ORIGINAL ARTICLE Segmented Filamentous Bacteria in a Defined Bacterial Cocktail Induce Intestinal Inflammation in SCID Mice Reconstituted with CD45RB high CD4+ T Cells Renata Stepankova, PhD,* Fiona Powrie, MD, PhD, † Olga Kofronova, PhD, ‡ Hana Kozakova, PhD,* Tomas Hudcovic, PhD,* Tomas Hrncir, MD,* Holm Uhlig, MD, PhD, † Simon Read, PhD, † Zuzana Rehakova, PhD,* Oldrich Benada, PhD, ‡ Pioter Heczko, MD, PhD, § Magda Strus, MD, PhD, § Paul Bland, PhD, P,¶ and Helena Tlaskalova-Hogenova, MD, PhD* Background: The aim was to analyze the influence of intestinal microbiota on the development of intestinal inflammation. We used the model of chronic inflammation that develops spontaneously in the colon of conventional severe combined immunodeficiency (SCID) mice restored with the CD45 RB high subset of CD4+T cells isolated from the spleen of normal BALB/c mice. Methods: A CD4+CD45RB high subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and transferred into immunodeficient SCID mice. Germ-free (GF) SCID mice or SCID mice monoassociated with Enterococcus faecalis, SFB (segmented filamentous bacteria), Fusobacterium mortiferum, Bacteroides distasonis, and in combi- nation Fusobacterium mortiferum + SFB or Bacteroides distasonis + SFB were used as recipients. SCID mice were colonized by a defined cocktail of specific pathogen-free (SPF) bacteria. Mice were evaluated 8 –12 weeks after the cell transfer for clinical and mor- phological signs of inflammatory bowel disease (IBD). Results: After the transfer of the CD4+CD45RB high T-cell sub- population to SCID mice severe colitis was present in conventional animals and in mice colonized with a cocktail of SPF microflora plus SFB. Altered intestinal barrier in the terminal ileum of mice with severe colitis was documented by immunohistology using antibodies to ZO-1 (zona occludens). Conclusions: Only SFB bacteria together with a defined SPF mixture were effective in triggering intestinal inflammation in the model of IBD in reconstituted SCID mice, while no colitis was detected in GF mice or in mice colonized either with SPF microflora or monoassociated only with SFB or colonized by Bacteroides distasonis + SFB or Fusobacterium mortiferum + SFB. (Inflamm Bowel Dis 2007;13:1202–1211) Key Words: colitis, animal model, inflammatory bowel disease, regulatory T cells, germ-free, microflora, SFB T he transfer of CD4+CD45RB high T cells, a predomi- nantly naive population from the periphery of normal BALB/c mice, to severe combined immunodeficiency (SCID) recipients (immunodeficient mice lacking T and B lympho- cytes) 1 caused a severe colon inflammation. 2,3 This model of Th1-mediated colitis had a similarity to inflammatory bowel disease (IBD) in humans, where Th1 responses are also elevated in the intestine of patients with Crohn’s disease. Colitis induced by the transfer of CD4+CD45RB high T ag- gressive cells can be prevented by cotransfer of the CD4+CD45RB low T cells or (CD4+CD25+) population. 4,5 A population of CD4+CD45RB low or CD4+CD25+ T cells from germ-free (GF) mice had a significantly lower ability to inhibit colitis compared to the same populations from mice with normal microflora. 5 Thus, the presence of commensal bacteria in the donor mice drives T cells into the antigen- experienced/memory T cell pool. 6,7 CD4+CD25+T cells play a major role in the maintenance of oral tolerance. Be- sides this CD4+CD25+ population, other populations of regulatory CD4+ T cells were described: Tr1 secreting high amounts of IL-10 and Th3 secreting TGF-. 8 Consequently, the control of inflammation during the first week after the cell transfer is the result of a balance between proinflammatory and Tr cells. 8 Received for publication December 7, 2006; accepted May 29, 2007. From the *Laboratory of Gnotobiology, Department of Immunology, Institute of Microbiology, Czech Academy of Sciences, Novy Hradek, Czech Republic, † Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, UK, ‡ Laboratory of Electron Microscopy, Insti- tute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic, § Jagiellonian University, Medical School, Cracow, Poland, P Dept. of Clin- ical Veterinary Science, University of Bristol, Bristol, UK, ¶ Dept. of Clinical Immunology, University of Gothenburg, Gothenburg, Sweden. Supported by European Union grant QLGI-1999-00050, Czech Grant Agency grant 303/06/0974, Grant Agency, Academy of Sciences of the Czech Republic grants A5020205 and S 500200572, and Institutional Re- search Concept No. AV0Z50200510. Reprints: Renata Stepankova, PhD, Laboratory of Gnotobiology, Institute of Microbiology, Novy Hradek, 549 22, Doly, Czech Republic (e-mail: stepankova.renata@seznam.cz). Copyright © 2007 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1002/ibd.20221 Published online 2 July 2007 in Wiley InterScience (www.interscience. wiley.com). 1202 Inflamm Bowel Dis ● Volume 13, Number 10, October 2007