Browsing by Author "Hirota, Simon"

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    A Pharmacodynamics Analysis of Glucocorticoid Receptor-Mediated Gene Expression in BEAS-2B Human Airway Epithelial Cells
    (2016) Joshi, Taruna; Giembycz, Mark; Newton, Robert; McKay, Derek; von der Weid, Pierre; Hirota, Simon; Cameron, Lisa
    International treatment guidelines recommend that inhaled glucocorticoids be used as a monotherapy to control mild-to-moderate asthma. However, a combination of an inhaled glucocorticoid with a long-acting β2-adrenoceptor agonist is recommended for managing moderate-to-severe asthma, which cannot be controlled by glucocorticoids alone. Increasing evidence supports the idea that glucocorticoids acting through the glucocorticoid receptor (GR) can attenuate inflammatory responses either by inducing anti-inflammatory genes (a process called transactivation) or by suppressing pro-inflammatory genes (a process called transrepression). Gene transactivation by glucocorticoids was initially thought to be responsible for causing the metabolic side-effects and, hence, is relatively understudied when compared to transrepression. However, it has become increasingly clear that transactivation plays an important role in the anti-inflammatory actions of glucocorticoids. Moreover, the extent to which clinically-relevant glucocorticoids are equivalent in their ability to promote gene expression is unclear. This thesis describes the first pharmacodynamic approach to evaluate the transactivation potential of a panel of glucocorticoids alone and in combination with indacaterol (Ind), a long-acting β2-adrenoceptor agonist. Pharmacodynamic analyses showed that magnitude of luciferase gene induction was agonist dependent (i.e. seven different glucocorticoids tested displayed varying degrees of agonism). In addition, there were significant differences in agonist potency and, more importantly, the relationship between GR occupancy and response. To complement the reporter studies, similar analyses were performed on four glucocorticoid-inducible candidate genes. Three of these (GILZ, p57kip2 and CRISPLD2) are genes with potential anti-inflammatory activity and a fourth gene, PDK4 is predicted to promote metabolic side-effects. Similar to the luciferase reporter system, the expression of these genes was agonist-dependent and displayed markedly different GR occupancy-response relationships. Furthermore, Ind, when combined with the seven GR agonists tested, synergistically enhanced transactivation, the magnitude of which was agonist and gene dependent. These studies demonstrate that when gene transactivation is used as a functional output, glucocorticoids used to treat asthma are not biologically-equivalent. It is proposed that these differences may be exploited to therapeutic advantage. Thus, the generation of gene expression ‘fingerprints’ in target and off-target human tissues may allow new GR agonists to be rationally designed for asthma with an improved therapeutic index.
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    A Primary Epithelial-Immune Cell Co-Culture Model to Investigate the Interactions between the Intestinal Epithelium and Innate Immune Cells in Response to Clostridioides difficile toxins
    (2024-01-04) Smith, Lauren; MacNaughton, Wallace; Hirota, Simon; Geuking, Markus; Arrieta, Marie-Claire
    Clostridioides difficile is a leading cause of healthcare-associated diarrhea in the United States, with symptoms ranging from mild diarrhea to toxic megacolon. Upon disruption of the colonic epithelium by C. difficile toxins A and B (TcdAB), epithelial cells are thought to signal to underlying immune cells including group-3 innate lymphoid cells (ILC3s) and macrophages to mount an effective immune response. ILC3s and macrophages both modulate pathogen defense (including C. difficile), tissue repair, and inflammation. ILC3s do so largely through interleukin- 22 (IL-22) secretion, and macrophages do so through phagocytosis and the secretion of a wide array of cytokines and chemokines. However, pathways by which the intestinal epithelium responds to C. difficile TcdAB to affect underlying immune cells, such as ILC3s and macrophages, remain poorly understood. We therefore hypothesized that the response to C. difficile toxins throughout the colonic epithelium and underlying tissue is modulated by interactions between the intestinal epithelium, ILC3s and IL-22, and macrophages. To test this hypothesis and address the limitations of cancer cell lines and single lineage models, we developed two novel primary epithelial cell-innate immune cell co-culture models to investigate epithelial-immune interactions in response to C. difficile toxins in vitro. Co-culture of monolayers with either MNK-3 (ILC3-like) cells or bone marrow derived macrophages did not influence monolayer permeability in response to TcdAB. However, in the process of characterizing these models, we established that MNK-3 cells and macrophages remained viable in co-culture and released relevant mediators with respective stimulation. While MNK-3 cells were not activated in co-culture, macrophages were activated by both monolayer media and epithelial presence in co-culture. With these novel experiments, we have provided groundwork for the further development of multi-lineage models.
