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Carcinogenesis Advance Access originally published online on August 27, 2007
Carcinogenesis 2007 28(12):2614-2623; doi:10.1093/carcin/bgm180
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Rapid reversal of interleukin-6-dependent epithelial invasion in a mouse model of microbially induced colon carcinoma

Theofilos Poutahidis1,5, Kevin M. Haigis2,6, Varada P. Rao1, Prashant R. Nambiar1, Christie L. Taylor1, Zhongming Ge1, Koichiro Watanabe1, Anne Davidson3,7, Bruce H. Horwitz4, James G. Fox1 and Susan E. Erdman1,*

1 Division of Comparative Medicine
2 Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA
3 Department of Medicine and Microbiology, Columbia University, 1130 St Nicholas Avenue, Room 918, New York, NY 10032
4 Immunology Research Division, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115, USA
5 Present address: Laboratory of Pathology, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece
6 Present address: Unit for Molecular Pathology, Center for Cancer Research, Massachusetts General Hospital, Bldg 149, Rm 7148, 13th Street, Charlestown, MA 02129, USA
7 Present address: Feinstein Institute for Medical Research, 350 Community Drive, Manhasset NY 11030, USA

* To whom correspondence should be addressed. Tel: +1 617 252 1804; Fax: +1 617 258 5708; Email: serdman{at}mit.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Supplementary material
 References
 
Chronic inflammation of mucosal surfaces renders them increasingly susceptible to epithelial cancers both in humans and mice. We have previously shown that anti-inflammatory CD4+CD45RBloCD25+ regulatory (Treg or TR) lymphocytes down-regulate inflammation and block development of bacteria-triggered colitis and colorectal cancer (CRC) in 129/SvEv Rag2–/– mice. Interestingly, TR cells collected from Interleukin (IL)-10-deficient cell donors not only failed to suppress carcinogenesis but instead promoted invasive mucinous colonic carcinoma with a strong gender bias expressing in male mice. We found we show that peritoneal invasion in this model is dependent on pleiotropic cytokine IL-6. Mucinous carcinoma arose rapidly and consistently after treatment with IL10–/– TR cells, which were found to express Foxp3+ and localize throughout tumor tissue. Carcinogenesis was rapidly reversible with transfer of wild type IL10-competent TR cells. Likewise, treatment with IL10-Ig fusion protein was sufficient to revert the lesions histologically, and restore inflammatory cytokine and oncogene expression to base line levels. These studies indicate an essential role for IL 6 in this CRC phenotype. Furthermore, immune-competent TR cells were important not only for preventing pathology but also for constructive remodeling of bowel following tumorigenic microbial insults. These data provide insights into etiopathogenesis of inflammation-associated epithelial invasion and maintenance of epithelial homeostasis.

Abbreviations: IBD, inflammatory bowel disease; Ig, immunoglobulin; IL, interleukin; PCR, polymerase chain reaction; PI, post-infection; Tgf, transforming growth factor; TNF-{alpha}, tumor necrosis factor {alpha}; TR or Treg, CD4+CD45RBloCD25+; wt, wild-type; CRC, colorectal cancer


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 Supplementary material
 References
 
Colorectal cancer (CRC) is among the leading cancer killers in USA and other developed countries (1). Mucinous adenocarcinoma is one type of CRC, that is characterized by abundant pools of mucin surrounded by fibrous stroma and cancerous glands. Mucinous CRC represents ~20% of colonic tumors in humans (2). In human patients, the mucinous lesions are found located most often on the right side of the colon. Molecular characterization studies have revealed that mutations of K-ras are more frequent in mucinous carcinomas in contrast to their non-mucinous counterparts (3). In general, mucinous carcinoma is less readily resected by surgery and carries a poorer prognosis compared with other types of CRC in humans (4,5).

We have shown that 129/SvEv recombinase-activating gene 2-deficient (Rag2–/–) mice, lacking mature lymphocytes, develop several different types of colon cancer associated with colitis, including mucinous CRC, following infection with a widespread enteric bacterial mouse pathogen Helicobacter hepaticus (6,7). The inflammatory bowel disease (IBD) and carcinoma that develop in H.hepaticus-infected Rag2–/– mice are abrogated by treatment with interleukin (IL)-10-competent CD4+CD45RBloCD25+ regulatory (Treg or TR) cells (7). Other studies using immune-deficient mice have revealed similar protective and therapeutic effects mediated by TR cells in mice with colitis (8,9).

Interestingly, adoptive transfer of CD4+CD45RBloCD25+ cells obtained from IL10-deficient donors fail to protect and instead exacerbate a malignant epithelial phenotype, such that 100% of recipient male Rag2–/– mice rapidly develop mucinous tumors in the ascending (right) and transverse colon that invade the peritoneal cavity (7). Mucinous adenocarcinomas in these mice match tumors in human patients according to a National Cancer Institute sponsored consensus report (10). Although it is clear from prior studies that the immune system and TR cells regulate epithelial cancer progression, the extent to which neoplastic epithelial invasion may be modulated and repaired by down-regulating inflammation has not been characterized.

Here we demonstrate that bacteria-triggered mucinous colonic carcinoma that arises rapidly and consistently in IL10–/– TR cell-recipient mice is dependent on IL-6 and is completely reversible by wild-type (WT) TR cells. Likewise, treatment with IL10-immunoglobulin (Ig) fusion protein is sufficient to revert the lesions histologically and restore inflammatory cytokine and oncogene expression levels to baseline. We propose that a signaling pathway comprised, at least in part, of IL-10 and IL-6 is pivotal in maintaining epithelial homeostasis and modulating epithelial invasion during bacterially driven inflammatory diseases.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 Supplementary material
 References
 
129/SvEv Rag2-deficient mice
All animals, whose health status was as described previously (6), were housed in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved facilities in static microisolator cages. 129/SvEv Rag2-deficient mice (originally obtained from Taconic Farms, German town, NY) and transforming growth factor (Tgf)β1-deficient Rag2-deficient mice (obtained from the Mouse Models of Human Cancer Consortium repository, Frederick, MD) were bred in-house to provide animals for these experiments. Experimental mice dosed with H.hepaticus were housed separately in a biocontainment area within the same animal facility. Studies used 10–12 mice per group in each experiment, with three or four repetitions (total n = 30–48 mice per group), unless otherwise specified. All experiments using animals were approved by the Institutional Animal Care and Use Committee.

Experimental infection
Helicobacter hepaticus (strain 3B1, American Type Culture Collection #51449) was grown under microaerobic conditions, prepared and confirmed pure as described elsewhere (6,11). Experimental mice received 0.2 ml of fresh inoculum by gastric gavage every other day for a total of three doses. The cecum and colon of mice were collected at necropsy and analysed by polymerase chain reaction (PCR) using H.hepaticus-specific primers to confirm Helicobacter status (6).

Adoptive transfer with lymphocytes
Rag2–/– recipients of wt or IL10-deficient CD4+CD45RBloCD25+ cells, collected from mice backcrossed at least 10 generations onto a 129/SvEv genetic background, underwent adoptive transfer 48–72 h prior to H.hepaticus infection (6,7). All the recipient mice in this study were male, based on the earlier observation that microbially induced cancer was exacerbated in male mice when compared with female mice of this strain (7). Recipient mice anesthetized with isofluorane were injected intravenously via the retro-orbital sinus with 3 x 105 cells suspended in 0.2 ml of Hank's balanced salt solution. Replicate experiments were conducted with two or three groups of similar size for select experiments.

