CD4+ CD25+ Regulatory T Lymphocytes Inhibit Microbially Induced Colon Cancer in Rag2-Deficient Mice
2003; Elsevier BV; Volume: 162; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)63863-1
ISSN1525-2191
AutoresSusan E. Erdman, Theofilos Poutahidis, Michal Tomczak, Arlin B. Rogers, Kathleen S. Cormier, Benjamin Plank, Bruce Horwitz, James G. Fox,
Tópico(s)Immunotherapy and Immune Responses
ResumoInflammatory bowel diseases, including ulcerative colitis and Crohn's disease, increase the risk of colorectal cancer in humans. It has been recently shown in humans and animal models that intestinal microbiota and host immunity are integral in the progression of large bowel diseases. Lymphocytes are widely believed to prevent bacterially induced inflammation in the bowel, and lymphocytes are also critical in protecting against primary tumors of intestinal epithelia in mice. Taken together, this raises the possibility that lymphocytes may inhibit colon carcinogenesis by reducing bacterially driven inflammation. To examine the role of bacteria, lymphocytes, and inflammatory bowel disease in the development of colon cancer, 129/SvEv Rag-2-deficient and congenic wild-type mice were orally inoculated with a widespread enteric mouse bacterial pathogen, Helicobacter hepaticus, or sham-dosed with media only. H. hepaticus-infected Rag2−/−, but not sham-dosed Rag2−/− mice, rapidly developed colitis and large bowel carcinoma. This demonstrated a link between microbially driven inflammation and cancer in the lower bowel and suggested that innate immune dysregulation may have an important role in inflammatory bowel disease and progression to cancer. H. hepaticus-infected wild-type mice did not develop inflammation or carcinoma showing that lymphocytes were required to prevent bacterially induced cancer at this site. Adoptive transfer with CD4+ CD45RBlo CD25+ regulatory T cells into Rag-deficient hosts significantly inhibited H. hepaticus-induced inflammation and development of cancer. These results suggested that the ability of CD4+ T cells to protect against intestinal cancer was correlated with their ability to reduce bacterially induced inflammatory bowel disease. Further, regulatory T cells may act directly on the innate immune system to reduce or prevent disease. These roles for T cells in protection against colon carcinoma may have implications for new modes of prevention and treatment of cancer in humans. Inflammatory bowel diseases, including ulcerative colitis and Crohn's disease, increase the risk of colorectal cancer in humans. It has been recently shown in humans and animal models that intestinal microbiota and host immunity are integral in the progression of large bowel diseases. Lymphocytes are widely believed to prevent bacterially induced inflammation in the bowel, and lymphocytes are also critical in protecting against primary tumors of intestinal epithelia in mice. Taken together, this raises the possibility that lymphocytes may inhibit colon carcinogenesis by reducing bacterially driven inflammation. To examine the role of bacteria, lymphocytes, and inflammatory bowel disease in the development of colon cancer, 129/SvEv Rag-2-deficient and congenic wild-type mice were orally inoculated with a widespread enteric mouse bacterial pathogen, Helicobacter hepaticus, or sham-dosed with media only. H. hepaticus-infected Rag2−/−, but not sham-dosed Rag2−/− mice, rapidly developed colitis and large bowel carcinoma. This demonstrated a link between microbially driven inflammation and cancer in the lower bowel and suggested that innate immune dysregulation may have an important role in inflammatory bowel disease and progression to cancer. H. hepaticus-infected wild-type mice did not develop inflammation or carcinoma showing that lymphocytes were required to prevent bacterially induced cancer at this site. Adoptive transfer with CD4+ CD45RBlo CD25+ regulatory T cells into Rag-deficient hosts significantly inhibited H. hepaticus-induced inflammation and development of cancer. These results suggested that the ability of CD4+ T cells to protect against intestinal cancer was correlated with their ability to reduce bacterially induced inflammatory bowel disease. Further, regulatory T cells may act directly on the innate immune system to reduce or prevent disease. These roles for T cells in protection against colon carcinoma may have implications for new modes of prevention and treatment of cancer in humans. In humans with inflammatory bowel disease (IBD), colon cancer arises from dysplastic epithelial foci.1Riddell RH Goldman H Ransohoff DF Appleman HD Fenoglio CM Haggitt RC Ahren C Correa P Hamilton SR Morson BC Sommers SC Yardley JH Dysplasia in inflammatory bowel disease: standardized classification with provisional clinical applications.Hum Pathol. 