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    Biofilm-derived Small Molecules Released from Mixed-species Cultures of Fusobacterium necrophorum and Porphyromonas levii Inhibit Bovine Neutrophil Functional Responses
    (2022-05-06) Lockhart, Joey Scott; Morck, Douglas Walter; Buret, Andre G; Harrison, Joe Jonathan; Hirota, Simon; De Buck, Jeroen Marc Daniel; James, Garth Alan
    Bacterial biofilms are surface adhered communities of microorganisms encased in a protective polysaccharide layer. These resilient structures present significant challenges for effective host immune cell clearance or clinical interventions, frequently resulting in recurrent infections in cattle, humans, and other animals. Fusobacterium necrophorum and Porphyromonas levii are two species of anaerobic, Gram-negative, opportunistic pathogens that have been described as etiological agents in a variety of necrotic, recalcitrant infections. With antimicrobial resistance on the rise, it is important to elucidate the reasons why biofilm-associated infections are so difficult to eradicate. Neutrophils are critical first responder cells that are recruited to a site of bacterial colonization to initiate the inflammatory response. In this dissertation research, an in vitro system for the growth of mixed-species anaerobic biofilms was employed to investigate bovine neutrophil responses to biofilm bacteria compared to planktonic cultures of the same two species. Neutrophils exposed to biofilm soluble factors generated reduced oxidative and chemotactic responses compared to neutrophils exposed to planktonic bacteria-generated products. Ultra-filtration revealed that a <3kDa molecule is differentially expressed in the bacterial supernatants and allows biofilm-bacteria to inhibit neutrophil responses. Intensive heat, protease, lipase, or nuclease treatment of the <3kDa fractions prior to exposure to bovine neutrophils did not alter the differential oxidative responses. This led to an investigation of small, heat stable compounds and our results suggest that the accumulation of protoporphyrin IX in the biofilm mode of growth attenuates neutrophil chemotaxis and reactive oxygen species production. This work provides evidence for a mechanism by which these biofilm bacteria evade host immune cells and understanding such mechanisms is important to reduce the persistent inflammation and negative clinical outcomes associated with chronic biofilm-mediated disease.
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    Early Life Regulation of TRPV1+ Nociceptors by the Microbiome: Implications for Pathological Pain?
    (2024-04-30) Abdullah, Nasser Salem; Altier, Christophe; Trang, Tuan; Hirota, Simon
    Pain is essential for the survival and wellbeing of organisms. Dysregulation in the pain pathway leads to pathological pain in part due to poor pain management stemming from a lack of understanding of the underlying mechanisms that lead to pathological pain. Pain is initiated by specialised primary afferent neurons called nociceptors. TRPV1+ nociceptors play a central role in multiple pathological pain conditions, including inflammatory pain, where their sensitization can lead to chronic pain. In this thesis, we shed light on how TRPV1+ nociceptors participate in chronic pain and the factors that contribute to their regulation. This thesis provides two studies divided into 3 chapters. The first study (Chapter 3) is focused on the role of TRPV1+ nociceptors in initiating chronic visceral pain. The second study (Chapter 4 and 5) is focused on understanding how the early life microbiome regulates the sensitivity of TRPV1+ nociceptors. In Chapter 3, we present findings indicating that in a murine model of colitis, TRPV1+ nociceptors activate spinal microglia leading to visceral hypersensitivity, demonstrating their essential role for the transition from acute to chronic pain in the context of colitis. In Chapter 4, we investigated the role of the early life microbiome on TRPV1+ nociceptor specification and pain sensitivity using germ-free mice and then germ-free mice colonized before or after weaning. We found that a lack of microbiome in early life leads to hyposensitivity to heat and capsaicin, The hyposensitive phenotype was not due to changes in nociceptor specification, innervation, or TRPV1 expression, but it correlated with a reduction in TRPV1 trafficking to the cell membrane. In Chapter 5, we investigated the underlying mechanisms for early life microbiome induced hyposensitivity and identified that the early life microbiome regulates the sensitivity of nociceptors and the trafficking of TRPV1 through regulating mast cell derived NGF. Altogether, this study demonstrates the central role of TRPV1+ nociceptors in inducing the transition to chronic pain and the important role of the early life microbiome in regulating the sensitivity of these nociceptors through mast cell derived NGF.