To obtain viable and highly purified cell populations, single-cell suspensions of CD4+CD25+ lymphocytes from spleen and mesenteric lymph nodes from Helicobacter-free WT or IL10-deficient 129/SvEv donor mice were prepared as described previously (6). Briefly, CD4-positive cells were isolated by using L3T4 Dynabeads (Dynal, Oslo, Norway). Cells were detached from the beads using mouse CD4 DETACHaBEAD (Dynal). CD45RBlo CD25+ cells were further isolated from the CD4+ population by labeling with anti-CD45RB antibodies (PharMingen, San Diego, CA) and anti-CD25 antibodies (PharMingen) and then purified by flow cytometry. For these studies, half of the donor mice were males and half were females. Re-analysis of these cells prior to transfer into mice indicated that they were >96% pure.

Foxp3 characterization
For flow cytometry, after being stained with anti-CD4-APC and anti-CD25-PE (Becton Dickinson Biosciences PharMingen), cells were fixed and permeabilized for intracellular staining with Foxp3- fluorescein isothiocyanate (BD Bioscience PharMingen) or isotype control fluorescein isothiocyanate-labeled antibody. A minimum of 50 000 events was collected for each sample. FACScalibur was used for data analysis. For immunohistochemistry, after deparaffinization, formalin-fixed sections were antigen retrieved with pepsin (Zymed, San Francisco, CA) for 10 min at 37°C and were labeled with rat monoclonal antibody recognizing mouse Foxp3 antigen (# 14-5773 rat anti-mouse; eBioscience, San Diego, CA). Primary antibody binding was detected with species-appropriate biotinylated secondary antibodies (Sigma chemical company), streptavidin peroxidase and 3,3-diaminobenzidieine (Vector Labs, Burlingame, CA). Immunohistochemical assays were performed on an automated immunostainer (i6000; Biogenex, San Ramon, CA).

Carcinoma intervention using adoptive transfer of lymphocytes
Rag2–/– recipients of IL10-deficient CD4+CD25+ cells underwent a second adoptive transfer of wt TR cells at 6–8 weeks after H.hepaticus infection, when mice had already developed mucinous carcinoma and localized peritoneal invasion, to determine ability of TR cells to induce cancer regression.

Subsets of Rag2–/– mice (n = 8 mice per group) that received H.hepaticus and IL10–/– deficient cells, as above, subsequently underwent adoptive transfer of a lower dose of wt TR cells at 6–8 weeks post-infection (PI) to ascertain and compare therapeutic potency. A dosage of 1.0 x 105 cells was predetermined to be the minimum effective dosage of lymphocytes in suppressing carcinoma.

In vivo ultrasound imaging
To confirm the presence of mucinous colonic masses prior to the onset of treatment, selected mice underwent imaging of the abdominal cavity using high-resolution ultrasound (VisualSonics, Toronto, Canada). Imaging was performed both before (at 8–12 weeks PI) and after (2 weeks) treatment with TR cells, using a 707B scan head, with isofluorane inhalant anesthesia.

Treatment with IL10-Ig fusion protein
To produce the IL10-Ig fusion protein, murine IL10 was fused to the IgG2a CH2-CH3 regions, mutated at the Fc receptor-binding site, using a PCR cloning strategy and the chimeric gene was cloned into an adenoviral vector and infectious virus generated (Ad-IL-10-Ig) as described previously (12,13). We determined that 150 ng/ml of IL-10-Ig was comparable with 1 ng/ml of recombinant IL10 in its ability to inhibit the production of IL-12 p40 and IP-10 by IL10-deficient macrophages (data not shown). Serum containing 2–5 µg of fusion protein was administered by intraperitoneal injection to mice with established invasive cancer, according to the data from other mice that were euthanized from the same cohort during initial studies, twice weekly for 7–10 days.

Determination of serum IL6 protein by enzyme-linked immunosorbent assay
To determine serum IL6 concentration in treated and untreated mice, a sandwich enzyme-linked immunosorbent assay was performed using Quantikine mouse IL6 kit (R&D Systems, MN) as per the manufacturer's instructions. The IL6 standard curve and sample concentrations were determined by measuring absorbance at 450 nm and after applying correction for plate background (Biotek Instruments, VT).

Treatment with cytokine-neutralizing antibodies
For neutralization of tumor necrosis factor {alpha} (TNF-{alpha}), Rag2–/–recipients of IL10–/– lymphocytes also infected with H.hepaticus were treated for 6–8 weeks PI, with anti-TNF-{alpha} antibody (clone XT-3; BioExpress, NH) at a dose of 200 µg per mouse (n = 8) intraperitoneally thrice weekly for 7–10 days as described previously (14). To determine whether IL6 is required to sustain colonic cancer, mice with established carcinoma (n = 8) were treated with 500 µg of rat anti-mouse IL6 (clone MP5-20F3; eBioscience) by intraperitoneal injection twice weekly for 1 week. Matched control mice (n = 8) received the same concentration of rat IgG1 (eBioscience).

Histologic evaluation
Formalin-fixed tissues were embedded in paraffin, cut at 5 µm and stained with hematoxylin and eosin. Lesions were scored by two pathologists blinded to sample identity. Hyperplastic and inflammatory lesions were graded on a scale of 0–4 with ascending severity as described previously (6,15). Intestinal epithelial dysplasia and neoplasia were graded using a scale of 0–4 based on a recently described grading scheme (6,10). Non-parametric data are presented as median score and range (in parentheses) for each group.

Epithelial purification
RNA from colonic epithelium was purified according to Whitehead et al. (16) with minor modifications. Briefly, colons were removed and flushed with ice-cold 1x phosphate-buffered saline. Colons were then opened lengthwise and incubated in 3 mM ethylenediaminetetraacetic acid, 0.05 mM dithiothreitol for 60 min on ice at 4°C. Following the 1 h incubation, tissues were rinsed once in cold 1x phosphate-buffered saline. New phosphate-buffered saline was added and the tissue was shaken vigorously to dislodge epithelium. In an independent assay, preparations were found to be >95% pure for intestinal epithelial cells.

Gene expression analysis
The samples for analyses of colonic epithelial genes were centrifuged to pellet the purified epithelia (as above), whereas other gene expression assays utilized snap-frozen 0.5–1.0 cm full-thickness sections of ascending colon. Supernatant was removed and replaced with 1 ml of Trizol (Invitrogen). RNA was isolated according to manufacturer's instructions. After Trizol, the RNA was further purified using Qiagen's RNeasy Kit. Colonic epithelial cDNA was produced from 1 µg of purified RNA using Superscript III reverse transcriptase (Invitrogen). For each treatment group, RNA was obtained from colons of at least three different animals per group for purified colonic epithelia and at least eight animals per group for whole bowel. Gene expression analysis was performed by TaqMan analysis (Applied Biosystems, Foster City, CA) on an ABI Prism 7000 Sequence Detection System. All expression assays were designed by ABI (Assays-on-Demand, www.appliedbiosystems.com).

