Browsing by Author "Dufour, Antoine"

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    Enhancing remyelination through modulation of phagocytosis and lipid recycling
    (2025-01-30) Wuerch, Emily Claire; Yong, Voon Wee; Ousman, Shalina; Dufour, Antoine
    Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), characterized by demyelination and neurodegeneration. MS pathology also includes the presence of inhibitory myelin debris. Current MS therapies primarily target inflammatory processes, such as immune cell infiltration, but fail to directly promote remyelination, representing a significant gap in treatment. Previous research has shown that niacin (vitamin B3) can promote remyelination in the lysolecithin-induced demyelination model by enhancing the phagocytic clearance of inhibitory myelin debris by macrophages and microglia. However, its therapeutic potential in experimental autoimmune encephalomyelitis (EAE), an inflammatory MS model, was previously unexplored. In the first part of my thesis, I tested the hypothesis that niacin administration could ameliorate EAE-induced clinical disability and neuropathology through immune modulation and enhanced remyelination. We found that niacin inconsistently ameliorated EAE clinical disease scores, and failed to promote remyelination. We propose that these outcomes may stem from the inability of niacin to modulate lymphocyte activity, a key driver of EAE pathology. Building on this work, I began investigating lipid recycling from foamy macrophages, as evidence suggests that microglia may be a source of cholesterol for oligodendrocytes during remyelination. Following demyelination, macrophages engulf myelin debris, including lipids, and develop a foamy, lipid-laden phenotype, characterized by increased inflammation and diminished capacity to clear additional toxic debris. We investigated strategies to improve phagocytosis and lipid recycling, hypothesizing that promoting lipid export from foamy macrophages could enhance neuronal and oligodendrocyte survival and facilitate remyelination. To test this hypothesis, we first characterized myelin phagocytosis by human and murine microglia and macrophages, and generated foamy macrophages through chronic exposure to myelin and inflammatory cytokines. To enhance lipid export, we treated foamy macrophages with cyclodextrins, compounds known to promote lipid clearance in other disease models. In collaboration with our chemistry team, we tested both a standard commonly-used commercial cyclodextrin and novel formulations. We also used the lysolecithin demyelination model to assess the therapeutic potential of cyclodextrins in an animal model of multiple sclerosis. Cyclodextrins facilitated lipid export from foamy macrophages in culture, and promoted a beneficial macrophage phenotype in lysolecithin lesions. Cyclodextrins also preserved axons and increased the presence of oligodendrocyte lineage cells in lesions. In conclusion, cyclodextrins offer a promising therapeutic strategy for people with MS by enhancing oligodendrocyte cell number and reducing neurodegeneration, addressing limitations of current therapies. Overall, this thesis explores several novel therapeutic approaches to modulate lipid metabolism and myelin debris clearance in MS, with the overarching goal of advancing remyelination therapies.
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    Evaluating Mass Spectrometry Approaches for Identifying Proteomic Changes in Maternal Urine in Pregnancy and Labour
    (2025-01-31) Sonali; Dufour, Antoine; Slater, Donna; MacDonald, Justin; Sycuro, Laura
    Preterm birth (PTB) is a critical global health issue, contributing to significant neonatal mortality and long-term complications. Almost half of the preterm births occur due to spontaneous preterm labour (PTL). Despite substantial research efforts, our knowledge of labour onset and progression leading to either preterm and term delivery is limited. With the intent of exploring more about the physiological processes of pregnancy and labour, we performed label-free shotgun proteomics on maternal urinary samples using two different modes of mass spectrometer – data- dependent acquisition (DDA) and data-independent acquisition (DIA). The goal was to assess what approach resulted in high quality and reproducible data by detecting and quantifying large number of proteins for a discovery study using urine samples. Comparative analysis of DDA and DIA demonstrated superior performance of DIA. DIA resulted in better data in terms of consistency of protein detection, retaining higher number of total proteins and uniquely detected proteins for downstream analysis and presence of fewer number of missing values. To investigate further, we expanded our discovery cohort and performed DIA proteomics. Proteomics data analysis revealed elevated levels of Defensin alpha 1 (DEFA1) and Kallikrein 1 (KLK1) in the Labour group while the Non Labour group showed elevated levels of Endosialin (CD248) and Leucine-rich alpha-2-glycoprotein (LRG1). To identify the proteomics changes with respect to gestational age, we compared Preterm Non Labour (PTNL) group with Term Non Labour (TNL) group. The PTNL group was found significantly enriched in Fatty acid binding protein 5 (FABP5) and Keratin type 1 cytoskeletal 16 (KRT16) while TNL group showed significant enrichment in Calmodulin (CALM1). Differential levels of aforementioned proteins seem to be associated with labour and changes with gestational age. Future studies are required to validate the observed trends in protein abundances in the study groups and to determine the potential of these proteins for prediction of preterm labour or preterm birth.
