Studies on the Interaction between Porcine Plasmacytoid Dendritic Cells and Porcine Reproductive and Respiratory Syndrome Virus

For over 20 years, porcine reproductive and respiratory syndrome (PRRS) has been the most troubling disease afflicting the pork industry. The main reason for this dissertation is that the pathogen responsible for this disease, PRRS virus (PRRSV), has developed the ability to subvert the immune response of its host. Evidence available at the outset of this project hinting that subversion of the immune system was a possibility, were the observations that PRRSV infection provoked a rapid humoral response characterized by the production of ineffective, non-immunizing antibodies and a cell-mediated immune response that is weak and protracted. The later was proposed to be result of a lack of adequate IFN-<;t production by the host, which is necessary to promote the development of protective anti-viral cell mediated immunity. Although this deficiency was attributed to non-translation of IFN-a gene transcripts produced in PRRSV-infected alveolar macrophages (AM), the most potent cell responsible for IFN-a. production in response to a viral infection, the plasmacytoid dendritic cell (PDC), had been largely ignored. Conceivably, this dismissal was based on the fact that a subset of AM is the principal target cell of PRRSV and that these cells readily support its replication in vitro. Because of the exuberant ability of PDC to produce IFN-a, the focus of this dissertation was directed instead to examine the porcine PDC. First, methodology was created that deployed the use of cell sorting instruments to allow isolation of a relatively homogeneous group of cells (CD4+CD17ztow) that phenotypically were likely to represent porcine PDC. Subsequently, these cells were indeed shown to be PDC based on their characteristic ability to rapidly secrete copious amounts of IFN-a. upon activation by the exposure to the coronavirus, transmissible gastroenteritis virus (TGEV) or to type A CpG-ODN, followed by a maturational change in the appearance of these cells from plasmacytoid to dendritic cell morphology. Based on their established phenotype, electronic gating on flow cytometric histograms in conjunction with multiple antibody stainings were used to further analyze these cells and reveal the existence of novel surface proteins including CD18, CD29, and CD44. However, the more impressive results were obtained with the purified PDC. For the first time, secretion of a variety of cytokines including IL-2, IL-8, and IFN-y, by activated porcine PDC could be demonstrated with a degree of certainty. Moreover, simple transcriptome patterns of resting and activated PDC could be compared and by doing so exemplify the tremendous induction of type I IFN gene expression by TGEV and a TLR9 agonist, type A CpG­ ODN (ODN19). In terms of immunobiology, probably the most important discovery was that in general PRRSV not only did not elicit an IFN-a response from PDC, but, actually impeded the typical induction of IFN-a expression by TGEV- or ODNl 9- stimulated PDC. Confirming that the inhibitory effect of PRRSV on PDC function also occurred upon in vivo exposure of PDCs to PRRSV validated the potential biological significance of these observations. Furthermore, the maturation of PDC was also impaired by PRRSV, as manifested by the lack of transformation of the PDC into dendritic cells and the de novo expression of CD80/86 upon to exposure to activating agents. Experiments aimed at determining the mechanism of inhibition suggest that the inhibitory effect PRRSV on PDC function is probably mediated by preventing the translocation of STAT-1 to the nucleus, thus preventing an increase in the level IRF-7 expression, which is required for optimal IFN-a production by PDC. The extent of the subversive effect of PRRSV on the activation of PDC was further appreciated by transcriptional analyses of PDC exposed to PRRSV as compared to their exposure to the porcine coronavirus TGEV. These studies revealed that the expression of a number of IFN-inducible genes was not stimulated by PRRSV. The unique nature of the subversive effect of PRRSV on porcine PDC was illustrated by the distinct transcriptional profile and cytokine production pattern exhibited by PDC exposed to PRRSV as compared to porcine AM exposed to the same virus. In the case of AM, the cells were not inhibited by PRRSV in their ability to produce IFN-a in response to TGEV. Finally, a ray of hope was obtained by the analyses of the outcome of the interaction between PDC and a mutant of PRRSV that does not inhibit IFN-a production. It is expected that this virus will provide information for the development of an effective second generation PRRSV vaccine