Statistical analyses
Analyses of colonic lesion scores were performed using a Mann–Whitney U non-parametric test for ordinal data. Comparisons of frequency of carcinoma between groups were performed using a two-sided Fisher's exact test. Statistical analysis of gene expression data was performed by Wilcoxon Rank Sum test using the Mstat computer program (http://mcardle.oncology.wisc.edu/mstat/).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 Supplementary material
 References
 
Infection with H.hepaticus bacteria triggers carcinoma
We demonstrated previously that H.hepaticus infection triggers colitis-associated carcinoma in 129/SvEv Rag2–/– mice (6,7). In that model, microbially triggered invasive epithelial lesions matched mucinous colonic carcinoma in humans according to a published consensus report on mouse models of colon cancer (10). We discovered that frequency and extent of epithelial invasion in mucinous carcinoma in Rag2–/– mice was accelerated and greatly exacerbated by prior adoptive transfer of CD4+CD45RBloCD25+ lymphocytes derived from donors lacking IL-10 (7) and that male mice were more susceptible to CRC than females (7). To determine whether carcinogenic effects of IL10-deficient lymphocytes require pathogenic bacterial challenge, as in some mouse models of IBD (6,9), we first compared H.hepaticus-infected and uninfected Rag2–/– recipients of IL10–/– TR cells. Starting as early as 6 weeks PI, colonic carcinoma developed only in animals infected with H.hepaticus (n = 10, Figure 1C; n = 12, Figure 1D). When examined at 6–8 weeks PI, >80% of male mice infected with H.hepaticus and receiving IL10–/– TR cells had >0.3 cm diameter locally invasive mucinous tumors in colon. In contrast, only minimal changes in epithelial morphology are found in uninfected recipients of IL10–/– TR cells (n = 10, Figure 1A; n = 10, Figure 1B). These findings match published data from other murine models (9,17–19) and suggest that risk for intestinal carcinoma and invasive sequellae may increase in susceptible humans after challenge with similar pathogenic bacteria.


Figure 1
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  		Fig. 1. Histopathology of bowel. Photomicrographs of ascending and transverse colon of (A) uninfected Rag2–/– mice (n = 10); (B) uninfected Rag2–/– mice + IL10-deficient CD4+CD25+ regulatory cells (n = 10); (C) Rag2–/– mice infected with Helicobacter hepaticus (n = 10); (D) Rag2–/– mice + IL10-deficient CD4+CD25+ cells, then infected with H.hepaticus, then examined at 6–8 weeks PI (n = 12); (E) Rag2–/– mice infected with H.hepaticus given wt IL10-competent regulatory cells (n = 10); (F) Rag2–/– mice given IL10-deficient CD4+CD25+ cells and infected with H.hepaticus, then given IL10-competent regulatory cells (n = 10); (G) Rag2–/– + IL10-deficient CD4+CD25+ cells infected with H.hepaticus, then given IL10-Ig (n = 10); (H) Rag2–/– mice given IL10-deficient CD4+CD25+ cells and infected with H.hepaticus, then treated with anti-IL6 antibody. There were significant increases in colitis and carcinoma in Rag2–/– mice treated with IL10-deficient CD4+CD25+ cells, but only after infection with H.hepaticus. When IL6 was neutralized or when IL10 was introduced, either through adoptive transfer of lymphocytes or treatment with fusion protein at 6 weeks PI, there were significant decreases in colonic inflammation and dysplasia, as well as evidence of regression of invasive neoplastic epithelia. 4X, H&E; bar = 250 µm

 
Colonic carcinoma in these mice demonstrated a highly invasive mucinous phenotype (Figure 1D) that appeared to arise from areas of severe colitis. Neutrophils were the predominant inflammatory cell type associated with both ulcerative and invasive epithelial lesions in these mice. The rapid (at 6–8 weeks PI) and uniform (>80% penetrance) phenotype of macroscopically evident carcinoma in male mice led us to utilize this mouse model to determine whether immune cells and cytokines may modulate epithelial invasion and provide insight into development and regression of certain types of tumors.

IL-10-deficient CD4+CD25+ cells express Foxp3 and localize in colonic tumors
We described previously that adoptive transfer with IL10-deficient regulatory (TR) cells rapidly induces colonic carcinoma, resembling that seen in H.hepaticus-infected Rag2–/– mice receiving pro-inflammatory CD4+CD45RBhi effector lymphocytes (7). However, from those studies, it was unclear whether CD4+CD45RBloCD25+ cells collected from IL10-deficient donors have a regulatory cell identity, as defined by the expression of the forkhead/winged-helix family member Foxp3 (20,21). In order to determine this, Foxp3 status of purified lymphocytes was examined in spleen tissue derived from IL10-deficient and wt mice, and found to be comparable (9.34 and 9.43% of CD4+ cells, respectively) and approximating the anticipated 10% frequency (21) in both genotypes of mice (Figure 2A and B). Interestingly, immunohistochemistry of colonic tissue revealed that recipients of IL10–/– TR cells had numerous Foxp3+ cells scattered throughout the mucinous carcinoma masses (Figure 2C), matching findings of Foxp3+ cells in IBD patients (22,23). In contrast, wt IL10-competent Foxp3+ cells, which effectively suppress IBD and carcinoma, were relatively infrequent and localized only within the lymphoid follicles (Figure 2D) of quiescent colonic mucosa. These data indicate that expression of Foxp3 in TR cells does not necessarily correlate with a normally functioning immune-suppressive phenotype.


Figure 2
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  		Fig. 2. IL10-deficient CD4+CD25+ TR cells are Foxp3+ and localize in tumors. Flow cytometry analysis plots of splenocytes from mice used as lymphocyte donors. Total splenocytes were harvested from groups of IL-10-deficient (A) and WT (B) mice (n = 4) and stained with CD4, CD25 and Foxp3 as described in Materials and methods and analysed by flow cytometry. The percent of CD4+ cells that express Foxp3 is shown in the upper right corner. Plots depict results from individual animals representative of each group (n = 4). (C and D) Foxp3-specific immunohistochemistry of colon sections from Helicobacter hepaticus-infected Rag2–/– mice that received CD4+ CD25+ cells derived from lymphocyte populations matching those shown in plot A (C) or plot B (D). The inflammatory infiltrate at the invasive front of the mucinous tumor shown in (C) includes a large number of IL10–/– cells showing strong Foxp3 immunoreactivity. Note, intense nuclear brown-colored signal in (C) inset. In contrast, transfer of IL10-competent WT CD4+ CD25+ cells prevent and suppress hepaticus-induced colitis and cancer (D) resulting in normalized colon with small numbers of Foxp3-positive cells. Those cells localized mainly in lymphoid follicles (D inset). 3,3-diaminobenzidine, hematoxylin counterstain. Bars, 100 µm (C and D); 25 µm (C and D insets).

 
IL-10-competent TR cells abrogate colitis and reverse epithelial invasion
We demonstrated previously that adoptive transfer with IL10-competent wild type (wt) regulatory (TR) cells inhibits development of carcinoma in H.hepaticus-infected Rag2–/– mice (7). However, from those studies, it was unclear whether competent TR cells are able to restore epithelial homeostasis in recipients with advanced carcinoma with localized invasion in the peritoneal cavity. In order to determine this, mice underwent subsequent adoptive transfer with 3 x 105 wt (IL10 competent) TR cells per recipient at 6–8 weeks after initial transfer of IL10–/– cells and infection with H.hepaticus. The timing of 6 weeks PI corresponds to mucinous carcinoma in >80% of mice receiving IL10–/– cells and H.hepaticus during four repetitions with at least 10 mice per group. When examined at 1 week after treatment with wt TR cells, mice with invasive tumors that received TR cells (n = 10) had significantly less colitis (P < 0.01) and cancer (P < 0.001) than untreated controls (n = 12) (Figure 1 and Table I).