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    High MUC2 Biosynthesis and Production Augment Pro-Inflammatory Responses in Colonic Goblet Cells
    (2023-09-11) Kim, Ariel Jayu; Chadee, Kris; McCafferty, Donna-Marie; Dufour, Antoine
    Goblet cells are specialized secretory epithelial cells whose primary function is to secrete mucins into the lumen of the gastrointestinal tract to form the mucus layer, which acts as a physical barrier separating luminal contents from the underlying epithelium. During amoebiasis, caused by the human intestinal protozoan parasite, Entamoeba histolytica, goblet cells hypersecrete and become depleted of MUC2 mucin, impairing the protective mucus barrier to allow the parasite to contact the colonic epithelium and induce a robust pro-inflammatory cytokine response. While colonic absorptive epithelial and immune cells have been shown to produce pro-inflammatory cytokines, it is not known if goblet cells do so as well in addition to mucin production. In this study, I investigated if the pro-inflammatory chemokine, CXCL8, was altered in WT and CRISPR/Cas9 MUC2KO human colonic goblet cells in response to E. histolytica. CXCL8 (IL-8) is a potent chemokine that recruits neutrophils to sites of inflammation, and symptomatic E. histolytica infections are marked by robust production of CXCL8 and subsequent neutrophil tissue infiltration. RT-qPCR and ELISA were used to analyze mRNA and protein expression in response to various stimuli. Specific pro-inflammatory pathway pharmacological inhibitors were used to enumerate defects in intracellular signaling, actinomycin D to inhibit transcription, and cycloheximide to inhibit translation. Diphenyleneiodonium chloride (DPI) was used to alleviate reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress and ROS production was measured by the 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) assay. WT goblet cells produced more CXCL8 and ROS than MUC2KO cells in response to amoebic stimulation. Whereas CXCL8 mRNA was reduced by inhibiting multiple pro-inflammatory signaling pathways in WT, only the MAPK/ERK pathway reduced transcription in MUC2KO cells. CXCL8 mRNA from WT was more stable than MUC2KO cells, and this stability was reduced when ER stress was alleviated. This study identified that metabolically stressed goblet cells regulate the expression of CXCL8 in response to E. histolytica by a ROS-dependent mechanism, activating multiple signalling pathways to stabilize transcripts and maximize pro-inflammatory responses against E. histolytica. Importantly, these results unraveled a previously uncharacterized dualistic role for goblet cells- mucus secretion and pro-inflammatory chemokine production- in innate host defence against E. histolytica.
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    Immune escape of multiple myeloma cells results from low miR29b and the ensuing epigenetic silencing of proteasome genes
    (2024-04-23) Leone, Patrizia; Malerba, Eleonora; Prete, Marcella; Solimando, Antonio G.; Croci, Giorgio A.; Ditonno, Paolo; Tucci, Marco; Susca, Nicola; Derakhshani, Afshin; Dufour, Antoine; De Re, Valli; Silvestris, Nicola; Racanelli, Vito
    Abstract Background Activation of CD28 on multiple myeloma (MM) plasma cells, by binding to CD80 and CD86 on dendritic cells, decreases proteasome subunit expression in the tumor cells and thereby helps them evade being killed by CD8+ T cells. Understanding how CD28 activation leads to proteasome subunit downregulation is needed to design new MM therapies. Methods This study investigates the molecular pathway downstream of CD28 activation, using an in vitro model consisting of myeloma cell lines stimulated with anti-CD28-coated beads. Results We show that CD28 engagement on U266 and RPMI 8226 cells activates the PI3K/AKT pathway, reduces miR29b expression, increases the expression of DNA methyltransferase 3B (DNMT3B, a target of miR29b), and decreases immunoproteasome subunit expression. In vitro transfection of U266 and RPMI 8226 cells with a miR29b mimic downregulates the PI3K/AKT pathway and DNMT3B expression, restores proteasome subunit levels, and promotes myeloma cell killing by bone marrow CD8+ T cells from MM patients. Freshly purified bone marrow plasma cells (CD138+) from MM patients have lower miR29b and higher DNMT3B (mRNA and protein) than do cells from patients with monoclonal gammopathy of undetermined significance. Finally, in MM patients, high DNMT3B levels associate with shorter overall survival. Conclusions Altogether, this study describes a novel molecular pathway in MM. This pathway starts from CD28 expressed on tumor plasma cells and, through the PI3K-miR29b-DNMT3B axis, leads to epigenetic silencing of immunoproteasome subunits, allowing MM plasma cells to elude immunosurveillance. This discovery has implications for the design of innovative miR29b-based therapies for MM.
