THE IMPACT OF VIRUS INFECTION IN DEREGULATION OF CYTOKINE PRODUCTION UPON SECONDARY BACTERIAL INFECTION

Dendritic cells (DCs) secrete cytokines such as interleukin-23 (IL-23) when stimulated with certain Toll-like receptor (TLR) agonists and infected with pathogens such as P. aeruginosa. IL- 23 is a proinflammatory cytokine that plays a critical role in the proliferation and differentiation of the IL-17 producing Th17- CD4 T helper cells. The lack of efficient cytokine production from antigen-presenting cells, such as DCs, can impact CD4 differentiation and thus impair the immune responses against pathogens. Clearance of some bacterial infections, such as Klebsiella pneumonia and Listeria monocytogenes has been shown to be dependent on the induction of IL-23 and therefore, deregulation of these cytokines as a direct result of virus infection may impede immune responses to secondary infections. Here, an inhibition of TLR ligand or P. aeruginosa-induced IL- 23 expression in Lymphocytic Choriomeningitis Virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs) has been demonstrated, indicating that an important function of these cells is disrupted during virus/bacterial coinfection. While production of TNF-α was unaffected in LPS stimulated cells, TNF-α was significantly inhibited in bacterium infected cells by LCMV. Type I IFN in LPS or LCMV infected cell was not detected and therefore, ruling out the possibility of cytokine suppression by Type I IFN. The production of IL-10 was high in BMDCs infected with LCMV and stimulated with LPS or bacteria. Analysis of multiple cytokines produced in this coinfection model demonstrated that LCMV infection impacts specific cytokine production upon LPS or bacterium infection, which may be important for bacterial clearance. This data is important for future immunotherapy use in viral/bacterial coinfection scenarios.

The role of vitamin D signalling in the interaction between mesenchymal mammary tumour cells and macrophages

The transition of epithelial-like tumour cells to those exhibiting mesenchymal characteristics (Epithelial-to-mesenchymal Transition; EMT) is an integral process in breast cancer metastasis. EMT can be promoted by Transforming growth factor-beta (TGF-β) which can be found at high levels in the tumour stroma. Tumour-associated macrophages (TAMs) can also induce EMT in breast cancer cells, which is one way that they promote breast cancer metastasis. Vitamin D signalling has been implicated in EMT suppression and plays a role in modulating macrophage differentiation and stimulating their anti-inflammatory functions. This project had two major aims. First, we aimed to create and verify a unique fluorescent reporter gene construct designed to evaluate the dynamics of EMT in real-time and at the single-cell level. While some components of this reporter system were successfully validated, work to complete the final reporter construct is ongoing. The second and main aspect of this project focused on exploring the ability of 1,25-dihydroxyvitamin D3 (1,25D3) to modulate the interaction between mesenchymal mammary tumour cells and TAMs. Unexpectedly, in short-term treatment (48 hours) studies of 4T1 murine mammary tumour cells, we observed that 1,25D3 and TGF-β signalling work together to increase expression of the mesenchymal markers, Snai1, Fn1, and Col1a1. 1,25D3 and TGF-β also synergistically activate transcription of the gene encoding the 1,25D3-catabolizing enzyme, Cyp24a1. The ability of 1,25D3 and TGF-β to enhance expression of these genes was diminished in a long-term treatment (14 days) of 4T1 cells, and this effect was accompanied by a decrease in cell proliferation. 1,25D3 may also cooperate with cytokines produced by normal macrophages and macrophages considered to be TAM-like. Conditioned media experiments revealed that in the presence of factors from normal macrophages, 1,25D3 enhanced expression of Fn1, and in the presence of factors from TAM-like macrophages, 1,25D3 enhanced expression of Fn1 and Cyp24a1. Rather than mitigating the interaction as hypothesized, 1,25D3 may exacerbate the tumour-promoting effects of the EMT-TAM relationship. Also, signalling pathways involved in the EMT-TAM relationship may synergize with 1,25D3 to upregulate Cyp24a1 expression. These findings are important for understanding the potential of vitamin D compounds to be used in the treatment of breast cancer.

