Skip to main content
Springer Nature Link
Log in

Recommendations for Improved Use of the Murine TNBS-Induced Colitis Model in Evaluating Anti-inflammatory Properties of Lactic Acid Bacteria: Technical and Microbiological Aspects

  • Inflammatory Bowel Disease
  • Published:
Digestive Diseases and Sciences Aims and scope Submit manuscript

Abstract

See More

Probiotic bacteria have been shown to exert promising beneficial effects in different types of intestinal disorders, including chronic inflammation. In this context, animal models of inflammatory bowel disease are useful in studying the possible prophylactic role of candidate probiotic strains. This study aimed at evaluating the critical technological and microbiological parameters as well as the robustness of the murine trinitrobenzene sulfonic acid (TNBS)–induced model of colitis, after intragastric administration of lactic acid bacteria (LAB) preparations. A standardized methodology was applied to assess the protective effect achieved by various bacterial concentrations and culture conditions of the reference strain Lactobacillus plantarum NCIMB 8826. Not only was protection found to vary in function in different levels of colitis, but also repeated experiments showed a clear bacterial dose-dependent attenuation of colitis. The physiological stage of bacteria was shown to impact as well, with substantial, mild, or reduced improvement of inflammatory scores for exponentially growing, stationary-phase, or killed bacteria, respectively. A recombinant strain, secreting murine interleukin-10 (IL-10) and previously reported to successfully treat colitis in two different models of murine colitis (dextran sulfate sodium [DSS] and IL-10-deficient mice), was used to validate the final experimental conditions. In conclusion, we identified and optimized some of the key parameters that need to be controlled in order to ensure reliable comparison of results generated over a long period of time or independent experiments. The recommendations for an improved model presented here will prove to be helpful for reproducible, independent comparison of the anti-inflammatory potential of wild-type or recombinant candidate probiotic strains, whether administered as pure cultures or as blends.

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

Access this article

Log in via an institution

Subscribe and save

Springer+
from $39.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

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

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Explore related subjects

Discover the latest articles, books and news in related subjects, suggested using machine learning.

References

  1. Chandran P, Satthaporn S, Robins A, Eremin O: Inflammatory bowel disease: dysfunction of GALT and gut bacterial flora (II). Surgeon 3:125–136, 2003

    Google Scholar 

  2. Rath HC, Schultz M, Freitag R, Dieleman LA, Li F, Linde HJ, Scholmerich J, Sartor RB: Different subsets of enteric bacteria induce and perpetuate experimental colitis in rats and mice. Infect Immun 69:2277–2285, 2001

    Article  CAS  PubMed  Google Scholar 

  3. Schultz M, Veltkamp C, Dieleman LA, Grenther WB, Wyrick PB, Tonkonogy SL, Sartor RB:Lactobacillus plantarum. 299V in the treatment and prevention of spontaneous colitis in interleukin-10-deficient mice. Inflamm Bowel Dis 8:71–80, 2002

    Google Scholar 

  4. Madsen KL, Doyle JS, Jewell LD, Tavernini MM, Fedorak RN: Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice. Gastroenterology 116:1107–1114, 1999

    Article  CAS  PubMed  Google Scholar 

  5. Guarner F, Malagelada JR: Role of bacteria in experimental colitis. Best Pract Res Clin Gastroenterol 17:793–804, 2003

    Article  CAS  PubMed  Google Scholar 

  6. Cong Y, Konrad A, Iqbal N, Elson CO: Probiotics and immune regulation of inflammatory bowel diseases. Curr Drug Targets Inflamm Allergy 2:145–154, 2003

    Article  CAS  PubMed  Google Scholar 

  7. Fedorak RN, Madsen KL: Probiotics and the management of inflammatory bowel disease. Inflamm Bowel Dis 10:286–299, 2004

    PubMed  Google Scholar 

  8. Strober W: Epithelial cells pay a Toll for protection. Nat Med 10:898–900, 2004

    Article  CAS  PubMed  Google Scholar 

  9. Rachmilewitz D, Katakura K, Karmeli F, Hayashi T, Reinus C, Rudensky B, Akira S, Takeda K, Lee J, Takabayashi K, Raz E: Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis. Gastroenterology 126:520–528, 2004

    Article  CAS  PubMed  Google Scholar 

  10. Dunne C, O'Mahony L, Murphy L, Thornton G, Morrissey D, O'Halloran S, Feeney M, Flynn S, Fitzgerald G, Daly C, Kiely B, O'Sullivan GC, Shanahan F, Collins JK: In vitro. selection criteria for probiotic bacteria of human origin: correlation with in vivo. findings. Am J Clin Nutr Suppl 73:386–392, 2001