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    Effect of Bacterial Species Associated with Major Depressive Disorder on Emotionality Behaviors, Microbiome Structure, and Metabolism in Gnotobiotic Mouse Models
    (2021-08-17) Cooke, Noah C.A.; McCoy, Kathy D; Taylor, Valerie H; Hirota, Simon; McGirr, Alexander; Sharkey, Keith A; Savchenko, Alexei
    Major depressive disorder (MDD) is a prevalent disease, and antidepressants lack efficacy in many patients, motivating investigation for new therapeutic modalities. Altered microbiome composition and metabolism have been implicated in MDD, but the specific species and metabolites involved, and their mechanisms of effect, remain elusive. We set out to test whether two bacterial species correlated with MDD alter emotionality-related behaviors in stress-naïve and chronic stress-exposed mice, when added to the stable defined moderately diverse mouse microbiome 2 (sDMDMm2) consortium. We hypothesized that Lachnospiraceae bacterium would increase, and Faecalibacterium prausnitzii would decrease, these behaviors in both conditions. Further, we hypothesized that these behavioural effects would coincide with effects on microbiome composition and metabolism. To assess emotionality-like behavior, we established a gnotobiotic behavioral system comprised of five tests and the chronic variable stress (CVS) model for studying depression. L. bacterium did not alter depressive and anxiety-like behaviors in the stress-naïve condition. In the CVS condition, an increase in anxiety-like behavior was seen in one assay, the elevated plus maze test, in one preliminary experimental replicate. Illumina 16S V4 amplicon sequencing revealed that L. bacterium significantly altered microbiome composition. Hydrophilic interaction liquid chromatography with mass spectrometry (HILIC-MS/MS) also revealed a significant effect of L. bacterium on microbiome and serum metabolism. This thesis offers a novel methodological approach to investigate causal relationships between bacterial species and emotionality-related behaviors, and to identify microbial candidates for microbiome-targeted therapies for MDD.Keywords: microbiome, Lachnospiriceae bacterium, Major Depressive Disorder, Metabolism.
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    Enteric Tuft Cell Responses and Host-Microbial Dynamics During Giardia muris Infection
    (2024-09-18) Sosnowski, Olivia Stefania; Buret, Andre; Hirota, Simon; McKay, Derek
    Enteric tuft cells constitute an important aspect of the host response to intestinal parasites. Tuft cells detect and respond to enteric parasites, secreting molecules such as IL-25, leading to the expansion of tuft and goblet cells to ultimately promote parasite clearance. The intestinal protozoan parasite Giardia disrupts the intestinal environment and leads to impaired barrier function. Infection is also associated with altered microbiota composition and function. This study characterised the tuft cell response in the murine small intestine after G. muris infection, identifying tuft and goblet cell hyperplasia during the clearance phase (day 21). G. muris infected mice also displayed increased expression of genes defining secretory epithelial cells (Aoth1), tuft cell-related genes (Dclk1, Tas2r130, Tas2r), and goblet cell-related genes (Muc2, Klf4) during early infection (day 4 and day 11). Increases in cell number and many of the changes in gene expression during G. muris infection were observed at both the site of active Giardia colonization (jejunum) and more distally (ileum). Tuft cell-deficient (Pou2f3-/-) mice had a lower trophozoite burden compared to wild-type (C57Bl/6) mice during early infection, demonstrating that tuft cells help facilitate G. muris colonization. To investigate other contributors to the tuft cell-Giardia crosstalk, the microbiota was considered since microbial derived products can modify tuft cell functions, Giardia can cause microbiota dysbiosis, and the microbiota can influence Giardia colonization. A fecal microbiota transplantation (FMT) using microbiota from naïve C57Bl/6 or Pou2f3-/- donors revealed that FMT from Pou2f3-/- donors prior to G. muris infection resulted in a lower parasite load than in mice that received FMT from C57Bl/6 donors, illustrating that the microbiota influences G. muris colonization. Upon evaluating if Giardia-modified microbiota (GMM) could elicit a tuft cell response, transplantation with small intestinal GMM 11 days after infection did not lead to changes in tuft or goblet cell number, thus the mechanism through which Giardia engages tuft cells requires further exploration. Collectively, this study characterises the tuft cell response to Giardia, identifies a novel role for tuft cells in promoting Giardia colonization, and provides insights into how the microbiota contributes to the dynamics between enteric tuft cells and parasitic infections.
