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 ERK and p38 pathways mediate monocyte necrosis and IL-1β release ind...
The ERK and p38 pathways mediate monocyte necrosis and IL-1β release induced by toxin A. Peripheral blood monocytes were isolated as described in Methods and incubated in medium alone, or the presence of the MEK inhibitor PD98059 (20 μM), or the p38 inhibitor SB203580 (10 μM) for 30 minutes. Then, C. difficile toxin A (40 nM) or S. aureus α-toxin, a pore-forming toxin (10 nM), was added to the medium, and monocytes were incubated for an additional 90–120 minutes until more than 90% of the monocytes were necrotic in the control group. (a) As observed by phase-contrast microscopy (×320), both toxins induced morphological features that characterized necrosis, including cell swelling, cytoplasmic translucence, and absence of nuclear fragmentation. PD98059 and SB203580 completely prevented the morphological features of necrosis induced by C. difficile toxin A, but not by S. aureus α-toxin. (b, c) Dose-dependent effect of PD98059 and SB203580 on necrosis (b) and IL-1β release (c). Necrotic monocytes were counted by phase-contrast microscopy, and IL-1β was measured by ELISA in the supernatant. PD98059 and SB203580 each prevented toxin A–induced necrosis and IL-1β release with an IC50 of 1 μM and 100 nM, respectively. However, they did not prevent necrosis or IL-1β release induced by S. aureus α-toxin. Means and SE of triplicates from a representative experiment are shown.