Journal of Molecular Medicine

, Volume 87, Issue 2, pp 169–180 | Cite as

Clostridium difficile toxin A promotes dendritic cell maturation and chemokine CXCL2 expression through p38, IKK, and the NF-κB signaling pathway

  • Jin Young Lee
  • Hyunah Kim
  • Mi Yeon Cha
  • Hong Gyu Park
  • Young-Jeon Kim
  • In Young Kim
  • Jung Mogg Kim
Original Article


Clostridium difficile toxin A causes acute colitis associated with intense infiltrating neutrophils. Although dendritic cells (DCs) play an important role in the regulation of inflammation, little is known about the effects of toxin A on the maturation and neutrophil-attracting chemokine expression in DCs. This study investigated whether C. difficile toxin A could influence the maturation of mouse bone-marrow-derived DCs and chemokine CXCL2 expression. Toxin A increased the DC maturation which was closely related to CXCL2 upregulation. Concurrently, toxin A activated the signals of p65/p50 nuclear factor kappa B (NF-κB) heterodimers and phospho-IκB kinase (IKK) in DCs. The increased DC maturation, CXCL2 expression, and neutrophil chemoattraction were significantly downregulated in the NF-κB knockout mice. In addition, toxin A activated the phosphorylated signals of mitogen-activated protein kinases (MAPKs), such as ERK, p38, and JNK. Of all three MAPK signals, p38 MAPK was significantly related to DC maturation. Thus, suppression of p38 activity using SB203580 and siRNA transfection resulted in the significant reduction of IKK activity, DC maturation, and CXCL2 upregulation by toxin A. These results suggest that p38 MAPK may lead to the activation of IKK and NF-κB signaling, resulting in enhanced DC maturation and CXCL2 expression in response to C. difficile toxin A stimulation.


Clostridium difficile toxin A Dendritic cells Maturation CXCL2 Mitogen-activated protein kinase 



We thank Dr. Martin F. Kagnoff for providing plasmids of murine β-actin internal standard, Dr. Hyung-Joo Kwon for the pMIP-2-luciferease plasmid, Dr. Kyoung-Ho Kim for performing the animal studies, and Han-Jin Lee for the excellent technical assistance. This work was supported by MRC Program from the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST; R13-2008-026-01000-0).

Supplementary material

109_2008_415_MOESM1_ESM.ppt (3.8 mb)
ESM 1 (PPT 3.75 MB)


