, Volume 35, Issue 4, pp 1487–1499 | Cite as

Metabolites of Lactobacillus plantarum 2142 Prevent Oxidative Stress-Induced Overexpression of Proinflammatory Cytokines in IPEC-J2 Cell Line

  • Erzsebet Paszti-Gere
  • Krisztina Szeker
  • Edina Csibrik-Nemeth
  • Rita Csizinszky
  • Andras Marosi
  • Orsolya Palocz
  • Orsolya Farkas
  • Peter Galfi


Probiotics have already proven beneficial effects in the treatment of several intestinal infections, but the underlying mechanisms how the probiotics can affect responses of porcine IPEC-J2 enterocytes to oxidative stress remained to be elucidated. The immunmodulatory effect of five bacterial strains (Lactobacillus plantarum 2142, Lactobacillus casei Shirota, Bifidobacterium animalis subsp. lactis BB-12, Bacillus amyloliquefaciens CECT 5940 and Enterococcus faecium CECT 4515) on 1 mM peroxide-triggered upregulation of interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) level was screened by q RT-PCR. Our data revealed that spent culture supernatant (SCS) of L. plantarum 2142 had significant lowering effect on IL-8 and TNF-α level with concomitant promoting activity on protective Hsp70 gene expression. According to our results, lactic acid (racemic, d- and l-lactic acid) and acetic acid produced by lactobacilli had no protective effect in quenching upregulation of proinflammatory cytokines. Furthermore, L. plantarum 2142-specific supernatant peptides were detected by gel electrophoresis and capillary zone electrophoresis.


IPEC-J2 probiotics oxidative stress proinflammatory cytokines Hsp70 



The research described here has been supported by the Hungarian Scientific Research Fund (grant OTKA nos. 76133 and 100701). We are indebted to Dr. Jody Gookin and Dr. Stephen Stauffer, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA for providing IPEC-J2 cells and for the valuable advice on handling them. PCR product sequencing support from Dr. Balázs Gereben (Institute of Experimental Medicine of the Hungarian Academy of Sciences, Laboratory of Endocrine Neurobiology) is also acknowledged. We also would like to thank Dr. Éva Gelencsér, Dr. Emőke Németh-Szerdahelyi and Katalin Háder-Sólyom (Central Food Research Institute, Food Safety Department, Unit of Biology, Budapest, Hungary) for their extensive support in peptide electrophoretic studies.


  1. 1.
    Meyer, T.N., C. Schwesinger, J. Ye, B.M. Denker, and S.K. Nigam. 2001. Reassembly of the tight junction after oxidative stress depends on tyrosine kinase activity. Journal of Biological Chemistry 276: 22048–22055.PubMedCrossRefGoogle Scholar
  2. 2.
    Seth, A., F.F. Yan, D.B. Polk, and R.K. Rao. 2008. Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism. AJP-Gastrointestinal and Liver Physiology 294: 1060–1069.CrossRefGoogle Scholar
  3. 3.
    Sleator, R.D., and C. Hill. 2008. New frontiers in probiotic research. Letters in Applied Microbiology 46: 143–147.PubMedCrossRefGoogle Scholar
  4. 4.
    Oelschlaeger, T.A. 2010. Mechanisms of probiotic actions- A review. International Journal of Medical Microbiology 300: 57–62.PubMedCrossRefGoogle Scholar
  5. 5.
    Ohashi, Y., and K. Ushida. 2009. Health-beneficial effects of probiotics: its mode of action. Animal Science Journal 80: 361–371.PubMedCrossRefGoogle Scholar
  6. 6.
    Langerholc, T., P.A. Maragkoudakis, J. Wollgast, L. Gradisnik, and A. Cencic. 2011. Novel and established intestinal cell line models—an indispensable tool in food science and nutrition. Trends in Food Science and Technology. doi: 10.1016/j.tifs.2011.03.010.
  7. 7.
    Wells, J.M. 2011. Immunomodulatory mechanisms of lactobacilli. Microbial Cell Factories 10(Suppl 1): S17.PubMedCrossRefGoogle Scholar
  8. 8.