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    Examining the activation of xenobiotic receptors using microbial metabolites and chemical ligands
    (2024-09-18) Shenoda, Eva Ibrahim Gorgy; Hirota, Simon; Nasser, Yasmin; McCafferty, Donna-Marie
    The aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) are key xenobiotic receptors involved in regulating chemical metabolism and detoxification. Traditionally, these receptors were known for mediating toxic responses by sensing and responding to chemicals. Recent research shows their role in maintaining gut homeostasis and regulating inflammation. However, the mechanisms by which they induce these responses are not clear. This thesis examines whether the activation of AhR and PXR in epithelial cells by microbial metabolites versus chemical ligands drives unique transcriptional responses and could explain differences in beneficial versus deleterious biological outcomes in the host. PXR and AhR were activated with indole-3-propionic acid (IPA) and indole-3-pyruvic acid (IPyA) as microbial metabolites, and pregnenolone 16α-carbonitrile (PCN) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as chemical ligands, respectively. We compared responses to these ligands, revealing significant gene expression differences. Further analysis showed both IPyA and TCDD activated genes involved in hypoxia-inducible factor (HIF) signaling and metabolism. IPyA upregulated genes for ATP synthesis and purine biosynthesis, while TCDD promoted genes related to cell cycle, cancer, and apoptosis. Given that AhR translocates to the nucleus upon activation, we hypothesized that the differences in transcriptomic responses and gene induction might be attributed to differences in nuclear translocation of AhR, with TCDD potentially inducing greater nuclear translocation compared to IPyA. To test this hypothesis, we established a cellular fractionation protocol for organoids. However, our results revealed no significant difference in AhR nuclear translocation between the two treatments, suggesting that other factors may affect gene expression in the AhR pathway. These findings highlight AhR and PXR's complex roles in gut health and inflammation, suggesting receptor activation can have both adverse and beneficial effects. This research enhances our understanding of AhR and PXR mechanisms and their potential for therapeutic strategies targeting gut disorders and inflammation.
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    Exploring the combinatorial effects of inflammatory stimuli and glucocorticoids on BIRC3 and BIRC2 expression: Differential regulation and functional investigations
    (2024-01-16) Thorne, Andrew James; Newton, Robert; Hirota, Simon; Yipp, Bryan
    Inflammation serves as a crucial innate mechanism in response to injury, infection, or harmful stimuli that activates the immune system and aims to restore homeostasis. Chronic inflammatory disorders like asthma present with ongoing inflammation whereby pulmonary epithelial cells release inflammatory mediators, intensifying airway inflammation, and worsening disease. Synthetic glucocorticoids represent the primary treatment for asthma by utilizing the anti-inflammatory properties of their endogenous counterparts to reduce inflammatory mediators. Their mechanism of action involves binding to the glucocorticoid receptor (GR) in cells and interacting with DNA elements to enhance anti-inflammatory gene expression. However, certain inflammatory genes play protective roles by promoting tissue repair or self-regulatory feedback mechanisms to limit inflammatory signaling. Some of these genes are spared the repressive effects of glucocorticoids and are even augmented, presumably for protection. The BIRC3 gene, while being associated with the inflammatory NF-κB pathway, is a gene upregulated in models of the pulmonary epithelium and in vivo by glucocorticoids. BIRC3, along with its family member BIRC2, may contribute to regulating NF-κB pathways, but also inhibit apoptosis, although their specific roles remain unclear. Consequently, the rationale of glucocorticoid-enhanced BIRC3 remain ambiguous. Given their similarities, BIRC3 and BIRC2 were both investigated, focusing on their expression, regulation, and the interactions between the GR and NF-κB at the gene promoter. These analyses detailed robust expression patterns differentiating BIRC3 and BIRC2 by their response to inflammatory cytokines and glucocorticoids over time. Promoter analysis revealed insights into possible GR and NF-κB interactions to explain the regulation of BIRC3 expression, thus providing an example of GR-NF-κB positive interactions leading to inflammatory gene expression. Further, investigations into the NF-κB pathway as a platform for BIRC2/3 function revealed the established canonical NF-κB pathway may challenge established dogma. This exploratory research highlighted the significance of TAK1 as central kinase within the NF-κB pathway. Moreover, possible redundancy between IKK1 and IKK2, and the presence of additional regulators within canonical NF-κB signaling may exist. While this study provided no greater insights for redundant, or individual roles for BIRC2/3 in NF-κB signaling or apoptosis, it facilitated the development of tools for future investigations.
