Induction of colitis by a CD4؉ T cell clone specific for a bacterial epitope Marika C. Kullberg*†, John F. Andersen‡, Peter L. Gorelick§, Patricia Caspar*, Sebastian Suerbaum¶ʈ, James G. Fox**, Allen W. Cheever††, Dragana Jankovic*, and Alan Sher* *Immunobiology Section and ‡Laboratory of Malaria and Vector Research, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; §Animal Health Diagnostic Laboratory, Laboratory Animal Sciences Program, National Cancer Institute, SAIC, Frederick, MD 21702; ¶Institute of Hygiene and Microbiology, University of Wu¨rzburg, D-97080 Wu¨rzburg, Germany; **Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139; and ††The Biomedical Research Institute, Rockville, MD 20852 Edited by Howard M. Grey, La Jolla Institute for Allergy and Immunology, San Diego, CA, and approved October 23, 2003 (received for review July 18, 2003) It is now well established that the intestinal flora plays an impor- IL-10 KO animals, but not simultaneously infected WT mice, tant role in the pathogenesis of inflammatory bowel disease (IBD). display marked inflammation in the cecum and colon (13). This However, whether bacteria serve as the sole target of the immune colitis is associated with a T helper 1 (Th1)-type cytokine response in this process or whether they act indirectly by triggering response to SHelAg, a soluble H. hepaticus Ag preparation, and an anti-self response is still unclear. We have previously shown treatment with anti-IL-12 mAb from the start of the infection or that specific pathogen-free IL-10-deficient (IL-10 KO) mice develop once disease is established reduces the inflammation as well as a T helper (Th1)-cytokine associated colitis after experimental the SHelAg-induced IFN-␥ production (13, 14). Together these infection with Helicobacter hepaticus. We here show that H. studies indicate a strong correlation between the Th1-type ؉ hepaticus Ag (SHelAg)-specific CD4 Th1 clones transfer disease to response to the bacterium and disease severity. H. hepaticus-infected T cell-deficient RAG KO hosts. Importantly, In the present study we have investigated the role of the uninfected recipients of the SHelAg-specific clones did not develop microbial-specific T cell response in the development of H. intestinal inflammation, and a control Schistosoma mansoni- hepaticus-induced colitis. In particular, we have addressed the specific Th1 clone did not induce colitis upon transfer to infected question of whether an aberrant T cell response directed exclu- RAG KO mice. The disease-inducing T cell clones recognized anti- sively against this bacterium is sufficient to trigger intestinal gen(s) (Ag) specifically expressed by certain Helicobacter species as inflammation. By transferring SHelAg-specific T cell clones into they responded when stimulated in vitro with H. hepaticus and T cell-deficient hosts, we demonstrate that colitis can develop as Helicobacter typhlonius Ag, but not when cultured with Ag prep- a result of an immune response directed solely against gut arations from Helicobacter pylori, various non-helicobacter bacte- bacteria. ria, or with cecal bacterial lysate from uninfected mice. Character- ization of the Ag specificity of one of the clones showed that it Materials and Methods reacts uniquely with a 15-mer peptide epitope on the flagellar Experimental Animals and Infections. Six- to 8-wk-old female spe- hook protein (FlgE) of H. hepaticus presented by I–Ab. Together, cific pathogen-free C57BL͞10 WT, C57BL͞10 IL-10 KO, our results demonstrate that colitis can be induced by clonal T cell C57BL͞10 RAG-2 KO (Taconic Farms), and C57BL͞6J SCID populations that are highly specific for target Ag on intestinal mice (The Jackson Laboratory) were used. The animals tested bacteria, suggesting that an aberrant T cell response directed negative for Ab to specific murine viruses and were free of against gut flora is sufficient to trigger IBD. Helicobacter species as assessed by PCR. All animals were housed in sterile microisolator cages at the National Institute of any inflammatory diseases are now thought to have a Allergy and Infectious Diseases (NIAID) animal facility under Mmicrobial etiology. For example, various bacteria and a study proposal approved by the NIAID Animal Care and Use viruses have been implicated in the development of peptic ulcers, Committee. reactive arthritis, arteriosclerosis, type 1 diabetes, and multiple Mice were inoculated intragastrically with 0.5 ml of an H. sclerosis. One of the best studied examples of the involvement of hepaticus suspension [‘‘NCI-Frederick isolate 1A’’ (15) isolated microbes in disease pathogenesis is inflammatory bowel disease from the same mouse colony as isolate Hh-1 (ATCC51449) (16)] (IBD). IBD is believed to be the result of an aberrant intestinal prepared to a McFarland turbidity standard of 1.0 in PBS mucosal immune response (1–3), and several reports have representing 2.45 ϫ 109 colony-forming units (cfu)͞ml. implicated a role for gut flora in