    Wells, J.M., R. Oriana, M. Meijerink, and P. van Baarlen. 2011. Epithelial crosstalk at the microbiota–mucosal interface. Proceedings of the National Academy of Sciences of the United States of America 18(1): 4607–4614.CrossRefGoogle Scholar
  9. 9.
    Karczewski, J., F.J. Troost, I. Konings, J. Dekker, M. Kleerebezem, R.J.M. Brummer, and J.M. Wells. 2010. Regulation of human epithelial tight junction proteins by Lactobacillus plantarum in vivo and protective effects on the epithelial barrier. Translational Physiology 298: G851–G859.Google Scholar
  10. 10.
    Klingberg, T.D., M.H. Pedersen, A. Cencic, and B.B. Budde. 2005. Application of measurements of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic activity. Applied and Environmental Microbiology 71(11): 7528–7530.PubMedCrossRefGoogle Scholar
  11. 11.
    Qin, H., Z. Zhang, X. Hang, and Y. Jiang. 2009. L. plantarum prevents Enteroinvasive Escherichia coli-induced tight junction proteins changes in intestinal epithelial cells. BMC Microbiology 9: 63.PubMedCrossRefGoogle Scholar
  12. 12.
    Ewaschuk, J.B., H. Diaz, L. Meddings, B. Diederichs, A. Dmytrash, J. Backer, M. Looijer-van Langen, and K.L. Madsen. 2008. Secreted bioactive factors from Bifidobacterium infantis enhance epithelial cell barrier function. AJP-Gastrointestinal and Liver Physiology 295: 1025–1034.CrossRefGoogle Scholar
  13. 13.
    Madsen, K., A. Cornish, P. Soper, C. McKaigney, H. Jijon, C. Yachimec, J. Doyle, L. Jewell, and C. De Simone. 2001. Probiotic bacteria enhance murine and human intestinal epithelial barrier function. Gastroenterology 121: 580591.CrossRefGoogle Scholar
  14. 14.
    Eckmann, L., M.F. Kagnoff, and J. Fierer. 1993. Epithelial cells secrete the chemokine interleukin-8 in response to bacterial entry. Infection and Immunity 61: 4569–4574.PubMedGoogle Scholar
  15. 15.
    Hobbie, S., L.M. Chen, R.J. Davis, and J.E. Galán. 1997. Involvement of mitogen-activated protein kinase pathways in the nuclear responses and cytokine production induced by Salmonella typhimurium in cultured intestinal epithelial cells. Journal of Immunology 159: 5550–5559.Google Scholar
  16. 16.
    McCormick, B.A., C.A. Parkos, S.P. Colgan, D.K. Carnes, and J.L. Madara. 1998. Apical secretion of a pathogen-elicited epithelial chemoattractant activity in response to surface colonization of intestinal epithelia by Salmonella typhimurium. Journal of Immunology 160: 455–466.Google Scholar
  17. 17.
    Wilson, M., R. Seymour, and B. Henderson. 1998. Bacterial perturbation of cytokine networks. Infection and Immunology 66: 2401–2409.Google Scholar
  18. 18.
    Lang, A., M. Lahav, E. Sakhnini, I. Barshack, H.H. Fiddler, B. Avidan, E. Bardan, R.R. Hershkowiz, S. Bar-Meir, and Y. Chowers. 2004. Allicin inhibits spontaneous and TNF-alpha induced secretion of proinflammatory cytokines and chemokines from intestinal epithelial cells. Clinical Nutrition 23: 1199–1208.PubMedCrossRefGoogle Scholar
  19. 19.
    Chowers, Y., L. Cahalon, M. Lahav, H. Schor, R. Tal, S. Bar-Meir, and M. Levite. 2000. Somatostatin through its specific receptor inhibits spontaneous and TNF-alpha- and bacteria-induced IL-8 and IL-1 beta secretion from intestinal epithelial cells. Journal of Immunology 165: 2955–2961.Google Scholar
  20. 20.