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    Genetic Manipulation of Bacteroides for Transient Colonization of Germ-free Mice
    (2022-04) Dong, Sharon; Geuking, Markus; Harrison, Joe; Hirota, Simon; Buret, Andre
    Human and murine hosts have a dynamic relationship with the microbes that colonize their bodily surfaces. Shifts in composition of the community of microbes, or their function, are implicated in many chronic and/or inflammatory conditions. There are currently gaps in understanding whether these shifts cause disease phenotypes or are artefacts of changed host conditions due to disease. The ability to tease apart these dynamics by generating bacterial tools that limited bacterial exposure of germ-free mice to a certain time-period was explored in this thesis. I target genes in synthesis pathways of two uniquely-bacterial amino acids, meso-diaminopimelic acid (mDAP) and D-Alanine (D-Ala) in a species of interest, Bacteroides thetaiotaomicron. These amino acids are incorporated into the peptidoglycan, or cell wall, of most bacteria, like B. thetaiotaomicron. This study builds on concepts from a previously developed E. coli strain called HA107, where disruption of these two synthesis pathways generated an auxotrophic mutant that grew if supplemented with said amino acids, however, did not proliferate and persist in germ-free murine gastrointestinal tracts. I designed deletion alleles that would exchange with the functional allele of target genes, and transferred the allelic exchange vector, pExchange, containing the deletion allele into B. thetaiotaomicron through conjugation. The double and triple gene deletion mutants failed to grow when not supplemented with mDAP and D-Ala. This mutation strategy may be adapted to genetically manipulate other notable Bacteroides species. The generated auxotrophic mutants from this study will be useful tools in answering questions regarding how timing or dosage of bacterial exposure affect health or disease outcomes in germ-free mice.
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    Mechanisms of Mycophenolic Acid-Induced Gastrointestinal Toxicity and Potential Therapeutic Interventions in Primary Mouse Colonic Organoids
    (2024-09-17) Mack-Bowles, Brenan; Greenway, Steven; Hirota, Simon; Lewis, Ian
    Mycophenolate Mofetil (MMF) is a commonly prescribed immunosuppressant that demonstrates important clinical relevance. However, MMF therapy is linked to frequent gastrointestinal (GI) side effects that limit its use. Little is known about the mechanisms underlying MMF-induced GI injury. Using a primary mouse colon organoid model, we have found that mycophenolic acid (MPA), the pharmacologically active metabolite of MMF, significantly alters intestinal barrier function and permeability through modulation of tight junctions. RNA sequencing revealed that MPA significantly disrupted pathways related to cell cycle regulation, DNA replication, cytoskeleton dynamics, and suppression of senescence. MPA was observed to significantly reduce cellular proliferation, which was ameliorated through guanosine supplementation. Addition of exogenous guanosine was also observed to significantly restore barrier function back to control levels. The guanosine studies presented in this thesis suggest MPA’s inhibition of nucleotide metabolism is not selective for lymphocytes but is broader than originally described. This work represents one of the first investigations of MPA using a colon organoid model, providing critical insights into the intracellular mechanisms of MPA-induced GI toxicity.
