Plasmacytoid dendritic cells (pDC) are a specialized group of circulating dendritic cells that respond to viral nucleic acids with Type I IFN production as well as other cytokine and chemokines. These pDC responses lead to the production of antiviral molecules and recruitment of defense cells. During zoonotic B virus infection, a simplex virus of the subfamily Alphaherpesviridae, our lab has observed that infected individuals who succumb to infection have little-to-no-antibody or cell-mediated defenses. To identify whether this was partly due to failure of pDCs to produce antiviral interferon responses or produce chemokine and cytokines, we tested the hypothesis that B virus modulates the IFN response during zoonotic infection by blocking pDC activation and subsequent IFN signaling pathways to circumvent host defenses, while these pathways remain intact in the macaque hosts. We showed that human pDCs respond to B virus through the production of IFN-a, IL-1a, IL-6, TNF-a, MIP-1a/b and IP-10. Human pDCs co-cultured with B virus infected fibroblasts produced fewer cytokines and at lower levels. The macaque response to B virus was measured using PBMCs, as there are no specific reagents available to enrich macaque pDCs. Human and macaque PBMCs produced IFN-a when exposed directly to B virus infected lysates. Co-cultures of PBMCs with B virus infected fibroblasts from both hosts failed to produce any significant amounts of IFN-a. To quantify the antiviral effects of PBMC induced IFN-a, we measured B virus titers after exposure to supernatants from B virus exposed PBMCs, PBMC co-cultures with infected fibroblasts and exogenous recombinant Type I IFN. Our data further suggest that B virus resistance was not due to virus specific blockade of the Type I IFN signaling pathway because STAT-1 was activated in infected fibroblasts when treated with Type I IFNs. These data demonstrate for the first time that B virus replication is unimpeded in the presence of any source of IFN-a in either host cell type. In conclusion, this dissertation shows that the IFN-a production by both hosts in response to B virus is similar and that IFN-a treatment of B virus infected fibroblasts did not reduce B virus replication.

PhD (Microbiology) / Department of Microbiology / Background Conversion of Cholesterol to 25-HydroxyCholesterol (25HC) by Cholesterol 25-hydroxylase (CH25H) has been shown to exert broad antiviral properties. Given its antiviral activities, CH25H is part of an increasingly appreciated connection between type I interferon (IFN-I) and lipid metabolism. Moreover, the details of this connection appear to differ in mouse and human cells. Nevertheless, the molecular basis for the induction of CH25H in humans is not known. Objective Elucidation of signaling and transcriptional events for induction of CH25H expression is critical to design therapeutic antiviral agents. Materials and methods: Wildtype THP-1 monocytic cell-line or THP-1 MyD88 Knockout cell-line were treated with PMA for 72 hours for differentiation into macrophages. Differentiated macrophages or Microglial cells were stimulated with either TLR-agonists, pro-inflammatory cytokine, or interferons, and CH25H mRNAs expression levels were measured by qPCR. Results In this study, we show that CH25H is induced by Zika virus infection or TLR stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines including 1L- 1, TNF-, and IL-6, and this induction depends on STAT-1 transcription factor. Additionally, we have observed that ATF3 weakly binds to the CH25H promoter, suggesting co-operation with STAT-1. However, ZIKV induced CH25H was independent of type I interferon. Conclusion This study has demonstrated for the first time that pro-inflammatory cytokines such as 1L-1, TNF-, and IL-6 induce CH25H expression. Moreover, this provides further understanding to the connection between innate immunity and sterol metabolism and encourages the exploration of cytokines in antiviral immunity. / NRF

No description available.

