Browsing by Author "Muruve, Daniel"
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- ItemOpen AccessA Non-Canonical Function for NLRP3 and AIM2 in Kidney Diseases(2017) Chung, Hyun Jae; Muruve, Daniel; Hollenberg, Morley; Altier, ChristopheNLRP3 and AIM2 are inflammasome-forming proteins that have been mostly studied in leukocytes. Canonical NLRP3 or AIM2 inflammasomes regulate cytokine maturation and pyroptosis via ASC and caspase-1 activation. Emerging studies demonstrate that NLRP3 or AIM2 is expressed in non-haematopoietic cells such as tubular epithelial cells (TEC) in the kidney. The central hypothesis of this thesis is that NLRP3 and AIM2 regulate host response to renal injury in the kidney. Primary mouse TEC lacking Nlrp3 displayed reduced caspase-8 activation downstream of the tumor necrosis factor (TNF) receptor and CD95. TNFα/cycloheximide treatment induced NLRP3/ASC/caspase-8 speck-like complex formation at the mitochondria during apoptosis. The assembly of NLRP3/ASC/caspase-8 specks was downstream of TNFR signaling and independent of caspase-1 or -11 activation. This data shows that NLRP3 and ASC form a conserved non-canonical platform for caspase-8 activation, independent of the inflammasome that regulates apoptosis within epithelial cells. Interestingly, AIM2 was detected primarily in podocytes in the glomerulus and distal tubules at a low level. In a mouse model of nephrotoxic serum (NTS)-mediated anti-GBM, Aim2-/- mice displayed increased glomerular cellular crescent (multilayered accumulation of activated parietal cells) formation, tubular injury and inflammation, and worse renal function compared to wild-type controls. In vitro outgrowth of podocyte lacking Aim2 was greater than wild-type and Aim2-/- podocytes did not express Nhps2 (podocin) mRNA, a podocyte maturation marker. Furthermore, AIM2 was found to augment transcriptional activity of Wilm’s tumour-1 that regulates Nphs1 expression. This data suggests that non-canonical AIM2 regulates podocyte maturation, proliferation and potentially podocyte-to-parietal cell trans-differentiation during cellular crescent formation in vivo. In a mouse model of kidney ischemia/reperfusion in vivo, a significant difference in tubular injury was not detected between wild type and Aim2-/- mice. In vitro, tubular Aim2 did not regulate caspase-8 activation during apoptosis, confirming a limited role for Aim2 in tubular cell death. However, Aim2 deficiency attenuated TGFβ-mediated Smad phosphorylation and αSMA induction. Overall, these data increase the understanding of NLRP3 and AIM2 biology in the kidney diseases and highlight overarching non-canonical roles for NLRP3 and AIM2 to regulate critical biological processes in the kidney independent of inflammasome activation
- ItemOpen AccessHsp90 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.
- ItemOpen AccessInvestigating the Stimulator of Interferon Genes Pathway as a Translational Immunogenic Therapy for Soft Tissue Sarcoma(2024-06-17) Hildebrand, Karys Maddison; Monument, Michael James; Jirik, Frank; Muruve, Daniel; Dufour, AntoineUndifferentiated pleomorphic sarcoma (UPS) is one of the most common, aggressive, and metastatic soft tissue sarcomas in adults. Generally, UPS are unresponsive to conventional chemotherapies or immunotherapies, which is believed to be attributed to the immunosuppressive tumor microenvironment (TME) of these malignancies. Preclinical studies in the murine KP model of UPS showed that intratumoral (i.t.) activation of the STimulator of INterferon Genes (STING) pathway using the murine STING agonist DMXAA, elicited immune mediated UPS clearance in 50-75% of treated mice. To assess the translational potential of STING immunotherapy, I tested the anti-tumor efficacy of three STING agonists in the KP model of UPS capable of activating both human and murine STING. Excitingly, E7766 emerged as a translational STING agonist which can shift the UPS TME towards an immunologically inflamed phenotype. Thirty-eight percent of E7766 treated mice eradicated their primary tumors which was CD8+ T-cell dependent. Of the mice that eradicated primary UPS tumors, 87.5% develop protective immunity against UPS re-challenge. Next, I investigated which cell types in the UPS TME engage in STING signaling following therapy. I found that STING expression in non-malignant host cells and not UPS cells is required to observe tumor eradication following STING immunotherapy. Single cell RNA sequencing of UPS tumors revealed that neutrophils are abundant cells in the TMEs of UPS tumors treated with DMXAA and E7766 at both timepoints. Myeloid cells were identified as the cell type with the highest interferon stimulated gene (ISG) score. Both DMXAA and E7766 maintain higher ISG scores in myeloid and lymphoid cells relative to control and CDN at the 1-week timepoint. Finally, I developed a novel gene therapy tool to explore forced expression of the constitutively active mutant hSTINGN154S protein using plasmid DNA. Using these tools, I transfected TAO1 UPS and HEK293T cells and confirmed the functional status of the hSTINGN154S protein’s expression in vitro. In summary, these data suggest that E7766 is an exciting therapeutic candidate for UPS, and further investigation into the importance and consequences of STING signaling in various UPS TME cell types is required to understand therapeutic mechanisms of this therapy.