1983; 14: 931-968Abstract Full Text PDF PubMed Scopus (1642) Google Scholar, 2Crawford JM The Gastrointestinal Tract. 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Indeed, enteric bacteria were required for lower bowel adenocarcinoma to develop in germ-free TCRβ-deficient mice,15Kado S Uchida K Funabashi H Iwata S Nagata Y Ando M Onoue M Matsuoka Y Ohwaki M Morotomi M Intestinal microflora are necessary for development of spontaneous adenocarcinoma of the large intestine in T-cell receptor beta chain and p53 double-knockout mice.Cancer Res. 2001; 61: 2395-2398PubMed Google Scholar and bacterial infection increased severity of cancer in Apcmin/+ mice.16Newman JV Kosaka T Sheppard BJ Fox JG Schauer DB Bacterial infection promotes colon tumorigenesis in Apc min/+ mice.J Infect Dis. 2001; 184: 227-230Crossref PubMed Scopus (136) Google Scholar Recombinase-activating gene (Rag)-1- or -2-deficient mice lack functional lymphocytes because of an inability to properly rearrange antigen receptors.17Shinkai Y Rathbun G Lam KP Oltz EM Stewart V Mendelsohn M Charron J Datta M Young F Stall AM Rag-2 deficient mice lack mature lymphocytes owing to inability to initiate V(D)J chain rearrangement.Cell. 1992; 68: 855-867Abstract Full Text PDF PubMed Scopus (2214) Google Scholar The absence of functional T and B cells, and the ability to transfer specified subsets of lymphocytes from immunocompetent mice, make this mouse a valuable model to study innate and adaptive immunity in the lower bowel. Rag-deficient mice have been used extensively as a host in adoptive transfer studies of colitis induced by CD4+CD45RBhi T cells. The role of T cells in induction of IBD has been widely studied;18Groux H Powrie F Regulatory T cells and inflammatory bowel disease.Immunol Today. 1999; 20: 442-445Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 19Singh B Read S Asseman C Malmstrom V Mottet C Stephens LA Stepankova R Tlaskalova H Powrie F Control of intestinal inflammation by regulatory T cells.Immunol Rev. 2001; 182: 190-200Crossref PubMed Scopus (443) Google Scholar however, T cells are not absolutely required for chronic inflammation in the colon.11Cahill RJ Foltz CJ Fox JG Dangler CA Powrie F Schauer DB Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus.Infect Immun. 1997; 65: 3126-3131PubMed Google Scholar, 20Engle SJ Hoying JB Boivin GP Ormsby I Gartside PS Doetschman T Transforming growth factor beta-1 suppresses nonmetastatic colon cancer at an early stage of tumorigenesis.Cancer Res. 1999; 59: 3379-3386PubMed Google Scholar Colitis in SCID and Rag-deficient mice could be induced by infection with a widespread enteric mouse bacterial pathogen, Helicobacter hepaticus, without the need for effector T lymphocytes.11Cahill RJ Foltz CJ Fox JG Dangler CA Powrie F Schauer DB Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus.Infect Immun. 1997; 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Based on evidence that lymphocytes were critical to preventing intestinal cancer, and that CD4+ CD45RBlo CD25+ T lymphocytes regulate inflammation in the lower bowel of mice, we hypothesized that these T cells may inhibit cancer by suppressing chronic microbially induced inflammation in the lower bowel. Inability to inhibit bacterially driven inflammation might explain the high frequency of lower bowel adenocarcinoma observed in Rag-deficient mice. Thus, we examined progression of H. hepaticus-induced inflammation and cancer in Rag-2-deficient mice, with and without adoptive transfer of CD4+ CD45RBlo CD25+ T lymphocytes. All mice were housed in Association for Assessment and Accreditation of Laboratory Animal Care-approved facilities in static microisolator cages. 