Browsing by Author "Hirota, Simon Andrew"

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    Antibiotic Reduction of Bacterial β-Glucuronidase Activity in the Murine Gut Prevents and Reverses Mycophenolate Mofetil-Induced Gastrointestinal Toxicity
    (2018-08-27) Taylor, Michael Robert; Greenway, Steven C.; Lewis, Ian A.; Hirota, Simon Andrew
    Mycophenolate mofetil (MMF) is an important immunosuppressive drug widely used post- transplantation, but whose use is often limited by gastrointestinal (GI) side effects including diarrhea, weight loss and colitis. The etiology of this GI toxicity has not been fully explained although we have shown in the mouse that an intact microbiome is required. This thesis developed and tested a potential mechanism, hypothesizing that bacterial conversion of the MMF metabolite mycophenolic acid glucuronide (MPAG) to mycophenolic acid (MPA) in the colon is responsible for the GI toxicity caused by MMF. Vancomycin both prevented and reversed MMF- induced GI toxicity and we characterized its effect on the intestinal microbiota in MMF-treated mice. Vancomycin eliminated many β-glucuronidase-producing bacteria, resulting in decreased hydrolysis of MPAG to MPA that was also associated with decreased weight loss and reduced colonic inflammation and injury. These findings outline a mechanism permitting interventions to improve MMF tolerance.
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    Antigen-specific CD4+ T-B cell interplay induces a robust, polyreactive systemic immunoglobulin response to commensal bacteremia.
    (2020-03-04) Koegler, Mia Elizabeth; Geuking, Markus B.; Peters, Nathan C.; Hirota, Simon Andrew; Jenne, Craig N.
    The impact of cognate CD4+ T cell help on the systemic antibody response during commensal bacteremia was assessed in detail in this thesis. To specifically evaluate cognate T cell-B cell interactions, we utilized a genetically modified commensal E. coli strain that expressed gp61, an additional T helper epitope, in its outer membrane ompC protein (E. coli ompC_gp61). Germ-free mice that were systemically primed with E. coli ompC_gp61 produced a significantly more robust E. coli-specific antibody response than mice that received the corresponding wild-type (WT) E. coli strain. The observed antibody response to E. coli ompC_gp61 appeared to be MHC II haplotype-dependent, as this phenomenon was reproducible in C57BL/6 mice (I-Ab) but not in BALB/c mice (I-Ad and I-Ed). Furthermore, mice adoptively transferred with gp61-specific SMARTA CD4+ T cells and later challenged with E. coli ompC_gp61 produced significantly more E. coli-specific IgM than recipient mice that were primed with WT E. coli. This finding suggests that the proportion of antigen-specific CD4+ T cells present during systemic immune priming may impact on class switch recombination and IgM+ memory formation. Finally, increasing gut microbiota complexity resulted in lower E. coli-specific antibody titers compared to germ-free mice in response to E. coli ompC_gp61 priming. However, non-primed mice with a more complex gut microbiota had higher total serum antibodies than their germ-free counterparts. Collectively, these data suggest that cognate CD4+ T cell help during commensal-induced bacteremia can orchestrate a potent, commensal-specific, and polyreactive antibody response. These findings shed new light on the systemic humoral immune response to bacteremia and could potentially be exploited to develop more effective and personalized vaccine strategies.