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    Pregnane X Receptor (PXR) modulates NLRP3 Inflammasome
    (2017-12-18) Hudson, Grace; Hirota, Simon; McCafferty, Donna-Marie; Chadee, Khrisendath
    Compounds released from the intestinal microbiota may play a role in maintaining mucosal homeostasis, but little is known about the receptors that sense and respond to these compounds. Recently, a cytosolic xenobiotic sensor, the pregnane X receptor (PXR), was identified as a receptor for microbial metabolites in the gastrointestinal (GI) tract. The PXR has been shown to play a protective role in the gut, with gene variants associated with IBD risk. Recent data suggest that the PXR may regulate innate immune signaling platforms, in a variety of tissues. In vascular endothelial cells, the PXR was shown to stimulate the expression of NLRP3, and initiate NLRP3 inflammasome activation. Interestingly, alterations in NLRP3 inflammasome function have been linked to IBD susceptibility. In the current thesis, we sought to characterize the role of the PXR in modulating the function of the NLRP3 inflammasome in macrophages, a key innate immune cell that contributes to host-defense and the regulation of intestinal mucosal homeostasis. Using the THP-1 cell line and mouse peritoneal macrophages, we found that PXR agonists stimulated caspase-1 activation, along with IL-1β processing and release. These responses were lost in cells lacking NLRP3 and blocked by selective inhibition of caspase-1. Furthermore, PXR-deficient cells failed to activate caspase-1 and release IL-1β in response to PXR agonist stimulation. Lastly, we found that PXR activation triggered ATP release, an effect that was responsible for inflammasome activation, as these responses were abolished by apyrase and P2X7 inhibition. Through this thesis, we demonstrated that the PXR activates the NLRP3 inflammasome through stimulating ATP release within a macrophage.
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    The regulation and function of viperin during rhinovirus infection
    (2022-12-19) Love, Michelle; Proud, David; Leigh, Richard; Grandvaux, Nathalie; Jenne, Craig; Hirota, Simon; Yipp, Bryan
    The airway epithelium is the primary site of rhinovirus replication and plays a pivotal role in orchestrating innate immune responses to respiratory viruses, including rhinovirus. Interfacing with inhaled material in the airway lumen, the airway epithelium experiences continuous onslaught from respiratory pathogens. To combat viral infection, cells of the airway epithelium express a range of pattern recognition receptors capable of recognizing diverse viral ligands. Detection of an infectious virus by one of these receptors rapidly upregulates an array of effector molecules, including pro-inflammatory cytokines, interferons and interferon stimulated genes. Viperin is one of the most highly upregulated interferon stimulated genes in rhinovirus infected airway epithelial cells. Viperin expression during viral infection has long been associated with direct antiviral effects across diverse viral infection and cell models, including in rhinovirus infected airway epithelial cells. Despite the long-standing knowledge that viperin expression helps to impedes rhinovirus replication in the airway epithelium, little is known about how it is upregulated in response to rhinoviruses or how viperin proceeds to impact airway epithelial cell biology during infection. In addition to confirming previously published findings demonstrating that viperin is induced in rhinovirus infected primary human airway epithelial cells and is dependent on viral replication, we also demonstrated induction of viperin in airway epithelial cells by interferons. We also highlight the importance of the type III interferon IL-29 as a potent inducer of viperin in the airway epithelium. Finally based on our findings we propose a novel feedback loop, according to this model detection of rhinoviruses by the cytosolic dsRNA receptor melanoma differentiation-associated protein 5 (MDA5), or IL-29 signalling, promotes IRF-1 mediated viperin transcription. Viperin then establishes a protein-protein interaction with MDA5 which results in enhanced MDA5 stability in RV infected cells. This leads to enhanced signalling through MDA5, as measured by increased CXCL10 gene expression and protein secretion. As we have also demonstrated that MDA5 signalling is involved in promoting viperin expression in response to RV we hypothesize that stabilization of MDA5 by viperin may also serves to amplify viperin production during infection.
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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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    Rhinovirus-Bacteria Co-Exposure Modulates CCL20 Induction From Human Bronchial Epithelial Cells
    (2016) Maciejewski, Barbara; Proud, David; Armstrong, Glen; Giembycz, Mark; Hirota, Simon
    Exacerbations of chronic obstructive pulmonary disease (COPD) are often triggered by infection by viral or bacterial pathogens, with human rhinovirus (HRV) and nontypeable Haemophilus influenzae (NTHI) among the most commonly detected pathogens. Patients who suffer from concurrent viral and bacterial infection have more severe exacerbations. CCL20 is a chemokine that attracts immature dendritic cells to the airways, acts as an antimicrobial, and so contributes to both innate and adaptive immune responses to infection. In this thesis, we determined what effect co-infection of human bronchial epithelial (HBE) cells with HRV and bacteria would have on CCL20 induction. HRV/bacterial co-infection resulted in synergistic CCL20 induction in response more than one HRV or bacterial strain. Synergistic CCL20 induction was transcriptionally regulated, and induction was inhibited by cigarette smoke extract (CSE). Impaired induction of CCL20 by CSE raises the prospect of weakened antimicrobial immunity during infections in smokers and patients with COPD.