The production of Nitric Oxide (NO) under physiological conditions has beneficial roles in acting as a key signaling component of many biological processes as well as having an anti-microbial effect. However its effects following excess production by the inducible NO pathway is potentially detrimental in the pathogenesis of chronic inflammation including sepsis and several other inflammatory diseases. Understanding the mechanisms that regulate the expression of the inducible nitric oxide synthase (iNOS) responsible for producing the excessive amounts of NO in disease states is therefore critical. In this regards, experiments were carried out to identify the signaling pathways that may mediate this process, focusing specifically on the JAK/STAT cascade. The reason for selecting the latter is because our research group, amongst others, has carried out extensive work investigating other signaling pathways, including the mitogen activated kinases (MAPK). Moreover, studies have also been carried out in an attempt to identify the critical role of JAK/STAT signaling for iNOS induction. These studies however failed to conclusively demonstrate whether, as with the MAPKs, the JAK/STATs may also play an essential role. Furthermore there is indeed controversy in the literature with researchers unable to agree whether expression of iNOS does require JAK/STAT activation. Thus, the aim of the project described in this thesis was to establish unequivocally whether activation of the JAK/STATs preceeds induction of iNOS. The studies were extended to L-arginine transport as well because the latter is widely reported to be induced in parallel with iNOS and substrate supply to iNOS may be critical for sustained NO production. Changes in transporter activity as well as their expression profiles were assessed. All experiments were carried out in either rat aortic smooth muscle cells (RASMCs) or in the J774 macrophage cell line. These cell types were selected because RASMCs are one of the prime targets for induced NO production in vascular inflammation and the macrophages are involved in host defence, acting in part through NO production. To establish the role of JAK/STATs, pharmacological and molecular approaches were used. Pharmacologically, two inhibitors were used and these were AG490 and JAK inhibitor I. The former is reported to be a selective JAK2 inhibitor and the other blocks all known JAK proteins. The potential of the GTPases to regulate the induction of iNOS was also examined using selective inhibitor known to regulate these proteins. In addition to these drugs, siRNA targeting JAK2 was also exploited and western blotting was extensively used to detect expression of various proteins including iNOS, native and phosphorylated JAK2 and TYK2. Changes in iNOS activity was monitored by determining nitrite production using the Griess assay and L-arginine transport was monitored using tritiated arginine (L-[3H]arginine). RASMCs were treated with a combination of LPS (100 µg/ml) and IFN- (100 U/ml) and the macrophages with LPS (1 µg/ml) to induce iNOS and transporter activity. Consistent with previous reports, the above treatment of both cell types resulted in the expression of iNOS, production of NO and enhanced transport of L-arginine. These effects were not affected by AG490 but blocked by JAK inhibitor I. Furthermore, although both cell types expressed the key JAKs (JAK2 and TYK2), neither of these proteins were phosphorylated under conditions of induced NO production. Moreover, siRNA experiments showed that JAK2 expression could be abolished without any significant change in NO production, confirming that at least JAK2 may not be required for this process. Whether TYK2 is involved still remains to be resolved as the phosphor-protein could not be detected. However the conclusive siRNA knockdown studies could not be carried out due to time and cost constraints. Apart from iNOS and NO production, changes in induced L-arginine transport were also not significantly affected under the experimental conditions described above suggesting that like with iNOS, induction of L-arginine transport is independent of at least JAK2. Interestingly however, STAT-1 was phosphorylated and this was blocked by JAK inhibitor I but not AG490. Thus, STAT-1 activation may be essential but its activation may be independent of the JAKs. One possible alternate upstream activator of STAT-1 may be the GTPases. Indeed these proteins have been indicated to phosphorylate STAT-1 independent of the JAKs. However, in this project, inhibition of the GTPase pathway enhanced NO production and L-arginine transport suggesting that the GTPases downregulate these processes. In conclusion, the studies carried out in this thesis have shown that induction of iNOS, NO production and L-arginine transport in both RASMCs and J774 macrophages are independent of JAK2 but require STAT-1 activation which may be phosphorylated independently of the JAKs. The role of other JAKs such as TYK2 although unlikely, will need to be resolved using a more specific approach such as siRNA.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.