Roles for inflammation and regulatory T cells in colon cancer

Abstract

Risk for developing cancer rises substantially as a result of poorly regulated inflammatory responses to pathogenic bacterial infections. Anti-inflammatory CD4(+) regulatory cells (T(REG)) function to restore immune homeostasis during chronic inflammatory disorders. It seems logical that T(REG) cells would function to reduce risk of inflammation-associated cancer in the bowel by down-regulating inflammation. It is widely believed, however, that T(REG) function in cancer mainly to suppress protective anticancer inflammatory responses. Thus roles for inflammation, T(REG) cells, and gut bacteria in cancer are paradoxical and are the subject of controversy. Our accumulated data build upon the "hygiene hypothesis" model in which gastrointestinal (GI) infections lead to changes in T(REG) that reduce inflammation-associated diseases. Ability of T(REG) to inhibit or suppress cancer depends upon gut bacteria and IL-10, which serve to maintain immune balance and a protective anti-inflammatory T(REG) phenotype. However, under poorly regulated pro-inflammatory conditions, T(REG) fail to inhibit and may instead contribute to a T helper (Th)-17-driven procarcinogenic process, a cancer state that is reversible by down-regulation of inflammation and interleukin (IL)-6. Consequently, hygienic individuals with a weakened IL-10- and T(REG)-mediated inhibitory loop are highly susceptible to the carcinogenic consequences of elevated inflammation and show more frequent inflammation-associated cancers. Taken together, these data help explain the paradox of inflammation and T(REG) in cancer and indicate that targeted stimulation of T(REG) may promote health and significantly reduce risk of cancer.

Figure 1

Inflammation modulates colon cancer progression in mice. Innate immune inflammatory response is sufficient for development of bacteria-triggered CRC in Rag2-deficient mice that entirely lack functional lymphocytes. Intramucosal carcinoma and invasion of isolated glands within the submucosa or muscle layers are readily observed in the colon of Rag2−/− mice two to three months after infection with pathogenic H. hepaticus. Mucinous carcinoma with transmural invasion is a uniform feature after six to eight months postinfection (A) and is the dominant histological type of CRC found in this model. Occasionally, other histological types were encountered, including poorly differentiated intramucosal “medullary” adenocarcinoma (B) and signet ring cell adenocarcinoma (C). Adoptive transfer with pro-inflammatory effector T (T_EFF) cells into H. hepaticus–infected mice increased the frequency and diversity of CRC, including adenosquamous carcinoma arising from the anus (D) and extending into the transverse colon. In contrast, anti-inflammatory therapy disrupted the carcinogenic network and even abolished established neoplastic invasion. Normal colon mucosa (E) results after adoptive transfer of anti-inflammatory regulatory T (T_REG) cells. Treatment of mice with anti-TNF-α or supplementation with IL-10-Ig fusion protein had similar therapeutic effects. T_REG cells require anti-inflammatory IL-10 to prevent pathology, as T_REG cells derived from IL-10–deficient donor mice instead greatly exacerbate frequency and the severity of mucinous CRC lesions (F), and peritoneal invasion was a consistent feature. Features of malignancy and neoplastic invasion were associated with increased local and systemic levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-17. Hematoxylin and eosin. Bars: A, E, and F, 250 µm; B and D, 100 µm; C, 50 µm.

