MAPK-Activated Protein Kinase 2 Contributes to Clostridium difficile-Associated Inflammation

Abstract

Clostridium difficile infection (CDI) results in toxin-induced epithelial injury and marked intestinal inflammation. Fecal markers of intestinal inflammation correlate with CDI disease severity, but regulation of the inflammatory response is poorly understood. Previous studies demonstrated that C. difficile toxin TcdA activates p38 kinase in tissue culture cells and mouse ilium, resulting in interleukin-8 (IL-8) release. Here, we investigated the role of phosphorylated mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2 kinase, pMK2), a key mediator of p38-dependent inflammation, in CDI. Exposure of cultured intestinal epithelial cells to the C. difficile toxins TcdA and TcdB resulted in p38-dependent MK2 activation. Toxininduced IL-8 and GRO␣ release required MK2 activity. We found that p38 and MK2 are activated in response to other actin-disrupting agents, suggesting that toxin-induced cytoskeleton disruption is the trigger for kinase-dependent cytokine response. Phosphorylated MK2 was detected in the intestines of C. difficile-infected hamsters and mice, demonstrating for the first time that the pathway is activated in infected animals. Furthermore, we found that elevated pMK2 correlated with the presence of toxigenic C. difficile among 100 patient stool samples submitted for C. difficile testing. In conclusion, we find that MK2 kinase is activated by TcdA and TcdB and regulates the expression of proinflammatory cytokines. Activation of p38-MK2 in infected animals and humans suggests that this pathway is a key driver of intestinal inflammation in patients with CDI.

Intestinal Inflammatory Biomarkers and Outcome in Pediatric Clostridium difficile Infections

“…In vitro studies have implicated p38 in C difficile infection pathophysiology, 37, 38 and we recently reported that the p38 pathway and its downstream kinase target MK2 are major components of the inflammatory response elicited by C. difficile toxins in cells, mice and adults. 19 We now complement these experimental and adult data by showing that p38 is activated in children with C difficile infection. Unfortunately, pp38 is not a sensitive biomarker, and is only elevated in a subset of children with C. difficile .…”

“…18 In vitro , C. difficile toxins activate the p38 pathway 19 and induce multiple pro-inflammatory cytokines and chemokines such as interleukin (IL)-8, IL-12, IL-18 and tumor necrosis factor (TNF)-α which may be responsible for host damage and many of the histopathologic features of severe illness. 18 …”

Pathogenesis of Clostridium difficile Infection and Its Potential Role in Inflammatory Bowel Disease

“…Thus, exposure of intestinal epithelial cells to toxin A leads to secretion of the potent neutrophil chemoattractant interleukin (IL)-8. 57,[70][71][72] Monocytes also express IL-8, and other cytokines, in response to toxins A and B. 65,73,74 The inflammasome is a proteolytic complex that responds to signals from bacterial pathogens and from stressed host cells.…”

Host recognition of Clostridium difficile and the innate immune response

“…In particular, p38 MAPK phosphorylation has been shown to be essential for NFκB activation in response to TcdA, and this is thought to occur as a result of mitochondrial oxygen radical generation [21-22]. Both toxins have also been shown to activate MAPK-activated protein kinase (MK2) downstream of p38 MAPK, and this pathway is essential for IL-8 expression [23]. Similarly, production of cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PEG 2 ), thought to be responsible for the fluid accumulation seen in response to toxins, is also dependent on p38 MAPK activation and signaling via mitogen- and stress-activated protein kinase (MSK-1) [24].…”