Tumor infiltrating CD4+ Th lymphocytes can support an effective host anti-tumor immune response against sporadic CRC [10]. Contrarily, these cells are critical for the maintenance of chronic inflammation in IBD patients and could promote IBD-associated CRC. The role of the Th1 and Th2 response in colitis-associated CRC was assessed by Osawa and coworkers [11]. By using an experimental model that mimics CD-associated CRC, in which mice received intrarectal administration of trinitrobenzene sulfonic acid (TNBS) and intraperitoneal injection of the carcinogen azoxymethane (AOM), these authors show that mice deficient in IFN-γ, develop significantly more neoplasms compared to wild-type mice and Th2-biased IL4−/− mice. Due to the high expression of Th2-derived cytokines (i.e., IL-4 and IL-5) observed in IFNγ−/− mice, the authors suggest that a Th2-dominant cytokine response may enhance CRC growth [11]. Indeed, the Th2 response correlates with sporadic CRC progression both in pre-clinical models and in humans [12,13]. Additionally, the Th2 response could directly participate in colitis-associated tumor initiation since Th2-related cytokines (i.e., IL-4 and IL-13) increase the expression of activation-induced cytidine deaminase (AID), an enzyme, which induces DNA mutation in cultured colonic epithelial cells [14]. Moreover, the increased susceptibility of IFNγ−/− mice to colitis-associated CRC could also rely on the reduced anti-tumor activity because IFN-γ activates cytotoxic NK and CD8+ T cells [15]. These observations in mice are consistent with the demonstration that the Th1 response associates with an improved prognosis in many cancers [16]. However, an exaggerated Th1 immune response does not necessarily associate with a reduced risk of CRC. This is for example seen in patients with colonic CD, who have an enhanced risk of CRC despite high levels of Th1 cytokines in the gut [17]. Recent advances in the pathogenesis of CD and the discovery that the inflamed gut of CD patients contains high levels of Th17-type cytokines (e.g., IL-17A, IL-21, IL-22) [18], which are mitogenic for epithelial cancer cells, could explain this apparent contradiction.

CD8+ T cells and natural killer T (NKT) cells are immune cell subsets with cytotoxic activity on cancer cells. Presentation of tumor-specific antigens by antigen-presenting cells to CD8+ T cells results in release of different cytotoxic molecules (e.g., perforin, granzyme A, granzyme B, TNF-related apoptosis-inducing ligand (TRAIL), Fas ligand) which can target and kill dysplastic and tumor cells [19]. Activated CD8+ T cells also produce IFN-γ, known to play a crucial role in amplifying the anti-tumor immune response [20]. Although infiltration of CD8+ T cells has been associated with an improved prognosis in many cancers, including CRC [21,22], data on the host immune response against IBD-related CRC are limited and somehow controversial. By comparing samples taken from patients with colitis-associated and sporadic CRC, Michael-Robinson and coworkers showed that, despite the fact that inflammation-associated lesions had an increased infiltration of CD8+ T cells, this was not associated with an improved prognosis [23]. This can be related to the fact that CD8+ T cells are implicated not only in cancer immunosurveillance but also in the pathogenesis of IBD. Indeed, increased infiltration with CD8+ T cells in the intestinal mucosa of active CD and UC correlates with elevated expression of perforin and granzyme which may sustain a tumor-promoting chronic inflammation [24]. In accordance with this hypothesis, by using a well-known experimental model of colitis-associated CRC, induced by AOM and dextran sodium sulfate (DSS), Waldner et al. reported that perforin-deficient mice developed a less severe colitis and fewer tumors than wild-type mice [25]. On the other hand, work from our laboratory showed that mice over-expressing Smad7 in T and NKT cells developed a severe colitis following AOM + DSS administration, which reduced the tumor burden. This protection was associated with increased expression of IFN-γ and increased accumulation of cytotoxic CD8+ T and NKT cells in the tumors and peritumoral areas [26]. A typical example of bi-functional immune cell subset in CRC growth is represented by NKT cells [27]. Besides cytotoxic activity, these cells can express Th1-, Th2- and Th17-related cytokines and regulate the immune response. Different and even the same NKT cell subsets can act as enhancers or suppressors of tumor immunity. Type I NKT cells, which express an invariant TCR-α chain (i.e., Vα24Jα18-Vβ11 in humans and Vα14Jα18-Vβ8 in mice), potentiate anti-tumor immunity by inducing IFN-γ expression [28], and tumor infiltration by Type I NKT cells in CRC patients has been positively correlated with disease-free survival [27]. Contrary, Type II NKT cells, which do not express this invariant TCR-α chain, associate with suppression of tumor immune surveillance in different cancer models [27]. Regarding IBD, activation of Type II NKT cells and the following production of the immunosuppressive cytokine IL-13 is a feature of the “atypical” Th2 response characterizing UC patients [29]. Additionally, an aberrant Type II NKT cell response was recently reported to directly contribute to intestinal inflammation in mice [30]. Although there is no conclusive evidence that Type II NKT cells contribute to colitis-associated CRC, it is tempting to speculate that activation of this cell subset in course of colitis may contribute to colon carcinogenesis by both dampening the host anti-tumor response and amplifying the ongoing inflammation.