  1. 1.
    Kelly CP, LaMont JT (1998) Clostridium difficile infection. Annu Rev Med 49:375–390PubMedCrossRefGoogle Scholar
  2. 2.
    Burakoff R, Zhao L, Celifarco AJ, Rose KL, Donovan V, Pothoulakis C, Percy WH (1995) Effects of purified Clostridium difficile toxin A on rabbit distal colon. Gastroenterology 109:348–354PubMedCrossRefGoogle Scholar
  3. 3.
    Bobak DA (2008) The molecular pathogenesis of Clostridium difficile-associated disease. Curr Infect Dis Rep 10:111–115PubMedCrossRefGoogle Scholar
  4. 4.
    Lee JY, Park HR, Oh YK, Kim YJ, Youn J, Han JS, Kim JM (2007) Effects of transcription factor activator protein-1 on interleukin-8 expression and enteritis in response to Clostridium difficile toxin A. J Mol Med 85:1393–1404PubMedCrossRefGoogle Scholar
  5. 5.
    Mahida YR, Makh S, Hyde S, Gray T, Borriello SP (1996) Effect of Clostridium difficile toxin A on human intestinal epithelial cells: induction of interleukin 8 production and apoptosis after cell detachment. Gut 38:337–347PubMedCrossRefGoogle Scholar
  6. 6.
    He D, Sougioultzis S, Hagen S, Liu J, Keates S, Keates AC, Pothoulakis C, Lamont JT (2002) Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation. Gastroenterology 122:1048–1057PubMedCrossRefGoogle Scholar
  7. 7.
    Kim JM, Kim JS, Jun HC, Oh YK, Song IS, Kim CY (2002) Differential expression and polarized secretion of CXC and CC chemokines by human intestinal epithelial cancer cell lines in response to Clostridium difficile toxin A. Microbiol Immunol 46:333–342PubMedGoogle Scholar
  8. 8.
    Kim JM, Lee JY, Yoon YM, Oh YK, Youn J, Kim YJ (2006) NF-κB activation pathway is essential for the chemokine expression in intestinal epithelial cells stimulated with Clostridium difficile toxin A. Scand J Immunol 63:453–460PubMedCrossRefGoogle Scholar
  9. 9.
    Steinman RM (1991) The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9:271–296PubMedCrossRefGoogle Scholar
  10. 10.
    Nakahara T, Moroi Y, Uchi H, Furue M (2006) Differential role of MAPK signaling in human dendritic cell maturation and Th1/Th2 engagement. J Dermatol Sci 42:1–11PubMedCrossRefGoogle Scholar
  11. 11.
    Sozzani S, Allavena P, Vecchi A, Mantovani A (2000) Chemokines and dendritic cell traffic. J Clin Immunol 20:151–160PubMedCrossRefGoogle Scholar
  12. 12.
    Bilsborough J, Viney JL (2004) Gastrointestinal dendritic cells play a role in immunity, tolerance, and disease. Gastroenterology 127:300–309PubMedCrossRefGoogle Scholar
  13. 13.
    Rimoldi M, Chieppa M, Salucci V, Avogadri F, Sonzogni A, Sampietro GM, Nespoli A, Viale G, Allavena P, Rescigno M (2005) Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nat Immunol 6:507–514PubMedCrossRefGoogle Scholar
  14. 14.
    Ausiello CM, Cerquetti M, Fedele G, Spensieri F, Palazzo R, Nasso M, Frezza S, Mastrantonio P (2006) Surface layer proteins from Clostridium difficile induce inflammatory and regulatory cytokines in human monocytes and dendritic cells. Microbes Infect 8:2640–2646PubMedCrossRefGoogle Scholar
  15. 15.
    Steinman RM (2007) Dendritic cells: understanding immunogenicity. Eur J Immunol 37(Suppl 1):S53–S60PubMedCrossRefGoogle Scholar
  16. 16.
    Niess JH, Reinecker HC (2006) Dendritic cells in the recognition of intestinal microbiota. Cell Microbiol 8:558–564PubMedCrossRefGoogle Scholar
  17. 17.
    Rescigno M, Martino M, Sutherland CL, Gold MR, Ricciardi-Castagnoli P (1998) Dendritic cell survival and maturation are regulated by different signaling pathways. J Exp Med 188:2175–2180PubMedCrossRefGoogle Scholar
  18. 18.