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    Characterization of Bacterial Depletion and Colitis-Associated Changes in the Gut Microbiota on Behaviour and Intestinal Physiology
    (2021-05-10) Vicentini, Fernando Augusto; Sharkey, Keith Alexander; Hirota, Simon Andrew; Pittman, Quentin Jerome; McCafferty, Donna-Marie; DeVinney, Rebekah; Gareau, Melanie
    The role of the microbiota in modulating host physiology is now widely accepted. Acting via the microbiota-gut-brain (MGB) axis, intestinal bacteria have been associated with the regulation of gastrointestinal tract and the central nervous system function, in both physiological and pathophysiological conditions, such as in inflammatory bowel disease (IBD). Patients with IBD have an increased incidence of anxiety and depression, which is accompanied by alterations in the microbiota composition. However, the link between changes in microbiota composition and behavioural abnormalities in the context of intestinal inflammation is unknown. Mechanisms that mediate the MGB axis in homeostasis have emerged but are still incomplete. We sought to characterize the effects of bacterial depletion on intestinal physiology and animal behaviour, and the role of colitis-associated microbiota in the modulation of behavioural abnormalities. Using mouse models of bacterial depletion (antibiotic treatment) and colitis (administration of dextran sulfate sodium), we investigated the role of the microbiota in intestinal physiology and behavioural outputs, and the potential mechanisms of these interactions. The major findings were: (1) bacterial depletion induced a reduction in despair behaviour in a sex-specific manner. The altered behaviour observed in male mice was normalized by activation of the Aryl hydrocarbon receptor, suggesting that this receptor is involved in modulation of behaviour via host-microbe interactions. (2) Bacterial depletion was linked to sex-independent changes in intestinal physiology, including reduced motility, altered secretion, and increased permeability. These changes were accompanied by a reduction in the numbers of enteric neurons, suggesting that microbiota influences the enteric nervous system integrity. Administration of bacterial-derived molecules, lipopolysaccharide and short-chain fatty acids, was associated with neuronal survival and neurogenesis, respectively. (3) Colitis induced changes in the intestinal bacteria composition, which was associated with behavioural abnormalities. Microbiota transfer of colitis-associated microbiota into recipient healthy mice was sufficient to induce behavioural alterations. Remarkably, no intestinal or neuroinflammation were observed in recipient mice. Collectively, these findings provide support to expand our understanding of the MGB axis under homeostatic and intestinal inflammatory conditions. Thus, a better understanding of this integrated network provides a rationale for the development of microbiota-centred therapeutic approaches for the improvement of health.
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    Characterizing Host Protective Immune Responses to the Parasitic Worm Heligmosomoides polygyrus
    (2020-05-05) Ariyaratne, Anupama; Finney, Constance A. M.; Hirota, Simon Andrew; Storey, Douglas G.
    Intestinal roundworms cause chronic debilitating disease in humans and livestock. The lack of vaccines and the emerging drug resistance only increase the need to better understand worm-host interactions. H. polygyrus larvae induce an intestinal granuloma response whereby immune cells, mostly alternatively activated macrophages and eosinophils, accumulate around the tissue encysted parasites. In vitro, these cells and antibodies immobilize, damage/kill worms within the granuloma. This is yet to be confirmed in vivo. We focused on understanding: the mechanisms underlying effective granulomas (chapter 1), the role of resistin like molecule (RELM) alpha, a protein shown to downregulate Th2 immunity (chapter 2), and eosinophils, an enigmatic cell type whose role in anti-worm immunity remains controversial (chapter 3). C57Bl/6 mice are susceptible to H. polygyrus infection. However, mice that are infected using a more natural approach (multiple low doses – (trickle) - as opposed to one large dose (bolus)), had fewer adult worms with more granulomas. Their granulomas had decreased RELM- expression, increased anti-parasitic RELM- and fibrotic gene expression as well as a stronger IgG1 accumulation. In the absence of RELM-, granulomas from trickle-infected mice remained more protective, with upregulated genes involved in eosinophil chemotaxis. Despite this increased eosinophil recruitment, the granulomas from RELM--/- mice were not as protective as those from wild type animals highlighting the need for RELM- for maximal granuloma efficiency. In WT mice, we found eosinophils accumulated around tissue encysted H. polygyrus during acute and chronic infections. The spleen, mesenteric lymph nodes and Peyer’s patches (PP) had increased eosinophilia, with resident and recruited eosinophil surface markers. In addition, eosinophils accumulated around the protozoan parasite Toxoplasma gondii indicating that intestinal eosinophils may represent a key cell type in immunity to different types of incoming parasites. T. gondii infection did not increase eosinophil numbers in the intestine or PP. However, eosinophils showed an inflammatory phenotype marked by increased MHC II and CD101 expression. These findings highlight that the granuloma is a key, complex site of anti-worm immunity and that trickle infection models provide us with a novel tool to study the mechanisms at play within granulomas.