- ItemOpen AccessNLRP3 in Renal Injury and Inflammation(2014-07-11) Vilaysane, Akosua; Muruve, DanielRenal cell injury, death and inflammation are hallmarks of chronic kidney disease (CKD). Unresolved inflammation can chronically damage renal tissue resulting in fibrosis and kidney failure. The Nlrp3 inflammasome has been implicated in the innate immune response to cellular injury and represents a potentially important pathway in the pathogenesis of CKD. The central hypothesis of this thesis is that NLRP3 plays a significant role in renal inflammation and chronic renal injury. In a mouse model of progressive renal injury and fibrosis (unilateral ureteral obstruction, UUO), Nlrp3 expression and inflammasome activation were increased in injured kidneys. Compared to wild-type counterparts, Nlrp3-/-mice undergoing UUO displayed reduced renal injury, fibrosis and inflammatory cytokine levels confirming an important role for Nlrp3 in experimental kidney disease. Bone marrow chimeras in the UUO model indicated that in addition to its inflammasome-forming capabilities in leukocytes, Nlrp3 may play a novel role in the renal epithelial compartment. Indeed, Nlrp3 expression was found in both human and mouse primary tubular epithelial cells. Unlike the inflammasome-forming capability of Nlrp3 in macrophages, renal tubular epithelial cells lacked the components of the inflammasome including caspase-1 and its substrate IL-1β. Rather, Nlrp3 primarily regulated caspase-8 activation and apoptosis in these cells. Experiments to elucidate the mechanism of Nlrp3- regulated caspase-8 activation and apoptosis suggested the involvement of Asc, but not inflammatory cytokines, caspase-1 or caspase-11. Furthermore, Nlrp3-/- tubular epithelial cells were found to have impaired mitochondrial ROS production that was linked to decreased caspase-8 activation during apoptosis. Taken together, results from this thesis identify a significant and multidimensional role for Nlrp3 in the pathogenesis of kidney disease. First, Nlrp3 inflammasome activation in macrophages releases cytokines such as IL-1β and IL-18 that cause renal inflammation, fibrosis and tubular epithelial cell apoptosis. Second, Nlrp3 expressed in tubular epithelial cells displays an intrinsic inflammasome-independent function to regulate caspase-8, mitochondrial ROS and apoptosis. These data identify Nlrp3 as an important player in renal injury and potential therapeutic target for CKD.
- ItemOpen AccessPathogenesis of Renal Fibrosis: a Role for Proteinase-activated Receptor-2(2013-01-28) Chung, Hyun Jae; Hollenberg, Morley; Muruve, DanielRenal fibrosis is the final manifestation of all progressive chronic kidney disease with a significant morbidity and mortality. However, the underlying mechanisms remain largely unknown. Proteinase-activated Receptor-2 (PAR2) is a G-protein-coupled receptor that is proteolytically activated by serine proteinases such as trypsin. In our in vitro studies, we found that stimulation of PAR2 in human proximal tubular cells with PAR2-activating peptide alone significantly upregulated expression of CCN2 and did so synergistically to augment Transforming growth factor-β (TGF-β)-induced CCN2 production. This synergy was reduced by MAPKinase inhibition. We also found that PAR2 deficiency in mice with unilateral ureteral obstruction (UUO) significantly reduced tubular injuries and fibrosis, and synthesis of renal collagen and α-smooth muscle actin at 7 days post UUO. Our findings demonstrate the potential contribution of PAR2 to renal injury and fibrosis at an early time point due to the ability of PAR2 to augment the production of a profibrotic cytokine.
