Browsing by Author "Beck, Paul"
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Item Open Access Body Composition Features in Colorectal Cancer: Clinical Outcomes and Biological Factors(2022-12-06) Armstrong, Victoria Suzanne; Bathe, Oliver Frank; MacLean, Anthony; Beck, Paul; Culos-Reed, NicoleBackground: Cancer is a global health burden and a leading cause of death worldwide. One obstacle to improving patient outcomes is the development of cancer cachexia, which is formally described as the loss of muscle tissue (with or without adipose tissue atrophy). The classical presentation of cachexia is low muscle mass (sarcopenia) which can be assessed by computed tomography (CT) image assessment techniques, but this may not embody all important body composition phenotypes. I hypothesize that cancer can induce multiple clinically significant body composition (BC) features beyond sarcopenia, such as myosteatosis and high subcutaneous fat density (dense sub-q). Methods: In a cohort of patients with stage I – IV colorectal cancer (CRC) (N = 319), BC features were measured using Slice-O-Matic CT image analysis techniques. The influence of BC phenotypes and other clinical parameters on overall survival (OS) outcomes was assessed using univariate Kaplan-Meier and multivariate Cox regression analyses. Findings were then validated on an external dataset of 960 patients with CRC. To explore the physiological basis of these BC features, I performed targeted serum proteomics using Luminex assays and untargeted shotgun tumor proteomics using liquid chromatography-mass spectrometry. Results: Overall, the three BC features (sarcopenia, myosteatosis and dense sub-q) appeared independent of one another in 60% of cases. Myosteatosis and dense sub-q were independent predictors of worse OS outcomes (P < 0.001, P = 0.050). Sarcopenia was influential on reducing OS when phenotypic overlap was removed (P = 0.002). Several perturbed protein correlates were found in relation to each BC phenotype, specifically with respect to inflammation and metabolic processes. For example, myosteatosis was independently associated with increased leptin, resistin and adipsin (P < 0.01 for all). Conclusion: Overall, this work demonstrates that colorectal cancer can lead to the development of several BC phenotypes beyond just muscle wasting. Distinct patterns of serum protein abundance suggested that each of the BC features represented biologically distinct manifestations.Item Open Access Characterization of NLRP3 in the Maintenance of Cardiac Tissue Homeostasis(2014-02-13) Bracey, Nathan; Duff, Henry; Beck, PaulChronic cardiovascular diseases are characterized by tissue destruction and ongoing inflammation that impairs organ function. This non-microbial and chronic inflammation is mediated by pattern recognition receptors (PRRs). NLRP3 is one intracellular PRR that regulates pro-inflammatory cytokine secretion in response to endogenous signals from dying cells. While much in known regarding NLRP3 regulation of cytokine secretion, its broader function in diverse cell populations during cardiac injury has not been explored. Using murine models, we sought to characterize the role and expression of NLRP3 in chronic cardiovascular disease. We discovered that in addition to its established roles in regulating cytokine secretion, Nlrp3-/- mice were protected against Angiotensin II-induced hypertensive cardiac fibrosis despite negligible leukocytic infiltrate, apoptosis and cytokine processing. These results suggested the possibility of non-canonical signalling mechanisms in non-immune cells. We further explored the possibility of cytokine-independent roles for NLRP3 with the use of primary human and murine cell culture, microscopy and in vitro reporter systems. We began looking in cardiac fibroblasts, which displayed significant NLRP3 expression in left ventricular tissue samples from human patients with heart failure. NLRP3 was induced by pro-fibrotic signalling during cardiac fibroblast differentiation to myofibroblast phenotype by TGFβ. We found that NLRP3 augmented TGFβ-induced receptor associated Smad (R-Smad2/3) phosphorylation, nuclear accumulation and transcriptional activity in a Nucleotide Binding Domain-dependent mechanism. Interestingly, these phenotypes appeared independent from canonical NLRP3 signalling through caspase-1, IL-1β and IL-18. The ability of NLRP3 to regulate diverse cellular processes suggested a potentially broad function in cellular physiology. We went on to establish that endogenous NLRP3 localized to mitochondrial structures in primary human cardiac fibroblasts, macrophages and epithelial cell lines. Finally, using live-cell imaging and