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Children with chronic inflammatory disease (CID) are at an increased risk for health complications including mental health issues, cancer, and cardiovascular disease. These complications have been linked to elevated levels of pro-inflammatory cytokines and lifestyle behaviours including low physical activity, and high sedentary time. Physical activity may represent a simple and effective strategy to modulate inflammation and subsequently improve health outcomes. However, the link between cytokines and movement behaviours in children with a CID remains poorly understood. Indeed, no studies to date have examined the link between a broad complement of inflammatory markers and patterns of movement behaviours in children with a CID. Therefore, the objectives of this study were to: (1) examine movement behaviours and inflammation in children with either cystic fibrosis (CF), juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), and type 1 diabetes (T1DM), (2) examine movement behaviours and inflammation in children with a CID compared with healthy controls and (3) examine the association between movement behaviours and inflammation profiles in children with a CID. We hypothesized that (1) children with a CID will have comparable movement profiles and inflammation, (2) children with a CID will have lower physical activity levels and higher levels of pro-inflammatory cytokines relative to healthy counterparts, and (3) children with a CID who are more physically active and engage in less sedentary time will have less inflammation. Boys and girls with a CID and controls wore an ActiGraph GT3X accelerometer around the waist during waking hours for 7 days. Outcomes of interest included, sedentary time, time spent in light physical activity (LPA), moderate-to-vigorous PA (MVPA), and total PA (TPA), determined using Evenson cut-points. After one week a fasted blood sample was collected to determine serum cytokines (TNFα, IL-23, IL-1β, IL-12, IL-6, IL- 17, TGFβ, IL-10) by multiplex assays and C-reactive protein by enzyme linked immunosorbent assay. A total of 132 participants (47% girls; age: 13.3±2.8 years), including JIA (N=27), IBD (N=21), CF (N=14), T1DM (N=18) and healthy controls (N=52), completed the study. Physical activity and inflammatory profiles were comparable between CF, JIA, IBD and T1DM groups. Children with a CID participated in 13.3 fewer mins/day [95% confidence interval 6.9, 101.2] (MVPA (F(1,113)=11.015, p=0.001) of MVPA relative to healthy controls and had comparable cytokine profiles. Physical activity did not predict inflammation in children with a CID. However, we know that physical activity has many beneficial cardiorespiratory and mental health effects. As such, it is still of interest to uncover any potential effects movement behaviours may have on our immune system. / Thesis / Master of Science (MSc) / The purpose of this study was to assess how physical activity, sedentary time, and inflammation are associated. Inflammation is an important part of our immune system that protects us from infection and disease; however, when inflammation goes unchecked, it can cause serious chronic inflammatory disease. We were interested in understanding if children with a chronic inflammatory disease had different levels of physical activity and sedentary time, or different levels of inflammation than healthy children with no medical conditions. We were also interested in understanding if physical activity or sedentary time were related to levels of inflammation in children with a chronic disease. We asked boys and girls between 7 and 17 years old to visit our lab twice. Some of our participants had a medical condition, including chronic kidney disease, cystic fibrosis, juvenile idiopathic arthritis, or inflammatory bowel disease and type 1 diabetes. We also invited a group of children who had no medical condition to participate. During their first visit, we measured their weight, height, pubertal status and gave them a physical activity monitor to wear for 7 days before coming back for their second visit. At their second visit we took a small blood sample that we used to measure immune proteins called cytokines, these proteins act like messengers to tell the immune system what to do. Some of them make inflammation worse, and others help to bring down levels of inflammation. We found that children who had a chronic inflammatory disease participated in less physical activity but had similar levels of sedentary time compared with healthy children. We also found that there were no differences in inflammation between children with a chronic disease and healthy children. Lastly, we found that physical activity and sedentary time were not related to inflammation levels. Although we did not find a relationship between physical activity and inflammation, we know that physical activity has many beneficial cardiorespiratory and mental health effects. As such, it is still of interest to uncover any potential effects movement behaviours may have on health outcomes.