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    Investigating 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, Antoine
    Undifferentiated 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.
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    Investigating versican as a primary inhibitor of remyelination in models of multiple sclerosis
    (2022-01) Jelinek, Emily; Yong, V. Wee; Dufour, Antoine
    The demyelination that characterizes multiple sclerosis (MS) lesions can be repaired to a degree in some individuals, though in many patients, this repair is inadequate. Remyelination has the potential to restore function in the central nervous system (CNS). In order to promote remyelination in MS lesions, the inhibition of oligodendrocyte precursor cell (OPC) recruitment and differentiation must be overcome. Targeting the deposited extracellular matrix (ECM) factors in the lesion to create a more permissive environment for OPCs is a promising therapeutic strategy. Versican, and likely its V1 isoform, has been identified as a prominent inhibitory chondroitin sulfate proteoglycan (CSPG) that is upregulated in the MS lesion. In this thesis, I sought to determine the outcome of the interaction between newly available purified V1 and OPCs in culture. I observed a robust inhibitory effect of V1 as a substrate and as a soluble treatment on OPCs in vitro. Using the lysolecithin (LPC) murine model of demyelination and remyelination, where versican upregulation in CNS lesions appears to be in macrophages/microglia from previous studies, I conditionally deleted versican from CCR2+ monocytes to evaluate the outcome on OPC presence in the lesion. The conditional versican knockout was dependent on the administration of tamoxifen and resulted in reduced V1 in the lesion compared to controls. With less V1 in the lesion, there was an increase in oligodendrocyte lineage cells during the documented period of remyelination in the LPC lesion. These results are promising in proposing a target for future therapeutic strategies to improve remyelination in MS lesions.
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    Lung Proteomics of Mice Infected with Influenza
    (2025-01-02) Subha, Maneesha; Jenne, Craig; Dufour, Antoine; Deniset, Justin
    Influenza A virus (IAV) infection causes an inflammatory reaction and activation of coagulation, which is often the basis for destruction of lung tissue. This study examines the changes to the of lungs from IAV-infected mice and assesses the effectiveness of Argatroban, a thrombin inhibitor, when given early in the infection. Pro-inflammatory and cell adhesion proteins were significantly upregulated in lung tissue on the first day after infection. As disease progressed, proteins related to coagulation, immunological signaling, and oxidative stress were more highly expressed in infected lungs. Argatroban treatment notably shifted the proteomic response on Day 1 and Day 5 post- infection, pointing to the ability of argatroban to control oxidative and inflammatory reactions during IAV infection, which may have consequences for maintaining lung function and reducing tissue damage brought on by the virus.
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    Mesenchymal progenitor cells from non-inflamed versus inflamed synovium post-ACL injury present with distinct phenotypes and cartilage regeneration capacity
    (2023-06-25) Krawetz, Roman J.; Larijani, Leila; Corpuz, Jessica M.; Ninkovic, Nicoletta; Das, Nabangshu; Olsen, Alexandra; Mohtadi, Nicholas; Rezansoff, Alexander; Dufour, Antoine
    Abstract Background Osteoarthritis (OA) is a chronic debilitating disease impacting a significant percentage of the global population. While there are numerous surgical and non-invasive interventions that can postpone joint replacement, there are no current treatments which can reverse the joint damage occurring during the pathogenesis of the disease. While many groups are investigating the use of stem cell therapies in the treatment of OA, we still don’t have a clear understanding of the role of these cells in the body, including heterogeneity of tissue resident adult mesenchymal progenitor cells (MPCs). Methods In the current study, we examined MPCs from the synovium and individuals with or without a traumatic knee joint injury and explored the chondrogenic differentiation capacity of these MPCs in vitro and in vivo. Results We found that there is heterogeneity of MPCs with the adult synovium and distinct sub-populations of MPCs and the abundancy of these sub-populations change with joint injury. Furthermore, only some of these sub-populations have the ability to effect cartilage repair in vivo. Using an unbiased proteomics approach, we were able to identify cell surface markers that identify this pro-chondrogenic MPC population in normal and injured joints, specifically CD82LowCD59+ synovial MPCs have robust cartilage regenerative properties in vivo. Conclusions The results of this study clearly show that cells within the adult human joint can impact cartilage repair and that these sub-populations exist within joints that have undergone a traumatic joint injury. Therefore, these populations can be exploited for the treatment of cartilage injuries and OA in future clinical trials.
