T cell status influences colon tumor occurrence in Min mice fed short chain fructo-oligosaccharides as a diet supplement

Fabrice Pierre, Pascale Perrin, Euphémie Bassonga, Francis Bornet1, Khaled Meflah and Jean Menanteau2

Institut National de la Santé et de la Recherche Médicale U419, Human Nutrition Research Center of Nantes, Institut de Biologie, 9 Quai Moncousu, F-44035 Nantes Cedex 01, France and
1 Eridania Béghin-Say, Vilvoorde Research and Development Centre, Nutrition and Health Service, Havenstraat 84, B-1800 Vilvoorde, Belgium


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have previously shown that addition of short chain fructo-oligosaccharides (indigestible carbohydrates) to food prevented colon tumors in C57BL/6-ApcMin/+ mice, a model for human colon cancer. As gut-associated lymphoid tissue was concomitantly developed, we suggested that the immune response generated by this food may interfere with carcinogenesis due to involvement of mucosal cells in the regulation of tissue homeostasis. In the present experiment, we tested whether T cell status may influence colon tumor formation in Min mice fed a food supplement of short chain fructo-oligosaccharides. Min mice depleted of CD4+ and CD8+ lymphocytes developed twice as many tumors as immunocompetent mice (0.8 as compared with 0.4, the mean number in 7-week-old Min mice when food supplementation began; P = 0.02). It is concluded that food supplementation with a substrate (a known prebiotic) fermented in the colon may stimulate a mechanism of immunosurveillance that would otherwise remain inefficient.

Abbreviations: IEL, intraepithelial lymphocytes; PBS, phosphate-buffered saline; sc-FOSs, short chain fructo-oligosaccharides.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most epidemiological studies have found a negative correlation between dietary intake of fiber and the incidence of colorectal adenoma and cancer, although this was not the case in a large recent series (13). Experimental data are also controversial, probably because the fibers used were heterogeneous and the protocols did not address the same sequence of carcinogenesis. We have recently shown that addition of a chemically well-defined indigestible carbohydrate (short chain fructo-oligosaccharides, sc-FOSs) to food reduced the incidence of colon tumors in Min mice (4), a relevant model for both hereditary and sporadic human colon cancer suitable for nutrition studies (5), whereas other types of fiber were inefficient. sc-FOSs concomitantly induced gut-associated lymphoid tissue, which suggests that immunosurveillance could be involved in this mechanism. sc-FOSs have been documented as a prebiotic that can enhance the population of lactic acid-producing bacteria in the large intestine, which in turn affect local and systemic immune responses. Although results are still controversial, various data in humans (6,7) and animal models (8,9) suggest that immune response is not restricted to cancers of viral origin. In the present experiment, we decided to use sc-FOS-fed Min mice as a model to emphasize the involvement of the immune system in the development of colon tumors. Although immunodeficient animal strains such as nude and scid mice are available, our investigations concerned a process likely to involve the gut flora, which excluded the use of these animal models. Instead, we chose to immunodeplete mice with antibodies and target T cells (CD4+ and CD8+), rather than NK cells, which do not affect the incidence of intestinal neoplasia in Min mice (10).

Our experiment shows that immunocompetent Min mice fed sc-FOSs exhibit a mean number of tumors equivalent to that already present at the start of food supplementation, whereas twice as many tumors developed in their CD4/CD8-depleted counterparts. It is concluded that T cells participate in a mechanism of colon tumor surveillance in Min mice fed sc-FOSs.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Six-week-old C57BL/6J-Min/+ mice (Min mice) were obtained from the Jackson Laboratory (Bar Harbor, ME). Five deliveries of nine animals each (n = 45) were divided randomly into three groups (13) upon reception and housed 1 group/cage.

Diets
All mice were fed the experimental powdered diet previously detailed (4), formulated to provide 5.8% (g/100 g) sc-FOS (glucose–fructosen, n <= 4; Actilight P®; Béghin Meiji Industries, Neuilly sur Seine, France), 19.4% protein (casein), 1.9% corn oil, 6.1% lard, 1.9% cellulose, 59.4% pregelatinized starch, 0.39% methionine, minerals and vitamins.

Protocol
Mice were 7 weeks old at the beginning of the nutritional experiment. Each group was fed a diet ad libidum in protected feeders that was renewed daily for 42 days.

Animals were weighed per cage every week throughout the experiment.