129/SvEv Rag2-deficient mice from Taconic Farms (Germantown, NY) were housed for 18 months with health status free of known murine viruses, Salmonella spp, Citrobacter rodentium, ecto- and endoparasites, and known murine Helicobacter spp. A Rag2-deficient breeding colony was established in this facility to provide mice for subsequent infection experiments. H. hepaticus (strain 3B1, no. 51449; American Type Culture Collection, Rockville, MD) was grown as described elsewhere.30Fox JG Dewhirst FE Tully JG Paster BJ Yan L Taylor NS Collins MJ Gorelick PL Ward JM Helicobacter hepaticus species. nov. a microaerophilic bacterium isolated form livers and intestinal mucosal scrapings from mice.J Clin Microbiol. 1994; 32: 1238-1245Crossref PubMed Google Scholar Cultures were examined by Gram's stain and phase microscopy for contaminants and subcultured on blood agar to confirm purity. Bacteria were resuspended in brucella broth at 108 bacteria/ml as confirmed by spectrophotometry.31Fox JG Yan L Shames B Campbell J Murphy JC Persistent hepatitis and enterocolitis in germfree mice infected with Helicobacter hepaticus.Infect Immun. 1996; 64: 3673-3681PubMed Google Scholar Experimental mice received 0.2 ml of fresh inoculum by gastric gavage every other day for three doses. At age 6 to 8 weeks, helicobacter-free Rag-2-deficient mice were dosed with H. hepaticus suspended in broth or sham-dosed with broth only. A cohort of aging 129/SvEv Rag-2-deficient mice remained untreated. The infected and control uninfected mice were housed in microisolator caging in different areas within the animal facility. Replicate experiments were conducted with three groups of similar size. In each experiment, half of the mice were male and half of the mice were female. Cecum and colon were collected at necropsy and analyzed by polymerase chain reaction to confirm experimental infection using H. hepaticus-specific primers.32Shames B Fox JG Dewhirst F Yan L Shen Z Taylor NS Identification of widespread Helicobacter hepaticus infection in feces in commercial mouse colonies by culture and PCR assay.J Clin Microbiol. 1995; 33: 2968-2972PubMed Google Scholar Helicobacter-free status was confirmed in controls using polymerase chain reaction with helicobacter genus-specific primers.33Fox J Dewhirst FE Shen Z Feng Y Taylor NS Paster BJ Ericson RL Lau CN Correa P Araya JC Roa I Hepatic Helicobacter species identified in bile and gallbladder from Chileans with chronic cholecystitis.Gastroenterology. 1998; 114: 755-763Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar To examine whether T cells modulate progression of cancer in H. hepaticus-infected Rag2 mice, we performed transfers of purified (as below) T lymphocytes from helicobacter-free 129/SvEv donors into Rag2-deficient mice. Half of the lymphocyte donors were male and half of the donors were female. Before experimental H. hepaticus infection, anesthetized mice were injected intravenously in the retro-orbital sinus with 2 × 105 T cells suspended in 0.2 ml of media using a 26-gauge needle. Mice were dosed 72 hours later with H. hepaticus, as described above. To obtain viable and highly purified populations of T cells for adoptive transfer, single cell suspensions from spleen and mesenteric lymph nodes from helicobacter-free 129/SvEv mice were prepared. CD4-positive cells were isolated by using L3T4 Dynabeads (Dynal, Oslo, Norway). Cells were detached from the beads using mouse CD4 DETACHaBEAD (Dynal). The purity of CD4+ cells isolated in this manner was >95%. CD45RBlo CD25+ cells were further isolated from the CD4+ population by labeling with anti-CD45RB-FITC antibodies (Pharmingen, La Jolla, CA) and anti-CD25-PE antibodies (Pharmingen) and then purified by flow cytometry. Purified cells were resuspended in RPMI with 10% fetal calf serum before injection as previously described.34Horwitz BH Scott ML Cherry SR Bronson RT Baltimore D Failure of Lymphopoiesis after adoptive transfer of NF-kB deficient fetal liver cells.Immunity. 