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    Characterizing the role of NR4A1 in the regulation of intestinal smooth muscle cell phenotype and function
    (2021-04-06) Szczepanski, Holly Elizabeth; Hirota, Simon Andrew; Newton, Robert; von der Weid, Pierre-Yves
    Intestinal fibrosis and stricture formation are common complications of Crohn’s disease (CD). Recently, smooth muscle hypertrophy and hyperplasia have gained greater recognition as a driver of stricture formation, rather than an increase in fibrosis alone. Despite advances in treatment of CD, current therapies do little to prevent or reverse strictures. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor (NR) that is anti-fibrotic in non-intestinal systems and exhibits anti-proliferative effects in smooth muscle cells (SMCs). NR4A1 gene variants have been associated with increased risk of IBD, however, mechanisms regulating NR4A1 expression and its role in intestinal SMC function have not been investigated. We therefore hypothesized that presence and activation of NR4A1 regulates cell proliferation and metabolism by modulating intestinal SMC phenotype. Primary intestinal SMCs isolated from Nr4a1+/+ and Nr4a1-/- mice and a commercially sourced human primary intestinal SMC line were used as in vitro models. We employed the chronic model of dextran sulfate sodium (DSS) and the SAMP1/YitFc model of spontaneous ileitis as in vivo models. In alignment with our hypothesis, NR4A1 presence and exposure to identified agonists, cytosporone-B (Csn-B) and 6-mercaptopurine (6-MP), regulated key cellular mechanisms involved in excessive smooth muscle hypertrophy and hyperplasia. NR4A1 expression was significantly induced in human colonic SMCs by platelet-derived growth factor (PDGF)-BB suggesting a potential negative feedback mechanism to control mitogen-induced SMC proliferation. Taken together, in this study, NR4A1 is shown to be a vital brake in intestinal SMC phenotypic modulation by limiting excessive proliferation and other associated characteristics that could contribute to pathogenic tissue remodelling observed in fibrostenotic CD.
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    Enhanced Induction of Epithelial-Derived IL-17C Following Bacteria and Rhinovirus Exposure
    (2019-05-15) Jamieson, Kyla Carol; Proud, David; Jenne, Craig N.; Hirota, Simon Andrew; Yipp, Bryan G.; McCoy, Kathy D.; Peebles, Ray Stokes
    Up to 80% of Chronic Obstructive Pulmonary Disease (COPD) exacerbations are associated with bacterial and/or viral pathogens, with bacteria-virus co-infections detected in up to 25% of exacerbations. These co-infections are associated with increased symptoms, increased systemic inflammation, longer hospital stays, and increased risk of hospital re-admission. Human rhinovirus (HRV) is the most common viral pathogens detected, while non-typeable Haemophilus influenzae (NTHI) and Pseudomonas aeruginosa (PAO) are among the most common bacterial pathogens identified. The airway epithelium is the first line of defence against these pathogens and responds by releasing proinflammatory cytokines and anti-microbial peptides. Interleukin (IL)-17C is a novel pro-inflammatory cytokine that is typically released from epithelial cells in response to bacteria, viral, or fungal pathogens, and in response to pro-inflammatory cytokines such as TNFα and IL-1β. In this thesis, we performed the first study to assess the involvement and functional role of IL-17C in bacteria-rhinovirus co-infections in human bronchial epithelial cells (HBECs). Bacteria-rhinovirus co-exposure for 24 hours induced significant, and synergistic, IL-17C gene expression and protein release. Synergistic IL-17C release was dependent on viral replication recognition sensors, RIG-I and MDA5, as well as NF-κB and p38 signalling. In an autocrine/paracrine manner, IL-17C acted on the airway epithelium to induce CXCL1, CXCL2, TFRC, and NFKBIZ gene expression, to induce CXCL1 protein release, and to promote HBEC-induced neutrophil recruitment. To assess how IL-17C is involved in the clinical context of COPD, HBECs were obtained via bronchial brushings from non-smokers, smokers with normal lung function, and patients with physician-diagnosed COPD and these cells were exposed to NTHI and HRV-1A concurrently. Interestingly, in response to concurrent NTHI and HRV-1A exposure, HBECs from COPD patients released significantly more IL-17C than cells from either non-smokers or healthy smokers, and HBECs from healthy smokers released significantly less IL-17C than non-smokers. Further, acute cigarette smoke extract exposure significantly reduced microbial-induced IL-17C release from cells from normal subjects. Using a morphologically-relevant well-differentiated HBEC model, IL-17C was predominantly released basolaterally, from apical cells, in response to HRV in a dose-, time-, and replication-dependent manner. High doses of NTHI could also induce basolateral IL-17C, however synergy was no longer achieved with NTHI+HRV-1A co-infections. Similar to monolayer culture, IL-17C acted on basal cells to induce significant basolateral release of CXCL1, providing physiological relevance for subsequent neutrophil recruitment. These results suggest that IL-17C acts to induce CXCL1 release and promote neutrophil recruitment to the site of bacteria or rhinovirus respiratory infections, however, this response is exaggerated in epithelial cells from COPD patients.