Figure 2

Systemic inflammatory status determines the outcome of intestinal polyposis in Apc^Min/+ mice. Intestinal adenomatous polyp lesions in Apc^Min/+ mice (A) typically do not progress to invasive adenocarcinoma. However, experimental manipulations that trigger a host inflammatory response rapidly induce mucinous carcinoma, as with (B) adoptive transfer of pro-inflammatory effector T (T_EFF) cells, or with (C) infection with pathogenic H. hepaticus. Features of malignancy and neoplastic invasion were associated with increased local and systemic levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-17. Multiplicity of polyps and intramucosal neoplasia and early neoplastic invasion were also increased by (D) depletion of CD25+ cells. Rag2−/−Apc^Min/+ mice, which completely lack lymphocytes, are at greater risk for malignant transformation of adenomas into invasive mucinous adenocarcinoma (E). Surprisingly, features of malignancy were increased in these mice, commensurate with increased levels of pro-inflammatory cytokines, even in the absence of overt inflammatory disease. The same anti-inflammatory treatments that rescued Rag2-deficient mice with colitis-associated CRC also lead to regression of adenomas and CRC in Apc^Min/+ mice. Morphology of a minute polyp (F) not only is representative of adenoma regression seen in Rag2-deficient Apc^Min/+ mice after treatment with regulatory T cells (T_REG), but it also typifies the effect of other anti-inflammatory treatments such as systemic neutralization of TNF-α or supplementation with IL-10-Ig fusion protein. Hematoxylin and eosin. Bars, 250 µm.

Figure 3

Colonic carcinoma depends upon inflammation and is reversible through restoration of immune homeostasis. Our studies in mouse models of intestinal cancer indicate that anti-inflammatory treatments not only prevent cancer from arising, but also abolish established neoplasms. These results suggest that tumor growth and survival may be more dependent upon inflammatory events and more readily reversible than previously thought. The reliance upon inflammatory cells and factors was demonstrated in mouse models of the typical colitis-associated cancer and in mouse models emulating sporadic colorectal cancer (CRC) in humans, as well. Modified from Poutahidis et al. (2007).

Figure 4

Proposed immune mechanisms for induction and prevention of cancer. Immune homeostasis is continually reinforced by an IL-10– and T_REG-mediated inhibitory loop that allows for protective acute inflammatory responses and then later restores homeostasis. Dysregulation of this host-protective process leads to chronic inflammation involving elevated levels of IL-6 and a Th-17 host response. Individuals with a weakened IL-10– and T_REG-mediated inhibitory loop have increased susceptibility to uncontrollable inflammation, IL-6, and IL-17, and are at increased risk for inflammation-associated cancers. Modified from Poutahidis et al. (2007).

Figure 5

Immunohistochemical detection of critical inflammatory cells and cytokines in mouse models of intestinal cancer. The mesenteric lymph nodes of H. hepaticus–infected, IL-10–deficient 129 strain mice show large numbers of IL-17+ cells residing mainly in paracortical and medullary areas. IL-17+ cells are histomorphologically consistent with macrophages and lymphocytes (A). Adoptive transfer of IL-10^−/− regulatory T cells into H. hepaticus–infected, Rag2-deficient mice rapidly promotes CRC with peritoneal invasion and simultaneously increases significantly the numbers of IL-17+ cells in lymph nodes (B). Neutrophils are found to be major sources of both TGF-β1(C) and IL-6 (D) within the colonic mucosa. Under these pro-inflammatory conditions, Foxp3+ cells are numerous in the mesenteric lymph nodes (E). Intratumoral Foxp3+ cells within adenomatous polyps of Apc^Min/+ mice were numerous within regressing polyps after anti-TNFα–treated Apc^Min/+ mice (F). 3,3-diaminobenzidine, hematoxylin counterstain. Bars: A–D, 50 µm; E–G, 25 µm.

Figure 6

Proposed carcinogenic and protective roles for gut bacteria in cancer. Chronic pathogenic intestinal bacterial infections trigger a well-established sequence of immune events leading to cancer. However, microbial exposures earlier in life also serve to reinforce IL-10– and T_REG-mediated protection from inflammatory disorders, which, in turn, conveys protection from inflammation-associated cancers later in life. Stringent hygiene practices deprive the immune system of routine stimulation by bacterial products and IL-10 needed for efficient immune function. Thus, individuals with a na¨ıve immune system and weakened IL-10– and T_REG-mediated inhibitory loop have increased susceptibility to uncontrolled inflammation and are at increased risk for inflammation-associated cancers. Insufficient early life microbial exposures in societies with stringent hygiene practices may serve to undermine protective immunity and lower the threshold for future carcinogenic events. Modified from Rao et al. (2007).