Clostridium difficile Toxin–Induced Inflammation and Intestinal Injury Are Mediated by the Inflammasome 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,* and PAUL L. BECK* Departments of *Medicine, ‡ Biochemistry and Molecular Biology, and ¶ Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada; § Department of Biochemistry, University of Lausanne, Epalinges, Switzerland; and Division of Infectious Diseases, Department of Biomedical Sciences, Tufts University, North Grafton, Massachusetts BACKGROUND & AIMS: 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 inflam- masome-dependent interleukin (IL)-1 production, which contributes to the pathogenesis of CDAD. METHODS: Macrophages exposed to TcdA and TcdB were assessed for IL-1 production, an indication of inflammasome activa- tion. Macrophages deficient in components of the inflam- masome were also assessed. Truncated/mutated forms of TcdB were assessed for their ability to activate the inflam- masome. The role of inflammasome signaling in vivo was assessed in ASC-deficient and IL-1 receptor antagonist– treated mice. RESULTS: TcdA and TcdB triggered inflam- masome activation and IL-1 secretion in macrophages and human mucosal biopsy specimens. Deletion of Nlrp3 de- creased, whereas deletion of ASC completely abolished, tox- in-induced IL-1 release. TcdB-induced IL-1 release re- quired 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. CONCLUSIONS: TcdA and TcdB trigger IL-1 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 af- forded the same level of protection that was observed in ASC / mice. These data suggest that targeting inflam- masome or IL-1 signaling may represent new thera- peutic targets in the treatment of CDAD. Keywords: TcdA; TcdB; IL-1; Caspase-1. C lostridium difficile–associated disease (CDAD), which in- cludes antibiotic-associated diarrhea and pseudomem- branous colitis, has increased substantially in hospitalized patients due to the emergence of hypervirulent strains that produce very high levels of toxins TcdA and TcdB. 1,2 In the United States, C difficile is a significant burden to the health care system with costs totaling $1 billion per year. 3 Current treatment strategies are failing; thus, new ther- apeutic targets must be identified for the treatment of CDAD. TcdA and TcdB are monoglucosyltransferases that cause severe intestinal injury by disrupting the intestinal epithelium and triggering the release of proinflammatory cytokines, including interleukin (IL)-1, 4–6 from resident mucosal immune cells. Both toxins contain a C-terminal binding domain, a large central translocation domain, and an N-terminal glucosyltransferase domain. 7 Once inside the epithelial cell, the toxins target the small guanosine triphosphatases of the Rho family, disrupting the epithelial actin cytoskeleton and tight junctions. 8 Furthermore, TcdA and TcdB induce significant inflam- matory responses that are believed to contribute to the pathogenesis of CDAD 3 ; however, the mechanisms by which these toxins activate the immune system have yet to be fully characterized. It has been proposed that the toxins work synergistically to cause disease; once TcdA has breached the intestinal epithelium, immune cells in the lamina propria are exposed to TcdB, triggering in- flammation and intestinal damage. 9 However, a recent report by Lyras et al suggested that TcdB alone was sufficient to induce intestinal inflammation and damage in an animal model of CDAD. 10 Although the mechanisms through which TcdA and TcdB compromise the intestinal barrier are well charac- terized, 11,12 little is known about how the innate immune Abbreviations used in this paper: CDAD, Clostridium difficile–asso- ciated disease; ELISA, enzyme-linked immunosorbent assay; IL, inter- leukin; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MPO, myeloperoxidase; NLR, nucleotide-binding domain leucine-rich repeat gene family; PMA, phorbol-12-myristate-13-acetate; TcdB-CBD, TcdB C-terminal binding domain mutant; TcdB-GTD, TcdB glucosyltrans- ferase domain-deleted; TcdB-GTDmut, mutant TcdB with inactivating point mutations (W102A and D287N) in the glucosyltransferase do- main. © 2010 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2010.04.005 BASIC– ALIMENTARY TRACT GASTROENTEROLOGY 2010;139:542–552
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