TRANSIENT LIPOPOLYSACCHARIDE-INDUCED CYTOKINE RESPONSES IN THE MATERNAL AND FETAL GUINEA PIG

The aim of this study was to further investigate the role of pro-inflammatory cytokines in the pathogenesis of fetal cererbral white matter injury associated with chorioamnionitis by charaterizing the time course of the cytokine response in the pregnant guinea pig following a maternal inflammatory insult. Chorioamnionitis increases the risk for fetal brain injury. In the guinea pig, a threshold maternal inflammatory response must be reached for significant fetal brain injury to occur. However, a previous study demonstrated that, by seven days after an acute maternal inflammatory insult, cytokine levels in both maternal and fetal compartments are not different from controls. The purpose of this study, therefore, was to test the hypothesis that a significant cytokine response occurs within the first seven days following an acute maternal inflammatory response. Pregnant guinea pigs (n=34) were injected intraperitoneally with 100µg/kg lipopolysaccharide (LPS) at 70% gestation and euthanized at 24 hours, 48 hours or 5 days following endotoxin exposure. Control animals were euthanized at 70% gestation without exposure. Concentrations of interleukin-6, interleukin 1-β and tumour necrosis factor-α (IL-6, IL-1β, TNF-α) were quantified in the maternal serum and amniotic fluid by enzyme-linked immunosorbent assay. IL-6 and IL-1β concentrations were elevated in the maternal serum at 24 hours and returned to control levels by five days. In the amniotic fluid, IL-6 peaked at 48 hours and IL-1β at 24 hours. TNF-α levels were not significantly increased. A single maternal LPS injection produces transient increases in cytokine concentrations in the maternal serum and amniotic fluid. This further implicates the cytokines as potential mediators of fetal white matter damage. Although this response might not be sufficient to produce the brain injury itself, it may initiate harmful pro-inflammatory cytokine cascades, which could even continue to harm the fetus following delivery. A human diagnostic protocol was developed to assess the use of serial serum biomarkers, including IL-6 and TNF-α, in the prediction of histological chorioamnionitis. Preliminary analysis of the pilot study suggests that certain biomarkers might be worthy of further investigation in a larger-scale study.

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.

IMPACT OF RNA VIRUSES ON THE REGULATION OF IL-23 IN MOUSE AND HUMAN MODELS OF INFECTION.

Interluekin-23 (IL-23) is a pro-inflammatory cytokine critical to the regulation of innate and adaptive immune responses. The main role for this cytokine is in the proliferation and differentiation of the IL-17 producing CD4 T helper cell, Th17. Virus infection deregulates IL-23 expression and function, but little is known about the mechanism behind this phenomena. Here, I demonstrate a reduction of Toll like receptor (TLR) ligand-induced IL-23 expression in lymphocytic choriomeningitis virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs), indicating that a function of these cells is disrupted during virus infection. I propose a mechanism of TLR ligand-induced IL-23 expression inhibition upon LCMV infection via the deactivation of p38, AP-1, and NF-κB. Further analysis revealed a direct relationship between LCMV infection with the IL-10 and SOCS3 expression. To understand IL-23 function, I characterized IL-23-induced JAK/STAT signalling pathway and IL-23 receptor expression on human CD4 T cells. My results demonstrate that IL-23 induces activation of p-JAK2, p-Tyk2, p-STAT1, p-STAT3, and p-STAT4 in CD4 T cells. For the first time I show that IL-23 alone induces the expression of its own receptor components, IL-12Rβ1 and IL-23Rα, in CD4 T cells. Blocking JAK2, STAT1, and STAT3 activation with specific inhibitors detrimentally effected expression of IL-23 receptor demonstrating that activation of JAK/STAT signalling is important for IL-23 receptor expression. I also addressed the effect of viral infection on IL-23 function and receptor expression in CD4 T cells using cells isolated from HIV positive individuals. These studies were based on earlier reports that the expression of IL-23 and the IL-23 receptor are impaired during HIV infection. I demonstrate that the phosphorylation of JAK2, STAT1, and STAT3 induced by IL-23, as well as IL-23 receptor expression are deregulated in CD4 T cells isolated from HIV positive individuals. This study has furthered the understanding of how the expression and function of IL-23 is regulated during viral infections.