flow cytometry we found that NLRP3 potentiated the production of mitochondrial reactive oxygen species (ROS), which are required for TGFβ signalling and differentiation. Our results propose for the first time a general function for NLRP3 in structural and professional immune cells. A role for NLRP3 upstream of ROS offers a potentially unifying explanation for NLRP3 cytokine-dependent and independent pathways, and provides novel insight into the pathogenesis of chronic inflammation and fibrosis in cardiovascular disease.Item Open Access Clostridium difficile infection susceptibility is controlled by alterations to the gut microbiota before and after antibiotic exposure(2014-07-30) Schenck, Louis Patrick; Beck, Paul; MacDonald, JustinClostridium difficile (Cdif) infections (CDI) cause devastating diarrheal disease. Small clinical trials have generated excitement for the use of fecal microbial transplants as a therapeutic option; however, the exact components of the microbiota needed for protection against CDI have remained elusive. C57Bl/6 mice from two vendors, Jackson (JAX) and Charles River (CRV), were exposed to antibiotics before gavage with Cdif spores. JAX mice developed severe CDI as evidenced by weight loss, histological damage, and increased pro-inflammatory cytokines, whereas CRV mice were resistant to CDI. There were significant differences in the gut microbiota between the mice, but CRV mice retained a high abundance of Parabacteroides after antibiotics. Following cohousing, JAX mice had less severe CDI and CRV mice had more severe CDI. Transfer of P. distasonis, a CRV commensal bacteria, was able to dampen effects of CDI in JAX mice. This study may lead to more targeted bacteriotherapy for the treatment and prevention of CDI.Item Open Access Crohn’s-like disease in a patient exposed to anti-Interleukin-17 blockade (Ixekizumab) for the treatment of chronic plaque psoriasis: a case report(2019-09-05) Smith, Matthew K; Pai, Jay; Panaccione, Remo; Beck, Paul; Ferraz, Jose G; Jijon, HumbertoAbstract Background Plaque psoriasis and inflammatory bowel disease (IBD) are both chronic immune-mediated inflammatory diseases with an overlapping genetic profile and have been linked in epidemiological studies. Psoriasis and IBD share similar components in their inflammatory pathways and animal and human studies have suggested a potential role for targeting interleukin (IL)-17 with novel antibody therapies in the treatment of these diseases. These studies, while promising for psoriasis, have been associated with deterioration in patients with IBD. Post-hoc analyses of clinical trials involving Ixekizumab revealed adverse outcomes in a small cluster of patients with IBD, prompting recommendations to monitor this population with the use of this drug. Case presentation Forty-two year old Caucasian male with treatment-refractory chronic plaque psoriasis who developed new onset diarrheal illness and rectal bleeding following a 12 week induction period with Ixekizumab (anti-IL-17 neutralizing antibody). Colonoscopy revealed severe ulceration throughout the ascending and transcending colon. Histopathology, combined with endoscopic findings, led to a diagnosis of Crohn’s-like colitis. The patient’s anti-IL-17 medication was discontinued and endoscopic remission was induced with the use of corticosteroids, escalated anti-TNF therapy and eventually anti IL-12/23 neutralizing antibody (ustekinumab). Conclusion Murine studies implicate IL-17 and the downstream effects of its inhibition, in the breakdown of the gut epithelial layer, the disruption of normal host immune responses and the propagation of intestinal inflammation. The increasing use of IL-17 inhibitors has led to reports of exacerbation and potential development of inflammatory bowel disease. While clinical trials have revealed clusters of new inflammatory bowel disease cases amongst psoriasis patients using an IL-17 inhibitor, there remains a lack of evidence to suggest a causal relationship. This is the first case report of de-novo severe Crohn’s-like IBD in association with the use of Ixekizumab requiring rescue with escalated dosing of anti-TNF therapy and highlights the importance of close monitoring in patients being treated with IL-17 inhibitors, especially in those patients with known risk factors for inflammatory bowel disease.Item Open Access Gut-derived Danger Signals Contribute to Hepatocellular Injury and Inflammation during Non-alcoholic Fatty Liver Disease(2016) Reid, Danielle; Eksteen, Bertus; Reimer, Raylene; Madsen, Karen; von der Weid, Pierre-Yves; Beck, Paul; McKay, Derek; Jenne, CraigNon-alcoholic fatty liver disease (NAFLD) has become the leading liver