Skip to main content

Advertisement

SpringerLink
Log in

The Effects of Intestinal LPS Exposure on Inflammatory Responses in a Porcine Enterohepatic Co-culture System

  • Published:
Inflammation Aims and scope Submit manuscript

Abstract

A porcine enterohepatic co-culture system, with primary hepatocytes as bottom layer and IPEC-J2 epithelial cells as upper layer, was developed to study the effects of lipopolysaccharides (LPS) on the gene expression profile of pro-inflammatory cytokines (interleukin-8 (IL-8) and tumor necrosis factor-α) and CYP enzymes (CYP1A1, CYP1A2, CYP3A29). The barrier integrity of IPEC-J2 cells was investigated by transepithelial electrical resistance measurements and by fluorescein isothiocyanate–dextran-based test. Basolateral IL-8 production was significantly elevated in LPS-treated IPEC-J2 and primary hepatocyte mono-cultures as well as in the co-culture system, in a dose-independent manner. The LPS-induced changes in the expression of the CYP1A2 and CYP3A29 genes in hepatocyte mono-cultures differed from those in co-culture after LPS treatment on the apical side of the IPEC-J2 cell layer. CYP1A2 was downregulated by the LPS treatment in mono-cultures but upregulated at 10 μg/ml LPS in co-culture; gene expression of CYP3A29 showed no significant LPS-induced change in the hepatocyte mono-culture but was significantly downregulated in co-culture. The newly established co-culture system capable of mimicking enterohepatic interplay in LPS-induced inflammatory responses in vitro can be used in the future for reliable screening of potential anti-inflammatory compounds.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

REFERENCES

  1. 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 & Technology 22: S11–S20.

    Article  CAS  Google Scholar 

  2. Oswald, I.P. 2006. Role of intestinal epithelial cells in the innate immune defence of the pig intestine. Veterinary Research 37: 359–368.

    Article  CAS  PubMed  Google Scholar 

  3. Burkey, T.E., K.A. Skjolaas, S.S. Dritz, and J.E. Minton. 2007. Expression of Toll-like receptors, interleukin 8, macrophage migration inhibitory factor, and osteopontin in tissues from pigs challenged with Salmonella enterica serovar Typhimurium or serovar Choleraesuis. Veterinary Immunology and Immunopathology 115: 309–319.

    Article  CAS  PubMed  Google Scholar 

  4. Didierlaurent, A., J.C. Sirard, J.P. Kraehenbuhl, and M.R. Neutra. 2002. How the gut senses its content. Cellular Microbiology 4: 61–72.

    Article  CAS  PubMed  Google Scholar 

  5. Backhed, F., S. Normark, E.K. Schweda, S. Oscarson, and A. Richter-Dahlfors. 2003. Structural requirements for TLR4-mediated LPS signalling: a biological role for LPS modifications. Microbes and Infection 5: 1057–1063.

    Article  CAS  PubMed  Google Scholar 

  6. Ogle, C.K., X.L. Guo, P.O. Hasselgren, J.D. Ogle, and J.W. Alexander. 1997. The gut as a source of inflammatory cytokines after stimulation with endotoxin. European Journal of Surgery 163: 45–51.

    CAS  Google Scholar 

  7. Feuerstein, G.Z., T. Liu, and F.C. Barone. 1994. Cytokines, inflammation, and brain injury: role of tumor necrosis factor-alpha. Cerebrovascular and Brain Metabolism Reviews 6: 341–360.

    CAS  PubMed  Google Scholar 

  8. Hassoun, H.T., B.C. Kone, D.W. Mercer, F.G. Moody, N.W. Weisbrodt, and F.A. Moore. 2001. Post-injury multiple organ failure: the role of the gut. Shock 15: 1–10.

    Article  CAS  PubMed  Google Scholar 

  9. Berschneider, H.M. 1989. Abstract of the Annual Meeting of the American Gastroenterological Association. Digestive Disease Week and the 90th annual meeting of the American Gastroenterological Association, 774. Washington, DC: Elsevier.

    Google Scholar 

  10. Schierack, P., M. Nordhoff, M. Pollmann, K.D. Weyrauch, S. Amasheh, U. Lodemann, J. Jores, B. Tachu, S. Kleta, A. Blikslager, et al. 2006. Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine. Histochemistry and Cell Biology 125: 293–305.

