p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis

Abstract

IntroductionClostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis in humans (1). Pathogenic strains of C. difficile release 2 large exotoxins: toxin A (308 kDa) and toxin B (269 kDa). In animal models, only toxin A is enterotoxic and causes fluid secretion, mucosa edema, and villous disruption by inducing massive acute inflammation with neutrophil infiltration. We have shown that blocking neutrophil extravasation using an anti-CD18 antibody prevented toxin A-induced enteritis and mucosal damage (2).Toxin A and toxin B show 63% homology (3) and share similar domains. Their COOH-terminal portions carry repeating sequences that may be involved in receptor binding. The NH 2 -terminal portions carry an enzymatic domain, which hydrolyzes UDP-glucose and transfers the glucose moiety to a conserved threonine residue of the small GTP-binding proteins Rho, Cdc42, and Rac (4-6). The small GTP-binding protein Rap is glucosylated only by toxin A (7). These covalent modifications inactivate Rho proteins, which in turn induce cytoskeleton disaggregation and cell rounding. Whether Rho glucosylation is involved in toxin A enterotoxicity and in vitro cytokine production is not known. Toxin A or toxin B were shown to block Rho-regulated signaling pathways, including basophilic cell activation (8), receptor signaling to phospholipase D (9), NF-κB activation by bradykinin (10), and mitogen-activated protein-kinase (MAP kinase) activation (11,12). Blockage of the above signal transduction pathways by C. difficile toxins is consistent with the role of Rho proteins in signaling to MAP kinases and NF-κB (13-15). However, it appears at variance with the known in vivo and in vitro inflammatory actions of toxin A.In cells of the monocyte lineage, C. difficile toxins stimulate inflammatory cytokine release, including TNF-α, IL-1β, . In addition, we have reported recently that both toxins induce necrosis in human monocytes and in THP-1 human monocytic cells. This cell-death pathway is associated with potassium depletion, caspase activation, and maturation and release of preformed IL-1β (19). The mechanisms whereby these toxins activate and kill monocytes are not known.In this study, we investigated the role of MAP kinases in IL-8 production, IL-1β release, and necrosis induced by toxin A in monocytic cells. MAP kinases regulate cell responses to growth factors and stress stimuli and transmit signals from the cell surface to the nucleus via 3 distinct but related pathways. These culminate in the selective activation of extracellular signal-related kinases (ERK), p38, and c-Jun NH 2 -terminal kinase (JNK) Clostridium difficile toxin A causes acute neutrophil infiltration and intestinal mucosal injury. In cultured cells, toxin A inactivates Rho proteins by monoglucosylation. In monocytes, toxin A induces IL-8 production and necrosis by unknown mechanisms. We investigated the role of mitogen-activated protein (MAP) kinases in these events. In THP-1 monocytic cells, toxin A activated the 3 main MAP kinase cascad...

Local injection of dsRNA targeting calcitonin receptor-like receptor (CLR) ameliorates Clostridium difficile toxin A-induced ileitis

“…It was unclear why CLR dsRNA decreased TxA-induced epithelial damage and neutrophil migration. After TxA binds to its receptor on colonocytes, ERK1/2, NF-κB, and p38 MAP kinase are activated, leading to increased expression of proinflammatory cytokines that activate sensory nerves and recruit monocytes, mast cells, and neutrophils (2,19). After a local injection of CLR dsRNA, TxA was unable to increase pERK1/2 or decrease NF-κB and IκBα protein expression in the cytosol.…”

“…TxA activates MAP kinase signaling pathways during inflammatory insults, with ERK and p38 mediating monocyte necrosis, IL-1β release, and IL-8 gene expression (19). We examined pERK1/2 and ERK1/2 levels by Western blot analysis.…”

Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-κB and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines

“…Other results also support a major role of MAPKs in IL-8 secretion. For example, the inhibitor of MAPK/ERK kinase, PD98059, significantly inhibited IL-8 release induced by Clostridium difficile toxin A in human monocytes (43) as well as that induced by H. pylori in gastric epithelial cells (20). We report here for the first time that MXF inhibits activation of the ERK induced by LPS and LPS-PMA in THP-1 cells.…”

Differential Regulation of Chemokines by IL-17 in Colonic Epithelial Cells

“…The CMV-␤gal plasmid had a bacterial ␤-galactosidase gene under control of the CMV immediate-early enhancer plus promoter (32). In other reporter constructs, luciferase transcription was under control of a fragment extending from Ϫ525 to ϩ 97 of the CXCL10 gene promoter (CXCL10/luciferase reporter; obtained from R. Ransohoff, Cleveland Clinic, Cleveland, OH) (33), a 1521-bp fragment of the CXCL8 gene promoter (CXCL8/luciferase reporter, obtained from A. Keates, Beth Israel Deaconess Hospital, Boston, MA) (34), and a 400-bp region of the I B␣ promoter (obtained from K. Yamamoto, University of California, San Francisco, CA) (35,36).…”

“…1, D-F). All six chemokine genes have at least one binding site for NF-B in their promoter elements (33)(34)(35)(36)(42)(43)(44), and in each case NF-B is thought to play an important role in the induction of the genes by proinflammatory cytokines, including TNF-␣. The effect of IL-17, TNF-␣, and IL-17 plus TNF-␣ on another well-studied NF-B target gene, that encoding I B␣, was also examined.…”