Autophagy, an intracellular degradation, and recycling process is essential in maintaining cellular homeostasis. Dysregulated autophagy is linked to the pathogenesis of various diseases, including inflammatory bowel disease (IBD) which consists of Crohn’s disease and ulcerative colitis. In IBD, enterochromaffin cell numbers and one of its main product serotonin (5-hydroxytryptamine; 5-HT) levels are elevated. Previously, we had shown that tryptophan hydroxylase 1 deficient (Tph1-/-) mice, with reduced gut 5-HT had decreased severity of colitis. Here, we showed that gut 5-HT plays a vital role in modulating autophagy and thus regulating gut microbial composition and susceptibility to intestinal inflammation. Tph1-/- mice, had upregulated colonic autophagy via the mammalian target of rapamycin pathway (mTOR), and decreased colitis severity. Tph1-/- mice after 5-HT replenishment, and serotonin reuptake transporter deficient (SERT-/-) mice, which have increased 5-HT levels, showed converse results. Deletion of intestinal epithelial cell-specific autophagy gene, Atg7, in Tph1-/- mice (DKO mice) abolished the protective effect of Tph1 deficiency in colitis, decreased the production of antimicrobial peptide, β-defensin 1 and promoted colitogenic microbiota. Furthermore, using cecal microbial transplantation, we found that the colitic microbiota of the DKO mice contributed to the increased severity of colitis. Supporting this pathway's translational importance, we uncovered that 5-HT treatment of peripheral blood mononuclear cells from both healthy volunteers and patients with Crohn’s disease inhibited autophagy via the mTOR pathway. Our results in this thesis emphasize the role of 5-HT-autophagymicrobiota axis in intestinal inflammation. Moreover, these findings suggest 5-HT as a novel therapeutic target in intestinal inflammatory disorders such as IBD that exhibit dysregulated autophagy. / Thesis / Doctor of Philosophy (PhD) / Approximately 0.7% of Canadians are currently affected with inflammatory bowel disease (IBD). The gut hormone serotonin, which regulates many normal functions, is elevated in gut inflammation. Reduced serotonin levels decrease the severity of inflammation. IBD pathology has been linked to a unique cell self-eating process called autophagy. Using cell lines, mice, and samples from IBD patients, we assessed the interactions between serotonin signaling and autophagy during gut inflammation. I found that an increase in serotonin levels enhances the severity of gut inflammation by inhibiting autophagy. We also established the connection between serotonin and autophagy in the intestinal epithelial cells, and how this modulates epithelial cell function. Furthermore, we demonstrated the establishment of an altered gut microbiota upon disruption of the serotonin-autophagy axis in the epithelial cells, which subsequently influenced gut inflammation severity. Thus, we identified one of the key triggers related to the pathogenesis and severity of IBD.

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Homocysteine, an intermediate metabolite biosynthesized from the methionine cycle, is a homologue of cysteine. Homocysteine differs from cysteine by an additional methylene group, which makes it more reactive. Elevated homocysteine level is a risk factor for cardiovascular disease and cerebrovascular disease, brain atrophy, neurodegenerative diseases and cognitive dysfunctions. Recent studies suggest a bi-directional relationship between homocysteine levels and immune-inflammatory activation. Our studies sought to determine if hyperhomocysteinemia affects cell infiltrates in the Central Nervous System (CNS). Inflammatory monocytes recruitment into the CNS and microglia proliferation have been shown in several inflammatory models, and Ly-6Chi CCR2+ monocytes have been shown to be the precursor for microglia. Based on these findings, we hypothesized that hyperhomocysteinemia (HHcy) would alter CNS infiltrate composition. We investigated whether HHcy affected the total mononuclear cells composition in the CNS. We also determined whether HHcy altered the inflammatory monocyte subsets composition in the CNS. In order to determine the effects of HHcy in the CNS mononuclear cells composition, we genotyped the mice, and isolated mononuclear cells from the CNS using percoll gradient method. Then we simultaneously stained the cells with three antibodies, PE-labeled anti-mouse CD11b, PE-Cy5-labeled anti-mouse CD45, and FITC-labeled anti-mouse Ly-6C and analyzed the samples by flow cytometry method. HHcy made no difference in the percentage of lymphocytes, infiltrating monocytes and microglia in the total CNS mononuclear cells, but within infiltrating monocytes, HHcy decreased Ly-6Clo and increased Ly-6Chi subsets. These findings demonstrate that HHcy has effects on the CNS mononuclear cell composition. In summary, HHcy decreased Ly-6Clo and increased Ly-6Chi subsets of infiltrating monocytes in the CNS. There is a potential role of HHcy in increasing inflammatory monocytes infiltration. / Pharmacology