T cell depletion
Thirty mice were first injected i.p. for three consecutive days with 0.5 mg purified monoclonal antibodies (5x3 animals, group 3) or phosphate-buffered saline (PBS) alone as a control (5x3 animals, group 2) and then once a week throughout the experiment. T cell hybridomas were obtained from the ATCC: GK 1.5 ATCC TIB 207 (CD4) and 2.43 ATCC TIB 210 (CD8). Supernatants from cultured hybridomas were purified on a protein G affinity column (PROSEP-G; Bioprocessing, Princeton, NJ).

Scoring of tumors
The mice were killed and the number and size of large intestine (colon and cecum) tumors were scored blind under a dissection microscope in refrigerated PBS.

Purification of intraepithelial lymphocytes (IEL)
IEL were prepared according to a previously published procedure (11). Cecum and colon were used as a single unit as these organs share identical IEL populations (11). Briefly, the large intestine was dissected, opened longitudinally, washed with PBS, cut into small pieces, washed with DMEM medium (Gibco, Grand Island, NY) and incubated for 30 min at 37°C in PBS containing 5 mM EDTA and 5 mM DL-dithiothreitol (Sigma Chemical Co., St Louis, MO), with stirring. Debris was sedimented and the supernatants collected. Cells were pelleted, suspended in 67% Percoll (Pharmacia Fine Chemicals, Piscataway, NJ), overlayed with 44% Percoll and centrifuged at 600 g for 30 min. IEL were recovered from the interface.

Purification of splenic lymphocytes
Freshly isolated spleens were perfused with RPMI medium (Gibco). Cells were recovered by centrifugation, washed with PBS containing 10% fetal calf serum and centrifuged on Ficoll (Seromed, Berlin, Germany) for 30 min at 500 g. Lymphocytes were collected from the interface and washed with PBS supplemented with 10% fetal calf serum.

Immunofluorescence staining and flow cytometric analyses
Briefly, 105 IEL or splenic lymphocytes were incubated in PBS/0.1% (w/v) gelatin with the appropriate conjugated antibody for 30 min at 4°C, washed with PBS/0.1% gelatin and fixed with PBS/1% formalin (w/v). Antigen expression was analyzed using a FACScan (Becton Dickinson, San Jose, CA) with CELLQuest software, drawing light scatter gates around live lymphocyte cell populations. The following antibodies from Pharmingen (San Diego, CA) were used: CD3-FITC (145-2C11), PE-CD4 (RM4-5) and Cy-chrome-CD8a (53-6.7).

Statistical analyses
Tumor counts were evaluated by Fisher's exact test and the {chi}2 test for trend in binomial proportions (13), both tests being two-sided and considered significant for P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal growth was unaffected by repeated injections of PBS or antibodies targeted to CD4+ and CD8+ T cells. The mean number of tumors was not influenced by PBS treatment, indicating that repeated injections into the peritoneum did not affect tumor occurrence per se. Thus, the 30 immunocompetent mice (groups 1 and 2) were subsequently considered as a single group and compared with the 15 immunodeficient mice (group 3). The depletion we obtained was both efficient at the local (IELs; Figure 1Go) and systemic (spleen cells) levels. The mean percentages of CD4+ and CD8+ were, respectively, 0.7 ± 0.4 and 16.9 ± 4.5 among the population of T cells (CD3+) within the IEL prepared from the large intestine of immunodeficient mice versus 3.3 ± 0.9 and 53.3 ± 4.5 for immunocompetent mice.



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Fig. 1. Fluorescence staining of CD4+ and CD8+ (among CD3+) colon intraepithelial lymphocytes of immunocompetent (A and B) and immunodeficient (C and D) Min mice fed sc-FOSs.

 
The mean number of tumors for immunocompetent mice fed sc-FOSs was 0.4 (Figure 1Go), a value corresponding to the mean number of tumors for 7-week-old Min mice (12), when diet supplementation began. When the animals were depleted of CD4 and CD8 T cells, an additional set of tumors developed within 44 days (mean 0.8 per mouse, significantly different from the immunocompetent group at P = 0.02 using Fisher's exact test) (Figure 2Go). In addition, there was an increase in the proportion of immunodeficient mice between the group of animals bearing no tumors (28%) and the group bearing two tumors (100%) (P < 0.05 with the {chi}2 test for trend). The size of the adenomas was not different between immunocompetent and immunodeficient groups (data not shown). Our findings, which are in good agreement with those of Hioki et al. (14), who studied a low risk diet in Apc knockout mice, indicate that food decreased the frequency rather than retarded the growth of polyp adenomas.