disease in North America. Associated with the obesity epidemic, it is estimated that NAFLD currently affects over seven million Canadians. In certain cases, individuals with NAFLD may develop a progressive, inflammatory liver disease known as non-alcoholic steatohepatitis (NASH) that may lead to cirrhosis or hepatocellular carcinoma. Currently, the etiology of NASH remains unclear and there are no treatment strategies available beyond intensive dietary and lifestyle modification. The goal of this dissertation was to examine if the activation of liver-resident macrophages, Kupffer cells, by gut-derived metabolites contributes to the release of pro-inflammatory mediators and the recruitment of effector immune cells to promote inflammation and hepatocellular damage in experimental NASH. We found that Kupffer cells are critical for initiating inflammation during NASH while the absence of KCs limits the recruitment of leukocytes into the liver and protects against inflammation. Moreover, we identified unique transcriptional differences in resident and recruited macrophage populations that reflect the dynamic changes we characterized in Kupffer cells and infiltrating monocyte-derived macrophages during NASH. It has been suggested that compromised gut barrier function associated with obesity may result in the translocation of microbial by-products into the liver and contribute to hepatocellular injury and inflammation observed in NASH. We strengthen this link between the gut and the liver through the identification of novel, gut-derived volatile organic compounds (VOCs) in portal circulation in mice with NASH that have the potential to directly disrupt cellular processes in KCs and result in the secretion of proinflammatory cytokines. Once in the liver, microbial by-products such as VOCs may be detected by KCs through innate pattern recognition receptors such as the NLRP3 inflammasome, lead to the activation of KCs and contribute to inflammation in the liver. We found that the expression of the NLRP3 inflammasome within KCs has a dual proinflammatory and protective role during the development of experimental steatohepatitis. These studies provide evidence that gut-liver crosstalk influencing the activation of Kupffer cells is central in the development of experimental NASH and should be a major target for the development of novel treatment strategies.Item Open Access Hsp90 Regulates the NLRP3 Inflammasome via the NF-kB Signaling Pathway(2020-04-17) Sparksman, Steven; Beck, Paul; Muruve, Daniel; MacDonald, Justin; McKay, Derek; Braun, JaniceAn over-reactive inflammatory response can lead to chronic inflammation and auto-immune disorders such as Crohn’s disease, ulcerative colitis or cancer. At the heart of the host’s inflammatory response is an immune cell intracellular sensor protein known as NLRP3 that regulates the cellular response to a wide range of PAMPS/DAMPS. NLRP3 has been characterized primarily as an inflammasome-forming protein in response to infection and injury. The inflammasome regulates IL-1β and IL-18 maturation leading to their subsequent secretion from the immune cell. Secretion of these cytokines recruits other immune cells and factors that leads to the resolution of the initiating infection or injury. Hsp90, with its co-chaperone SGT1, was shown to be required for NLRP3 inflammasome activation via a direct protein-protein interaction. An Hsp90-SGT1 interaction was suggested to stabilize NLRP3 prior to inflammasome activation allowing the sensing of PAMPS/DAMPS; however, the mechanism, timing and sequence of events of this interaction have yet to be shown experimentally. Thus, the central hypothesis of this thesis is that Hsp90 regulates the activation of the NLRP3 inflammasome by stabilizing NLRP3 via direct protein-protein interactions. Treatment with DMAG, an Hsp90 inhibitor, blocked canonical NLRP3 function in differentiated THP-1 immune cells. However, we found no evidence that Hsp90-SGT1 was involved in protein-protein interactions with NLRP3. Instead, experiments revealed that DMAG attenuated IL1β gene transcription but did not interfere with translocation of the transcription factor, NF-kB to the nucleus. This suggests Hsp90 regulates the NLRP3 inflammasome by regulating transcription of NLRP3 inflammasome component genes. This project has revealed new insights for Hsp90 in the inflammatory response and suggests Hsp90 as a credible target for chronic inflammatory disorders.Item Open Access Investigating the Functional Role of Aquaporin 3 in Intestinal Epithelial Cells(2016) Morgan, Elizabeth Ellen; MacNaughton, Wallace; McKay, Derek; Beck, Paul; von der Weid, Pierre-YvesWater is the most abundant molecule