Chronic inflammation follows defined phases of induction, inflammation, and resolution. The resolution phase requires cycloxygenase-2 (COX-2) activity. This study aims to address what other molecules are required for a functional resolution phase. We demonstrated that in murine collagen-induced arthritis the transcription factor, PPARgamma plays a role in the resolution phase. Inhibition of COX-2 activity results in fewer PPARgamma positive cells in the arthritic synovium. Treatment with a PPARgamma antagonist, SR202, alone, also disrupts the process of resolution. PPARgamma antagonist treatment results in a decrease in eNOS phosphorylation within the arthritic synovium. These observations indicate that PPARgamma may function to regulate eNOS activity. The source of pro-resolving nitric oxide is eNOS but not, iNOS. The effect of COX-2 inhibition on the resolution phase is ameliorated by injection of a PGE2 analog. Restoration of PGE2 levels results in an increase in PPARgamma positive cells in the arthritic synovium which correlates with this restoration of resolution. Thus, this study provides in vivo evidence for the pro-resolving role of PPARgamma and its relationship with PGE2 and eNOS. / Microbiology and Immunology

There is a clear need for testing targeted drug carrier systems in a more realistic microenvironment where both biochemical interactions and shear forces are present. This is critical both for understanding of the molecular mechanisms involved in this process and during the drug discovery process. Current in vitro models of the leukocyte adhesion cascade cannot be used for real-time studies of the entire leukocyte adhesion cascade including rolling, adhesion and migration in a single assay. In this study, we have developed and validated a novel bioinspired microfluidic device (bMFD) and used it to test the hypothesis that blocking of specific steps in the adhesion/migration cascade significantly affects other steps of the cascade. The bMFD consists of an endothelialized microvascular network in communication with a tissue compartment via a 3 µm porous barrier. Human neutrophils in bMFD preferentially adhered to activated human endothelial cells near bifurcations with rolling and adhesion patterns in close agreement with in vivo observations. Treating endothelial cells with monoclonal antibodies to E-selectin or ICAM-1 or treating neutrophils with wortmannin reduced rolling, adhesion, and migration of neutrophils to 60%, 20% and 18% of their respective control values. Antibody blocking of specific steps in the adhesion/migration cascade (e.g. mAb to E-selectin) significantly downregulated other steps of the cascade (e.g. migration). This novel in vitro assay provides a realistic human cell based model for basic science studies, identification of new treatment targets, selection of pathways to target validation, and rapid screening of candidate agents. / Mechanical Engineering

Obesity and insulin resistance are characterized by elevated pro-inflammatory proteins in the blood and immune cell accumulation in the visceral adipose tissue. Resident leukocytes release tumor necrosis factor α (TNFα) and other inflammatory cytokines which stimulate adipocyte lipolysis, recruit leukocytes to adipose tissue, promote pro-inflammatory immune cell polarization, facilitate oxidative stress, and activate intracellular kinases which dull insulin signaling cascades in metabolic tissues. Immune cell mediated dysregulation of stromal and parenchymal cells has raised suspicion that insulin resistance is an immune disorder initiated by activated white blood cells with over-nutrition. Efforts to improve pathological metabolism by reducing inflammation have yielded mixed results in humans and animal models. The role of inflammation and immune cell accumulation in the visceral fat (VF) in the progression of insulin resistance remains presently debated. There is, however, a consensus that identifying the triggers for obesity and impaired insulin signaling is of the utmost importance. The goal of this report is to identify dietary fat absorption as a key initiator of inflammatory action and insulin desensitization which may be dampened by reducing immune cell accumulation in adipose tissue. To explore how lean, healthy organisms become obese and insulin resistant, we examined the inflammatory consequences of isocaloric but variable macronutrient loads in the VF of lean mice. Mice were administered single liquid meals composed of low-fat (10% fat) or high-fat (60% fat) diet and observed by intravital microscopy to quantify leukocyte-endothelium interactions in mesenteric postcapillary venules (MPCV) 1, 2, 3, and 4 hours after oral gavage. Leukocyte rolling and leukocyte adhesion were transiently elevated within 1 hour after feeding and returned to baseline levels 4 hours later. Endothelial cell surface expression of P-selectin (Psel), a rapidly activated cell adhesion molecule (CAM), confirmed that high-fat feeding induced Psel dependent leukocyte rolling through the VF microcirculation. Furthermore, leukocyte accumulation in the VF was modestly increased by a single high-fat meal (HFM). Repetitive high-fat diet (HFD) consumption for 24 hours prolonged elevated leukocyte-endothelium interactions and promoted neutrophil accumulation in the VF. The neutrophilic enzyme myeloperoxidase (MPO), a producer of the chlorinating agent hypochlorous acid, increased in abundance and activity in the VF of HFM fed mice. Elevated leukocyte-endothelium interactions, leukocyte infiltration, and MPO activity in VF were not observed in Psel deficient (Psel-/-) mice following lipid