    Google Scholar 

  11. McCarthy J, O'Mahony L, O'Callaghan L, Sheil B, Vaughan EE, Fitzsimons N, Fitzgibbon J, O'Sullivan GC, Kiely B, Collins JK, Shanahan F: Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance. Gut 52:975–980, 2003

    Article  CAS  PubMed  Google Scholar 

  12. Shanahan F: Probiotics and inflammatory bowel disease: from fads and fantasy to facts and future. Br J Nutr Suppl 88:5–9, 2002

    Google Scholar 

  13. Hart AL, Stagg AJ, Kamm MA: Use of probiotics in the treatment of inflammatory bowel disease J Clin Gastroenterol 36:111–119, 2003

    Article  PubMed  Google Scholar 

  14. Gionchetti P, Amadini C, Rizzello F, Venturi A, Palmonari V, Morselli C, Romagnoli R, Campieri M: Probiotics role in inflammatory bowel disease. Dig Liver Dis 34:58–62, 2002

    Article  Google Scholar 

  15. Sugimoto K, Hanai H, Tozawa K, Aoshi T, Uchijima M, Nagata T, Koide Y: Curcumin prevents and ameliorates trinitrobenzene sulfonic acid-induced colitis in mice. Gastroenterology 123:1912–1922, 2002

    Article  CAS  PubMed  Google Scholar 

  16. Neurath MF, Fuss I, Kelsall BL, Stuber E, Strober W: Antibodies to interleukin 12 abrogate established experimental colitis in mice. J Exp Med 182:1281–1290, 1995

    Article  CAS  PubMed  Google Scholar 

  17. Strober W, Fuss IJ, Blumberg RS: The immunology of mucosal models of inflammation. Annu Rev Immunol 20:495–549, 2002

    Article  CAS  PubMed  Google Scholar 

  18. Pizarro TT, Arseneau KO, Bamias G, Cominelli F: Mouse models for the study of Crohn's disease. Trends Mol Med 9:218–222, 2003

    Article  CAS  PubMed  Google Scholar 

  19. Fiorucci S, Antonelli E, Distrutti E, Del Soldato P, Flower RJ, Clark MJ, Morelli A, Perretti M, Ignarro LJ: NCX-1015, a nitric-oxide derivative of prednisolone, enhances regulatory T cells in the lamina propria and protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice. Proc Natl Acad Sci USA 99:15770–15775, 2002

    Article  CAS  PubMed  Google Scholar 

  20. Ameho CK, Adjei AA, Harrison EK, Takeshita K, Morioka T, Arakaki Y, Ito E, Suzuki I, Kulkarni AD, Kawajiri A, Yamamoto S: Prophylactic effect of dietary glutamine supplementation on interleukin 8 and tumour necrosis factor alpha production in trinitrobenzene sulphonic acid induced colitis. Gut 41:487–493, 1997

    CAS  PubMed  Google Scholar 

  21. Cherbut C, Michel C, Lecannu G: The prebiotic characteristics of fructooligosaccharides are necessary for reduction of TNBS-induced colitis in rats. J Nutr 133:21–27, 2003

    CAS  PubMed  Google Scholar 

  22. Holma R, Salmenpera P, Lohi J, Vapaatalo H, Korpela R: Effects of Lactobacillus rhamnosus. GG and Lactobacillus reuteri. R2LC on acetic acid-induced colitis in rats. Scand J Gastroenterol 36:630–635, 2001

    Google Scholar 

  23. Kennedy RJ, Hoper M, Deodhar K, Kirk SJ, Gardiner KR: Probiotic therapy fails to improve gut permeability in a hapten model of colitis. Scand J Gastroenterol 35:1266–1271, 2000

    CAS  PubMed  Google Scholar 

  24. Gionchetti P, Rizzello F, Venturi A, Brigidi P, Matteuzzi D, Bazzocchi G, Poggioli G, Miglioli M, Campieri M: Oral bacteriotherapy as maintenance treatment in patients with chronic pouchitis: a double-blind, placebo-controlled trial. Gastroenterology 119:305–309, 2000

    Article  CAS  PubMed  Google Scholar 

  25. Rachmilewitz D, Karmeli F, Takabayashi K, Hayashi T, Leider-Trejo L, Lee J, Leoni LM, Raz E: Immunostimulatory DNA ameliorates experimental and spontaneous murine colitis. Gastroenterology 122:1428–1441, 2002

    Article  CAS  PubMed  Google Scholar 

  26. Obermeier F, Dunger N, Strauch UG, Grunwald N, Herfarth H, Scholmerich J, Falk W: Contrasting activity of cytosin-guanosin dinucleotide oligonucleotides in mice with experimental colitis. Clin Exp Immunol 134:217–224, 2003

