Unifying roles for regulatory T cells and inflammation in cancer

Abstract

Activities of CD4(+) regulatory (T(REG)) cells restore immune homeostasis during chronic inflammatory disorders. Roles for T(REG) cells in inflammation-associated cancers, however, are paradoxical. It is widely believed that T(REG) function in cancer mainly to suppress protective anticancer responses. However, we demonstrate here that T(REG) cells also function to reduce cancer risk throughout the body by efficiently downregulating inflammation arising from the gastrointestinal (GI) tract. Building on a "hygiene hypothesis" model in which GI infections lead to changes in T(REG) that reduce immune-mediated diseases, here we show that gut bacteria-triggered T(REG) may function to inhibit cancer even in extraintestinal sites. Ability of bacteria-stimulated T(REG) to suppress cancer depends on interleukin (IL)-10, which serves to maintain immune homeostasis within bowel and support a protective antiinflammatory T(REG) phenotype. However, under proinflammatory conditions, T(REG) may fail to provide antiinflammatory protection and instead contribute to a T helper (Th)-17-driven procarcinogenic process; a cancer state that is reversible by downregulation of inflammation. Consequently, hygienic individuals with a weakened IL-10 and T(REG)-mediated inhibitory loop are highly susceptible to the carcinogenic consequences of elevated IL-6 and IL-17 and show more frequent inflammation-associated cancers. Taken together, these data unify seemingly divergent disease processes such as autoimmunity and cancer and help explain the paradox of T(REG) and inflammation in cancer. Enhancing protective T(REG) functions may promote healthful longevity and significantly reduce risk of cancer.

Figure 1

Interleukin-10-dependent functions of regulatory T cells restore immune homeostasis in 129 strain Rag2-deficient mice. (a) Gene expression of IL-6 and IL-17 were elevated (p < 0.05) within colonic tissues of infected recipients of IL10-deficient Treg cells. For comparison of mRNA levels, the target mRNA was normalized to that of the housekeeping gene GAPDH. Numbers on the y-axis represent mean fold change of target mRNA levels in reference to the control levels (B6 wt, defined as 0, standard deviation represented by solid bars). mos = age in months on necropsy. *p values were compared with the control. (b) Immunohistochemical staining illustrates overexpression of IL-17 in the mesenteric lymph nodes of mice lacking IL-10. The aberrant immunostaining pattern observed in IL-10−/− animals after infection with H. hepaticus can be appreciated by comparison with tissues of wt mice (top row). The carcinogenic effect was isolated to IL-10-deficient lymphocytes and was correlated with overexpression of IL-17. 3,3-Diaminobenzidine, hematoxylin counterstain. Bars: 25 μm. (c) Overview of interrelated pathways of inflammation involving Treg cells, IL-6 and IL-17 contributing to cancer development and growth. (d) Purified T_REG cells of differing dosages or genotypes functioned differently, but these differences are not the result of differential expansion or recruitment of cells within lymph nodes. Each plot indicates % of MLN cells in the live cell gate that express CD4. The second number represents the % of CD4+ cells that expressed Foxp3.

Figure 2

Gut bacteria-triggered T_REG cells protect against intestinal and mammary neoplasia and increase survival. (a) Levels of TNF-α and IL-17 were significantly (p < 0.01) elevated in female Min mice. Serum cytokine levels of 5 months old C57Bl/6 wt (blue bars) and APC^Min/+ mice (green bars) were measured using the Bioplex assay system. Statistically significant higher levels of TNF-α, IL-9 and IL-17 were detected in APC^Min/+ mice. *p < 0.05; **p < 0.01. (b) T_REG cells from “hygienic” Treg cell donors failed to reduce gene expression of IL-6 and IL-17. Levels of IL-6 and IL-17 were elevated (p < 0.05) within ileal and mammary tissues of recipients of T_EFF cells. (c) Adoptive transfer of purified T_EFF cells cause highly infiltrative mammary adenosquamous carcinoma in female APC^Min/+ mice. Foxp3+ cells (right panel arrow) locate within tumor-associated inflammatory cell foci at the margins of tumors. The right panel provides a higher magnification of the same field. 3,3-Diaminobenzidine, hematoxylin counterstain. Bars: left panel: 100 μm; right panel: 25 μm. (d) Survival curve illustrating increased lifespan in APC^Min/+ mouse recipients of T_REG cells from H. hepaticus-infected syngeneic wt donor mice. For the survival curve, mice were humanely euthanized using institutional criteria (i.e., poor body condition score, large tumor size) or when exhibiting other signs of distress.

Figure 3

Neutralization of TNF-α restores epithelial and immune homeostasis in FVB strain HER2/neu mice. To directly test requirements for inflammation in mammary cancer, we used anti-TNF-α treatment in HER2/neu mice. We selected 6-month-old HER2/neu mice with a small but established tumor burden. Anti-TNF-α was delivered by intraperitoneal injection ×3 weekly for 10 days and then underwent necropsy immediately afterward. Mammary tumor counts were based on grossly evident tumor nodules in mammary tissue on necropsy and then compared between groups by unpaired Student’s t test. Mammary tumor volumes (cm³) were estimated based on dimensions of solid tumor tissue (excluding fluid-filled cysts) on necropsy and then were compared between groups using Mann–Whitney U analyses. (a) Blockade of TNF-α led to decreased tumor burden with significantly (p < 0.05) reduced volume and a trend (p = 0.07) toward fewer tumors in 6-month-old female HER2/neu mice. (b) Foxp3+ cells in untreated or sham mice were located within lymph nodes and inflammatory foci adjacent to, but not inside, mammary tumors. Treatment with anti-TNF-α significantly increased frequency of Foxp3+ cells within tumors. Similarly, anti-TNF-α treatment reduced (p < 0.05) the numbers of IL-17+ cells in mammary tumors (shown in right panels). Cell counts were performed as described in Material and Methods section. 3,3-Diaminobenzidine, hematoxylin counterstain. Bars: 25 μm.

Figure 4

Summary of tumor outcomes in different mouse models of cancer. Mouse models that mimic cancer in humans were consistent with the “hygiene hypothesis” and connected seemingly divergent disease phenotypes including autoimmunity and cancer. Bacteria-triggered IL-10-dependent functions of T_REG cells protect from inflammation-associated cancer.

Figure 5

Conceptual overview of immune factors contributing to cancer development and growth under hygienic host conditions. Under normal physiological conditions, T_REG responses are beneficial to the host by reinforcing protective acute inflammation and then afterward regain protection from pathologic sequellae of chronic inflammation. During a proinflammatory microbial challenge, elevated levels of IL-6 upregulate a T helper type (Th)-17 host response that contributes to cancer growth and invasion. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]