    Article  CAS  PubMed  Google Scholar 

  11. Paszti-Gere, E., E. Csibrik-Nemeth, K. Szeker, R. Csizinszky, O. Palocz, O. Farkas, and P. Galfi. 2013. Lactobacillus plantarum 2142 prevents intestinal oxidative stress in optimized in vitro systems. Acta Physiologica Hungarica 100: 89–98.

    Article  CAS  PubMed  Google Scholar 

  12. Arce, C., M. Ramirez-Boo, C. Lucena, and J.J. Garrido. 2010. Innate immune activation of swine intestinal epithelial cell lines (IPEC-J2 and IPI-2I) in response to LPS from Salmonella typhimurium. Comparative Immunology, Microbiology and Infectious Diseases 33: 161–174.

    Article  CAS  PubMed  Google Scholar 

  13. Paszti-Gere, E., E. Csibrik-Nemeth, K. Szeker, R. Csizinszky, C. Jakab, and P. Galfi. 2012. Acute oxidative stress affects IL-8 and TNF-alpha expression in IPEC-J2 porcine epithelial cells. Inflammation 35: 994–1004.

    Article  CAS  PubMed  Google Scholar 

  14. Paszti-Gere, E., K. Szeker, E. Csibrik-Nemeth, R. Csizinszky, A. Marosi, O. Palocz, O. Farkas, and P. Galfi. 2012. Metabolites of Lactobacillus plantarum 2142 prevent oxidative stress-induced overexpression of proinflammatory cytokines in IPEC-J2 cell line. Inflammation 35: 1487–1499.

    Article  CAS  PubMed  Google Scholar 

  15. Angrisano, T., R. Pero, S. Peluso, S. Keller, S. Sacchetti, C.B. Bruni, L. Chiariotti, and F. Lembo. 2010. LPS-induced IL-8 activation in human intestinal epithelial cells is accompanied by specific histone H3 acetylation and methylation changes. BMC Microbiology 10: 172.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Hanson, P.J., A.P. Moran, and K. Butler. 2011. Paracellular permeability is increased by basal lipopolysaccharide in a primary culture of colonic epithelial cells; an effect prevented by an activator of Toll-like receptor-2. Innate Immunity 17: 269–282.

    Article  CAS  PubMed  Google Scholar 

  17. Yu, L.C., A.N. Flynn, J.R. Turner, and A.G. Buret. 2005. SGLT-1-mediated glucose uptake protects intestinal epithelial cells against LPS-induced apoptosis and barrier defects: a novel cellular rescue mechanism? FASEB Journal 19: 1822–1835.

    Article  CAS  PubMed  Google Scholar 

  18. Morgan, E.T. 1997. Regulation of cytochromes P450 during inflammation and infection. Drug Metabolism Reviews 29: 1129–1188.

    Article  CAS  PubMed  Google Scholar 

  19. Sewer, M.B., D.R. Koop, and E.T. Morgan. 1997. Differential inductive and suppressive effects of endotoxin and particulate irritants on hepatic and renal cytochrome P-450 expression. Journal of Pharmacology and Experimental Therapeutics 280: 1445–1454.

    CAS  PubMed  Google Scholar 

  20. Morgan, E.T. 2001. Regulation of cytochrome p450 by inflammatory mediators: why and how? Drug Metabolism and Disposition 29: 207–212.

    CAS  PubMed  Google Scholar 

  21. Haller, D., C. Bode, W.P. Hammes, A.M. Pfeifer, E.J. Schiffrin, and S. Blum. 2000. Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures. Gut 47: 79–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Westendorf, A.M., D. Fleissner, W. Hansen, and J. Buer. 2010. T cells, dendritic cells and epithelial cells in intestinal homeostasis. International Journal of Medical Microbiology 300: 11–18.

    Article  CAS  PubMed  Google Scholar 

  23. 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(Suppl 1): S4–14.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Mahler, G.J., M.L. Shuler, and R.P. Glahn. 2009. Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability. Journal of Nutritional Biochemistry 20: 494–502.