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    The Cost of Cystic Fibrosis Care in Canada: A Scoping Review
    (2024) Desai, Leena; Bomersback, Taryn; Galante, Gary; Bjornson, Candice; Hirota, Simon
    Background: Cystic fibrosis (CF) contributes a significant economic burden on individuals, health systems and society. This economic impact is highly relevant in a single-payer, publicly funded health system such as Canada. CFTR modulator therapy has been shown in large clinical trials to reduce the burden of disease in the pediatric population. Currently 17% of patients at the Alberta Children’s Hospital in Calgary, AB do not qualify for any CFTR modulator therapy, presumably resulting in an incremental cost to the system. Precision medicine tools such as organoid models may offer an opportunity for n-of-1 trials for individual patients who are currently not approved for therapy. Rationale: In order to better understand the health and economic impact of cystic fibrosis in Canada, we wish to identify direct, indirect and intangible costs of cystic fibrosis care in Alberta and Canada, and highlight the higher rate of heterogeneity amongst our southern Alberta CF population which predisposes to higher cost of care due to lack of approval to currently available therapies. Aim and Objective: This protocol describes the purpose of a scoping review which is to identify the most accurate estimation of the cost of cystic fibrosis care in Canada, and gather comprehensive data on how the costs of CF care (including direct, indirect and intangible costs) are defined and measured in Canada. Methodology: The protocol is developed using the JBI (Joanna Briggs Institute) guidance for scoping reviews and based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) Checklist, both of which will also guide its reporting. We will search PUBMED and OVID/MEDLINE for peer-reviewed English-language publications and grey literature search of relevant databases (ProQuest Dissertation and Theses, Canadian Health Research Collection) and organizational websites (Health Canada, Government of Canada, Cystic Fibrosis Foundation of Canda, Canadian Agency for Drugs and Technology in Health (CADTH)).
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    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, Simon
    Genome-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.
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    Understanding the Invasion Dynamics of Mycobacterium avium subspecies Paratuberculosis (MAP)
    (2022-09-20) Baruta, Grace M; Hirota, Simon; Colarusso, Guiseppina; De Buck, Jeroen MD;McKay, Derek M; Morck, Douglas W
    Paratuberculosis is a global infectious disease caused by the bacterium, Mycobacterium avium subspecies Paratuberculosis (MAP). Infection of ruminants with MAP can trigger progressive wasting disease characterized by granulomatous lymphadenitis, enteritis, and severe intestinal pathology potentially leading to death of the animal. The role of MAP infection within innate immune cells, including macrophages, in the small intestine has been extensively examined; however, the mechanisms of MAP entrance into the mucosal barrier of the small intestine are poorly defined. Additionally, there is a potential zoonotic risk of MAP linked to Crohn’s Disease (CD), but these claims have yet to be substantiated. Due to this and the economic burden and welfare impact on the livestock industry, a deeper understanding of the initial mechanisms of MAP uptake within the host is crucial to revealing novel treatment strategies. While the current paradigm suggests MAP travels to the small intestine, where it gains entry through the epithelium, the exact cellular tropism/-mechanism(s) of entry are not well defined. Therefore, we developed an enteroid system to visualize entrance of MAP into distinct cells of the intestinal epithelium using a GFP-expressing MAP strain. We sought to test the hypothesis that MAP uptake occurs via M cells through receptor mediated transcytosis.To evaluate this hypothesis, we generated a novel mouse enteroid-derived monolayer with functional M cells capable of transcytosis. MAP was detected mainly within M cells, more notably with the fibronectin pre-incubation. Integrin-blocking peptides decreased this M cell tropism. With this novel model system developed, we were able to examine MAP-host pathogen interactions, which will provide insight into blocking MAP entry at its initial point. Beyond this, our model will add valuable information to the field of mouse enteroid-derived monolayer techniques and can be used in the future to study alternate intestinal pathogens. In the process of characterizing a mouse enteroid-derived monolayer with M cells for use in establishing a model for MAP invasion dynamics, we discovered unique roles for RANKL/RANK signalling regarding M cell differentiation and the intestinal epithelium.