We recently reported that intestinal-specific overexpression of the POZ-ZF transcription factor Kaiso produced two prominent phenotypes in 1-year old mice: Kaiso transgenic (KaisoTg) mice presented with chronic intestinal inflammation, and an increase in secretory cell types – a trait typical of Notch signalling inhibition. Despite these findings however, the factor(s) responsible for Kaiso-mediated inflammation and secretory cell increases had not been elucidated. The primary goal of this thesis was to begin filling in this knowledge gap, by shedding mechanistic insight on Kaiso’s role in governing these two prominent phenotypes. First, we elucidated Kaiso’s role in the Notch signalling pathway and found that Kaiso inhibited the expression of the Notch1 receptor, and its ligand Dll-1, but promoted the expression of the Jagged-1 ligand. We postulated that the Kaiso-mediated reduction in Dll-1 might be responsible for the increase in secretory cell types, whereas Kaiso-mediated regulation of Jagged-1, which is dispensable for cell fate decisions, may be implicated in colon cancer progression. Importantly, we also found that Kaiso’s effects on Notch pathway inhibition occurred prior to the onset of chronic intestinal inflammation. Our analyses of the chronic inflammatory phenotype in KaisoTg mice demonstrated that Kaiso overexpression drives pathogenic neutrophil-specific recruitment (as evidenced by increases in neutrophil-specific enzymatic activity, the formation of crypt abscesses, and augmented expression levels of the neutrophils-specific chemokine, MIP2); an increase in the pore-forming Claudin-2; reduction of the cell adhesion protein E-cadherin; and abnormal intestinal epithelial repair mechanisms. Together, these findings imply that the pathogenesis of Kaiso-mediated intestinal inflammation is a multi-factorial process. A secondary goal of this thesis was to initiate studies to elucidate how the Kaiso binding partner, Znf131, might play a role Kaiso-mediated transcriptional regulation. We found that Znf131 indirectly associated with several Kaiso target genes, including Cyclin D1 (CCND1). Importantly, Znf131 activated a minimal CCND1 promoter previously shown to be inhibited by Kaiso. Moreover, Kaiso overexpression attenuated Znf131-mediated transcriptional activation and Znf131 expression in intestinal cells. Together, these findings hint that Znf131 and Kaiso may exert opposing biological functions, which may have implications in Kaiso-mediated intestinal homeostasis and disease. / Thesis / Doctor of Philosophy (PhD)

N/A, thesis is written in chapters. / Thesis / Master of Science (MSc)

Crohn’s disease (CD) is an inflammatory bowel disease characterised by chronic inflammation with a complex pathophysiology involving host, environmental, and microbial factors. The intestinal microbiota is an important regulator of inflammation within the intestine, and a disruption of the interplay between gut bacteria and host immunity is a key factor in CD development. Intestinal inflammation itself is known to cause changes to the intestinal physiology that affect the ability of various bacteria to survive. Additionally, certain environmental risk factors for CD such as antibiotics are also known for their ability to impact the intestinal microbiota. CD is associated with various changes in the intestinal microbiome including increased colonisation with a group of bacteria known as adherent-invasive Escherichia coli (AIEC). The purpose of this study is to investigate how AIEC interact with antibiotics and intestinal inflammation in vivo. Multiple classes of antibiotics were found to increase the colonisation of AIEC and to increase its persistence. These antibiotics caused a loss diversity in the intestinal microbiome, but this did not explain the increased infectivity of AIEC. Antibiotic-induced inflammation was found to produce metabolites that benefitted AIEC growth in the intestine and similar results were found with chemically-induced inflammation. These results show that AIEC can benefit from both antibiotics and other sources of inflammation through inflammation-derived metabolites, which contributes to a greater understanding of the interactions between AIEC and CD. / Thesis / Doctor of Philosophy (PhD) / Crohn’s disease is a type of inflammatory bowel disease that affects many young adults in Canada. It causes a wide range of symptoms including nausea, pain, and diarrhea. While the disease can be treated with surgery and medications, it is considered incurable and affects most individuals for life. The exact cause of Crohn’s disease is not known, but it is thought to be caused by a combination of factors including genetics, environmental exposures, and changes in the number and types of bacterial species in the intestine. Intestinal bacteria play an important role in preventing inflammation in the intestine. An unusual strain of bacteria called adherent-invasive E. coli is found more commonly in Crohn’s disease patients than in healthy individuals. This strain does not cause the disease on its own, but it may interact with other environmental factors that are also associated with Crohn’s disease, such as taking antibiotics. Antibiotic use is a risk factor for developing Crohn’s disease later in life and antibiotics have previously been shown to promote the growth of other E. coli strains in the intestine. In a mouse model of Crohn’s disease, we found that antibiotics made mice more vulnerable to infection with this E. coli strain. This increased vulnerability was because the antibiotics caused inflammation, and we also found that other sources of inflammation benefitted this E. coli strain. These findings help us understand how gut bacteria and other Crohn’s disease risk factors might interact to cause the disease.