    Article  CAS  PubMed  Google Scholar 

  27. Pathmakanthan S, Li CK, Cowie J, Hawkey CJ: Lactobacillus plantarum. 299: beneficial in vitro. immunomodulation in cells extracted from inflamed human colon. J Gastroenterol Hepatol 19:166–173, 2004

    Article  PubMed  Google Scholar 

  28. Sanders ME: Probiotics. Food Technol 53:67–75, 1999

    Google Scholar 

  29. Molin G: Probiotics in foods not containing milk or milk constituents, with special reference to Lactobacillus plantarum. 299v. Am J Clin Nutr Suppl 73:380–385, 2001

    Google Scholar 

  30. Cebeci A and Gürakan C: Properties of potential probiotic Lactobacillus plantarum. strains. Food Microbiol 20:511–518, 2003

    Google Scholar 

  31. Muller-Alouf H, Grangette C, Goudercourt D, Reveneau N, Mercenier A: Comparative cytokine inducing pattern of lactic acid bacteria used for mucosal vaccine development. Immunol Lett 69:33, 1999

    Google Scholar 

  32. Vesa T, Pochart P, Marteau P: Pharmacokinetics of Lactobacillus plantarum. NCIMB 8826, Lactobacillus fermentum. KLD, and Lactococcus lactis. MG 1363 in the human gastrointestinal tract. Aliment Pharmacol Ther 14:823–828, 2000

    Article  CAS  PubMed  Google Scholar 

  33. Pavan S, Desreumaux P, Mercenier A: Use of mouse models to evaluate the persistence, safety, and immune modulation capacities of lactic acid bacteria. Clin Diagn Lab Immunol 10:696–701, 2003

    Article  CAS  PubMed  Google Scholar 

  34. Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E: Treatment of murine colitis by Lactococcus lactis. secreting interleukin-10. Science 289:1352–1355, 2000

    Article  CAS  PubMed  Google Scholar 

  35. Schotte L, Steidler L, Vandekerckhove J, Remaut E: Secretion of biologically active murine interleukin-10 by Lactococcus lactis.. Enzyme Microb Technol 10:761–765, 2000

    Google Scholar 

  36. Wallace JL, MacNaughton WK, Morris GP, Beck PL: Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 96:29–36, 1989

    CAS  PubMed  Google Scholar 

  37. Desreumaux P, Dubuquoy L, Nutten S, Peuchmaur M, Englaro W, Schoonjans K, Derijard B, Desvergne B, Wahli W, Chambon P, Leibowitz MD, Colombel JF, Auwerx J: Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies. J Exp Med 193:827–838, 2001

    CAS  Google Scholar 

  38. Lindsay J, Van Montfrans C, Brennan F, Van Deventer S, Drillenburg P, Hodgson H, Te Velde A, Sol Rodriguez Pena M: IL-10 gene therapy prevents TNBS-induced colitis. Gene Ther 9:1715–1721, 2002

    Google Scholar 

  39. Campos FG, Waitzberg DL, Logulo AF, Cukier C, Soares SR, Oliveira TS, Torrinhas RS, Plopper C, Terra RM, Lotierzo PH, Habr-Gama A: Technical and histological standardization of experimental colitis with trinitrobenzenosulfonic acid (TNBS). Rev Hosp Clin Fac Med Sao Paulo 52:180–186, 1997

    CAS  PubMed  Google Scholar 

  40. Mahler M, Bristol IJ, Leiter EH, Workman AE, Birkenmeier EH, Elson CO, Sundberg JP: Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis. Am J Physiol 274:544–551, 1998

    Google Scholar 

  41. Savendahl L, Underwood LE, Haldeman KM, Ulshen MH, Lund PK: Fasting prevents experimental murine colitis produced by dextran sulfate sodium and decreases interleukin-1 beta and insulin-like growth factor I messenger ribonucleic acid. Endocrinology 138:734–740, 1997

    Article  CAS  PubMed  Google Scholar 

  42. Lindsay JO, Ciesielski CJ, Scheinin T, Brennan FM, Hodgson HJ: Local delivery of adenoviral vectors encoding murine interleukin 10 induces colonic interleukin 10 production and is therapeutic for murine colitis. Gut 52:363–369, 2003

    CAS  PubMed  Google Scholar 

  43. Corazza N, Eichenberger S, Eugster HP, Mueller C: Nonlymphocyte-derived tumor necrosis factor is required for induction of colitis in recombination activating gene (RAG)2(-/-) mice upon transfer of CD4(+)CD45RB(hi) T cells. J Exp Med 190:1479–1492, 1999