    Article  CAS  PubMed  Google Scholar 

  25. Heredi-Szabo, K., H. Glavinas, E. Kis, D. Mehn, G. Bathori, Z. Veres, L. Kobori, O. von Richter, K. Jemnitz, and P. Krajcsi. 2009. Multidrug resistance protein 2-mediated estradiol-17beta-D-glucuronide transport potentiation: in vitro–in vivo correlation and species specificity. Drug Metabolism and Disposition 37: 794–801.

    Article  CAS  PubMed  Google Scholar 

  26. Begue, J.M., C. Guguen-Guillouzo, N. Pasdeloup, and A. Guillouzo. 1984. Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell type. Hepatology 4: 839–842.

    Article  CAS  PubMed  Google Scholar 

  27. Harimoto, M., M. Yamato, M. Hirose, C. Takahashi, Y. Isoi, A. Kikuchi, and T. Okano. 2002. Novel approach for achieving double-layered cell sheets co-culture: overlaying endothelial cell sheets onto monolayer hepatocytes utilizing temperature-responsive culture dishes. Journal of Biomedical Materials Research 62: 464–470.

    Article  CAS  PubMed  Google Scholar 

  28. Brand, R.M., T.L. Hannah, C. Mueller, Y. Cetin, and F.G. Hamel. 2000. A novel system to study the impact of epithelial barriers on cellular metabolism. Annals of Biomedical Engineering 28: 1210–1217.

    Article  CAS  PubMed  Google Scholar 

  29. Choi, S.H., M. Nishikawa, A. Sakoda, and Y. Sakai. 2004. Feasibility of a simple double-layered coculture system incorporating metabolic processes of the intestine and liver tissue: application to the analysis of benzo[a]pyrene toxicity. Toxicology in Vitro 18: 393–402.

    Article  CAS  PubMed  Google Scholar 

  30. Castell-Auvi, A., M.J. Motilva, A. Macia, H. Torrell, C. Blade, M. Pinent, L. Arola, and A. Ardevol. 2010. Organotypic co-culture system to study plant extract bioactivity on hepatocytes. Food Chemistry 122: 775–781.

    Article  CAS  Google Scholar 

  31. Puviani, A.C., C. Ottolenghi, B. Tassinari, P. Pazzi, and E. Morsiani. 1998. An update on high-yield hepatocyte isolation methods and on the potential clinical use of isolated liver cells. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 121: 99–109.

    Article  CAS  Google Scholar 

  32. Meng, F.Y., Z.S. Chen, M. Han, X.P. Hu, X.X. He, Y. Liu, W.T. He, W. Huang, H. Guo, and P. Zhou. 2010. Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination. World Journal of Gastroenterology 16: 1660–1664.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Repetto, G., A. del Peso, and J.L. Zurita. 2008. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature Protocols 3: 1125–1131.

    Article  CAS  PubMed  Google Scholar 

  34. Anderson, J.M., and C.M. Van Itallie. 2009. Physiology and function of the tight junction. Cold Spring Harbor Perspectives in Biology 1: a002584.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Seki, E., and B. Schnabl. 2012. Role of innate immunity and the microbiota in liver fibrosis: crosstalk between the liver and gut. Journal of Physiology 590: 447–458.

    Article  CAS  PubMed  Google Scholar 

  36. Ilan, Y. 2012. Leaky gut and the liver: a role for bacterial translocation in nonalcoholic steatohepatitis. World Journal of Gastroenterology 18: 2609–2618.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Richardson, T.A., M. Sherman, L. Antonovic, S.S. Kardar, H.W. Strobel, D. Kalman, and E.T. Morgan. 2006. Hepatic and renal cytochrome p450 gene regulation during Citrobacter rodentium infection in wild-type and toll-like receptor 4 mutant mice. Drug Metabolism and Disposition 34: 354–360.

    Article  CAS  PubMed  Google Scholar 

  38. Warren, G.W., S.M. Poloyac, D.S. Gary, M.P. Mattson, and R.A. Blouin. 1999. Hepatic cytochrome P-450 expression in tumor necrosis factor-alpha receptor (p55/p75) knockout mice after endotoxin administration. Journal of Pharmacology and Experimental Therapeutics 288: 945–950.

    CAS  PubMed  Google Scholar 

  39. Siewert, E., R. Bort, R. Kluge, P.C. Heinrich, J. Castell, and R. Jover. 2000. Hepatic cytochrome P450 down-regulation during aseptic inflammation in the mouse is interleukin 6 dependent. Hepatology 32: 49–55.