- ItemOpen AccessSociodemographic associations with abnormal estimated glomerular filtration rate (eGFR) in a large Canadian city: a cross-sectional observation study(2018-08-09) Ma, Irene; Guo, Maggie; Muruve, Daniel; Benediktsson, Hallgrimur; Naugler, ChristopherAbstract Background Chronic kidney disease (CKD) is often asymptomatic in its early stages but is indicated and is diagnosed with an estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73m2. Certain sociodemographic groups are known to be at risk for CKD, but it is unclear if there are strong associations between these at risk groups with abnormal eGFR test results in Canada. Using only secondary laboratory and Census data, geospatial variation and sociodemographic associations with abnormal eGFR result rate were investigated in Calgary, Alberta. Methods Secondary laboratory data from all adult community patients who received an eGFR test result were collected from Calgary Laboratory Service’s Laboratory Information System, which is the sole supplier of laboratory services for the large metropolitan city. Group-level sociodemographic variables were inferred by combining laboratory data with the 2011 Canadian Census data. Poisson regression and relative risk (RR) were used to calculate associations between sociodemographic variables with abnormal eGFR. Geographical distribution of abnormal eGFR result rates were analyzed by geospatial analysis using ArcGIS. Results Of the 346,663 adult community patients who received an eGFR test result, 28,091 were abnormal (8.1%; eGFR < 60 ml/min/1.73m2). Geospatial analysis revealed distinct geographical variation in abnormal eGFR result rates in Calgary. Women (RR = 1.11, P < 0.0001), and the elderly (age ≥ 70 years; P < 0.0001) were significantly associated with an increased risk for CKD, while visible minority Chinese (RR = 0.73, P = 0.0011), South Asians (RR = 0.67, P < 0.0001) and those with a high median household income (RR = 0.88, P < 0.0001) had a significantly reduced risk for CKD. Conclusions Presented here are significant sociodemographic risk associations, and geospatial clustering of abnormal eGFR result rates in a large metropolitan Canadian city. Using solely publically available secondary laboratory and Census data, the results from this study aligns with known sociodemographic risk factors for CKD, as certain sociodemographic variables were at a higher risk for having an abnormal eGFR test result, while others were protective in this analysis.
- ItemOpen AccessStructural Dissection and Catalytic Properties of the NLRP (Nucleotide-Binding Domain and Leucine-Rich Repeat-Containing Gene Family, Pyrin Domain Containing) Family of Inflammatory Proteins(2022-09-19) Sandall, Christina F.; MacDonald, Justin; Muruve, Daniel; Lees-Miller, SusanInflammasomes are high molecular weight hetero-oligomeric protein complexes nucleated by innate immune cytosolic pattern recognition receptors (PRRs) including the NOD-like receptors (NLRs). A subset of NLR proteins include those with N-terminal Pyrin domains (NLRPs) which play critical roles in the detection and response to both endogenous and exogenous danger signals. The NLRP3 inflammasome is the most highly studied and contributes to a multitude of inflammatory and autoimmune conditions. Thus, this work provides a more detailed understanding of NLRP3 activation mechanisms that will be essential for the rationalised development of future pharmacological interventions. First, the impact of orientation and linkage on NLRP3 capture with immobilised ATP demonstrates ATP binds this protein with an exposed phosphate tail and buried adenine ring. Decreased competitive recovery with free ATP indicated NLRP3 underwent a conformational change upon ATP binding. Additionally, P-linked ATP Sepharose provides a strategy for capture of the entire NLRP family and enrichment of NLRP3 containing samples for mass spectrometry analyses. Next, the ATP hydrolysis kinetics of NLRP proteins and hyperactive NLRP3 disease mutant R262W were evaluated with GFP nano-trap beads and a bioluminescent ATPase assay. NLRP proteins displayed distinct ATPase kinetic profiles, suggesting variable ATP sensitivity and kinetics of assembly for some NLRP proteins. Classical Michaelis-Menten kinetics were observed for NLRP1, 3 and 12, and positive Hill cooperativity was revealed for NLRP3R262W as well as NLRP6 and 7. Furthermore, NLRP3 inhibitors targeting ATPase activity demonstrated promising results in blocking inflammasome activation. Next, the impacts of NLRP3 phosphorylation at Ser295 on protein structure, ATP binding & inflammasome activation were evaluated in silico. These results suggest that modifications to the NACHT domain are conveyed globally and result in variable nucleotide hydrolysis and inflammasome activation. Finally, unique mechanisms of ATP and ADP binding and the detailed structural impacts were illuminated by molecular dynamics simulations with NLRP3. NLRP3-ADP simulations indicate high protein stability and few global rearrangements, while NLRP3-ATP binding was thermodynamically favourable, induced protein flexibility, and resulted in global structural rearrangements that were initiated from the NACHT domain. To conclude, a detailed mechanism of ATP induced structural changes provided a basis for rationale design of NLRP3 inhibitors, and a region of NLRP3 in a cleft between the HD2 and LRR domains was proposed for pharmacological targeting.
