Browsing by Author "Hirota, Simon A."

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    Exaggerated IL-15 and Altered Expression of foxp3+ Cell-Derived Cytokines Contribute to Enhanced Colitis in Nlrp3−/− Mice
    (2016-08-17) Hirota, Simon A.; Ueno, Aito; Tulk, Sarah E.; Becker, Helen M.; Schenck, L. Patrick; Potentier, Mireille S.; Li, Yan; Ghosh, Subrata; Muruve, Daniel A.; MacDonald, Justin A.; Beck, Paul L.
    The pathogenesis of Crohn’s disease (CD) involves defects in the innate immune system, impairing responses to microbes. Studies have revealed that mutations NLRP3 are associated with CD. We reported previously that Nlrp3−/− mice were more susceptible to colitis and exhibited reduced colonic IL-10 expression. In the current study, we sought to determine how the loss of NLRP3 might be altering the function of regulatory T cells, a major source of IL-10. Colitis was induced in wild-type (WT) and Nlrp3−/− mice by treatment with dextran sulphate sodium (DSS). Lamina propria (LP) cells were assessed by flow cytometry and cytokine expression was assessed. DSS-treated Nlrp3−/− mice exhibited increased numbers of colonic foxp3+ T cells that expressed significantly lower levels of IL-10 but increased IL-17. This was associated with increased expression of colonic IL-15 and increased surface expression of IL-15 on LP dendritic cells. Neutralizing IL-15 in Nlrp3−/− mice attenuated the severity of colitis, decreased the number of colonic foxp3+ cells, and reduced the colonic expression of IL-12p40 and IL-17. These data suggest that the NLRP3 inflammasome can regulate intestinal inflammation through noncanonical mechanisms, providing additional insight as to how NLRP3 variants may contribute to the pathogenesis of CD.
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    Intestinal microbiota shapes gut physiology and regulates enteric neurons and glia
    (2021-10-26) Vicentini, Fernando A.; Keenan, Catherine M.; Wallace, Laurie E.; Woods, Crystal; Cavin, Jean-Baptiste; Flockton, Amanda R.; Macklin, Wendy B.; Belkind-Gerson, Jaime; Hirota, Simon A.; Sharkey, Keith A.
    Abstract Background The intestinal microbiota plays an important role in regulating gastrointestinal (GI) physiology in part through interactions with the enteric nervous system (ENS). Alterations in the gut microbiome frequently occur together with disturbances in enteric neural control in pathophysiological conditions. However, the mechanisms by which the microbiota regulates GI function and the structure of the ENS are incompletely understood. Using a mouse model of antibiotic (Abx)-induced bacterial depletion, we sought to determine the molecular mechanisms of microbial regulation of intestinal function and the integrity of the ENS. Spontaneous reconstitution of the Abx-depleted microbiota was used to assess the plasticity of structure and function of the GI tract and ENS. Microbiota-dependent molecular mechanisms of ENS neuronal survival and neurogenesis were also assessed. Results Adult male and female Abx-treated mice exhibited alterations in GI structure and function, including a longer small intestine, slower transit time, increased carbachol-stimulated ion secretion, and increased intestinal permeability. These alterations were accompanied by the loss of enteric neurons in the ileum and proximal colon in both submucosal and myenteric plexuses. A reduction in the number of enteric glia was only observed in the ileal myenteric plexus. Recovery of the microbiota restored intestinal function and stimulated enteric neurogenesis leading to increases in the number of enteric glia and neurons. Lipopolysaccharide (LPS) supplementation enhanced neuronal survival alongside bacterial depletion, but had no effect on neuronal recovery once the Abx-induced neuronal loss was established. In contrast, short-chain fatty acids (SCFA) were able to restore neuronal numbers after Abx-induced neuronal loss, demonstrating that SCFA stimulate enteric neurogenesis in vivo. Conclusions Our results demonstrate a role for the gut microbiota in regulating the structure and function of the GI tract in a sex-independent manner. Moreover, the microbiota is essential for the maintenance of ENS integrity, by regulating enteric neuronal survival and promoting neurogenesis. Molecular determinants of the microbiota, LPS and SCFA, regulate enteric neuronal survival, while SCFA also stimulates neurogenesis. Our data reveal new insights into the role of the gut microbiota that could lead to therapeutic developments for the treatment of enteric neuropathies. Video abstract
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    Recruitment of α4β7 monocytes and neutrophils to the brain in experimental colitis is associated with elevated cytokines and anxiety-like behavior
    (2022-04-04) Cluny, Nina L.; Nyuyki, Kewir D.; Almishri, Wagdi; Griffin, Lateece; Lee, Benjamin H.; Hirota, Simon A.; Pittman, Quentin J.; Swain, Mark G.; Sharkey, Keith A.