Investigation of the effect of intrauterine inflammation and infection on fetal brain injury using human and animal models

In recent years, increased focus has been placed on the role of intrauterine infection and inflammation in the pathogenesis of fetal brain injury leading to neurodevelopmental disorders such as cerebral palsy. At present, the mechanisms by which inflammatory processes during pregnancy cause this effect on the fetus are poorly understood. Our previous work has indicated an association between experimentally-induced intrauterine infection, increased proinflammatory cytokines, and increased white matter injury in the guinea pig fetus. In order to further elucidate the pathways by which inflammation in the maternal system or the fetal membranes leads to fetal impairment, a number of studies investigating aspects of the disease process have been performed. These studies represent a body of work encompassing novel research and results in a number of human and animal studies. Using a guinea pig model of inflammation, increased amniotic fluid proinflammatory cytokines and fetal brain injury were found after a maternal inflammatory response was initiated using endotoxin. In order to more closely monitor the fetal response to chorioamnionitis, a model using the chronically catheterized fetal ovine was carried out. This study demonstrated the adverse effects on fetal white matter after intrauterine exposure to bacterial inoculation, though the physiological parameters of the fetus were relatively stable throughout the experimental protocol, even when challenged with intermittent hypoxic episodes. The placenta is an important mediator between mother and fetus during gestation, though its role in the inflammatory process is largely undefined. Studies on the placental role in the inflammatory process were undertaken, and the limited ability of proinflammatory cytokines and endotoxin to cross the placenta are detailed herein. Neurodevelopmental disorders can be monitored in animal models in order to determine effective disease models for characterization of injury and use in therapeutic strategies. Our characterizations of postnatal behaviour in the guinea pig model using motility monitoring and spatial memory testing have shown small but significant differences in pups exposed to inflammatory processes in utero. The data presented herein contributes a breadth of knowledge to the ongoing elucidation of the pathways by which fetal brain injury occurs. Determining the pathway of damage will lead to discovery of diagnostic criteria, while determining the vulnerabilities of the developing fetus is essential in formulating therapeutic options.

Structural and functional studies of bacterial protein tyrosine kinases

While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. Studies of close to 20 homologs of bacterial protein tyrosine (BY) kinases have inaugurated a blooming new field of research, all since just the end of the last decade. These kinases are key regulators in the polymerization and exportation of the virulence-determining polysaccharides which shield the bacterial from the non-specific defenses of the host. This research is aimed at furthering our understanding of the BY kinases through the use of X-ray crystallography and various in vitro and in vivo experiments. We reported the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5Å resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting evidences, we proposed a unique activation mechanism for BY kinases in Gram-negative bacteria. The phosphorylation of tyrosine residue Y574 at the active site and the specific interaction of P-Y574 with a previously unidentified key arginine residue, R614, unblock the Etk active site and activate the kinase. Both in vitro kinase activity and in vivo antibiotics resistance studies utilizing structure-guided mutants further support the novel activation mechanism. In addition, the level of phosphorylation of their C-terminal Tyr cluster is known to regulate the translocation of extracellular polysaccharides. Our studies have significantly clarified our understanding of how the phosphorylation status on the C-terminal tyrosine cluster of BY kinases affects the oligomerization state of the protein, which is likely the machinery of polysaccharide export regulation. In summary, this research makes a substantial contribution to the rapidly progressing research of bacterial tyrosine kinases.

Relationship between abnormal maternal inflammation and insulin resistance during pregnancy

Abnormal maternal inflammation during pregnancy is linked to complications such as preeclampsia and fetal growth restriction. There is growing evidence that insulin resistance is also associated with a heightened inflammatory state, and is linked to pregnancy complications such as gestational diabetes. This study tested the hypothesis that abnormal inflammation during pregnancy is causally linked to elevations in blood glucose and insulin resistance. To induce a state of abnormal systemic inflammation, bacterial lipopolysaccharide (LPS) was administered to pregnant rats on gestational days (GD) 13.5-16.5. Dams treated with LPS exhibited an abnormal immune response characterized by an elevation in white blood cells, which was linked to reduced fetal weight and increased glucose levels over pregnancy. Abnormal inflammation is characterized by increased levels of circulating pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF) and interleukin-6, which contribute to insulin resistance by inhibiting the insulin signalling pathway. TNF in particular induces a serine phosphorylation (pSer307) of insulin receptor substrate 1 (IRS-1). In our model, insulin resistance was assessed by measuring the extent of pSer307 of IRS-1 and total IRS-1 expression in skeletal muscle, as well as changes in metabolic parameters and pancreas tissue morphology associated with insulin resistance. LPS-treated dams exhibited a significant reduction in IRS-1 expression, elevation in fasting glucose levels, and reduction in insulin sensitivity indices. There were also biologically relevant increases in fasting plasma insulin levels and insulin resistance indices, but not pSer307 of IRS-1 and pancreatic islet size. To determine whether inflammation plays a role in reducing insulin signalling and the other changes associated with LPS administration, etanercept, a TNF antagonist, was administered on GDs 13.5 and 15.5 prior to LPS injections. With the exception of IRS-1 expression, in rats treated with etanercept all of the measured parameters remained at the levels observed in saline controls, indicating a link between abnormal inflammation and insulin resistance. The results of this study support the practice of monitoring the inflammatory conditions of the mother prior to and during pregnancy, and support further investigation into the potential use of anti-inflammatory agents during pregnancy in women at risk of insulin resistance and gestational diabetes.