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    Metaproteomic profiling of fungal gut colonization in gnotobiotic mice
    (2022-02-22) Pettersen, Veronika K.; Dufour, Antoine; Arrieta, Marie-Claire
    Abstract Background Eukaryotic microbes can modulate mammalian host health and disease states, yet the molecular contribution of gut fungi remains nascent. We previously showed that mice exclusively colonised with fungi displayed increased sensitivity to allergic airway inflammation and had fecal metabolite profiles similar to germ-free mice. This marginal effect on the host metabolome suggested that fungi do not primarily use metabolites to modulate the host immune system. Methods To describe functional changes attributed to fungal colonisation, we performed mass spectrometry-based analyses of feces (Label-Free Quantitative; LFQ) and the small intestine (labeling with Tandem Mass Tag; TMT) of gnotobiotic mice colonised with defined consortia of twelve bacterial species, five fungal species, or both. We also evaluated the effect of microbiome perturbances on the metaproteome by analysing feces from mouse pups treated with an antibiotic or antifungal. Results We detected 6675 proteins in the mice feces, of which 3845 had determined LFQ levels. Analysis of variance showed changes in the different gnotobiotic mouse groups; specifically, 46% of 2860 bacterial, 15% of 580 fungal, and 76% of 405 mouse quantified proteins displayed differential levels. The antimicrobial treatments resulted in lasting changes in the bacterial and fungal proteomes, suggesting that the antimicrobials impacted the entire community. Fungal colonisation resulted in changes in host proteins functional in innate immunity as well as metabolism, predicting specific roles of gut fungi on host systems during early developmental stages. Several of the detected fungal proteins (3% of 1492) have been previously reported as part of extracellular vesicles and having immunomodulating properties. Using an isobaric labelling TMT approach for profiling low abundant proteins of the jejunal tissue, we confirmed that the five fungal species differentially impacted the host intestinal proteome compared to the bacterial consortium. The detected changes in mouse jejunal proteins (4% of 1514) were mainly driven by metabolic proteins. Conclusions We used quantitative proteomic profiling of gnotobiotic conditions to show how colonisation with selected fungal species impacts the host gut proteome. Our results suggest that an increased abundance of certain gut fungal species in early life may affect the developing intracellular attributes of epithelial and immune cells.
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    N-terminomics/proteomics investigation of Calpain-2 substrates
    (2022-04-21) Anowai, Anthonia; Dufour, Antoine; Hollenberg, Morley; Robbins, Stephen
    Calpains are a family of intracellular cysteine proteases that require calcium for activation. Calpain-1 and -2, first described as the classical calpains due to their ubiquitous expression, are part of this larger 15-member family in humans and share similar patterns of expression and structural domains. Despite suggestive indication of a role for calpains in different cell functions in disease and health, it is unclear the repertoire of substrates that are targeted by calpains 1 and 2 in macrophages. To investigate this hypothesis, we focused on calpain-2 and performed N-terminomics/TAILS experiment in calpain-2 knockout human THP-1 monocytic cell lines to identify new substrates. We analyzed the proteomics and Nterminomics data using bioinformatics and pathway enrichment tools (STRING-db (https://string-db.org) and Metascape (https://metascape.org/gp/index.html)). We validated proteolytically processed substrates of calpain-2. We have shown that calpain-2 has novel targets spleen tyrosine kinase (SYK) in THP-1 cells when activated by calcium and may play a role in regulating kinase.