Analysis of parameters of haemostasis in pre-eclamptic, normotensive pregnant and non-pregnant women.

Pregnancy is characterized by a state of heightened coagulation, which is exacerbated in pathological conditions such as pre-eclampsia (PET). PET is further associated with abnormal maternal inflammation and increased circulating microparticles (MP); however, a mechanistic link between these pathological features has never been established. It is proposed in this thesis that abnormal maternal inflammation is causally linked to pro-coagulant trophoblast MP shedding via a mechanism mediated by the pro-inflammatory cytokine tumour necrosis factor alpha (TNF), thereby contributing to maternal coagulopathies associated with PET. Using thromboelastography (TEG) and standard laboratory tests, haemostatic function was evaluated in PET and normotensive subjects at delivery and post-partum. Furthermore, the effects of the menstrual cycle and oral contraceptive (OC) use on haemostatic function were assessed in non-pregnant subjects in order to understand their influence on post-partum haemostasis. Plasma TNF and pro-coagulant MP levels were evaluated in the pregnant subjects. Using chorionic villi explants from human term placentas, MPs were quantified after TNF administration. The pro-coagulant potential of placental MPs was evaluated by TEG by spiking whole-blood with medium containing MPs from chorionic villi. TEG identified increased whole-blood coagulability in PET subjects at delivery, demonstrating its increased sensitivity over standard laboratory tests at identifying haemostatic alterations associated with PET. Haemostatic alterations were normalized by six weeks post-partum. TEG also identified cyclic haemostatic variations associated with OC use. Chorionic villi treated with TNF (1 ng/ml) shed significantly more MPs than untreated placentas. MPs from chorionic villi increased the coagulability of whole-blood. Together, results provide evidence supporting the concept that abnormal maternal inflammation is causally linked to the development of maternal coagulopathies in pregnancy complications. Moreover, TEG may be superior to standard laboratory tests in evaluating haemostasis in pregnant and non-pregnant subjects.

Optimizing The Nasal Allergen Challenge Model For The Study Of Allergic Rhinitis

Background The Allergic Rhinitis Clinical Investigator Collaborative (AR-CIC) uses a Nasal Allergen Challenge (NAC) model to study the pathophysiology of AR and provides proof of concept for novel therapeutics. The NAC model needs to ensure optimal participant qualification, allergen challenge, clinical symptoms capture and biological samples collection. Repeatability of the protocol is key to ensuring unbiased efficacy analysis of novel therapeutics. The effect of allergen challenge on IL-33 gene expression and its relation to IL1RL1 receptor and cytokine secretion was investigated. Methods Several iterations of the NAC protocol was tested, comparing variations of qualifying criteria based on the Total Nasal Symptom Score (TNSS) and Peak Nasal Inspiratory Flow (PNIF). The lowest allergen concentration was delivered and TNSS and PNIF recorded 15 minutes later. Participants qualified if the particular criteria for the protocol were met, otherwise the next higher allergen concentration (4-fold increase), was administered until the targets were reached. Participants returned for a NAC visit and received varying allergen challenge concentrations depending on the protocol, TNSS/PNIF were recorded at 15 minutes, 30 minutes, 1 hour, and hourly up to 12 hours, a 24 hour time point was added in later iterations. Repeatability was evaluated using a 3-4week interval between screening, NAC1, and NAC2 visits. Various biomarker samples were collected. Results A combined TNSS and PNIF criterion was more successful in qualifying participants. The cumulative allergen challenge (CAC) protocol proved more reliable in producing a robust clinical and biomarker response. Repeatability of the CAC protocol was achieved with a 3-week interval between visits, on a clinical and biological basis. IL-33 cytokine is an important biomarker in initiating the inflammatory response in AR in humans. IL-33 and IL1RL1 expression might employ a negative feedback mechanism in human nasal epithelial cells. Comparing the clinical and biological response to ragweed vs cat allergen challenge, proved the CAC protocol’s suitability for use employing different allergens. Conclusion The AR-CIC’s CAC protocol is an effective method of studying AR, capable of generating measurable and repeatable clinical and biomarker responses, enabling better understanding of AR pathophysiology and ensuring that any change would be purely due to medication under investigation in a clinical trial setting.