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Simon A Hirota,
Vadim Iablokov,
Sarah E Tulk,
L Patrick Schenck,
Helen Becker,
Jimmie Nguyen,
Samir Al Bashir,
Tanis C Dingle,
Austin Laing,
Jianrui Liu, Yan Li,
Jeff Bolstad,
George L Mulvey,
Glen D Armstrong,
Wallace K Macnaughton,
Daniel A Muruve,
Justin A Macdonald,
Paul L Beck
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ABSTRACT: Clostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, Toxin A (TcdA) and Toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell increasing intestinal permeability, triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically-generated 'ileal loop'. This modeI is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ of C. difficile infection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation, neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI including IL-6, IFN-gamma and IL-1beta. Finally we were able to demonstrate that TcdA was much more potent at inducing colonic injury than TcdB; but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.
Infection and immunity 10/2012; · 4.21 Impact Factor
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Simon A Hirota,
Jeffrey Ng,
Alan Lueng,
Maitham Khajah,
Ken Parhar, Yan Li,
Victor Lam,
Mireille S Potentier,
Kelvin Ng,
Misha Bawa,
Donna-Marie McCafferty,
Kevin P Rioux,
Subrata Ghosh,
Ramnik J Xavier,
Sean P Colgan,
Jurg Tschopp,
Daniel Muruve,
Justin A MacDonald,
Paul L Beck
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ABSTRACT: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1β and IL-18.
In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis.
Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1β, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-β. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic β-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota.
Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations.
Inflammatory Bowel Diseases 06/2011; 17(6):1359-72. · 4.86 Impact Factor
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Akosua Vilaysane,
Justin Chun,
Mark E Seamone,
Wenjie Wang,
Rick Chin,
Simon Hirota, Yan Li,
Sharon A Clark,
Jurg Tschopp,
Kiril Trpkov,
Brenda R Hemmelgarn,
Paul L Beck,
Daniel A Muruve
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ABSTRACT: Inflammation significantly contributes to the progression of chronic kidney disease (CKD). Inflammasome-dependent cytokines, such as IL-1β and IL-18, play a role in CKD, but their regulation during renal injury is unknown. Here, we analyzed the processing of caspase-1, IL-1β, and IL-18 after unilateral ureteral obstruction (UUO) in mice, which suggested activation of the Nlrp3 inflammasome during renal injury. Compared with wild-type mice, Nlrp3(-/-) mice had less tubular injury, inflammation, and fibrosis after UUO, associated with a reduction in caspase-1 activation and maturation of IL-1β and IL-18; these data confirm that the Nlrp3 inflammasome upregulates these cytokines in the kidney during injury. Bone marrow chimeras revealed that Nlrp3 mediates the injurious/inflammatory processes in both hematopoietic and nonhematopoietic cellular compartments. In tissue from human renal biopsies, a wide variety of nondiabetic kidney diseases exhibited increased expression of NLRP3 mRNA, which correlated with renal function. Taken together, these results strongly support a role for NLRP3 in renal injury and identify the inflammasome as a possible therapeutic target in the treatment of patients with progressive CKD.
Journal of the American Society of Nephrology 10/2010; 21(10):1732-44. · 9.66 Impact Factor
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Jeffrey Ng,
Simon A Hirota,
Olaf Gross, Yan Li,
Annegret Ulke-Lemee,
Mireille S Potentier,
L Patrick Schenck,
Akosua Vilaysane,
Mark E Seamone,
Hanping Feng,
Glen D Armstrong,
Jurg Tschopp,
Justin A Macdonald,
Daniel A Muruve,
Paul L Beck
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ABSTRACT: Clostridium difficile-associated disease (CDAD) is the leading cause of nosocomial diarrhea in the United States. C difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system. In the present study, we hypothesize that TcdA and TcdB trigger inflammasome-dependent interleukin (IL)-1beta production, which contributes to the pathogenesis of CDAD.
Macrophages exposed to TcdA and TcdB were assessed for IL-1beta production, an indication of inflammasome activation. Macrophages deficient in components of the inflammasome were also assessed. Truncated/mutated forms of TcdB were assessed for their ability to activate the inflammasome. The role of inflammasome signaling in vivo was assessed in ASC-deficient and IL-1 receptor antagonist-treated mice.
TcdA and TcdB triggered inflammasome activation and IL-1beta secretion in macrophages and human mucosal biopsy specimens. Deletion of Nlrp3 decreased, whereas deletion of ASC completely abolished, toxin-induced IL-1beta release. TcdB-induced IL-1beta release required recognition of the full-length toxin but not its enzymatic function. In vivo, deletion of ASC significantly reduced toxin-induced inflammation and damage, an effect that was mimicked by pretreatment with the IL-1 receptor antagonist anakinra.