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    Examining the Role of NLRP3 in Intestinal Homeostasis and Fibrosis
    (2018-07-24) Tjong, Jessica Ria; Beck, Paul L.; Muruve, Daniel A.; Hirota, Simon Andrew
    Fibrosis is the most common cause of surgery in Crohn’s disease (CD) and the mechanisms of fibrosis in CD are not well understood. Nucleotide-binding oligomerization domain (NOD)-like receptors, including NLRP3, are cytosolic protein sensors involved in inflammatory pathways and implicated in CD pathogenesis. NLRP3 has been shown to drive fibrosis in various tissues, including the heart, kidney, lungs, liver and pancreas; however, its role in intestinal fibrosis is currently unknown. The main hypothesis of this project is that NLRP3 mediates intestinal fibrosis. Primary intestinal myofibroblasts isolated from Nlrp3-/- mice had reduced response to pro-fibrogenic cytokine TGFβ compared to wildtype (WT), with lower phosphorylation of downstream signalling protein Smad2, and decreased induction of connective tissue growth factor (CTGF), as well as decreased migration. Loss of NLRP3 in intestinal myofibroblasts also led to increased resistance to cell death during serum deprivation. In an in vivo model of chronic colitis, Nlrp3-/- mice had significantly worse disease and susceptibility compared to WT. However, both Nlrp3-/- and WT mice had similar levels of intestinal fibrosis. Relevance to the human disease was further highlighted by the finding that NLRP3 transcript levels in mucosal colonic biopsies from patients with CD were significantly increased, and increased expression correlated with inflammation. Overall, our data suggest that loss of NLRP3 leads to reduced response to TGFβ in colonic myofibroblasts, and that NLRP3 may potentiate TGFβ beta signalling in the gut. Further insights into the mechanisms of intestinal fibrosis will significantly impact the development of new tools that will help with assessing and treating patients with CD complications.
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    Intestinal Homeostasis: The role of Indole-3-propionic acid (IPA) in mucosal homeostasis and repair
    (2020-05) Nieves, Kristoff Michael; Hirota, Simon Andrew; Geuking, Markus B.; Lu, Cathy; von der Weid, Pierre Yves; Chadee, Khrisendath
    Failure to resolve inflammation is often associated with the complications of Crohn’s Disease (CD). The pregnane X receptor (PXR), a xenobiotic receptor, is recognized for its role in suppressing inflammation and has recently been shown to influence fibrogenesis in the liver. In the intestine, PXR-signaling can be influenced by the microbial tryptophan metabolite indole-3- propionic acid (IPA), which can modulate intestinal inflammation, in turn influencing fibrogenesis, resolution and healing. This suggests that the gut microbiota could modulate mucosal homeostasis and resolution of inflammation via microbial metabolites. We therefore hypothesized that the microbial metabolite IPA through its activation of the PXR would act as a negative regulator of fibrosis. Using a gnotobiotic mouse model revealed, contrary to our hypothesis that activation of the PXR with the microbial metabolite IPA did not attenuate fibrosis. However, IPA did increase survival in the gnotobiotic mice to DSS-induced colitis. Additionally, fecal microbiota composition analysis revealed that IPA induces protection against the depletion of commensal Bacteroides spp. induced by DSS. Taken together, this data provides a foothold of knowledge into the relationship between the microbiota and host and highlights the potential for alternative means of the treatment of IBD and its complications.
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    Investigating pulmonary vascular B cells
    (2019-04-26) Podstawka, John; Yipp, Bryan G.; Hirota, Simon Andrew; Kelly, Margaret Mary; Flannigan, Kyle L.