in human cells and biological systems, and aquaporin (AQP) proteins have an important role in facilitating transepithelial transport of water. Aquaporin 3 (AQP3) is an aquaglyceroporin that is permeable to water and small solutes, and is expressed in intestinal epithelial cells. Inflammatory bowel diseases are associated with barrier dysfunction and altered absorption and secretion. Given that AQPs are involved in these processes, and are decreased in IBD, we sought to better understand the importance of AQP3 in epithelial cells of the gastrointestinal tract. Our studies show that in the absence of AQP3, HT29 adenocarcinoma cells exhibit impaired growth which is due to decreased proliferation caused by an arrest in G2/M phase of the cell cycle, and not increased rates of cell death. These results improve the understanding of the functional role and importance of AQP3 in intestinal epithelial cell homeostasis.Item Open Access On the Optimization of Clostridioides difficile Diagnostics Through RT-PCR Cycle Threshold Defined Zones of Disease Probability(2022-02-02) Doolan, Cody Patrick; Pillai, Dylan; Beck, Paul; Spackman, EldonClostridioides difficile is an opportunistic pathogen with a large burden of disease and no gold standard test. Quantitative polymerase chain reaction (qPCR) offers excellent sensitivity but overcalls clinical C. difficile infections (CDI) due to the prevalence of colonization. The hypothesis of this thesis is that the CDI qPCR results can be titrated to determine clinical CDI more accurately and aid in predicting disease severity. A cross-sectional study was conducted on suspected CDI patients evaluating if qPCR cycle threshold (Ct) can be correlated to probability of CDI. Latent class analysis (LCA) was employed with observed variables including four commercial qPCR tests, toxin detection by enzyme immunoassay, toxigenic culture, fecal calprotectin, and clinical diagnosis. Three defined zones as a function of qPCR cycle threshold (Ct) were identified: CDI likely (>90% probability), CDI equivocal (<90% and >10%), CDI unlikely (<10%). A model comprising toxigenic culture, clinical diagnosis, and toxin EIA demonstrated the best fitness. The following Ct cut-offs for 4 commercial test platforms delineated CDI probability zones: GeneXpert®: 24.00, 33.61; Simplexa®: 28.97, 36.85; Elite MGB®: 30.18, 37.43; and BD Max™: 27.60, 34.26. A prospective cohort study was conducted to investigate if these zones can be further correlated to indicators of severe CDI. Primary diagnosis, demographic data and indicators of disease severity were captured: white blood cell, creatinine, albumin, C-reactive protein, and hospital length of stay. A sub analysis was conducted evaluating a subset of the patient population attempting to isolate patients whose clinical variables were most influenced by CDI. No significant correlations were found between the clinical variables investigated and Ct values or Ct zones. This work establishes a method of using deployed diagnostics to allow clinicians to reduce overdiagnosis of CDI. Decreasing false positives could have broad impacts, increase targeted treatments, and decrease antibiotics. The average cost attributed to CDI for one patient is estimated at $11,917. LCA models predict that qPCR confirmation overdiagnoses patients in Calgary by at least 20.9%. If CDI confirmation were reduced by 20.9% this could equate to massive savings; Foothills Medical Center alone could save over $929,000 annually with no additional investment in laboratory infrastructure.Item Open Access Protecting the gut against Clostridium difficile: A role for Keratinocyte growth factor(2014-07-18) Alhassan, Basmah Faris; Beck, Paul; MacDonald, JustinClostridium difficle (Cdiff) infection (CDI) causes severe colitis via its toxins: toxin A and toxin B (TcdAB), inducing barrier disruption, inflammation and cell death. Current treatments are failing and the need to search for new targets is urgent. Several host factors have shown to modulate CDI in animals and patients. Intestinal growth factors are a major part of the mucosal host response in the gut. Among them, keratinocyte growth factor (KGF) has been shown to be protective in many colitis models. In this thesis, the protective role of KGF was demonstrated against Cdiff toxin injury. In vitro, KGF protected Caco-2 cells from barrier disruption and cell death induced by TcdAB. Exogenous KGF administration protected mice from acute intestinal toxin damage. Interestingly, KGF deletion did not impact the acute toxin-induced colitis in mice; however, endogenous KGF was essential for normal recovery from TcdAB-induced colitis as KGF−/− mice demonstrated impaired