p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis
Michel Warny, Andrew C. Keates, Sarah Keates, Ignazio Castagliuolo, Jeff K. Zacks, Samer Aboudola, Amir Qamar, Charalabos Pothoulakis, J. Thomas LaMont, Ciarán P. Kelly
Michel Warny, Andrew C. Keates, Sarah Keates, Ignazio Castagliuolo, Jeff K. Zacks, Samer Aboudola, Amir Qamar, Charalabos Pothoulakis, J. Thomas LaMont, Ciarán P. Kelly
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p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis

Abstract

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 cascades within 1 to 2 minutes. Activation of p38 was sustained, whereas stimulation of extracellular signal-regulated kinases and c-Jun NH2-terminal kinase was transient. Rho glucosylation became evident after 15 minutes. IL-8 gene expression was reduced by 70% by the MEK inhibitor PD98059 and abrogated by the p38 inhibitor SB203580 or by overexpression of dominant-negative mutants of the p38-activating kinases MKK3 and MKK6. SB203580 also blocked monocyte necrosis and IL-1β release caused by toxin A but not by other toxins. Finally, in mouse ileum, SB203580 prevented toxin A–induced neutrophil recruitment by 92% and villous destruction by 90%. Thus, in monocytes exposed to toxin A, MAP kinase activation appears to precede Rho glucosylation and is required for IL-8 transcription and cell necrosis. p38 MAP kinase also mediates intestinal inflammation and mucosal damage induced by toxin A.

Authors

Michel Warny, Andrew C. Keates, Sarah Keates, Ignazio Castagliuolo, Jeff K. Zacks, Samer Aboudola, Amir Qamar, Charalabos Pothoulakis, J. Thomas LaMont, Ciarán P. Kelly

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The p38 inhibitor SB203580 prevents IL-8 synthesis induced by C. diffici...
The p38 inhibitor SB203580 prevents IL-8 synthesis induced by C. difficile toxin A. (a) THP-1 cells (106/mL) were preincubated with various concentrations of the p38 inhibitor SB203580 for 30 minutes. Then, cells were stimulated for 4 hours with C. difficile toxin A (100 nM) or LPS (1 μg/mL). SB203580 blocked IL-8 release induced by toxin A (IC50, 20 nM), but did not prevent IL-8 release mediated by LPS. (b) Peripheral blood monocytes were preincubated with SB203580 (10 μM) for 30 minutes and then stimulated for 4 hours with C. difficile toxin A (2 nM) or LPS (1 μg/mL). SB203580 abrogated toxin A–induced IL-8 release, but did not affect LPS-induced IL-8 production. Means and SE are shown (n = 3; AP = 0.002). (c) THP-1 cells (106/mL) were preincubated with 10 μM SB203580 for 30, 60, or 90 minutes, or with 10 μM SB202474, an inactive analogue, for 60 minutes. Then, cells were stimulated for 4 hours with C. difficile toxin A (100 nM) or LPS (1 μg/mL). Inhibition of LPS-induced IL-8 release by SB203580 was observed when preincubated for 60 minutes, whereas 30 minutes was sufficient to prevent toxin A–induced IL-8 release. SB202474 was inactive (n = 2; AP < 0.01).