    Abstract Background Behavioral comorbidities, such as anxiety and depression, are a prominent feature of IBD. The signals from the inflamed gut that cause changes in the brain leading to these behavioral comorbidities remain to be fully elucidated. We tested the hypothesis that enhanced leukocyte–cerebral endothelial cell interactions occur in the brain in experimental colitis, mediated by α4β7 integrin, to initiate neuroimmune activation and anxiety-like behavior. Methods Female mice treated with dextran sodium sulfate were studied at the peak of acute colitis. Circulating leukocyte populations were determined using flow cytometry. Leukocyte–cerebral endothelial cell interactions were examined using intravital microscopy in mice treated with anti-integrin antibodies. Brain cytokine and chemokines were assessed using a multiplex assay in animals treated with anti-α4β7 integrin. Anxiety-like behavior was assessed using an elevated plus maze in animals after treatment with an intracerebroventricular injection of interleukin 1 receptor antagonist. Results The proportion of classical monocytes expressing α4β7 integrin was increased in peripheral blood of mice with colitis. An increase in the number of rolling and adherent leukocytes on cerebral endothelial cells was observed, the majority of which were neutrophils. Treatment with anti-α4β7 integrin significantly reduced the number of rolling leukocytes. After anti-Ly6C treatment to deplete monocytes, the number of rolling and adhering neutrophils was significantly reduced in mice with colitis. Interleukin-1β and CCL2 levels were elevated in the brain and treatment with anti-α4β7 significantly reduced them. Enhanced anxiety-like behavior in mice with colitis was reversed by treatment with interleukin 1 receptor antagonist. Conclusions In experimental colitis, α4β7 integrin-expressing monocytes direct the recruitment of neutrophils to the cerebral vasculature, leading to elevated cytokine levels. Increased interleukin-1β mediates anxiety-like behavior.
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    The Cellular Hypoxic Response upon Giardia Duodenalis Infection
    (2024-08-08) DeMichele, Emily; Buret, Andre G.; Hirota, Simon A.; Finney, Constance; Beck, Paul
    Oxygen tension plays an integral role in gastrointestinal homeostasis. When oxygen concentrations become suboptimal (hypoxia), mammalian cells rely on the hypoxia-inducible factor (HIF) to combat cellular stress and augment glycolytic flux. In the gastrointestinal epithelium, this cellular response is paramount as the luminal facing epithelial cells exist in a state of physiologic hypoxia that can be further altered by pathogens. Although growing evidence suggests tissue and blood protozoan parasites can activate HIF in a mammalian host, much remains to be understood in the context of enteric protozoan infections. This study uncovered the role of HIF-1α and associated epithelial hypoxic signature upon Giardia duodenalis infection, a leading cause of diarrheal disease worldwide. Coculture experiments were carried out using Caco-2 colonic epithelial cells infected with a human isolate of G. duodenalis (GSM) in normoxic (21% O2) or hypoxic (1% O2) conditions. Under normoxic conditions, infected cells displayed a time-dependent increase in HIF-1α protein expression, the oxygen-dependent subunit of HIF. This observation was concomitant with increased expression of HIF-target genes, including those responsible for combatting cellular stress (i.e., VEGFA, GADD45A, ANKRD37), and augmenting glycolytic flux (i.e., HK2, LDHA). Intracellular metabolomic analysis of normoxic infected cells revealed simultaneous increases in glucose-6-phosphate and lactate, the metabolic outputs of HK2 and LDHA, respectively. Under hypoxic conditions, HIF-1α stabilization was not significantly different compared to uninfected hypoxic control cells. However, HIF-target genes were still upregulated, albeit to a lesser degree compared to normoxic infected cells. Importantly, the metabolome of infected epithelial cells differed greatly between normoxic and hypoxic conditions, highlighting the influential role of oxygen during G. duodenalis infection. After 24 hours, HIF-1α stabilization and alterations to HIF-target gene expression were no longer detected. These findings indicate G. duodenalis induces a hypoxic response driven by HIF-1α stabilization in normoxic intestinal epithelial cells, while simply exacerbating this cellular response in hypoxic conditions. The stabilization of HIF-1α in the absence of oxygenic stress highlights a novel metabolic cell rescue mechanism in response to enteropathogens.