recovery after 24-48 hours post TcdAB exposure. Findings from this study may lead to identifying a cause for the variability in clinical response among patients with CDI as well as new therapeutic targets for this devastating disease.Item Open Access Regulation of Intestinal Epithelial Thymic Stromal Lymphopoietin Gene Expression by Retinoic Acid Receptor Alpha(2021-08-23) Mahmood, Ramsha; Jijon, Humberto; Beck, Paul; Hirota, Simon; McCafferty, Donna-Marie; Dufour, AntoineInflammatory bowel disease is characterized by chronic inflammation of the gastrointestinal tract. The pathogenesis is thought to be due to a dysregulated immune response to intestinal microbiota. Approximately 15% of the risk is genetically linked and approximately 85% is attributed to environmental exposures. Dietary factors like retinoic acid (RA), a vitamin A metabolite, have been linked to the onset of IBD by influencing intestinal immune function. RA induces Tregs and inhibits the actions of proinflammatory Th-17 cells. We have previously described decreased CD103+ DCs numbers in the intestinal compartments of RARα-deficient mice (RARαvillin mice). We sought to generate an experimental system to identify signaling pathway(s) or mechanism(s) that might be governing these effects, specifically RA signaling, as this data suggested there might be contributing factors intrinsic to IECs. We chose to establish a knockout cell line using the CRISPR/Cas9 system, given its affordability and efficiency compared to other in vitro models, and used it to study the effects of RAR⍺ ablation in IECs. Thus, our hypothesis was that RA signaling regulates the expression of lymphokines and other immune mediators (e.g., TSLP) by IECs, which then modulate the intestinal immune compartment. We hypothesized TSLP could be contributing to the decrease in CD103+ DCs as it is an important cytokine involved in TH2-type immunity and plays a key role in the maintenance of peripheral CD4+ T cell homeostasis by modulating the activation/maturation of myeloid cells. We used the CRISPR/Cas9 system to examine the effects of RARα ablation and its role in regulating intestinal epithelial TSLP expression. We found that TSLP expression is controlled by RARα in IECs where it may act as a repressor of TSLP promoter transactivation. This suggests an important role for RA signaling on myeloid/T cell function via effects on TSLP gene expression.Item Open Access Role of ING1b in Breast Cancer Promotion(2017) Bhunia, Pritha; Riabowol, Karl; Beck, Paul; Demetrick, DougINGs or Inhibitor of Growth proteins act as type II tumor suppressors in epithelial cancers. The senescence associated secretory phenotype (SASP) is a phenomenon where senescent cells secrete a defined group of proteins including various proinflammatory cytokines. Cancer-associated fibroblasts (CAFs) are another component of the tumor microenvironment secreting various growth factors, proinflammatory cytokines and proteases. The proinflammatory cytokines secreted by CAFs and senescent cells as SASP cause chronic inflammation, which is one of the hallmarks of cancer. Although generally described as a tumor suppressor, our lab has recently reported that higher ING1 levels in the stroma correlate with poor survival outcome in breast cancer patients. To better understand this, we examined the effects of altering ING1b levels in fibroblasts. We find that ING1b increases the secretion of IL8, IL6, CXCL1 and CCL7 (also secreted by CAFs and in the SASP). This most likely occurs by ING1b activating NF-κB. Factors secreted by fibroblasts in response to ING1b also enhances the growth and migration capacity of several lines of breast cancer cells and also induce a cancer like phenotype of migration and growth in normal breast epithelial cells.Item Open Access The Cellular Hypoxic Response upon Giardia Duodenalis Infection(2024-08-08) DeMichele, Emily; Buret, Andre G.; Hirota, Simon A.; Finney, Constance; Beck, PaulOxygen tension plays an integral role in gastrointestinal homeostasis. When oxygen concentrations become suboptimal (hypoxia), mammalian cells rely on the hypoxia-inducible factor (HIF) to combat cellular stress and augment glycolytic flux. In the gastrointestinal epithelium, this cellular response is paramount as the luminal facing epithelial cells exist in a state of physiologic hypoxia that can be further altered by pathogens. Although growing evidence suggests tissue and blood protozoan parasites can activate HIF in a mammalian host, much remains to be understood in the context of enteric protozoan infections. This study uncovered the role of HIF-1α and associated epithelial hypoxic signature upon Giardia duodenalis infection, a