overload. To ascertain if MPO is required for sustained endothelial activation, leukocyte-endothelium interactions and leukocyte infiltration were monitored in high-fat fed MPO deficient (MPO-/-) mice. Similar to the Psel-/- mice, MPO-/- mice were protected from the inflammatory effects of high-fat feeding. Our data supports postprandial hyperlipemia as an inducer of transient and Psel dependent inflammatory reactions that are sustained by prolonged HFD consumption. To study whether early phase inflammatory interventions granted late phase metabolic improvements, wild-type (WT), Psel deficient (Psel-/-), and MPO deficient (MPO-/-) C57BL/6 mice were given ad libitum access to LFD (10% fat) or HFD (60% fat) for 12-16 weeks. All mouse groups given HFD became obese. Prolonged HFD consumption sustained elevated leukocyte-endothelium interactions in MPCVs and was accompanied by increased local and systemic TNFα in WT mice. High-fat fed WT mice were hyperglycemic, hyperinsulinemic, glucose intolerant, and insulin resistant compared to LFD fed controls. Psel-/- mice were protected from leukocyte-endothelium interactions as well as local and systemic TNFα accumulation despite extended HFD consumption. Surprisingly, high-fat fed Psel-/- mice were equally hyperglycemic, hyperinsulinemic, glucose intolerant, and insulin resistant as the inflamed, high-fat fed WT mice. MPO-/- mice were also protected from elevated systemic TNFα and gained slightly less weight than the other high-fat fed groups. While MPO-/- mice were hyperglycemic and glucose intolerant, they did have improved insulin stimulated glucose clearance. The data presented in this report demonstrates the pro-inflammatory nature of postprandial hyperlipemia and the insulin desensitizing nature of prolonged HFD consumption. Ablation of VF immune cell accumulation by Psel deletion is not sufficient for improving insulin signaling or glycemic control, which is consistent with prior reports. Deletion of MPO, however, did result in slightly less obesity and marginally improved insulin signaling. We conclude that while immune cell accumulation in the VF contributes to the progression of insulin resistance, it is not a prerequisite for metabolic pathology development. / Physiology

Atherosclerosis, considered a chronic inflammatory disease, is the underlying mechanism for several cardiovascular diseases. Hyperlipidemia is the number one risk factor for atherogenesis. Caspase-1 is an inflammatory caspase, which can be activated by the metabolic stresses through pathogen associated molecular patterns (PAMPs)-recognition receptors, (PRR) recognition and inflammasome assembly. Activated caspase-1 can initiate inflammation in multiple ways. Thus, regulating inflammasome components expression is essential to control caspase-1 activation and its subsequent inflammatory processes. I hypothesized that the readiness of inflammasome component expression for caspase-1 activation in tissues is an index for inflammation privilege. Endothelial cells (EC) which are the innermost layer of the vessel and are the critical gatekeeper for monocyte migration. The first step of atherogenesis is activation of ECs, which allows monocyte adhesion and migration into the sub-endothelial layer. I also hypothesized that caspase-1 can sense hyperlipidemia and regulate EC activation and inflammation during early atherogenesis. I first determined the expression profiles of inflammasome components, pro-inflammatory caspases and PRRs is different among tissues, and cardiovascular tissues express relative less PRRs via a database-mining method. According to the readiness of inflammasome components, tissues could be classified into three tiers. The first tier consists of tissues with constitutively expressed inflammasomes. The second tier of tissues includes potentially inducible expression of one inflammasome component. The third tier of tissues has inducible expression of at least two inflammasome components. This three-tier model can be applied to determine the inflammation privilege of tissues in response to pro-inflammatory stimuli. I also demonstrated that hyperlipidemia induced caspase-1 expression and activation in aorta along with the atherogenesis in apolipoprotein E (ApoE)-/- mice with high fat (HF) diet, experimentally. We then generated the ApoE-/-/Casp-1-/- double knockout mice, and found that the ApoE-/-/Casp-1-/- mice contained significantly less atherosclerotic lesion in aortic sinus and less cytokine and chemokine expression in aortic tissues compared with ApoE-/- mice. ApoE-/-/Casp-1-/- mice also had less CD11b+/F4/80- neutrophil and CD11b+/F4/80+ monocyte recruitments into aorta compared with ApoE-/- mice. However, the percentage of monocyte subsets in peryphery blood remained at the same level in between ApoE-/- mice and ApoE-/-/Casp-1-/- mice. I then proposed that perhaps the caspase-1 activation in vascular cells, in ECs played the essential role of controling monocyte migraion. My in vitro data demonstrated that oxidized low density lipoprotein (ox-LDL) and its componnents could induced caspase-1 activation in human aortic ECs (HAECs) through ROS pathway which then led to EC activation and pyroptotic cell death. Deficiency of caspase-1 in aortic EC attenuated hyperlipidemia induced EC activation and inflammtion. Mechanically, I found that caspase-1 deficiency accumulated an anti-atherogenic protein, Sirt-1 in the aorta. Collectively, our data suggested that caspase-1/inflammasome in ECs can sense hyperlipidemia, become activated, drive EC activation, and promote monocyte recruitment and early atherosclerosis. / Pharmacology