    The lung is a vital organ which is required for gas exchange. The anatomy of the lung itself, immune molecules, and leukocytes provide the host with protection from harmful debris and pathogenic stimuli. A component of pulmonary host defense is neutrophils which are found within the lung capillary network. Within the pulmonary capillaries, there is a population of neutrophils which remains adherent to the vascular endothelium for extended periods of time and exhibits marginated behaviour. While pulmonary vascular neutrophils are important in the acute immune response of the lung, they tend to exhibit an aged and pro-inflammatory phenotype. While inflammation within the lung is necessary for host defence and is a part of wound healing, cellular infiltration of the lung significantly compromises oxygen delivery and carbon dioxide removal. Therefore, pulmonary inflammation must be tightly controlled and regulated. We previously determined that pulmonary vascular B cells regulate neutrophils via CD18-mediated interactions, labelling them MHCII+AnnexinV+, and that in the absence of B cells, the lung will develop pathological fibrotic interstitial lung disease. In this body of work, we determined that pulmonary intravascular B cells can marginate which allows them to specialize in regulating lung neutrophils. These B cells exhibit a mature, naïve, conventional B2 cell phenotype, and engage in marginated behaviour which can be mediated by CD49e or via CXCR5/CXCL13. Blocking CD49e decreased the amount B cell-neutrophil interactions taking place within the lung capillaries, and providing exogenous CXCL13 or neutralizing endogenous CXCL13 would respectively increase and decrease the amount of interactions. Moreover, we demonstrated an intratracheal injection of CXCL13 increased the number of neutrophils which exhibited an MHCII+AnnexinV+ phenotype; indicating increased regulation via B cell interactions. Thus, we acquired novel insight into how lung intravascular B cells are engaging in prolonged interactions with lung neutrophils; interactions which were previously defined as regulatory and important for maintaining inflammatory homeostasis.
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    Microscale Tissue Engineering and Contributors of the Cellular Niche
    (2019-05-31) Al-Ani, Abdullah; Ungrin, Mark D.; Biernaskie, Jeff A.; Kallos, Michael S.; Rancourt, Derrick E.; Hirota, Simon Andrew; Gratzer, Paul F.
    The behaviour of cells is modulated by their microenvironment or ‘niche’. While cellular therapies offer promising curative solutions for many diseases, the efficacy of transplanted cells is often hampered by a suboptimal microenvironment. One strategy to overcome this limitation is to reconstruct the niche of the cells of interest prior to transplantation. The central aim of this thesis is to develop novel tissue engineering approaches to further understand and reconstitute the cellular niche. While these approaches were specifically validated in the retinal and islet systems, they were also designed to be easily implemented in other biological systems. One project showcases a novel scaffold-free, scalable and injectable retinal pigment epithelium (RPE) microtissue for minimally-invasive transplantation. While RPE transplantation holds great potential to cure various retinal degenerative diseases, cells transplanted as cellular suspension exhibit suboptimal survival and function. Conversely, transplanting RPE as coherent cellular sheets has yielded better outcomes, but they are complex to transplant and produce at large scale. Our RPE microtissues were designed to capture the benefit of both approaches: namely, simplicity of production and transplantation, as well as enhanced performance. We found that our RPE microtissue exhibited superior cellular behaviour in terms of gene expression and in vitro function when compared to standard adherent culture. Another project presents a unique approach to produce transplants with a reconstituted cellular niche. This approach aims to repopulate the niche by incorporating finite amounts and proportions of niche cells into transplantable constructs. Using it enabled us to produce size-controlled pseudoislet constructs that contained various proportions of mesenchymal stem cells (MSCs), fibroblast and endothelial cells, and to quantitatively evaluate their in vitro performance. Further, applying this approach led us to discover more than one favourable condition that yielded improvements in islet cell performance in vitro. While the islets of Langerhans were used for biological validation, the approach was designed to be broadly applicable to various biological systems. In sum, this thesis offers several novel approaches for scientists to better understand and enhance the cellular niche. The simplicity, accessibility and scalability of these approaches render them suitable for both scientific applications and clinical translation.