Chronic, low-grade inflammation is an underlying condition across a globally increasing number of debilitating diseases. These diseases include obesity, atherosclerosis, and diabetes and their resultant low-grade inflammation can be effectivity modeled with low dose stimulants such as lipopolysaccharide (LPS). While the innate immunity plays a significant role in fighting infectious disease, an initial exposure to low dose LPS hinders secondary infection clearance and pre-disposes murine models for fatal sepsis. Neutrophils are the most prevalent circulating innate immune cell and their homotypic aggregation, or swarming, is a key mechanism in clearing pathogens greater than 20 μm in size. We hypothesize that neutrophil swarming ability is altered when in a low dose LPS primed state; potentially leading to an overall altered innate immune response in the face of infection. However, an in vitro model does not currently exist to reliably quantify and compare neutrophil swarms across treatment groups. Here we propose a novel model utilizing fungal zymosan coated beads as a uniform target to which neutrophils may swarm. / Master of Science / White blood cells are critical for our body’s ability to fight off infection. The pathogens that cause infections come in many forms including fungus, viruses, and bacteria. However, in many debilitating inflammatory diseases such as heart disease and obesity, chronic inflammation prevents one’s white blood cells from being able to properly fight off infection. In order to study white blood cell function without the variability that is analogous to living pathogens, we propose a model system that simulates an artificial pathogen target where both the target and the surrounding environment can be precisely controlled. This system can then be used to study white blood cell function, specifically how it may be impacted under inflammatory conditions.

Neutrophils are the innate immune system's first line of defense in response to an infection. During an infection in the tissue, chemical cues called chemoattractants are released, which signal neutrophils to exit circulation and enter the tissue. Once in the tissue, neutrophils directionally migrate in response to the chemoattractant and toward the site of infection in a process called chemotaxis. At the site of infection, they initiate antimicrobial responses to clear the infection and resolve inflammation, restoring homeostasis. However, neutrophils are exposed to multiple chemoattractants and must prioritize these signals in order to correctly migrate to the appropriate site. The ability of neutrophils to properly undergo chemotaxis in the presence of infection and inflammation is crucial for resolution of inflammation and pathogen clearance. It has been recently shown that when pre-conditioned with bacterial endotoxin (LPS), innate immune function can become dysregulated. Neutrophils start to display altered antimicrobial response as well as dysfunctional migration patterns. This behavior has been seen in patients with sepsis, where a person's immune system overreacts to an infection, leading to systemic inflammation throughout the body, causing tissue damage, multiple organ failure, and in many cases, death. We explore the effects of inflammation on neutrophil migratory patterns and decision-making within chemotaxis. Additionally, to understand how inflammation within disease impacts chemotaxis, we measure the difference between neutrophils from healthy individuals and those from septic patients. We approached this using a combination of experimental and computational techniques. We developed a microfluidic assay to measure neutrophil decision-making in a competitive chemoattractant environment between an end-target (fMLP) and intermediary (LTB4) chemoattractant. Additionally, we probed for the expression level of molecules related to neutrophil chemotaxis. We also built a system of ordinary differential equations to model the dynamics of the molecular interactions underlying neutrophil chemotaxis. Our results showed that when neutrophils were induced into a highly inflammatory state, they prioritized pro-inflammatory signals over pro-resolution signals and displayed dysfunctional migration patterns. Similarly, neutrophils from patients with sepsis also displayed dysregulated migration patterns. This aberrant neutrophil chemotaxis may be implicated in the pathogenesis of sepsis, where accumulation of neutrophils in off-target organs is often seen. These results shed light onto the directional migratory decision-making