    Yamamoto, K., R. Kushima, O. Kisaki, Y. Fujiyama, and H. Okabe. 2003. Combined effect of hydrogen peroxide induced oxidative stress and IL-1 on IL-8 production in CaCo-2 cells (a human colon carcinoma cell line) and normal intestinal epithelial cells. Inflammation 27: 123–128.PubMedCrossRefGoogle Scholar
  21. 21.
    Alzoghaibi, M.A., S.W. Walsh, A. Willey, D.R. Yager, A.A. Fowler, and M.F. Graham. 2004. Linoleic acid induces interleukin-8 production by Crohn's human intestinal smooth muscle cells via arachidonic acid metabolites. AJP-Gastrointestinal and Liver Physiology 286: 528–537.CrossRefGoogle Scholar
  22. 22.
    Coconnier, M.H., V. Lievin, M. Lorrot, and A.L. Servin. 2000. Antagonistic activity of Lactobacillus acidophilus LB against intracellular Salmonella enterica serovar Typhimurium infecting human enterocyte-like Caco-2/TC-7 cells. Applied and Environmental Microbiology 66: 1152–1157.PubMedCrossRefGoogle Scholar
  23. 23.
    Candela, M., F. Perna, P. Carnevali, B. Vitali, R. Ciati, P. Gionchetti, F. Rizello, M. Campieri, and P. Brigidi. 2008. Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells: adhesion properties, competition against enteropathogens and modulation of IL-8 production. International Journal of Food Microbiology 125: 286–292.PubMedCrossRefGoogle Scholar
  24. 24.
    Zhang, L., N. Li, R. Caicedo, and J. Neu. 2005. Alive and dead Lactobacillus rhamnosus GG decrease tumor necrosis factor-α-induced interleukin-8 production in Caco-2 cells. Journal of Nutrition 135: 1752–1756.PubMedGoogle Scholar
  25. 25.
    McCracken, V.J., T. Chun, M.E. Baldeón, S. Ahrné, G. Molin, R.I. Mackie, and H.R. Gaskins. 2002. TNF-alpha sensitizes HT-29 colonic epithelial cells to intestinal lactobacilli. Experimental Biology and Medicine 227(8): 665–670.PubMedGoogle Scholar
  26. 26.
    Ko, J.S., H.R. Yang, J.Y. Chang, and J.K. Seo. 2007. Lactobacillus plantarum inhibits epithelial barrier dysfunction and interleukin-8 secretion induced by tumor necrosis factor-alpha. World Journal of Gastroenterology 13(13): 1962–1965.PubMedGoogle Scholar
  27. 27.
    Pathmakanthan, S., C.K. Li, J. Cowie, and C.J. Hawkey. 2004. Lactobacillus plantarum 299: beneficial in vitro immunomodulation in cells extracted from inflamed human colon. Journal of Gastroenterology and Hepatology 19(2): 166–173.PubMedCrossRefGoogle Scholar
  28. 28.
    Santoro, M.G. 2000. Heat shock factors and the control of the stress response. Biochemical Pharmacology 59(1): 55–63.PubMedCrossRefGoogle Scholar
  29. 29.
    Borges, J.C., and C.H. Ramos. 2005. Protein folding assisted by chaperones. Protein & Peptid Letters 12(3): 257–261.CrossRefGoogle Scholar
  30. 30.
    Musch, M.W., M.J. Ciancio, K. Sarge, and E.B. Chang. 1996. Induction of heat shock protein 70 protects intestinal epithelial IEC-18 cells from oxidant and thermal injury. American Journal of Physiology. Cell Physiology 270(2): C429–C436.Google Scholar
  31. 31.
    Malago, J.J., E. Nemeth, J.F.J.G. Koninkx, P.C.J. Tooten, S. Fajdiga, and J.E. van Dijk. 2010. Microbial products from probiotic bacteria inhibit Salmonella enteritidis 857-induced IL-8 synthesis in Caco-2 cells. Folia Microbiologica 55(4): 401–408.PubMedCrossRefGoogle Scholar
  32. 32.