    Article  CAS  PubMed  Google Scholar 

  44. Waidmann M, Bechtold O, Frick JS, Lehr HA, Schubert S, Dobrindt U, Loeffler J, Bohn E, Autenrieth IB: Bacteroides vulgatus. protects against Escherichia coli.-induced colitis in gnotobiotic interleukin-2-deficient mice. Gastroenterology125:162–177, 2003

    Article  PubMed  Google Scholar 

  45. Vowinkel T, Kalogeris TJ, Mori M, Krieglstein CF, Granger DN: Impact of dextran sulfate sodium load on the severity of inflammation in experimental colitis. Dig Dis Sci 49:556–564, 2004

    Article  CAS  PubMed  Google Scholar 

  46. Gardiner KR, Erwin PJ, Anderson NH, Barr JG, Halliday MI, Rowlands BJ: Colonic bacteria and bacterial translocation in experimental colitis. Br J Surg 80:512–516, 1993

    CAS  PubMed  Google Scholar 

  47. Shibolet O, Karmeli F, Eliakim R, Swennen E, Brigidi P, Gionchetti P, Campieri M, Morgenstern S, Rachmilewitz D: Variable response to probiotics in two models of experimental colitis in rats. Inflamm Bowel Dis 8:399–406, 2002

    PubMed  Google Scholar 

  48. Osman N, Adawi D, Ahrne S, Jeppsson B, Molin G: Modulation of the effect of dextran sulfate sodium-induced acute colitis by the administration of different probiotic strains of Lactobacillus. and Bifidobacterium.. Dig Dis Sci 49:320–327, 2004

    Article  CAS  PubMed  Google Scholar 

  49. Castagliuolo I, Galeazzi F, Ferrari S, Elli M, Brun P, Cavaggioni A, Tormen D, Sturniolo GC, Morelli L, Palu G. Beneficial effect of auto-aggregating Lactobacillus crispatus. on experimentally induced colitis in mice. FEMS Immunol Med Microbiol 43:197–204, 2005

  50. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R: A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 98:694–702, 1990

    CAS  PubMed  Google Scholar 

  51. Ishizuka K, Sugimura K, Homma T, Matsuzawa J, Mochizuki T, Kobayashi M, Suzuki K, Otsuka K, Tashiro K, Yamaguchi O, Asakura H. Influence of interleukin-10 on the interleukin-1 receptor antagonist/interleukin-1 beta ratio in the colonic mucosa of ulcerative colitis. Digestion 63:22–27, 2001

    Google Scholar 

  52. Elson CO: Experimental models if intestinal inflammation: new insights into mechanisms of mucosal homeostasis. In. Mucosal Immunology. Ogro PL, Lamm ME, Bienenstock J, et al. (eds). San Diego, CA, Academic Press, 1999, pp 1007–1024

  53. Scheinin T, Butler DM, Salway F, Scallon B, Feldmann M: Validation of the interleukin-10 knockout mouse model of colitis: antitumour necrosis factor-antibodies suppress the progression of colitis. Clin Exp Immunol 133:38–43, 2003

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Bactériologie des Ecosystémes, Institut Pasteur de Lille, Lille, France

    Benoit Foligné PhD, Sophie Nutten PhD, Véronique Dennin, Denise Goudercourt, Annick Mercenier PhD & Bruno Pot PhD

  2. Alimentary Pharmabiotic Center, UCC, Cork, Ireland

    Lothar Steidler PhD

  3. Department of Molecular Biomedical Research, Ghent University, Belgium

    Lothar Steidler PhD

  4. IBL Ecosystèmes, Institut Pasteur de Lille, 1 rue du Pr Calmette, BP 245, Lille, 59019, France

    Bruno Pot PhD

Authors
  1. Benoit Foligné PhD
    View author publications

    Search author on:PubMed Google Scholar

  2. Sophie Nutten PhD
    View author publications

    Search author on:PubMed Google Scholar

  3. Lothar Steidler PhD
    View author publications

    Search author on:PubMed Google Scholar

  4. Véronique Dennin
    View author publications

    Search author on:PubMed Google Scholar

  5. Denise Goudercourt
    View author publications

    Search author on:PubMed Google Scholar

  6. Annick Mercenier PhD
    View author publications

    Search author on:PubMed Google Scholar

  7. Bruno Pot PhD
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Bruno Pot PhD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Foligné, B., Nutten, S., Steidler, L. et al. Recommendations for Improved Use of the Murine TNBS-Induced Colitis Model in Evaluating Anti-inflammatory Properties of Lactic Acid Bacteria: Technical and Microbiological Aspects. Dig Dis Sci 51, 390–400 (2006). https://doi.org/10.1007/s10620-006-3143-x

Download citation

  • Received:

  • Accepted:

  • Issue date:

  • DOI: https://doi.org/10.1007/s10620-006-3143-x

Key Words