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  		Table I. Histopathology scores in colon according to treatment

 
A second cohort of H.hepaticus-infected recipients of IL10–/– TR cells was examined at a later interval of 12 weeks PI when frequency of mucinous peritoneal invasion was 100% (10/10). Use of high-resolution ultrasound imaging allowed in vivo visualization of intestinal tumors (Figure 3A) before (Figure 3B) and 1 week after (Figure 3F) treatment with wt TR cells. Postmortem examination confirmed normal bowel morphology and epithelial histology (Figure 3C–E) at 14 days after transfer of wt TR cells. The individual animal shown in Figure 3 had restored appetite and activity and gained 9 g (from 18.5 to 27.8 g) of body weight during the 14 days after transfer of wt TR cells. Antitumor-suppressive effects of wt TR cells were prolonged such that other mice examined up to 18 months after wt TR cell transfer had no evidence of carcinoma (n = 4; data not shown). Taken together, these data indicate that IL10-competent TR cells not only prevent carcinoma but also suppress epithelial invasion and promote re-epithelialization following microbially induced mucosal injury.


Figure 3
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  		Fig. 3. Regression of mucinous adenocarcinoma in colon after treatment with wt regulatory T cells. Images depicting restoration of normal intestinal morphology at 2 weeks after transfer of 3 x 105 IL10-competent TR cells. (A) Transverse colon of Rag2–/– mice receiving IL10-deficient TR cells and infected with Helicobacter hepaticus, shown here at 12 weeks PI (n = 10) corresponding to pre-treatment status of bowel. Mucinous colonic carcinoma invading peritoneal cavity was present in 100% (10/10) of male mice at 12 weeks PI. The area in the box is shown in the higher magnification inset. Note, neoplastic epithelium within mucin pool and juxtaposed remnants of peritoneal fat. (B) In vivo imaging of abdominal cavity using high-resolution ultrasound of a Rag2–/– mouse with IL10–/– TR cells and also infected with H.hepaticus (as in A), examined prior to treatment at 12 weeks PI. Note right kidney (open arrow) and colonic mucinous mass (white arrow). (C) Entire bowel (cecum and colon) of the same mouse as in (B) upon necropsy at 2 weeks after treatment with wild-type TR cells. (D and E) Photomicrographs from different areas of the transverse part of the colon shown in (C). The overview of the largest part of transverse colon is highly suggestive for the absence of neoplastic lesions (D). Multiple serial sectioning of the paraffin block containing the whole bowel shown in (C) revealed only one area in the entire large bowel showing minute neoplastic epithelium and mucin pools within submucosa (arrow head in E). Those structures most probably represent remnants after regression of the tumor shown in (B). (F) In vivo high-resolution ultrasound image of the abdominal cavity of the same mouse as in (B) at 1 week after adoptive transfer of wild-type T regulatory cells. (A, D and E): hematoxylin and eosin; Bars, (A and D) = 1000 µm; (A) inset = 25 µm and (E) = 250 µm.

 
Although TR cells in this study were derived from naive helicobacter-free donors, epithelial ulceration was not seen in any (0/40) recipients of 3.0 x 105 wt TR cells. Prior knowledge that microbes or microbial products enhance survival and proliferation of TR cells prompted us to question whether prior microbial challenges in donor mice may further enhance anti-neoplastic protection of transferred TR cells in this model. To test this possibility, we adapted a titration assay (14,19) utilizing a suboptimal lower dose of 1 x 105 cells per recipient of CD4+CD45RBloCD25+ wt TR cells. This lower dose regimen of TR cells was transferred from donors either uninfected or previously infected with H.hepaticus 8 weeks earlier separately into Rag2–/– recipients of IL10–/– TR cells (n = 8) at the time of infection with H.hepaticus. We found that TR cells prepared from H.hepaticus-infected donors (supplementary figure is available at Carcinogenesis Online) were significantly (P < 0.05) more effective at inhibiting inflammation-associated bowel pathology than those derived from naive donors. While mucinous differentiation was typically absent from recipients of the lower dosage of cells from either infected or naive cell donors, an important contrast between treatment groups was the complete lack of epithelial ulceration among mice receiving TR cells from H.hepaticus-exposed cell donors. There was extensive ulceration remaining only in bowel of mice that received the lower dose of naive TR cells, which were collected from mice not previously exposed to Helicobacter sp bacteria. Taken together, this suggests that IL-10-competent hosts benefit from prior enteric bacterial challenges through enhanced ability of TR cells to prevent epithelial ulceration and neoplastic sequellae.

IL-10 reverses epithelial invasion and restores epithelial homeostasis
We next examined whether exogenous IL10 was sufficient to reverse malignancy and restore epithelial homeostasis in recipients of IL10–/– cells. Mice with established mucinous colonic carcinoma at 6–8 weeks PI were dosed twice weekly intraperitoneally with recombinant IL10-IgG2a fusion protein (IL10-Ig). A similar fusion protein was shown previously to exhibit IL10-like activity in vitro and in vivo (24). Colonic tissues were then examined at 1 week after onset of treatment. Recipients of IL10-Ig fusion protein (n = 10) had minimal colitis (P < 0.01) and no invasive cancer (Figure 1G and Table I), when compared with untreated mice or recipients of isogenic sham antibody alone (n = 8; data not shown). Assessment of oncogene expression using qRT–PCR (TaqMan) revealed that K-ras was up-regulated in H.hepaticus-infected mice with carcinoma (Figure 4), matching data from humans with mucinous CRC (3), and that K-ras returned to baseline after treatment with IL10-Ig fusion protein (Figure 4). These data suggest that IL10 restores epithelial homeostasis in mice through down-regulation of bacteria-triggered carcinogenic pro-inflammatory cytokines.


Figure 4
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  		Fig. 4. Relative mRNA levels of IL-6, TNF-{alpha} and K-ras. In each sample, the target mRNA was normalized to that of the ‘house-keeping’ gene Gapdh. Numbers represent mean fold change of the individual mRNA levels in reference to the control group (defined as 0 for no change) with _ representing the standard deviation. Expression of IL6, but not TNF-{alpha}, was significantly correlated with neoplastic epithelial invasion in this mouse model when examined at 4–6 weeks PI. Expression of K-ras was also correlated with neoplastic epithelial invasion, and treatment with IL10-Ig fusion protein returned oncogene expression to baseline in this mouse model. Bars, standard deviations. P values using the unpaired Student's t-test: *P < 0.05; **P < 0.01 and ***P < 0.001.