leading cause of diarrheal disease worldwide. Coculture experiments were carried out using Caco-2 colonic epithelial cells infected with a human isolate of G. duodenalis (GSM) in normoxic (21% O2) or hypoxic (1% O2) conditions. Under normoxic conditions, infected cells displayed a time-dependent increase in HIF-1α protein expression, the oxygen-dependent subunit of HIF. This observation was concomitant with increased expression of HIF-target genes, including those responsible for combatting cellular stress (i.e., VEGFA, GADD45A, ANKRD37), and augmenting glycolytic flux (i.e., HK2, LDHA). Intracellular metabolomic analysis of normoxic infected cells revealed simultaneous increases in glucose-6-phosphate and lactate, the metabolic outputs of HK2 and LDHA, respectively. Under hypoxic conditions, HIF-1α stabilization was not significantly different compared to uninfected hypoxic control cells. However, HIF-target genes were still upregulated, albeit to a lesser degree compared to normoxic infected cells. Importantly, the metabolome of infected epithelial cells differed greatly between normoxic and hypoxic conditions, highlighting the influential role of oxygen during G. duodenalis infection. After 24 hours, HIF-1α stabilization and alterations to HIF-target gene expression were no longer detected. These findings indicate G. duodenalis induces a hypoxic response driven by HIF-1α stabilization in normoxic intestinal epithelial cells, while simply exacerbating this cellular response in hypoxic conditions. The stabilization of HIF-1α in the absence of oxygenic stress highlights a novel metabolic cell rescue mechanism in response to enteropathogens.Item Open Access The Effects of Smoking on Autophagy in Transformed Cell Lines and Monocyte-derived Dendritic Cells of NOD2 Genotyped Inflammatory Bowel Disease Patients(2016) chenoo, Shem; Ghosh, Subrata; Beck, Paul; Kaplan, Gilaad; Hirota, SimonGenome-wide association studies have identified around 200 genes associated with inflammatory bowel disease (IBD) among which 3 susceptible genes including NOD2, ATG16L1 and IRGM are found to increase risk of Crohn’s Disease (CD) and altering the metabolic pathway known as autophagy. The aim of this study is to look at how smoking which is detrimental in CD but beneficial to ulcerative colitis (UC) affects autophagy in both cell lines as well as primary cells. HeLa cells and THP-1 like macrophages as well as monocyte-derived dendritic cells were treated with cigarette smoke extract (CSE) which is a surrogate of smoking at different concentrations for 24h. Cells were treated with rapamycin or muramyl dipeptide (MDP) and autophagy was assessed by microscopy and Western blotting. CSE significantly inhibits autophagy in HeLa cells and THP-1 like macrophages through both the mTOR and NOD2 mediated autophagic pathways.Item Open Access The Human Interleukin-4 Treated Macrophage and Epithelial Wound Repair(2018-04-17) Jayme, Timothy; McKay, Derek; Beck, Paul; Jijon, Humberto; Yates, Robin; Hanington, PatrickThe murine interleukin-4 (IL-4) treated alternatively activated macrophage (M(IL4)) is known to have key roles in wound repair. Given that mucosal wound healing is the best indicator of long term remission for patients with inflammatory bowel disease (IBD), adoptive transfer of the M(IL4) in mice has been extensively studied as a therapeutic, and its beneficial outcome may be induced, in part, by M(IL4)s inducing wound healing. However, research has been extremely limited in translating these findings into human studies. Furthermore, helminth-derived soluble products can induce and enhance the M(IL4) phenotype in both mice and humans, however, its ability to enhance M(IL4) function is unknown. Therefore, my MSc research was designed to test the hypotheses that: 1) the human blood-derived M(IL4) can be an autologous cell immunotherapy for IBD by inducing mucosal wound repair; and, 2) in vitro treatment of M(IL4)s with an extract of the rat tapeworm, Hymenolepis diminuta (HdE), can enhance its capacity to wound repair. To my knowledge this is the first time that a human blood-derived M(IL4) (CD206highCCL18+CD14low) has been reported to promote colonic epithelial wound repair in an in vitro scratch assay: an ability dependent on TGF and prostaglandins. Furthermore, cryopreservation of human M(IL4)s, which could be an important process if used therapeutically, did not lose their ability to promote wound repair, although decreased M(IL4) markers were noted. Monocytes from patients with inactive Crohn’s disease or ulcerative colitis could be differentiated into M(IL4)s with the capacity to promote epithelial wound repair. Interestingly, M(IL4)s from patients