- ItemOpen AccessThe Role of Endothelial Focal Adhesion Kinase (FAK) and FAK Related Non-Kinase (FRNK) in Leukocyte Recruitment(2017-01) Sharma, Ritu; Deans, Julie; DeVinney, Rebekah; Muruve, DanielEndothelial cells form the first barrier that leukocytes have to breach in order to reach the site of inflammation. Focal adhesions are the cellular structures that connect the endothelial cytoskeleton to the extracellular matrix. Focal adhesion kinase (FAK) is a signalling protein that is localized to focal adhesions and becomes phosphorylated during leukocyte recruitment. The main aim of this thesis research was to test the hypothesis that FAK is required for leukocyte transmigration. We used in vitro models of TNF-α - and IL-4-mediated inflammation to examine the recruitment and transmigration of neutrophils and eosinophils, respectively. We observed shear-independent loss of FAK in the proximity of neutrophil transmigration. Downregulation of FAK by siRNA, or disruption of FAK signalling by overexpression of FRNK (FAK-related non kinase, an inhibitor of FAK) decreased neutrophil transmigration without interfering with the TNF-α signalling pathway, implicating a functional role for FAK during neutrophil transmigration. In contrast, downregulation of FAK had no effect on eosinophil transmigration. Surprisingly, FRNK overexpression reduced eosinophil transmigration and this was shown to occur independently of FAK. FRNK blocked eosinophil recruitment and transmigration by preventing transcription and protein expression of IL-4 mediated VCAM-1 and CCL26. Interestingly, the FAK inhibitor FAK-II also blocked VCAM-1 and CCL26 expression through an unknown mechanism that we propose involves enhanced availability of the scaffolding domains of inactivated FAK. Finally, we attempted to determine how FRNK regulates expression of VCAM-1. Phosphorylation and nuclear localization of the transcription factor STAT6 were unaffected by FRNK. We found that, in addition to STAT6, IL-4 also regulates expression of GATA6, which induced VCAM-1 expression, and that loss of GATA6 attenuated eosinophil recruitment and transmigration. FRNK reduced IL-4-induced transcription of GATA6 but not its translation. Although we were unable to determine how FRNK regulates VCAM-1, endogenous FRNK was shown to be regulated by IL-4, suggesting an anti-inflammatory role for FRNK in an IL-4 model of leukocyte recruitment.
- ItemOpen AccessThe Role of NLRP3 in Shiga toxin Induced Inflammation(2018-01-08) Bondzi-Simpson, Nana Adom; Muruve, Daniel; Ho, May; Chadee, Krisendath; Armstrong, Glen D.Enterohemorrhagic Escherichia coli (EHEC) is a pathogen that causes severe colitis. Shiga toxins (Stx) are a major virulence factor for EHEC and Stx-induced inflammation and cell death play a critical role in the development of EHEC-related disease. Therefore, the goal of this project was to examine the mechanisms by which Stx activates proinflammatory cytokine IL-1β and cell death in human macrophages. Stx induced ROS-dependent IL-1β secretion and pyroptosis in human THP-1 macrophages but not murine macrophages that lacked the Stx receptor CD77. Stx triggered the assembly of NLRP3, ASC and caspase-1 containing inflammasomes and genetic deletion of NLRP3 abolished Stx-induced IL-1β maturation and pyroptosis. Stx preferentially induced expression the endoplasmic reticulum stress receptor IRE1α. Pharmacological inhibition of IRE1α significantly reduced Stx-induced mitochondrial ROS production, IL-1β, and pyroptosis. These data suggest that Stx activate the IRE1α arm of the ER stress pathway upstream of mitochondrial ROS to trigger the NLRP3 inflammasome.