    Article  CAS  PubMed  Google Scholar 

  40. Dickmann, L.J., S.K. Patel, D.A. Rock, L.C. Wienkers, and J.G. Slatter. 2011. Effects of interleukin-6 (IL-6) and an anti-IL-6 monoclonal antibody on drug-metabolizing enzymes in human hepatocyte culture. Drug Metabolism and Disposition 39: 1415–1422.

    Article  CAS  PubMed  Google Scholar 

  41. Chaluvadi, M.R., R.D. Kinloch, B.A. Nyagode, T.A. Richardson, M.J. Raynor, M. Sherman, L. Antonovic, H.W. Strobel, D.L. Dillehay, and E.T. Morgan. 2009. Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of Citrobacter rodentium. Drug Metabolism and Disposition 37: 366–374.

    Article  CAS  PubMed  Google Scholar 

  42. Behnia, K., S. Bhatia, N. Jastromb, U. Balis, S. Sullivan, M. Yarmush, and M. Toner. 2000. Xenobiotic metabolism by cultured primary porcine hepatocytes. Tissue Engineering 6: 467–479.

    Article  CAS  PubMed  Google Scholar 

  43. Kojima, M., M. Sekimoto, and M. Degawa. 2010. Androgen-mediated down-regulation of CYP1A subfamily genes in the pig liver. Journal of Endocrinology 207: 203–211.

    Article  CAS  PubMed  Google Scholar 

  44. Kojima, M., M. Sekimoto, and M. Degawa. 2008. A novel gender-related difference in the constitutive expression of hepatic cytochrome P4501A subfamily enzymes in Meishan pigs. Biochemical Pharmacology 75: 1076–1082.

    Article  CAS  PubMed  Google Scholar 

  45. Hyland, K.A., D.R. Brown, and M.P. Murtaugh. 2006. Salmonella enterica serovar Choleraesuis infection of the porcine jejunal Peyer’s patch rapidly induces IL-1beta and IL-8 expression. Veterinary Immunology and Immunopathology 109: 1–11.

    Article  CAS  PubMed  Google Scholar 

  46. Nygard, A.B., C.B. Jorgensen, S. Cirera, and M. Fredholm. 2007. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Molecular Biology 8: 67.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS

The research described here was supported by the Hungarian Scientific Research Fund (OTKA grants no. 100701 and no. 105718). The study was sponsored by the TÁMOP-4.2.2.B-10/1 and TÁMOP-4.2.1.B-11/2/KMR-2011-0003 projects. This paper is dedicated to Prof. Dr. Péter Gálfi on the occasion of his 60th birthday for establishment of the IPEC-J2 and hepatocyte co-culture system. We are indebted to Dr. Jody Gookin (Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA) for providing the IPEC-J2 cells. Special thanks have to be given to Hedvig Fébel (Research Institute for Animal Breeding and Nutrition, Herceghalom, Hungary) for providing the animals used for hepatocyte isolation. The excellent assistance of Janka Petrilla and Melinda Szabó (Faculty of Veterinary Science, Szent István University, Budapest, Hungary) is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Department of Pharmacology and Toxicology, Faculty of Veterinary Science, Szent István University, István u. 2, Budapest, 1078, Hungary

    Erzsebet Paszti-Gere, Orsolya Farkas, Orsolya Palocz, Gyorgy Csiko & Peter Galfi

  2. Department of Physiology and Biochemistry, Faculty of Veterinary Science, Szent István University, Budapest, Hungary

    Gabor Matis, Anna Kulcsar & Zsuzsanna Neogrady

Authors
  1. Erzsebet Paszti-Gere
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gabor Matis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Orsolya Farkas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anna Kulcsar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Orsolya Palocz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gyorgy Csiko
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zsuzsanna Neogrady
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peter Galfi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Erzsebet Paszti-Gere.

Additional information

Erzsebet Paszti-Gere and Gabor Matis have equal contribution to the manuscript.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Paszti-Gere, E., Matis, G., Farkas, O. et al. The Effects of Intestinal LPS Exposure on Inflammatory Responses in a Porcine Enterohepatic Co-culture System. Inflammation 37, 247–260 (2014). https://doi.org/10.1007/s10753-013-9735-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10753-013-9735-7

KEY WORDS

Search

Navigation