 
IL-6 is associated with a reversible neoplastic epithelial phenotype
We hypothesized that uncontrolled up-regulation of pro-inflammatory cytokines (14,25–27), such as TNF-{alpha} and IL-6, contribute to inflammation-associated carcinogenesis. Indeed, treatment with TNF-{alpha}-neutralizing antibody is sufficient to significantly (P < 0.05) reduce epithelial invasion and restore epithelial homeostasis in H.hepaticus-infected Rag2–/– recipients of IL10–/– cells (n = 8), when compared with sham antibody-injected matched control mice (n = 8), as shown previously in other immune-deficient mouse models (14). However, a downstream pleiotropic cytokine, IL6, has also clearly been linked with neoplastic epithelial invasion in colon (28,29) and other sites in humans and mice. To further investigate roles for bacteria-triggered inflammatory cytokines, we examined pro-inflammatory cytokine gene expression using qRT–PCR in H.hepaticus-infected mice at 6–8 weeks PI. Significant elevations in IL6 (Figure 4) but not TNF-{alpha} gene expression were observed among groups of mice with rapidly invading epithelia. Adoptive transfer with wt TR cells suppressed pathology and pro-inflammatory cytokine expression; in contrast, supplementation with regulatory cells lacking IL-10 up-regulated expression of IL-6 (Figure 4) and increased frequency of neoplastic invasion (Figure 1). In addition, IL6 protein was elevated (n = 4; µ = 61.4 pg/ml) in sera of H.hepaticus-infected mice with colitis and cancer, but not in Helicobacter-free counterparts (n = 4; µ = 4.28 pg/ml). Taken together, these data indicate that localized and systemic up-regulation of cytokine IL-6 was linked with an invasive epithelial phenotype following intestinal microbial infection.

Epithelial oncogene expression is associated with a reversible neoplastic phenotype
Disruption of Tgfβ signaling (30) is a common feature of CRC in humans (2). Given that the mucinous colonic phenotype of our mice is similar to that reported previously in H.hepaticus-infected Tgfβ1-knockout mice (31,32), we sought to determine whether Tgfβ signaling was disrupted after pathogenic bacterial infection in our model. We analysed the expression of Tgfβ pathway members (30) in H.hepaticus-infected Rag2–/– recipients of IL10–/– cells using qRT–PCR on purified colonic epithelia. We found that Tgfβ1 was significantly over-expressed in mice 4–6 weeks after infection with H.hepaticus (Figure 5). Treatment with IL10-Ig led to normalized expression of Tgfβ1. We also analysed the expression of TgfβRI, TgfβRII and Smad4 in purified colonic epithelia. The expression of TgfβRI was not significantly affected by infection with H.hepaticus. In contrast, expressions of TgfβRII and Smad4 were significantly increased in mice infected with H.hepaticus. The over-expression of Tgfβ1, TgfβRII and Smad4 may be compensatory for a defect further downstream in the signaling pathway. This hypothesis is consistent with the finding that TgfβRII and Smad4 are also over-expressed in H.hepaticus-infected Tgfβ1-knockout mice that develop mucinous colonic carcinoma (Figure 5).


Figure 5
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  		Fig. 5. Gene expression analysis. The relative expression of genes in purified colonic epithelium was measured using qRT–PCR (TaqMan) analyses. The expression level of each gene was normalized to that of Tbp and then compared with its expression in uninfected Rag2–/– mice. Treatment groups are as follows: uninfected Rag2–/– mice + IL10-deficient regulatory cells (white); Rag2–/– mice + IL10-deficient regulatory cells infected with Helicobacter hepaticus (black); Rag2–/– mice + IL10-deficient regulatory cells infected with H.hepaticus then given IL10-Ig fusion protein (light gray) and Tgfβ1–/– Rag2–/– infected with H.hepaticus (dark gray). There is a 5.8-fold increase in the expression of Tgfβ1 in animals infected with H.hepaticus relative to uninfected mice. The expression of TgfβRII and Smad4 was also slightly higher in infected animals, whereas TgfβRI did not change. The expression of c-Myc was significantly higher in Tgfβ1–/–; Rag2–/– mice infected with H.hepaticus, but was unchanged in mice with WT Tgfβ1. In contrast, the expression of Bcl3 was significantly higher in animals infected with H.hepaticus. The oncogene whose expression was most affected by H.hepaticus infection was Pim1, with a 20-fold increase in expression level. This increase in Pim1 expression is coincident with a very large increase in IL6 expression. Asterisks denote statistical significance when compared with uninfected Rag2–/– mice (*P < 0.05 and **P < 0.01). There is a 5.8-fold increase in the expression of Tgfβ1 in animals infected with H.hepaticus relative to uninfected mice. The expression of TgfβRII and Smad4 was also slightly higher in infected animals, whereas TgfβRI did not change. The expression of c-Myc was significantly higher in Tgfβ1–/–; Rag2–/– mice infected with H.hepaticus, but was unchanged in mice with WT Tgfβ1. In contrast, the expression of Bcl3 was significantly higher in animals infected with H.hepaticus. The oncogene whose expression was most affected by H.hepaticus infection was Pim1, with a 20-fold increase in expression level. This increase in Pim1 expression is coincident with a very large increase in IL6 expression. Asterisks denote statistical significance when compared with uninfected Rag2–/– mice (*P < 0.05 and **P < 0.01).

 
We postulated that other epithelial oncogenes downstream of IL-10 and Tgfβ may contribute to the neoplastic phenotype in H.hepaticus-infected mice. When examined at 6–8 weeks PI, purified colonic epithelia from H.hepaticus-infected mice had significant increases in the expression of Pim1, but not Bcl3 or c-Myc (Figure 5). The expression of Bcl3 is only slightly elevated in infected mice when compared with Pim1, which demonstrated a 20-fold increase in expression level after infection. Interestingly, changes in Pim1 gene expression were evident throughout the entire colonic epithelium and not restricted to the invasive carcinomas. It is noteworthy that IL6 and Pim1 are similarly dysregulated in Rag2–/– mice entirely lacking Tgfβ1 (Figure 5) that are also highly susceptible to H.hepaticus-induced mucinous carcinoma (31). The over-expression of IL6 and oncogene Pim1 is especially intriguing given the proposed roles for these factors in prostate cancer in men (33), and our prior finding that male mice are predisposed to H.hepaticus-induced CRC.

The balance between IL-6, IL-10 and Tgfβ predicts epithelial phenotype
We hypothesized that bacteria-triggered up-regulation of IL-6 contributes to a neoplastic colonic epithelial phenotype in helicobacter-infected male animals. To determine whether IL10 restores epithelial homeostasis through down-regulation of IL6, we examined mice that received IL10-Ig fusion protein. Recipients of IL10-Ig fusion protein demonstrated recovery of normalized IL6 expression in colonic tissue (Figure 4), within days of the onset of treatment (data not shown), and coincident with reversion to normal epithelial morphology. Likewise, elevated serum IL6 protein levels returned to near baseline following 1 week of treatment with IL10-Ig (n = 4; µ = 11 pg/ml). This matches prior data showing that IL 10 down-regulates IL 6 in colitis in humans and mice (13,34–36) and led us to propose a mechanistic overview as shown in Figure 6.


Figure 6
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  		Fig. 6. Proposed mechanistic overview. Pathogenic gut bacteria trigger pro-inflammatory cytokines including TNF-{alpha} and IL-6. IL-10 down-regulates bacteria-triggered IL-6 and prevents development of carcinoma. Whether wt TR cells simply secrete IL-10 or require IL-10 for recruitment and anti-inflammatory functions is undetermined.

 
It was next tested whether treatment with IL6-neutralizing antibodies restores epithelial homeostasis. Treatment with anti-IL6 antibody alone induced significant (P < 0.05) regression of mucinous carcinoma (Figure 1H and Table I) in colonic epithelia of H.hepaticus-infected mice. Treatment with IL 6-neutralizing antibody significantly (P < 0.01) down-regulated epithelial K-ras and Pim 1 gene expression (data not shown), which suggests that epithelial oncogene expression in mice is readily modulated by bacterially induced pro-inflammatory gene expression, and that oncogene expression is readily reversible through activities of anti-inflammatory cells or cytokines.