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Fig. 2. Mean number of colon tumors in immunocompetent (n = 30) and immunodepleted (CD4 and CD8 T lymphocytes) (n = 15) Min mice fed sc-FOSs for 42 days beginning at the age of 7 weeks. Values differ significantly at P = 0.02 between the two groups. Vertical lines indicate the standard deviation and the horizontal line the number of tumors in Min mice at 7 weeks of age.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As we previously showed, sc-FOSs as a diet supplement prevented the occurrence of colon tumors in Min mice. Since the number of tumors at day 42 for the immunocompetent mice was the same as at the start of the experiment, sc-FOSs may have impeded the appearance of new tumors, cured pre-existing tumors or generated a mixture of both mechanisms, the former hypothesis being the more probable.

For the first time, this study shows that sc-FOSs may provide an immunocompetent host with a mechanism of tumor surveillance, operative against spontaneously arising colon tumors. As we had previously shown that sc-FOSs did not reduce the occurrence of tumors in the small intestine (4), immunosurveillance was specifically generated in the colon and implicated the local immune system. As most {gamma},{delta}-receptor-bearing intraepithelial lymphocytes are CD4 CD8 (11) and thus not affected by depletion, it is unlikely that these cells were the main effector subset. Immunosurveillance appeared to be specifically generated by the diet since the Min phenotype was independent of the immune system, as shown by Dove et al. (10) and Dudley et al. (15).

As immunosurveillance is limited to the colon, it is likely that the flora plays an important direct and/or indirect role, although the molecule(s) involved (a fermentation product such as butyrate; 16), the specific flora, whether both parameters act in synergy and its (their) receptor in the mucosa still need to be identified. From studies with germ-free mice, Dove et al. (10) concluded that microbial flora in general is not necessary for the Min phenotype, but suggested that particular species can exert an effect. This would account for the difference in tumor multiplicity between conventional animals used in their experiment and their standard Min colony. Wasan et al. (5) also noted that microbial avoidance influenced tumor multiplicity in Min colonies housed in their facilities.

Other mechanisms account for the protective effect of probiotics and prebiotics depicted in other models of colon cancer (1720), depending on the stage of carcinogenesis, the nature of the carcinogen, the experimental set-up and the specific properties of the agent tested. However, the response of the gut-associated immune system to dietary interventions should also be considered.

The concept of immunosurveillance against tumors of non-viral origin has been seriously questioned, since immunodeficient animals and humans have failed to develop a higher rate of spontaneous cancers. It is possible that cancers escape immune clearance by a variety of mechanisms involving the suppression of immune function (anergy) or the killing of effector cells (Fas-mediated counter-attack) (2123). However, recent data based on large populations of immunosuppressed humans document the abnormal occurrence of tumors of non-viral origin in privileged locations (6,7). Surprisingly, the immune system seems capable of equivocal interference with cancerogenesis, acting as a promotor or antagonist depending on the organ concerned (6). This could relate to the level of immunogenicity of each kind of tumor and the different local density of lymph follicles. Immunogenicity should be considered in its broadest sense as the representation of the nature and intensity of direct or indirect interactions between the tumor cell and its environment, which may determine the nature of the immune response (immunofacilitation versus immunosurveillance) (24). It has been suggested that some effectors, such as IFN-{gamma} (10) or sodium butyrate (25), may promote tumor surveillance by enhancing tumor immunogenicity. In these circumstances, it is feasible to favor the delivery or expression of molecules that can reverse anergy or control immune cell apoptosis and thus stimulate a mechanism of immunosurveillance that would otherwise remain inefficient (10,14). In this context, the possibility that some indigestible carbohydrates from food stimulate immune rejection of nascent tumors offers new prospects for the prevention of colon cancer and opportunities for a better understanding of local immune response to colon cancer.


    Acknowledgments
 
We are grateful to Nadya Ruellan and Alain Maisonneuve for animal care and to Drs J. Le Pendu and F.M. Vallette for discussions. This work was supported by Eridania Béghin-Say (Nutrition and Health Service, Vilvoorde, Belgium), Nestlé (Research Center, Lausanne, Switzerland), the Ligue Départementale de Lutte contre le Cancer de Loire-Atlantique (Nantes, France) and the Association pour la Recherche sur le Cancer (Paris, France). F.P. is a recipient of a fellowship from the Association pour la Recherche sur le Cancer.


    Notes
 
2 To whom correspondence should be addressed. Email: menanteau{at}nantes.inserm.fr Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 18, 1999; revised June 15, 1999; accepted June 15, 1999.