    Nemeth, E., S. Fajdiga, J. Malago, J. Koninkx, P. Tooten, and J. van Dijk. 2006. Inhibition of Salmonella-induced IL-8 synthesis and expression of Hsp70 in enterocyte-like Caco-2 cells after exposure to non-starter lactobacilli. International Journal of Food Microbiology 112(3): 266–274.PubMedCrossRefGoogle Scholar
  33. 33.
    Son, D.O., H. Satsu, and M. Shimizu. 2005. Histidine inhibits oxidative stress- and TNF-alpha-induced interleukin-8 secretion in intestinal epithelial cells. FEBS Letters 579: 4671–4677.PubMedCrossRefGoogle Scholar
  34. 34.
    Ohland, C.L., and W.K. Macnaughton. 2010. Probiotic bacteria and intestinal epithelial barrier function. AJP-Gastrointestinal and Liver Physiology 298: 807–819.CrossRefGoogle Scholar
  35. 35.
    Yan, F., H. Cao, T.L. Cover, R. Whitehead, M.K. Washington, and D.B. Polk. 2007. Soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth. Gastroenterology 132: 562–575.PubMedCrossRefGoogle Scholar
  36. 36.
    Chon, H., B. Choi, G. Jeong, E. Lee, and S. Lee. 2010. Suppression of proinflammatory cytokine production by specific metabolites of Lactobacillus plantarum 10hk2 via inhibiting NF-κB and p38 MAPK expressions. Comparative Immunology, Microbiology & Infection Diseases 33: 41–49.CrossRefGoogle Scholar
  37. 37.
    Bernet-Camard, M.F., V. Lievin, D. Brassart, J.R. Neeser, A.L. Servin, and S. Hudault. 1997. The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo. Applied and Environmental Microbiology 63: 2747–2753.PubMedGoogle Scholar
  38. 38.
    Coconnier, M.H., V. Lievin, M.F. Bernet-Camard, S. Hudault, and A.L. Servin. 1997. Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB. Antimicrobial Agents and Chemotherapy 41: 1046–1052.PubMedGoogle Scholar
  39. 39.
    Silva, M., N. Jacobus, C. Deneke, and S.L. Gorbach. 1987. Antimicrobial substance from a human Lactobacillus strain. Antimicrobial Agents and Chemotherapy 31: 1231–1233.PubMedCrossRefGoogle Scholar
  40. 40.
    Samuvel, D.J., K.P. Sundararaj, A. Nareika, M.F. Lopes-Virella, and Y. Huang. 2009. Lactate boosts TLR4 signaling and NF-kappaB pathway-mediated gene transcription in macrophages via monocarboxylate transporters and MD-2 up-regulation. Journal of Immunology 182: 2476–2484.CrossRefGoogle Scholar
  41. 41.
    Jensen, J.C., C. Buresh, and J.A. Norton. 1990. Lactic acidosis increases tumor necrosis factor secretion and transcription in vitro. Journal of Surgical Research 49: 350–353.PubMedCrossRefGoogle Scholar
  42. 42.
    Douvdevani, A., O. Abramson, A. Tamir, A. Konforty, N. Isakov, and C. Chaimovitz. 1995. Commercial dialysate inhibits TNF alpha mRNA expression and NF-kB DNA-binding activity in LPS-stimulated macrophages. Kidney International 47: 1537–1545.PubMedCrossRefGoogle Scholar
  43. 43.
    Kellum, J.A., M. Song, and J. Li. 2004. Lactic and hydrochloric acids induce different patterns of inflammatory response in LPS-stimulated RAW 264.7 cells. AJP-Regulatory. Integrative and Comparative Physiology 286: 686–692.CrossRefGoogle Scholar
  44. 44.