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    Prostaglandin Metabolites and Human Labour
    (2024-05-29) Wood, Eilidh; Slater, Donna; Hemberger, Myriam; Dufour, Antoine; Shearer, Jane
    Preterm birth is a major cause of neonatal morbidity and mortality worldwide. The etiology of preterm birth, particularly those preterm births preceded by spontaneous preterm labour, is largely unknown. Prostaglandins and other lipid mediators in the eicosanoid family, have been implicated in the processes of term and preterm labour, although exactly which eicosanoids are involved and their exact roles in these processes has yet to be elucidated. To this end, we initially investigated the eicosanoid profile of discovery cohorts of maternal serum and urine. Analyses in first trimester serum revealed decreased levels of lipoxygenase pathway eicosanoids early in pregnancy in individuals with subsequent preterm birth. In 2nd and 3rd trimester urine, we found that term labour was associated with decreasing urinary tetranor prostaglandin metabolites (t-PGFM, t-PGEM, and t-PGDM), potentially suggesting altered prostaglandin metabolism with term labour. To interrogate the urine data further, we investigated levels of prostaglandin metabolites in a larger set of n=308 maternal urine samples and found that prostaglandin F2a and PGE2 metabolites (PGFM and PGEM, respectively) were increased with term labour, but unchanged leading up to labour onset. Finally, we investigated regulation of the prostaglandin metabolizing enzyme, 15-PGDH, by IL-1B and CRH and demonstrated that neither 15-PGDH levels nor PGE2 metabolism was significantly affected by either treatment in a chorionic explant model. In conclusion, eicosanoids and their metabolites appear to be involved in the labour process, although future work is required to determine the factors that regulate their production and metabolism and the utility of these lipid mediators as biomarkers for labour.
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    Proteolytic Regulation of Proteoglycan 4 in Inflammation
    (2023-05-05) Das, Nabangshu Shekhar; Dufour, Antoine; Schmidt, Tannin; Krawetz, Roman; Rezansoff, Alexander
    Proteoglycan 4 (PRG4) is an extracellular macromolecule that is synthesized and secreted by cells lining surfaces of multiple tissues. While it has traditionally been studied and described as a boundary lubricant, recent evidence suggests that it can bind to and affect downstream signaling of several cell surface receptors, including toll-like receptors (TLRs) that are involved in regulating inflammatory responses. Although previous studies have shown that proteolysis of PRG4 reduces its boundary lubricating ability in vitro compared to intact PRG4, the effect of proteolysis on inflammatory signaling remained uncharacterized. Furthermore, while differential levels of PRG4 expression have been associated with various inflammatory conditions, such as osteoarthritis, the role of PRG4 in maintaining inflammatory signaling during normal aging under homeostatic conditions is still unclear. The objectives of this thesis were to 1- Determine expression of PRG4 in global proteomes during inflammation, and 2- Characterize of proteolytic processing of PRG4 and the mechanism and effects in OA and 3- Examine the effects of PRG4 on the global proteomes during age-related joint inflammation. Global proteome analysis revealed PRG4 expression levels changed locally during inflammation as demonstrated by a reduction in the PRG4 expression in the tears while an increase in the saliva of Sjogren’s syndrome patients comparing to healthy individuals. These differential levels of PRG4 expression were associated with differential expression profiles of different signaling molecules (i.e., proteases) associated with multiple inflammatory pathways including neutrophil degranulation both of the tissue environment. With respect to proteolytic susceptibility of PRG4 during inflammation, this study revealed that the proinflammatory serine protease, tryptase β, cleaves PRG4 altering its functional properties with respect to lubrication and inflammation: it reduces the ability to provide boundary lubrication and increases the ability to activate NF-κB-mediated inflammation through the TLR pathway. In a destabilization of medial meniscus (DMM) model of osteoarthritis (OA) in rat knee joint, differential colocalization of tryptase β and PRG4 in knee joints was associated with the development of OA and disease progression. Treatment with an intra-articular injection of exogenous PRG4 was able to resolve joint inflammation and OA phenotypes in the post-DMM rat knee by restoring the expression of PRG4 on articular cartilage and tryptase β by cartilage chondrocytes. In the absence Prg4, chondrocytes in murine knee cartilage express elevated level of proteases, particularly tryptase β and Htra-1, along with concurrent development of neutrophil-like phenotypes during aging. The neutrophil-like phenotype of chondrocytes is associated with loss of joint integrity in Prg4-/- mice. Overall, this thesis demonstrates the importance of expression levels and structural integrity of PRG4 in maintaining homeostasis through regulating inflammation, provides greater insights into the complex interplay between PRG4, proteases and inflammation, and provide the foundation and motivation for the development of new treatments for inflammatory diseases.