1997; 6: 765-772Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar Reanalysis of these cells before transfer into mice indicated that purity was >99%. Host spleens were collected at the time of necropsy, and single cell suspensions were prepared. Cells were stained with anti-CD3 and anti-CD4 antibodies and analyzed by flow cytometry. The percentage of CD3+CD4+ cells in the spleens of these animals ranged from 0 to 7%. CD3+CD4+ T cells made up less than 1% of the splenocytes in 3 of the 17 mice that received CD4+CD45RBloCD25+ cells. One (1 of 17) mouse had no evidence of CD4+ cells in the spleen on necropsy. Formalin-fixed tissues were embedded in paraffin, cut at 5 μm, and stained with hematoxylin and eosin. Lesions were scored by a pathologist blinded to sample identity. The cecal and colonic lesions were scored on the basis of size and frequency of hyperplastic and inflammatory lesions on a scale of 0 to 4 with ascending severity (0, none; 1, minimal; 2, mild; 3, moderate; and 4, severe) modified from Berg and colleagues.35Berg DJ Davidson N Kuhn R Muller W Menon S Holland G Thompson-Snipes L Leach MW Rennick D Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses.J Clin Invest. 1996; 98: 1010-1020Crossref PubMed Scopus (978) Google Scholar Epithelial dysplasia and neoplasia were graded using a scale of 0 to 4 based on a recently described scheme:36Schempler RJ Riddell RH Kato Y Borchard F Cooper HS Dawsey SM Dixon MF Fenoglio-Preiser CM Flejou JF Geboes K Hattori T Hirota T Habashi M Iwafuchi M Iwashita A Kim YI Kirchner T Klimpfinger M Koike M Lauwers GY Lewin KJ Oberhuber G Offner G Price AB Ribio CA Shimizu M Shimoda T Sipponen P Solcia E Stolte M Watanabe H Vamabe H The Vienna Classification of gastrointestinal epithelial neoplasia.Gut. 2000; 47: 251-255Crossref PubMed Scopus (1771) Google Scholar, 37Hamilton SR Aaltonen LA Pathology and genetics of tumors of the digestive system.World Health Organization Classification of Tumors. IARC Press, Lyon2000Google Scholar, 38Schempler RJ Kato Y Stolte M Diagnostic criteria for gastrointestinal carcinomas in Japan and Western countries: proposal for a new classification of gastrointestinal epithelial neoplasia.Gastroenterol Hepatol. 2000; Oct 15: G49-G57Crossref Scopus (108) Google Scholar grade 0, normal (Figure 1; A to C); 1, mild dysplastic changes (Figure 1; D to F); 2, low-grade adenoma/dysplasia (Figure 1; G to I); 3, high-grade adenoma/dysplasia, carcinoma in situ, or intramucosal carcinoma (Figure 1, J to O; Figure 2, A and B; Figure 3A); and 4, invasive carcinoma (Figure 4; A to C). Figure 1, Figure 2 show the grading system and depict the spectrum of dysplastic and neoplastic lesions observed in this study. Data are compiled from three replicate experiments with statistically similar results. Nonparametric data are presented as median score and range (in parentheses) for each group.Figure 2Histopathological features of dysplasia and carcinoma including a mucinous cyst lined by highly dysplastic epithelium (A), intramucosal carcinoma (B), and submucosal invasion (C). Original magnifications: ×40 (A), ×100 (B, C).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3H. hepaticus-induced severe typhlitis (A) and colitis (C) in Rag-2-deficient mice at 2 months after infection. There is severe mucosal and submucosal inflammation, marked epithelial hyperplasia, loss of goblet cells, and surface epithelial cell necrosis and erosions. Note the irregularities of crypt architecture in A (tortuosity, branching, cystic dilatation) indicative of dysplasia. The inset depicts the mixed inflammatory cells including predominantly macrophages and eosinophils. Normal histology of cecum (B) and colon (D) from uninfected Rag-2-deficient mice at 12 months after infection. H&E; original magnifications, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Invasive adenocarcinoma of cecum (A) and colon (B and C), H&E. A: Well-differentiated neoplastic glands invaded into the submucosa. B: Invasive adenocarcinoma arising in an area of severe colitis and dysplasia. C: Higher magnification of the carcinoma illustrated in B, demonstrating invasion into the muscle layer; note the irregular shape of malignant glands and a mucin pool. Immunohistochemical staining of colon (D, E) and cecum (F, G) of uninfected (D, F) and H. hepaticus-infected (E, G) Rag-2-deficient mice for Ki-67 (D, E), and cytokeratins (F, G). Diaminobenzidine, Gill's hematoxylin counterstain. E: Increased proliferative activity and expansion of the crypt proliferative zone toward the surface epithelium. G: Cytokeratin immunolabeling highlights the presence of small nests and single epithelial cells invading into the lamina propria and submucosa. Original magnifications: ×40 (A, B, C, D, E, F, G); ×100 (H, I).