with active Crohn’s disease and ulcerative colitis were more variable in their response to IL-4, and impaired M(IL4) activation (CD206lowCCL18+CD14low) correlated with an impaired ability to promote epithelial wound repair. This suggests that in vivo, M(IL4)s may have an important role in the resolution of inflammation by inducing mucosal wound repair in patients with active IBD. Finally, I could not find any direct evidence, in vitro, that HdE enhances the capacity of M(IL4)s to promote wound repair. Having defined the ability of the monocyte-derived M(IL4)s from healthy volunteers and patients with IBD to promote epithelial wound healing, my research lays the foundation for a more extensive investigation of the anti-inflammatory/pro-resolution activity of the human M(IL4), which could be used as an autologous transplant in a personalized medicine approach to IBD or other enteropathies.Item Open Access The Role of Protease-Activated Receptor-2 During Wound Healing in Intestinal Epithelial Cells(2017) Fernando, Elizabeth; MacNaughton, Wallace; Hollenberg, Morley; Beck, Paul; Waterhouse, ChrisThe intestinal epithelial barrier is a single layer of epithelial cells that functions to regulate absorption and secretion, in addition to protecting our bodies from the contents of the intestinal lumen. When the barrier becomes damaged, uncontrolled passage of bacterial and antigenic factors generates an immune response that can result in intestinal inflammation. One principal step in the resolution of inflammation is epithelial barrier healing, which stops the entry of inflammatory triggers. The mechanisms of intestinal epithelial wound healing are not completely understood, especially in the context of proteases and their receptors. The intestinal epithelial barrier is exposed to numerous proteases originating from luminal bacteria, host immune cells, and proteases expressed by the epithelial cells themselves. It was recently shown that activation of protease-activated receptor-2 (PAR2) on epithelial cells induced the expression of cyclooxygenase-2 (COX-2), which has protective functions in the gastrointestinal tract. It was hypothesized that PAR2-induced COX-2 could enhance wound healing in intestinal epithelial cells. In the first part of this study, PAR2-induced COX-2 was characterized using western blotting and ELISA techniques to perform time-course and dose-response experiments. Actinomycin D was used to determine that PAR2-induced COX-2 was transcriptionally regulated. Potential components of the PAR2-COX-2 signaling pathway, including Rac1 and CUX1, were studied using pharmacological inhibitors and siRNA. However, both Rac1 and CUX1 were not involved in the PAR2-COX-2 signaling pathway. In the second part of this study, the Caco2 cell model was used for epithelial wound healing. Contrary to our hypothesis, PAR2 activation inhibited wound healing, independently of COX-2 activity. The inhibition of wound healing was due to reduced migration associated with a PAR2-mediated reduction in lamellipodia formation at the wound edge, and an increase in E-cadherin expression surrounding the wound. Conversely, when wound healing was investigated in T84 intestinal epithelial cells, PAR2 activation was found to enhance wound healing through increased cell migration, with opposite effects on actin dynamics and E-cadherin expression compared to the data obtained from Caco2 cells. These findings represent a novel effect of PAR2 activation on the mechanisms of epithelial cell wound healing that could influence the resolution of intestinal inflammation.Item Open Access Vitamin D induces the release of IL-1b via activation of the NLRP3 inflammasome(2012-07-30) Tulk, Sarah; MacDonald, Justin; Beck, PaulThe Crohn’s disease pathogenesis has been proposed to involve a hypoactive innate immune response and impaired acute inflammatory response. Recently, vitamin D3 has been shown to induce expression of the innate immune receptor, NOD2. Herein, PMA-differentiated THP-1 cells were treated with the active form of vitamin D3, 1,25(OH)2D3, and assessed for release of the pro-inflammatory cytokine IL-1β. We present novel findings that 1,25(OH)2D3 induces the release of IL-1β from PMA-differentiated THP-1 cells. Further, circulating vitamin D3, 25(OH)D3, was also able to induce the IL-1β response. Both 1,25(OH)2D3-induced and 25(OH)D3-induced IL-1β release were found to be dependent on the NLRP3 inflammasome. These novel findings support a beneficial role for vitamin D3 in Crohn’s disease; by boosting basal IL-1β secretion from macrophages, vitamin D3 could help counteract the impaired innate immune and acute inflammatory responses seen in this disease.