    Gerhard R, Tatge H, Genth H, Thum T, Borlak J, Fritz G, Just I (2005) Clostridium difficile toxin A induces expression of the stress-induced early gene product RhoB. J Biol Chem 280:1499–1505PubMedCrossRefGoogle Scholar
  19. 19.
    Kim H, Rhee SH, Kokkotou E, Na X, Savidge T, Moyer MP, Pothoulakis C, LaMont JT (2005) Clostridium difficile toxin A regulates inducible cyclooxygenase-2 and prostaglandin E2 synthesis in colonocytes via reactive oxygen species and activation of p38 MAPK. J Biol Chem 280:21237–21245PubMedCrossRefGoogle Scholar
  20. 20.
    Yeh CY, Lin CN, Chang CF, Lin CH, Lien HT, Chen JY, Chia JS (2008) C-terminal repeats of Clostridium difficile toxin A induce production of chemokine and adhesion molecules in endothelial cells and promote migration of leukocytes. Infect Immun 76:1170–1178PubMedCrossRefGoogle Scholar
  21. 21.
    Chae S, Eckmann L, Miyamoto Y, Pothoulakis C, Karin M, Kagnoff MF (2006) Epithelial cell IκB-kinase beta has an important protective role in Clostridium difficile toxin A-induced mucosal injury. J Immunol 177:1214–1220PubMedGoogle Scholar
  22. 22.
    Jefferson KK, Smith MF Jr, Bobak DA (1999) Roles of intracellular calcium and NF-kappa B in the Clostridium difficile toxin A-induced up-regulation and secretion of IL-8 from human monocytes. J Immunol 163:5183–5191PubMedGoogle Scholar
  23. 23.
    Hu JH, Chen T, Zhuang ZH, Kong L, Yu MC, Liu Y, Zang JW, Ge BX (2007) Feedback control of MKP-1 expression by p38. Cell Signal 19:393–400PubMedCrossRefGoogle Scholar
  24. 24.
    Kim JM, Lee JY, Yoon YM, Oh YK, Kang JS, Kim YJ, Kim KH (2006) Bacteroides fragilis enterotoxin induces cyclooxygenase-2 and fluid secretion in intestinal epithelial cells through NF-κB activation. Eur J Immunol 36:2446–2456PubMedCrossRefGoogle Scholar
  25. 25.
    Mashreghi MF, Klemz R, Knosalla IS, Gerstmayer B, Janssen U, Buelow R, Jozkowicz A, Dulak J, Volk HD, Kotsch K (2008) Inhibition of dendritic cell maturation and function is independent of heme oxygenase 1 but requires the activation of STAT3. J Immunol 180:7919–7930PubMedGoogle Scholar
  26. 26.
    Sohn WJ, Lee KW, Lee Y, Han JH, Choe YK, Kim DS, Kwon HJ (2005) Pyrrolidone dithiocarbamate-induced macrophage inflammatory protein-2 gene expression is NF-kappa B-independent but c-Jun-dependent in macrophage cell line RAW 264.7. Mol Immunol 42:1165–1175PubMedCrossRefGoogle Scholar
  27. 27.
    Kim JM, Lee JY, Kim YJ (2008) Inhibition of apoptosis in Bacteroides fragilis enterotoxin-stimulated intestinal epithelial cells through the induction of c-IAP-2. Eur J Immunol 38:2190–2199PubMedCrossRefGoogle Scholar
  28. 28.
    Kim JM, Kim JS, Kim YJ, Oh YK, Kim IY, Chee YJ, Han JS, Jung HC (2008) Conjugated linoleic acids produced by Lactobacillus dissociates IKK-gamma and Hsp90 complex in Helicobacter pylori-infected gastric epithelial cells. Lab Invest 88:541–552PubMedCrossRefGoogle Scholar
  29. 29.
    Kim JM, Kim JS, Lee LY, Kim YJ, Youn HJ, Kim IY, Chee YJ, Oh YK, Kim N, Jung HC, Song IS (2007) Vacuolating cytotoxin in Helicobacter pylori water-soluble proteins upregulates chemokine expression in human eosinophils via Ca2+ influx, mitochondrial reactive oxygen intermediates, and NF-κB activation. Infect Immun 75:3373–3381PubMedCrossRefGoogle Scholar
  30. 30.
    Kim JM, Jung HY, Lee JY, Youn J, Lee CH, Kim KH (2005) Mitogen-activated protein kinase and activator protein-1 dependent signals are essential for Bacteroides fragilis enterotoxin-induced enteritis. Eur J Immunol 35:2648–2657PubMedCrossRefGoogle Scholar
  31. 31.