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    Proteomic and transcriptomic analysis of clinical and in vitro flow-exposed breast cancer samples reveals pathways and markers relevant to postpartum breast cancer.
    (2024-01-13) Stibbards-Lyle, Maya; Rinker, Kristina; Dufour, Antoine; Shemanko, Carrie; Ungrin, Mark
    Mechanical forces are an important, yet poorly understood, influence on the metastatic capability of breast cancer cells. They exist in a dynamic interplay with the structural and immune aspects of the cancer microenvironment. Mammary gland involution is a remodeling process that occurs post-lactation and is believed to be responsible for poor outcomes observed in postpartum breast cancer. Fluid shear stress is well-linked to tissue remodeling and is likely increased during this stage of the postpartum period. In this thesis, a bioreactor system was combined with hormonal treatment to expose breast cancer cells to some of the conditions relevant to postpartum involution. We initially validated the model by studying the morphology and viability of cells upon exposure to fluid shear stress and involution and confirming the presence of relevant protein markers. Using machine learning-based analysis of publicly available RNA-seq datasets, we identified numerous genes and pathways upregulated in both postpartum and flow-exposed conditions. We further validated these findings using proteomic analysis of flow-exposed MDA-MB-231 breast cancer cells, which identified similar proteins and pathways to the initial transcriptomic analysis. These included pathways linked to inflammation, wound healing, cell migration, and extracellular matrix organization, which are all pathways critically involved in the action of mammary gland involution. Further investigation revealed that matrix metalloproteinases, a key promoter of the remodeling activities and tumorigenic effect of involution, are significantly upregulated upon exposure to fluid flow. Proteomic analysis of MCF-7 cells treated with hormones associated with lactation revealed markers and pathways related to immune-mediated progression of breast cancer. Finally, we established the utility of our model by validating that the protein expression of chemokine C-X-C motif ligand 13 (CXCL13), which was upregulated in our transcriptomics analysis and is a well-established marker of breast cancer metastasis in young patients, is upregulated in response to flow-exposure. This suggests that our model serves as an efficient method to identify relevant breast cancer biomarkers related to the mechanism of mammary gland involution. To our knowledge, these studies are the first to integrate hormonal and fluid shear stress influences in the context of breast cancer, and to suggest a link between fluid shear stress and mammary gland involution. They serve as an initial proof of concept, indicating that fluid shear stress could serve as a powerful tool in furthering our understanding of postpartum breast cancer and identification of relevant biomarkers for this unique cohort.
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    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, Antoine
    Inflammatory 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.
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    To define the interactome of neuronal primary cilia
    (2021-09-17) Whitmore, Brandon Alexander; Guo, Jiami; Dufour, Antoine; Huang, Peng; Schriemer, David; Mains, Paul
    Primary cilia are projections of the plasma membrane on almost all mammalian cells in the body, including neurons. The purpose of primary cilia is to act as a cellular antenna due to the unique membrane composition of the primary cilia which is abundant in different receptors, like GPCRs and RTKs, and ion channels. Dysfunction of cilia signaling gives rise to a class of diseases known as ciliopathies. Ciliopathies present with brain structural and functional deficits and have been implicated in intellectual disabilities, Autism Spectrum Disorder, and Schizophrenia. While a number of ciliopathies have been determined, there is a gap in the knowledge of the mechanisms of some ciliopathies due to an incomplete understanding of the primary cilia proteome, especially within the brain. Understanding the primary cilia proteome may determine potential gene candidates that have been implicated in ciliopathies. The gap in the knowledge of the neuronal ciliary proteome arises from the difficulty in purifying mammalian primary cilia. Different proteomics techniques have been used to begin to study the primary cilia composition within kidney cells; however no techniques have been applied to neurons to study the proteome of neuronal primary cilia. The brain and kidneys respond to different extracellular cues and have different environments, indicating that neuronal primary cilia should have a unique membrane composition and respond to distinct extracellular cues compared to primary cilia of other cells. To identify the unique membrane composition of neuronal primary cilia I developed an in vitro proximity labeling method to isolate ciliary proteins from primary neuron cultures. From this work, I was able to create the first list of potential neuronal primary cilia proteins. As an alternative method to understand the membrane composition, I identified a number of signaling molecules that affect primary cilia morphology and impact signaling within cilia. These results will be used to begin constructing an interactome of neuronal primary cilia signaling.