TcdA and TcdB trigger IL-1beta release by activating an ASC-containing inflammasome, a response that contributes to toxin-induced inflammation and damage in vivo. Pretreating mice with the IL-1 receptor antagonist anakinra afforded the same level of protection that was observed in ASC-/- mice. These data suggest that targeting inflammasome or IL-1beta signaling may represent new therapeutic targets in the treatment of CDAD.
Gastroenterology 08/2010; 139(2):542-52, 552.e1-3. · 11.68 Impact Factor
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Simon A Hirota,
Kyla Fines,
Jeffrey Ng,
Danya Traboulsi,
Josh Lee,
Eikichi Ihara, Yan Li,
William G Willmore,
Daniel Chung,
Melanie M Scully,
Thomas Louie,
Shaun Medlicott,
Manigandan Lejeune,
Kris Chadee,
Glen Armstrong,
Sean P Colgan,
Daniel A Muruve,
Justin A MacDonald,
Paul L Beck
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ABSTRACT: Clostridium difficile is the leading cause of nosocomial infectious diarrhea. Antibiotic resistance and increased virulence of strains have increased the number of C difficile-related deaths worldwide. The innate host response mechanisms to C difficile are not resolved; we propose that hypoxia-inducible factor (HIF-1) has an innate, protective role in C difficile colitis. We studied the impact of C difficile toxins on the regulation of HIF-1 and evaluated the role of HIF-1alpha in C difficile-mediated injury/inflammation.
We assessed HIF-1alpha mRNA and protein levels and DNA binding in human mucosal biopsy samples and Caco-2 cells following exposure to C difficile toxins. We used the mouse ileal loop model of C difficile toxin-induced intestinal injury. Mice with targeted deletion of HIF-1alpha in the intestinal epithelium were used to assess the effects of HIF-1alpha signaling in response to C difficile toxin.
Mucosal biopsy specimens and Caco-2 cells exposed to C difficile toxin had a significant increase in HIF-1alpha transcription and protein levels. Toxin-induced DNA binding was also observed in Caco-2 cells. Toxin-induced HIF-1alpha accumulation was attenuated by nitric oxide synthase inhibitors. In vivo deletion of intestinal epithelial HIF-1alpha resulted in more severe, toxin-induced intestinal injury and inflammation. In contrast, stabilization of HIF-1alpha with dimethyloxallyl glycine attenuated toxin-induced injury and inflammation. This was associated with induction of HIF-1-regulated protective factors (such as vascular endothelial growth factor-alpha, CD73, and intestinal trefoil factor) and down-regulation of proinflammatory molecules such as tumor necrosis factor and Cxcl1.
HIF-1alpha protects the intestinal mucosa from C difficile toxins. The innate protective actions of HIF-1alpha in response to C difficile toxins be developed as therapeutics for C difficile-associated disease.
Gastroenterology 03/2010; 139(1):259-69.e3. · 11.68 Impact Factor
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ABSTRACT: Nitric oxide (NO) is an important mediator of intestinal inflammation. Inducible NO synthase (iNOS) is the main source of NO in inflammation. Because iNOS is ubiquitously expressed, our aim was to determine which cellular source(s) of iNOS plays the central role in mediating intestinal inflammation.
Chimeric lines were produced via bone marrow (BM) transplantation following irradiation. Colitis was induced with dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS). The severity of colitis and markers of inflammation were assessed in standard fashion. Leukocyte recruitment was assessed by intravital microscopy.
The irradiated chimeric lines with iNOS-/- BM-derived cells were markedly more resistant to both DSS- and TNBS-induced injury. Resistance to DSS-induced colitis was lost when wild-type (wt) BM was used to reconstitute iNOS-/- mice. Neutrophils were the main source of iNOS in DSS-induced colitis. iNOS-/- chimeric lines had decreased colonic macrophage inflammatory protein 1beta and tumor necrosis factor alpha expression and increased levels of the protective growth factor, keratinocyte growth factor. LPS-mediated leukocyte recruitment was reduced in iNOS-/- mice, and there were marked changes in the inflammatory cell infiltrates between the chimeric lines with iNOS-/- vs wt BM-derived cells. Furthermore, the lamina propria CD4 +ve cells from chimeric lines with iNOS-/- BM-derived cells had reduced intracellular cytokine expression.
iNOS produced by BM-derived cells plays a critical role in mediating the inflammatory response during colitis. Cell-specific regulation of iNOS may represent a novel form of therapy for patients with inflammatory bowel disease.
Gastroenterology 05/2007; 132(5):1778-90. · 11.68 Impact Factor