   Discussion
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We show here that subserosal epithelial invasion resembling mucinous colonic adenocarcinoma in humans arises from bacterially triggered inflammation and depends upon effects of IL-6. Supplementation with IL10-competent regulatory TR cells inhibited ulceration and invasion and restored epithelial homeostasis through interrelated activities of IL 10 and Tgfβ signaling. The rapid restoration of epithelial integrity after neoplastic invasion indicated that TR cell-mediated remodeling and ability to restore and maintain epithelial homeostasis (6,37,38) may be more extensive than previously thought. In human patients, similar mucinous tumors carry a poorer prognosis than other types of CRC (4,5). Thus, harnessing constructive endogenous host immune-modulatory capabilities may provide adjunct therapies for IBD-associated CRC and similar neoplastic processes.

Induction of colonic tumors in these mice required pathogenic intestinal bacteria and was greatly exacerbated by the addition of IL10-deficient CD4+CD45RBloCD25+ TR cells. In this situation, IL-6 was significantly up-regulated by addition of Foxp3+ IL10–/– lymphocytes rather than suppressed as with the wt IL10-competent TR cell counterparts. The finding that TR cells collected from IL10–/– donors demonstrated Foxp3 (21) reveals that Foxp3 expression does not uniformly equate with immune-suppressive function in lymphocytes. The observation that IL10-deficient Foxp3+ TR cells accumulated in tumors matches the data in patients with intestinal and other cancers (39). In contrast, wt IL10-competent Foxp3+ cells were identified only in lymphoid follicles of quiescent colonic mucosa (because prevention of colitis and CRC after wt TR cells approached 100%). However, unpublished data from our laboratory indicate that normally functioning wt TR cells may also accumulate in H.hepaticus-induced colonic tumors in Rag2–/– mice. For example, Foxp3+ wt TR cells accumulate at early time points during the tumor regression process (data not shown). Importantly, not all physiologically relevant IL10-secreting regulatory cells express Foxp3 (40). Indeed, peripherally recruited IL10-dependent CD45RBloCD25-Treg subsets may not express Foxp3 but are shown to be potent down-regulators of H.hepaticus-induced IBD (19). Future studies will examine whether regulatory cells with bacterial antigen specificity (19) may provide enhanced antitumor protection. These cells may act synergistically with CD25+ cells to optimize epithelial wound repair (40). CD4+CD25+ cells utilized for the present experiments are derived from spleen and mesenteric lymph nodes of adult mice and are likely to include diverse regulatory subsets.

In the context of cancer as ‘a wound that does not heal’ (41), the present data indicate that constructive interplay between TR cells and epithelia are essential for epithelial cancer prevention. This is not surprising given the earlier findings (6,38) showing that TR cells are important for intestinal homeostatis. In this capacity, TR cells function, at least in part, through coordinated activities of cytokines IL 10 and Tgfβ1 (34) that are otherwise linked with epithelial wound repair (30,42,43). There is considerable prior evidence identifying complex and interrelated roles for IL 10 and Tgfβ among lymphocytes (44) and in epithelia (45) in mice with colitis. Within gut epithelial cells, IL 10 was shown previously to be required for normal Tgfβ signaling and SMAD-complex translocation following intestinal bacterial infection with colitogenic Enterococcus fecalis (45). Thus, H.hepaticus infection may trigger a similar Tgfβ signaling blockade within enteric epithelia of Rag2-deficient mice mimicking the neoplastic phenotype in Tgfβ1-deficient (31) and SMAD3-deficient (46,47) mice. An interesting possibility is that IL 10 expression in lymphocytes is required for proper signaling and elaboration of Tgfβ in our model. The precise relationships between IL 10 and Tgfβ within the tumor microenvironment remain unclear and are the subject of future investigations.

It is interesting that murine mutants with genetic disruptions in the Tgfβ signaling pathway, i.e. deficiency in Tgfβ1 (31) or SMAD3 (47), are also highly susceptible to H.hepaticus-induced colitis and neoplastic invasion. It is probable that the carcinoma phenotype in these models also arises from insufficiently down-regulated inflammatory response to bacterial challenge, i.e. cytokines such as IL-6, and downstream oncogenes such as K-ras rather than bacteria burden per se. Indeed, earlier data showed that H.hepaticus colonization levels were not significantly different between untreated Rag2–/– mice and recipients of competent TR cells or their IL10-deficient counterparts (7,9). The present finding of elevated TβRII gene expression matches the molecular signature of tumors within pancreas, uterus and other sites in humans (48), raising the possibility of a similar etiopathogenesis.

Neutralization of TNF-{alpha} activity alone is sufficient to suppress carcinogenesis in this and other murine models (14,25), indicating that TNF-{alpha} over-expression is necessary to sustain carcinoma. In chronic inflammatory diseases such as IBD and arthritis, it is proposed that prolonged elevations in TNF-{alpha} arising from bowel induce thymic and peripheral TR cell insufficiency—leading to further deterioration of health due to unmitigated systemic elevations in inflammatory factors (49). Therapeutic interventions in this setting would then focus upon strategies that promote or restore immune homeostasis in the bowel. Population-based cancer prevention may focus upon enhancing anti-inflammatory functions and potency of relevant regulatory cell subsets. Considering the relatively short half-life for IL-10 fusion protein (13), exogenous IL-10 supplementation is perhaps better suited for selective interventions to stabilize a dysregulated bowel.

The present data support a model that subscribes to the activation of downstream cytokines, such as IL-6, rather than TNF-{alpha} per se, that are directly linked with the invasive phenotype. Along these lines, one potential therapeutic target is the STAT3 signaling molecule, which appears pivotal in IL-6-mediated progression of malignancy (36,50,51). Carcinogenic effects of IL-6 may also be modulated by oxidative stress and other effects of neutrophils, which are prevalent in invasive epithelial foci (7) and shown previously to be highly responsive to up-regulation of IL 6 (52). Not discounting the risk of neutropenia, therapeutic blockade of IL 6 may be less disruptive to host-protective immunity than neutralization of TNF-{alpha} or supplementation with IL10-Ig for treatment of arthritis and other systemic immune disorders. Gut bacteria-triggered elevations in circulating IL 6 levels, as documented in the present mice, may destructively impact systemic immune status and carcinogenic processes in distant organs. IL 6 may contribute to generalized immune impairment through inhibition of anti-inflammatory functions of TR cells (29,34,36,50). Beyond IL-6-mediated disruption of Tgfβ expression in T lymphocytes (50), IL-6 may drive TR cells toward a pathogenic T helper (Th)-17 phenotype (36).

IL-10, whether integrated with activities of regulatory cells or not, appears to be fundamental in directly down-regulating TNF-{alpha} and IL-6 (see Figure 6), in addition to stabilizing Tgfβ signaling (45), during constructive wound repair and prevention of cancer. Whether TR cells simply secrete IL10 in this setting or also require IL10 for proper development and recruitment of cells with anti-inflammatory phenotype is unclear. It has been shown elsewhere that IL-10 facilitates recruitment of peripheral CD4+ cells to a regulatory phenotype that suppresses ongoing inflammatory processes (40,53). In spite of overwhelming evidence that IL 10 down-regulates pathogenic sequellae of gut microbial infections (35), roles for IL-10 in cancer development and progression are far from clear. For example, IL-10 is also a well-documented suppressor of antitumor immunity (36).