human IBD (3–5). Moreover, studies in experimental models have clearly demonstrated that Antigens. SHelAg was prepared as described (13) and, in certain intestinal flora play a crucial role in IBD pathogenesis because experiments, boiled for 5 min (17). Following the same protocol, specific pathogen-free or germ-free animals develop minimal or Ag was prepared from the following bacteria from the American no disease (6–9). Type Culture Collection: Escherichia coli (ATCC25922), Kleb- Among potential mechanisms by which microbes trigger in- siella pneumoniae (ATCC13883), Pseudomonas aeruginosa flammatory diseases are molecular mimicry, bystander activa- (ATCC27853), Proteus vulgaris (ATCC13315), Staphylococcus tion or epitope spreading, and superantigen activation of T cells (10–12). A key question in the field of IBD is whether the tissue-damaging response is directed against the gut flora itself This paper was submitted directly (Track II) to the PNAS office. or whether it represents an autoimmune reaction triggered by Abbreviations: Ag, antigen; FlgE, flagellar hook protein; IBD, inflammatory bowel disease; microbial costimulation. Although data from experimental mod- OMP, outer membrane protein; SHelAg, soluble Helicobacter hepaticus Ag; Treg, T regu- els suggest that bacterial antigen (Ag) are the initial trigger of T latory cell; KO, knockout; Th1, T helper 1; TCR, T cell receptor; CBL, cecal bacterial lysate. cell activation, it is not clear whether an antibacterial T cell Data deposition: The sequence reported in this paper has been deposited in the GenBank response by itself is sufficient to induce IBD, thus leaving open database (accession no. AJ583505). the question of target Ag(s) and T cell fine specificity. †To whom correspondence should be addressed. E-mail: [email protected]. We have studied a model of IBD involving Helicobacter ʈPresent address: Institute of Medical Microbiology and Hospital Epidemiology, Hannover hepaticus infection of specific pathogen-free IL-10 knockout Medical School, D-30625 Hannover, Germany. (KO) mice. Within weeks after inoculation with this bacterium, © 2003 by The National Academy of Sciences of the USA 15830–15835 ͉ PNAS ͉ December 23, 2003 ͉ vol. 100 ͉ no. 26 www.pnas.org͞cgi͞doi͞10.1073͞pnas.2534546100 Downloaded by guest on September 26, 2021 epidermidis (ATCC12228), Staphylococcus aureus SHelAg (1–10 ␮g͞ml), various bacterial Ag preparations, CBL, (ATCC25923), and Helicobacter muridarum (ATCC51212), or recombinant H. hepaticus proteins, or synthetic peptides. In from Helicobacter typhlonius (18), and Helicobacter pylori (strain some experiments, splenocytes were cultured overnight (107 per G27, cagAϩ) (19). Cecal bacterial lysate (CBL) and Schistosoma ml) in medium, SHelAg (10 ␮g͞ml), or CBL (200 ␮g͞ml), mansoni soluble worm antigen preparation were prepared as irradiated, and washed before addition to wells. When indicated, described (20, 21). Synthetic 15-mer peptides (96–98% pure) 10 ␮g͞ml anti-CD4 (GK1.5), anti-CD8 (2.43), or anti-class II were purchased from Multiple Peptide Systems (San Diego). MHC mAb (M5͞114) were added to cultures. Supernatants were The sequence of the peptide with stimulatory activity was collected after 48–72 h, and IFN-␥ was measured by ELISA confirmed by Edman degradation. using mAb from BD Biosciences. Cloning and Expression of H. hepaticus Flagellar Hook Protein (FlgE). Flow Cytometric Analysis. T cell clones were analyzed for T cell ␤ The flgE gene was amplified from H. hepaticus genomic DNA by receptor (TCR) V expression by using mAb from BD Bio- using gene-specific primers (Table 1, which is published as sciences: phycoerythrin anti-CD4 (RM4-5), Cy-chrome anti- ␤ ␤ ␤ ␤ ␤ ͞ ␤ supporting information on the PNAS web site) designed based TCR V (H57-597), FITC anti-V 2, V 3, V 4, V 5.1 5.2, V 6, ␤ ␤ ͞ ␤ ␤ ␤ ␤ ␤ ␤ ␤ on the H. hepaticus genome sequence (22). PCR products were V 7, V 8.1 8.2, V 8.3, V 9, V 10b, V 11, V 12, V 13, V 14, ␤ cloned into the pCR 4-TOPO vector (Invitrogen). Miniprep or V 17a. DNA was cut with NdeI and XhoI, and inserts were ligated into the expression vector pET17b (Novagen). Recombinant proteins Transfer of T Cell Clones into RAG KO Mice. Twelve to 25 days after ϫ 7 were expressed in E. coli strain BL21(DE3) pLys S (Invitrogen). Ag stimulation, T cell clones (4 10 per mouse) were trans- Isolated inclusion bodies were solubilized (20 mM Tris⅐HCl, pH 8͞10 mM DTT͞6 M guanidinium hydrochloride) and proteins purified by gel filtration (Superdex 200, 20 mM Tris⅐HCl, pH 8͞0.1 M NaCl͞3 M urea). Purified proteins were dialyzed against Tris-buffered saline followed by distilled water. The purity of recombinant proteins was verified by SDS͞PAGE. Generation of T Cell Clones. T cell lines were established from 4-wk H. hepaticus-infected anti-CD8-treated IL-10 KO mice (1 mg of mAb 2.43 i.p. per week). Briefly, mesenteric lymph node cells (3 ϫ 106 per well) were cultured in 24-well plates with 50 ng͞ml SHelAg in complete medium (17).