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    The Role of Protease-Activated Receptor-2 in Colonic Epithelial Wound Healing
    (2019-06-27) Yatigalpoththe, Mahesha N. S.; MacNaughton, Wallace Keith; McCafferty, Donna-Marie; Hirota, Simon Andrew; von der Weid, Pierre Yves
    The intestinal barrier function relies on the presence of a single layer of epithelial cells that selectively absorb nutrients and water while limiting intestinal content and gut microbiota into the lumen. When epithelial barrier function is compromised, unregulated translocation of microbiota and microbial products into the mucosa can result in intestinal inflammation as seen in IBD patients. Effective resolution of inflammation requires elimination of inflammatory triggers and this is partly dependant on epithelial wound healing. The intestine is constantly exposed to proteases originating from a variety of sources and these proteases can activate protease-activated receptors (PARs). The mechanism of intestinal epithelial wound healing is not completely understood, particularly in the context of proteases and PARs. It was recently shown that PAR2 activation transactivates epidermal growth factor receptor (EGFR) and also induces COX-2 expression in colonic epithelial cells. Based on this, it was hypothesized that PAR2-induced EGFR transactivation and COX-2 expression drive colonic epithelial wound healing. In the first part of the study, CMT-93 cells were characterized for PAR2 expression and PAR-2 induced COX-2 expression. PAR2 is primarily expressed in the plasma membrane with punctate immunoreactivity in the cytoplasm. Furthermore, the majority of PAR2 expression appeared to be basolateral. PAR2 activation by 2fLI (2-furoyl-LIGRLO-NH2) induced Ca2+ signaling. COX-2 expression was characterized using western blotting and PAR2 activation did not induce COX-2 expression in CMT93 cells. The effect of PAR2 activation in wound healing was investigated in the second part of the study. PAR2 activation induced wound healing and the treatment with the COX-2 selective inhibitor (NS-398) did not affect the PAR-2 induced wound healing, indicating that PAR-2 induced wound healing does not depend on COX-2 activity. The treatments with the EGFR tyrosine kinase inhibitor (PD153035), broad-spectrum matrix metalloproteases (MMP) inhibitor (GM6001) or Src tyrosine kinase inhibitor (PP2) inhibited PAR2-induced wound healing. These results suggest EGFR activity is required for PAR2-induced wound healing and PAR2 activation possibly transactivates EGFR via-matrix metalloproteinases and Src tyrosine kinase activity. In conclusion, this study shows the contribution of PAR-2 in epithelial wound healing, which could subsequently aid in the resolution of intestinal inflammation.
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    Xenobiotic receptor regulation of Clostridium difficile associated inflammation and tissue damage
    (2017) Erickson, Sarah Lindsay; Hirota, Simon Andrew; Yipp, Bryan; Giembycz, Mark
    The current treatments available for C. difficile infections (CDI) aims to eliminate C. difficile bacteria, however they do not treat the inflammation and tissue damage triggered by the virulence factors, TcdA and TcdB. Indeed, enhanced chemokine/cytokine expression during CDI is associated with poor clinical outcomes. The xenobiotic receptor, pregnane X receptor (PXR) can be activated by antibiotics which are known to target C. difficile. The PXR has also been reported to attenuate intestinal inflammation by inhibiting NFκB signaling. Therefore we hypothesized that targeting the PXR would be an effective strategy to reduce C. difficile toxin-induced inflammation and tissue damage. Activation of the PXR attenuated C. difficile toxin-induced inflammatory mediator release in Caco-2 intestinal epithelial cells (IECs) and in mouse enteroid derived colonic monolayers. In vivo studies demonstrated that PXR activation using the pharmacological agonist pregnenolone-16-α-carbonitrile (PCN) attenuated the expression of pro-inflammatory mediators and significantly reduced neutrophil infiltration to the colonic lamina propria following toxin challenge. Additionally, when compared to wild type mice, PXR-/- mice challenged with toxin displayed a hyper-susceptible phenotype with significant eosinophilic inflammation and increased pro-inflammatory gene expression. Contrary to our hypothesis, this phenotype was not mediated by the loss of the PXR in the epithelium, as determined by the use of a PXRfl/fl x villin cre+ knockout mouse. Taken together, these data will help us expand our knowledge of the PXR’s role in host-pathogen interactions, and its potential to be modulated for the treatment of inflammation and tissue damage in CDI.