of neutrophils exposed to inflammatory signals. Understanding these mechanisms may lead to the development of pro-resolution therapies that correct the neutrophil compass and reduce off-target organ damage. / Doctor of Philosophy / Neutrophils are innate immune cells that act as the first line of defense toward an infection. During an infection, chemical signals are released, stimulating neutrophils to migrate toward that specific site of infection. Once the cells are in the tissue, they can clear the pathogen and resolve inflammation. However, when neutrophils are migrating in the tissue, they are overwhelmed with multiple signals, directing them toward different sites. These signals must be prioritized by the cell so they can properly migrate toward the correct location. It has been recently shown that neutrophils that have been preconditioned into inflammatory states will display dysfunctional migration patterns. They are unable to migrate to the site of infection and instead migrate to healthy tissue, where they can cause damage. This has been shown in patients with sepsis, which is a condition where a person's immune system overreacts to an infection, causing inflammation throughout the body, leading to tissue damage and multiple organ failure. Our work explores the impact of inflammation on neutrophil migration patterns and the ability of the cell to properly prioritize when stimulated by multiple chemical signals. Additionally, we look at how neutrophils from healthy individuals differ from neutrophils from patients with sepsis, to understand how inflammation within disease impacts cellular migration. We approach this both experimentally and computationally. We designed a microfluidic assay to measure neutrophil migration in the presence of two competing chemical signals. We also measured the expression levels of molecules relevant to cell migration. We also built a mathematical model to investigate the molecular interactions underlying these processes. These results shed light on how inflammation impacts neutrophil migration and its role in inflammatory diseases.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production and deposition of nuclear self-antigen-containing immune complexes. Epigenetic factors, including altered microRNA (miRNA) expression, may contribute to aberrant immune cell function in SLE. miRNAs are small, noncoding RNAs that bind to the 3’ untranslated region of target mRNAs resulting in post-transcriptional gene modulation. IL-6, an inflammatory cytokine overproduced by mesangial cells in SLE, contains a potential binding site for miR-let-7a. In order to examine if alterations in miR-let-7a expression can influence inflammatory mediator production in SLE, we isolated mesangial cell miRNAs from 8 and 32- week-old female New Zealand Black/White (NZB/W) mice. We found miR-let-7a expression was significantly increased in the mesangial cells of pre-diseased and actively diseased NZB/W mice compared to age-matched female New Zealand White (NZW) controls. Overexpression of miR-let-7a in vitro increased IL-6 production in LPS/IFN-γ-stimulated mesangial cells compared to the stimulated control. Due to the crucial role of miR-let-7a in cell division and inflammation, we investigated miR-let-7a-mediated proliferation and NFκB activation in J774A.1 macrophages and MES 13 mesangial cells in vitro. Cell proliferation, retinoblastoma protein (Rb) phosphorylation, and NFκB activation were increased in cells transfected with miR-let-7a and stimulated with LPS/IFN-γ. Expression and production of the cell cycle inhibitor E2F5 was decreased in stimulated cells overexpressing miR-let-7a. We found that the cell cycle promoter E2F2 and NFκB target the miR-let-7a promoter. Next we sought to determine alterations in iii specific disease-associated miRNAs in female NZB/W mice treated with hydroxychloroquine (HCQ) or prednisone (PRED) for 12 weeks beginning at 20 weeks-of-age. We found that treatment with HCQ or PRED induced unique changes to miRNA expression in multiple tissues. In order to identify specific miRNAs as disease-modifying agents and not merely disease correlates, further in vitro analyses confirmed HCQ or PRED-mediated inhibition of miRNAs is critical to alter the inflammatory response. Taken together, our results suggest that overexpression of miR-let-7a may contribute to hyperplasia and the proinflammatory response in SLE. Our studies indicate that lupus therapeutics may work, in part, by altering the expression of disease-associated miRNAs in immune cells and the urine. / Ph. D.