    Crow MK (2006) Modification of accessory molecule signaling. Semin Immunopathol 27:409–424CrossRefGoogle Scholar
  32. 32.
    Randolph GJ, Ochando J, Partida-Sánchez S (2008) Migration of dendritic cell subsets and their precursors. Annu Rev Immunol 26:293–316PubMedCrossRefGoogle Scholar
  33. 33.
    Bozic CR, Gerard NP, von Uexkull-Guldenband C, Kolakowski LF Jr, Conklyn MJ, Breslow R, Showell HJ, Gerard C (1994) The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization. J Biol Chem 269:29355–29358PubMedGoogle Scholar
  34. 34.
    Kobayashi Y (2008) The role of chemokines in neutrophil biology. Front Biosci 13:2400–2407PubMedCrossRefGoogle Scholar
  35. 35.
    Castagliuolo I, Kelly CP, Qiu BS, Nikulasson ST, LaMont JT, Pothoulakis C (1997) IL-11 inhibits Clostridium difficile toxin A enterotoxicity in rat ileum. Am J Physiol 273:G333–G341PubMedGoogle Scholar
  36. 36.
    Richmond A (2002) NF-kappa B, chemokine gene transcription and tumour growth. Nat Rev Immunol 2:664–674PubMedCrossRefGoogle Scholar
  37. 37.
    Colobran R, Pujol-Borrell R, Armengol MP, Juan M (2007) The chemokine network. I. How the genomic organization of chemokines contains clues for deciphering their functional complexity. Clin Exp Immunol 148:208–217PubMedCrossRefGoogle Scholar
  38. 38.
    Pothoulakis C (2000) Effects of Clostridium difficile toxins on epithelial cell barrier. Ann N Y Acad Sci 915:347–356PubMedCrossRefGoogle Scholar
  39. 39.
    Thanos D, Maniatis T (1995) NF-κB: a lesson in family values. Cell 80:529–532PubMedCrossRefGoogle Scholar
  40. 40.
    Baeuerle PA, Henkel T (1994) Function and activation of NF-κB in the immune system. Annu Rev Immunol 12:141–179PubMedGoogle Scholar
  41. 41.
    Chio CC, Chang YH, Hsu YW, Chi KH, Lin WW (2004) PKA-dependent activation of PKC, p38 MAPK and IKK in macrophage: implication in the induction of inducible nitric oxide synthase and interleukin-6 by dibutyryl cAMP. Cell Signal 16:565–575PubMedCrossRefGoogle Scholar
  42. 42.
    Park KJ, Gaynor RB, Kwak YT (2003) Heat shock protein 27 association with the IκB kinase complex regulates tumor necrosis factor alpha-induced NF-κB activation. J Biol Chem 278:35272–35278PubMedCrossRefGoogle Scholar
  43. 43.
    Tam MA, Rydström A, Sundquist M, Wick MJ (2008) Early cellular responses to Salmonella infection: dendritic cells, monocytes, and more. Immunol Rev 225:140–162PubMedCrossRefGoogle Scholar
  44. 44.
    Herrmann TL, Morita CT, Lee K, Kusner DJ (2005) Calmodulin kinase II regulates the maturation and antigen presentation of human dendritic cells. J Leukoc Biol 78:1397–1407PubMedCrossRefGoogle Scholar
  45. 45.
    Connolly SF, Kusner DJ (2007) The regulation of dendritic cell function by calcium-signaling and its inhibition by microbial pathogens. Immunol Res 39:115–127PubMedCrossRefGoogle Scholar
  46. 46.
    Tamura T, Tailor P, Yamaoka K, Kong HJ, Tsujimura H, O'Shea JJ, Singh H, Ozato K (2005) IFN regulatory factor-4 and -8 govern dendritic cell subset development and their functional diversity. J Immunol 174:2573–2581PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Jin Young Lee
    • 1
  • Hyunah Kim
    • 1
  • Mi Yeon Cha
    • 1
  • Hong Gyu Park
    • 1
  • Young-Jeon Kim
    • 2
  • In Young Kim
    • 3
  • Jung Mogg Kim
    • 1
  1. 1.Department of MicrobiologyHanyang University College of MedicineSeoulSouth Korea
  2. 2.Department of BiotechnologyJoongbu UniversityGeumsanSouth Korea
  3. 3.Department of Biomedical EngineeringHanyang University College of MedicineSeoulSouth Korea

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