    Watanabe, T., H. Nishio, T. Tanigawa, H. Yamagami, H. Okazaki, K. Watanabe, K. Tominaga, Y. Fujiwara, N. Oshitani, T. Asahara, K. Nomoto, K. Higuchi, K. Takeuchi, and T. Arakawa. 2009. Probiotic Lactobacillus casei strain Shirota prevents indomethacin-induced small intestinal injury: involvement of lactic acid. AJP-Gastrointestinal and Liver Physiology 297: 506–513.CrossRefGoogle Scholar
  45. 45.
    Dietl, K., K. Renner, K. Dettmer, B. Timischl, K. Eberhart, C. Dorn, C. Hellerbrand, M. Kastenberger, L.A. Kunz-Schughart, P.J. Oefner, R. Andreesen, E. Gottfried, and M.P. Kreutz. 2010. Lactic acid and acidification inhibit TNF secretion and glycolysis of human monocytes. Journal of Immunology 184: 1200–1209.CrossRefGoogle Scholar
  46. 46.
    Peracaula, R., S. Barrabes, A. Sarrats, P.M. Rudd, and R. de Llorens. 2008. Altered glycosylation in tumours focused to cancer diagnosis. Disease Markers 25: 207–218.PubMedGoogle Scholar
  47. 47.
    Sambuy, Y., I. De Angelis, G. Ranaldi, M.L. Scarino, A. Stammati, and F. Zucco. 2005. The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biology and Toxicology 21: 1–26.PubMedCrossRefGoogle Scholar
  48. 48.
    Hidalgo, I.J. 2001. Assessing the absorption of new pharmaceuticals. Current Topics in Medicinal Chemistry 1: 385–401.PubMedCrossRefGoogle Scholar
  49. 49.
    Cencic, A., and T. Langerholc. 2010. Functional cell models of the gut and their applications in food microbiology—A review. International Journal of Food Microbiology 141: 4–14.CrossRefGoogle Scholar
  50. 50.
    Nemeth, E., A. Halasz, A. Barath, and P. Galfi. 2007. Influence of lactic acid bacteria and their spent culture supernatant on hydrogen peroxide-induced interleukin-8 synthesis and necrosis of Caco-2 cells. Food and Agriculture Immunology 18: 95–105.CrossRefGoogle Scholar
  51. 51.
    Schierack, P., M. Nordhoff, M. Pollmann, K.D. Weyrauch, S. Amasheh, U. Lodemann, J. Jores, B. Tachu, S. Kleta, A. Blikslager, K. Tedin, and L.H. Wieler. 2006. Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine. Histochemistry and Cell Biolology 125: 293–305.CrossRefGoogle Scholar
  52. 52.
    Paszti-Gere, E., E. Csibrik-Nemeth, K. Szeker, Cs Jakab, and P. Galfi. 2011. Acute oxidative stress affects IL-8 and TNF-α expression in IPEC-J2 porcine epithelial cells. Inflammation DOI:. doi: 10.1007/s10753-011-9403-8.
  53. 53.
    Rairakhwada, D., J.W. Seo, M.Y. Seo, O. Kwon, S.K. Rhee, and C.H. Kim. 2009. Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens. Journal of Industrial Microbiology and Biotechnology 37(2): 195–204.PubMedCrossRefGoogle Scholar
  54. 54.
    Xu, Q., T. Yajima, W. Li, K. Saito, Y. Ohshima, and Y. Yoshikai. 2006. Levan (β-2, 6-fructan), a major fraction of fermented soybean mucilage, displays immunostimulating properties via Toll-like receptor 4 signalling: induction of interleukin-12 production and suppression of T-helper type 2 response and immunoglobulin E production. Clinical and Experimental Allergy 36(1): 94–101.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Erzsebet Paszti-Gere
    • 1
  • Krisztina Szeker
    • 1
  • Edina Csibrik-Nemeth
    • 1
  • Rita Csizinszky
    • 1
  • Andras Marosi
    • 1
  • Orsolya Palocz
    • 1
  • Orsolya Farkas
    • 1
  • Peter Galfi
    • 1
  1. 1.Department of Pharmacology and Toxicology, Faculty of Veterinary SciencesSzent István UniversityBudapestHungary

Personalised recommendations