The finding that TR cells induce regression of carcinoma in this setting also conflicts with other data showing that regulatory T cells inhibit beneficial anti-cancer inflammatory responses (39,54). Whether outcomes differ due to varying etiopathogeneses, diverse regulatory T cell subsets, divergent tumor sites or host immune competency—as present studies utilize Rag2-deficient mice otherwise lacking lymphocytes—is unclear. It will be interesting to test whether supplementation with TR cells is able to rescue a mucinous colon carcinoma phenotype in other mouse models such as H.hepaticus-infected SMAD3-deficient mice that have a full repertoire of lymphocytes (47), as has been shown with TR cells in ApcMin/+ mice (37). In immune-competent humans, it is probable that dietary, hormonal and stress factors all contribute—along with bacterial triggers and ensuing immune dysregulation—to a multi-factorial process leading to cancer.

The over-expression of oncogene Pim1 in colonic carcinoma is intriguing, given its proposed role in prostate cancer in men (33), and the increased susceptibility of male mice to H.hepaticus-induced CRC in this model (7). In human male patients, IL 6 up-regulation is linked with prostatitis and prostate cancer and IL 6 is known to regulate Pim1 (33). This is the first report connecting expression of Pim1 with colitis-associated colon cancer. Reversible up-regulation of epithelial oncogenes correlates with the histologic status of the colonic epithelium in our model and suggests that sustained over-expression of oncogenes may be required for cancer. Chin et al. (55) previously demonstrated that sustained over-expression of oncogenic H-ras is required to maintain melanoma growth in transgenic mice (55). In the present model, expression of K-ras was dependent upon bacteria-triggered cytokine IL 6. Targeted deletion of candidate oncogenes such as Kras and Pim1 is needed to determine whether oncogene expression is coincident or required for tumor maintenance in inflammation-associated carcinoma.

In summary, we have demonstrated that invasive colonic carcinoma is rapidly reversible through IL-10-mediated restoration of epithelial homeostasis. The ability of competent TR cells to normalize epithelial signaling and restore epithelial homeostasis substantiates links between host immunity, epithelial homeostasis and malignancy. Adoptive transfer of IL10-competent regulatory cells also improved overall body condition and activity level of recipient mice, perhaps providing insights into links with systemic health. The finding that microbially triggered colitis induces universal up-regulation of IL-6 highlights possible roles for IL-6-mediated inflammatory responses throughout the body. Because dysregulation of IL-6 is a frequent feature of invasive malignancies, it will be important to consider and target immune-mediated effects among the steps initiating or modulating cancer and associated neoplastic invasion in humans.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
Supplementary material
 References
 
To S.E.E. (R01CA108854), to J.G.F. (R01CA67529 and R01AI51404), to B.H.H. and S.E.E. (R01AI052267), to T.P. (EU and GMNERA Pythagoras II 80860), to J.G.F. and S.E.E. (P01CA26731) and to J.G.F. (P30 ES02109 and T32RR07036).


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Supplementary material
References
 
Supplementary material can be found at http://carcin.oxfordjournals.org/


   Acknowledgments
 
We want to express our gratitude to Shilu Xu and Nancy S.Taylor for assistance with culturing H.hepaticus. We thank Erica B.Jarmon for technical assistance with the mice. We appreciate the help of Dr Marshall S.Horwitz with purification of the adenovirus vector. We also thank Glenn A.Paradis and Michael J.Jennings for assistance with cell sorting, and Kathy Cormier, Jeff Bajko, Erinn Stefanovitch and Chakib Boussahamian for assistance with histology and immunohistochemistry. Finally, we thank Sue Liang and Elaine Robbins for assistance with the manuscript preparation. K.H. is a Robert Black Fellow of the Damon Runyon Cancer Research Foundation.

Conflict of interest statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 Supplementary material
 References
 

