR-2, TLR-3, TLR-4, TLR-9 and nucleotid oligomerization domain 2 (Nod2) agonists to the microparticles. These microparticles are used in vivo (Experiment 6) and in vitro (Experiment 7). In Experiment 6, we have used a M. gallisepticum challenge model in chickens to test the effect of the pathogen mimicking microparticles. We have set up test groups of chickens (10 chickens per group) that were treated with the immunomodulatory microparticles orally 14 days prior to the M. gallisepticum challenge or after the challenge. The chickens were challenged with M. gallisepticum Rlow, a highly pathogenic strain of M. gallisepticum. Fourteen days after the challenge, the chickens were euthanized and examined for pathological lesions. Samples from different organs were taken for culture for M. gallisepticum as well as histopathology. In the challenge experiment, we have examined and compared the effects of PRR agonists, M. gallisepticum and M. gallinarum membranes, and the immunoaffinity-purified antigens with or without PRR agonist molecules. We have also examined the effect of the different post-transcriptional modifications of the M. gallisepticum antigens on the immune response. In Experiment 7, we have used the pathogen mimicking microparticles to study their effect in vitro with peripheral blood mononuclear cells (PBMC) and dendritic cells. We have induced monocytes with IL-4 and GM-CSF (granulocyte, macrophage colony stimulating factor) to obtain dendritic cells. We have been able to demonstrate that the microparticles interact with the cells of the innate immune system, such as PBMC and dendritic cells. We have assayed the activation of these cells by testing the level of pro-inflamatory cytokine, TNF-α and anti-inflamatory cytokine, IL-10 induction using ELISA. We have labeled the microparticles with fluorescein and used flow cytometry to show interaction with dendritic cells. We have shown that the microparticles used as an immunomodulator induce changes that are hallmarks of dendritic cells maturation, such as increase in the expression of MHCII (major histocompatibility complex) molecules and CD86 molecules. These were assayed by flow cytometry. Since the dendritic cells are the link between the innate and adaptive immunity, we have been able to show that the microparticles are able to influence both the innate and adaptive immune response.