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cell proliferation and invasion were determined using monoclonal antibodies directed against Ki-67 (BD Biosciences, San Diego, CA), and pan-cytokeratin (AE1/AE3; DAKO, Carpinteria, CA), respectively. Standard immunohistochemistry was performed on an i6000 autostainer (Biogenex, San Ramon, CA). Briefly, formalin-fixed tissues on positively charged slides were deparaffinized and rehydrated. Antigen retrieval was performed with microwave heat in pH 6 citrate buffer (DAKO) for Ki-67, or with 5 minutes of protease digestion at 37°C using 10 mg/ml of proteinase K (Roche Molecular Systems, Indianapolis, IN) for cytokeratin. Endogenous peroxidases were quenched with two rounds of 3% H2O2 in phosphate-buffered saline for 5 minutes each. For immunohistochemistry the ARK kit (DAKO) was used. Signal was detected with diaminobenzidine and tissues were counterstained with Gill's hematoxylin. One-cm segments of cecum and colon near the junction were harvested immediately after mice were euthanized and the segments were snap-frozen in liquid nitrogen. Frozen specimens were homogenized into Tri-reagent (Molecular Research Center, Cincinnati, OH) and RNA prepared per the manufacturer's instructions. RNase protection analyses were performed on 20 μg of total RNA using RiboQuant Multi-Probe Template Sets (Pharmingen). Intensities of the protected fragments were quantitated by phosphorimager analysis and each cytokine was normalized to GAPDH internal controls. Analyses of cecal and colonic lesion scores were performed using a Mann-Whitney U nonparametric test for ordinal data. Cytokine data were analyzed using a two-tailed t-test. To determine whether enteric microbial status influenced frequency or distribution of lower bowel cancer in Rag-deficient mice, we first examined unmanipulated helicobacter-free Rag-2-deficient mice. Uninfected 129/SvEv Rag-2-knockout mice examined at 12 months of age (n = 16) and 15 months of age (n = 10) had no or minimal inflammation in the cecum [0 (0 to 0)] or colon [0 (0 to 1)], no epithelial hyperplasia in the cecum [0 (0 to 0)] or colon [0 (0 to 0)], and no dysplasia or cancer in the cecum [0 (0 to 0)] or colon [0 (0 to 0)]. To evaluate the ability of H. hepaticus to induce bowel lesions in Rag-2-deficient mice, mice were dosed with H. hepaticus and examined for histological changes in the cecum and colon. In H. hepaticus-infected Rag-2-deficient mice, there was significantly increased epithelial hyperplasia in the cecum [4 (3 to 4); P = 0.0001] and colon [4 (2 to 4); P = 0.0001] associated with inflammation in the cecum [4 (3 to 4); P = 0.0001] and colon [4 (2 to 4); P = 0.0001] (Figure 3; A to D) compared with uninfected controls (Table 1). Figure 3 demonstrates the contrast between inflammation and hyperplasia in H. hepaticus-infected and uninfected Rag-deficient mice. Inflammation was most severe in the lamina propria, with extension into the submucosa in advanced cases, and was comprised chiefly of macrophages and granulocytes. Macrophages and eosinophils predominated in approximately equal numbers, whereas neutrophils were less frequent and associated primarily with microabscessation (Figure 3C, inset). The hyperplastic changes were characterized by mild to marked elongation and thickness of the colonic crypts (Figure 1D, Figure 3C), epithelial hypercellularity, and increased numbers of mitotic figures per high-power field.Table 1Comparison of Bowel Disease in H. hepaticus-Infected and Uninfected Rag-2-Deficient MiceH. hepaticus-free Rag2-deficient (controls)H. hepaticus-infected Rag2-deficient2 months post-dose n = 12 score (range)6 months post-dose n = 7 score (range)1 month PI n = 8 score (range)2 months PI n = 16 score (range)4 months PI n = 12 score (range)6 months PI n = 17 score (range)8 months PI n = 12 score (range)Cecum Inflammation0 (0–1)*Median score (range).0 (0–0)3.5 (2–4)4 (3–4)4 (4–4)4 (4–4)4 (4–4)P = 0.0001‡Mann-Whitney U test, comparison between H. hepaticus-infected and sham-dosed Rag2-deficient mice at 2 months after-infection.P = 0.0000§Mann-Whitney U test, comparison between H. hepaticus-infected and sham-dosed Rag2-deficient mice at 6 months after-infection. 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