  1. American Cancer Society. US Public Health Service. US Cancer Facts and Figures, (2004). ACS.
  2. Ming S. Malignant epithelial tumors of the intestines. In: Pathology of the Gastrointestinal Tract—Ming S-C, Goldman H, eds. (1998) Baltimore, MD: Williams & Wilkins. 855–898.
  3. Hanski C. Is mucinous carcinoma of the colorectum a distinct genetic entity? Br. J. Cancer (1995) 72:1350–1356.[Web of Science][Medline]
  4. Okuno M, et al. Mucinous colorectal carcinoma: clinical pathology and prognosis. Am. Surg. (1988) 54:681–685.[Web of Science][Medline]
  5. Minsky BD, et al. Colloid carcinoma of the colon and rectum. Cancer (1987) 60:3103–3112.[CrossRef][Web of Science][Medline]
  6. Erdman SE, et al. CD4+CD25+ regulatory t lymphocytes inhibit microbially-induced colon cancer in Rag2-deficient mice. Am. J. Pathol. (2003) 162:691–702.[Abstract/Free Full Text]
  7. Erdman SE, et al. CD4+CD25+ regulatory lymphocytes require interleukin 10 to interrupt colon carcinogenesis in mice. Cancer Res. (2003) 63:6042–6050.[Abstract/Free Full Text]
  8. Powrie F. T cells in inflammatory bowel disease: protective and pathogenic roles. Immunity (1995) 3:171–174.[CrossRef][Web of Science][Medline]
  9. Maloy K, et al. CD4+CD25+ Tr cells suppress innate immune pathology through cytokine-dependent mechanisms. J. Exp. Med. (2003) 197:111–119.[Abstract/Free Full Text]
  10. Boivin GP, et al. Pathology of mouse models of intestinal cancer: consensus report and recommendations. Gastoenterology (2003) 124:762–777.[CrossRef][Web of Science][Medline]
  11. Fox JG, et al. Helicobacter hepaticus species.nov. a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J. Clin. Micro. (1994) 32:1238–1245.[Abstract/Free Full Text]
  12. Mihara M, et al. CTLA4Ig inhibits T cell-dependent B-cell maturation in murine systemic lupus erythematosus. J. Clin. Invest. (2000) 106:91–101.[Web of Science][Medline]
  13. Tomczak M, et al. Inhibition of inflammatory responses by IL-10 depends upon the presence of the p50/p105 subunit of NF-{gamma}B1. J. Immunol. (2006) 177:7332–7339.[Abstract/Free Full Text]
  14. Rao VP, et al. Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice. Cancer Res. (2006) 66:7395–7400.[Abstract/Free Full Text]
  15. Berg DJ, et al. Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4(+) TH1-like responses. J. Clin. Invest. (1996) 98:1010–1020.[Web of Science][Medline]
  16. Whitehead RH, et al. Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans. Gastroenterology (1999) 117:858–865.[CrossRef][Web of Science][Medline]
  17. Maloy KJ, et al. Cure of innate intestinal immune pathology by CD4+CD25+ regulatory T cells. Immunol. Lett. (2005) 97:189–192.[CrossRef][Web of Science][Medline]
  18. Kullberg MC, et al. Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12- and gamma interferon-dependent mechanism. Infect. Immun. (1998) 66:5157–5166.[Abstract/Free Full Text]
  19. Kullberg MC, et al. Bacteria-triggered CD4+ T regulatory cells suppress Helicobacter hepaticus-induced colitis. J. Exp. Med. (2002) 196:505–1115.[Abstract/Free Full Text]
  20. Hori S, et al. Control of regulatory T cell development by the transcription factor Foxp3. Science (2003) 299:1057–1061.[Abstract/Free Full Text]
  21. Banham AH, et al. FOXP3+ regulatory T cells: current controversies and future perspectives. Eur. J. Immunol. (2006) 36:2832–2836.[CrossRef][Web of Science][Medline]
  22. Maul J, et al. Peripheral and intestinal regulatory CD4+ CD25 (high) T cells in inflammatory bowel disease. Gastroenterology (2005) 128:1868–1878.[CrossRef][Web of Science][Medline]
  23. Ling KL, et al. Increased frequency of regulatory T cells in peripheral blood and tumour infiltrating lymphocytes in colorectal cancer patients. Cancer Immun. (2007) 7:7.[Medline]
  24. Zheng XX, et al. Administration of a noncytolytic IL10-Fc in murine models of lipopolysaccharide-induced septic shock and allogeneic islet transplantation. J. Immunol. (1995) 154:5590–5600.[Abstract]
  25. Rao VP, et al. Proinflammatory CD4+ CD45RB(hi) lymphocytes promote mammary and intestinal carcinogenesis in Apc(Min/+) mice. Cancer Res. (2006) 66:57–61.[Abstract/Free Full Text]
  26. Luo JL, et al. Inhibition of NF-kappaB in cancer cells converts inflammation-induced tumor growth mediated by TNFalpha to TRAIL-mediated tumor regression. Cancer Cell (2004) 6:297–305.[CrossRef][Web of Science][Medline]
  27. Balkwill F, et al. Cancer: an inflammatory link. Nature (2004) 431:405–406.[CrossRef][Medline]
  28. Smith PC, et al. Interleukin 6 and prostate cancer progression. Cytokine Growth Factor Rev. (2001) 12:33–40.[CrossRef][Web of Science][Medline]
  29. Scheller J, et al. Interleukin-6 trans-signalling in chronic inflammation and cancer. Scand. J. Immunol. (2006) 63:321–329.[CrossRef][Web of Science][Medline]
  30. Massague J, et al. TGFb signlaing in growth control, cancer, and heritable disorders. Cell (2000) 103:295–309.[CrossRef][Web of Science][Medline]
  31. Engle SJ, et al. Elimination of colon cancer in germ-free transforming growth factor beta 1-deficient mice. Cancer Res. (2002) 62:6362–6366.[Abstract/Free Full Text]
  32. Engle SJ, et al. Transforming growth factor beta-1 suppresses nonmetastatic colon cancer at an early stage of tumorigenesis. Cancer Res. (1999) 59:3379–3386.[Abstract/Free Full Text]
  33. Kim O, et al. Synergism of cytoplasmic kinases in IL6-induced ligand-independent activation of androgen receptor in prostate cancer cells. Oncogene (2004) 23:1838–1844.[CrossRef][Web of Science][Medline]
  34. Powrie F, et al. Regulating the regulators. Science (2003) 299:1030–1031.[Abstract/Free Full Text]
  35. Moore KW, et al. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. (2001) 19:683–765.[CrossRef][Web of Science][Medline]
  36. Lin WW, et al. A cytokine-mediated link between innate immunity, inflammation, and cancer. J. Clin. Invest. (2007) 117:1175–1183.[CrossRef][Web of Science][Medline]
  37. Erdman SE, et al. CD4+CD25+ regulatory lymphocytes induce regression of intestinal tumors in ApcMin/+ mice. Cancer Res. (2005) 65:3998–4004.[Abstract/Free Full Text]
  38. Rodriguez-Molinero A, et al. Tissue homeostasis and cancer. Med. Hypotheses (2007) 68:1333–1341.[CrossRef][Web of Science][Medline]
  39. Curiel TJ, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. (2004) 10:942–949.[CrossRef][Web of Science][Medline]
  40. O'Garra A, et al. IL-10-producing and naturally occurring CD4+ Tregs: limiting collateral damage. J. Clin. Invest. (2004) 114:1372–1378.[CrossRef][Web of Science][Medline]
  41. Dvorak HF. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N. Engl. J. Med. (1986) 315:1650–1659.[Web of Science][Medline]
  42. Martin P. Wound healing—aiming for perfect skin regeneration. Science (1997) 276:75–81.[Abstract/Free Full Text]
  43. Crowe MJ, et al. Review of case definitions for nosocomial infection—towards a consensus. Presentation by the Nosocomial Infection Surveillance Unit (NISU) to the Hospital Infection Liaison Group, subcommittee of the Federation of Infection Societies (FIS). J. Hosp. Infect. (1998) 39:3–11.[CrossRef][Web of Science][Medline]
  44. Fuss IJ, et al. The Interrelated roles of TGF-b and IL10 in the regulation of experimental colitis. J. Immunol. (2001) 168:900–908.[Web of Science]
  45. Ruiz PA, et al. IL-10 gene-deficient mice lack TGF-beta/Smad signaling and fail to inhibit proinflammatory gene expression in intestinal epithelial cells after the colonization with colitogenic Enterococcus faecalis. J. Immunol. (2005) 174:2990–2999.[Abstract/Free Full Text]
  46. Maggio-Price L, et al. Dual infection with Helicobacter bilis and Helicobacter hepaticus in p-glycoprotein-deficient mdr1a–/– mice results in colitis that progresses to dysplasia. Am. J. Pathol. (2005) 166:1793–1806.[Abstract/Free Full Text]
  47. Maggio-Price L, et al. Helicobacter infection is required for inflammation and colon cancer in SMAD3-deficient mice. Cancer Res. (2006) 66:828–838.[Abstract/Free Full Text]
  48. Pasche B. Role of transforming growth factor beta in cancer. J. Cell. Physiol. (2001) 186:153–168.[CrossRef][Web of Science][Medline]
  49. Faubion WA, et al. Colitis is associated with thymic destruction attenuating CD4+CD25+ regulatory cells in the periphery. Gastroenterology (2004) 126:1759–1770.[CrossRef][Web of Science][Medline]
  50. Becker C, et al. TGF-beta suppresses tumor progression in colon cancer by inhibition of IL-6 trans-signaling. Immunity (2004) 21:491–501.[CrossRef][Web of Science][Medline]
  51. Corvinus FM, et al. Persistent STAT3 activation in colon cancer is associated with enhanced cell proliferation and tumor growth. Neoplasia (2005) 7:545–555.[CrossRef][Web of Science][Medline]
  52. Hurst SM, et al. Il-6 and its soluble receptor orchestrate a temporal switch in the pattern of leukocyte recruitment seen during acute inflammation. Immunity (2001) 14:705–714.[CrossRef][Web of Science][Medline]
  53. Vieira PL, et al. IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells. J. Immunol. (2004) 172:5986–5993.[Abstract/Free Full Text]
  54. Blattman JN, et al. Cancer immunotherapy: a treatment for the masses. Science (2004) 305:200–205.[Abstract/Free Full Text]
  55. Chin L, et al. Essential role for oncogenic Ras in tumour maintenance. Nature (1999) 400:468–472.[CrossRef][Medline]
Received July 5, 2007; revised July 26, 2007; accepted July 27, 2007.


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