CD4+ T cells expressing CD25 and the master transcription factor Foxp3 (CD4+CD25+Foxp3+), termed regulatory T cells (Tregs), exert immunosuppressive effects both via direct cell–cell interactions and through the release of the cytokines IL-10 and TGF-β [31]. Whereas Tregs are important in limiting autoimmune diseases and inflammatory responses, the suppression of the immune system may strongly hamper host immune surveillance against tumors. The pro-tumoral role of Tregs in the progression of established tumors is well accepted and Foxp3 expression has been associated with a poor prognosis in many cancer types [32]. Contrarily, in patients bearing CRC, infiltration of Tregs associates with a favorable prognosis [33]. Consistently, a study by Erdman and coworkers showed that Tregs reduce tumor growth in ApcMin/+ mice, which spontaneously develop intestinal lesions [34]. The role of Tregs in inflammation-associated CRC remains elusive. Given the potent immunoregulatory function Tregs exert on immune responses and inflammation, it is conceivable that these cells may help prevent and/or delay inflammation-mediated tumor growth. In line with this hypothesis, a recent work by Sugai and colleagues suggested that Tregs exert anti-tumor activity in colitis-associated CRC [35]. These authors showed that mice deficient for Runx3, a protein required for proper differentiation and function of Tregs, are more susceptible to develop colitis and inflammation-related colonic tumors compared to wild-type mice [35]. However, further investigation is needed to address the role of Tregs in IBD-related CRC.

Cells of the innate immune system, such as neutrophils, NK cells, DC and macrophages infiltrate colitis-associated CRC. Whereas the contribution of innate immunity in CRC initiation through oxidative stress is well accepted [1], the role of these cells in colitis-related CRC growth has recently started to be unveiled. A link between innate immunity and this neoplasia is suggested by the demonstration that Toll-like receptors (TLRs) are involved in inflammation-related carcinogenesis. TLRs are a family of membrane-bound receptors mainly expressed by cells of the innate immune system and able to recognize microbe specific molecules or endogenous stress signals. TLRs play a major role in maintaining gut homeostasis, and polymorphisms in the genes encoding TLRs are associated with an increased risk of IBD [36]. Additionally, TLR4 is up-regulated in intestinal epithelial cells of patients with active IBD [37] and TRL4-driven signals induce ROS production and mitogenic molecules (e.g., Prostaglandin E2) [38]. Experimental models of colitis-associated CRC showed that the presence and recognition of the gut microbiota are required for inflammation-associated carcinogenesis [38]. Fukata and coworkers report that TLR4 is over-expressed in UC-associated CRC and, by using the AOM + DSS mouse model, these authors showed that TLR4-deficient mice are largely protected against the development of tumors as compared to wild-type mice [39]. More recently, the same authors suggested that innate immune signaling by TLR4 could shape the inflammatory microenvironment to sustain CRC cell growth [40]. Contrarily, myeloid differentiation factor 88 (MyD88), a molecule critical for TLR intracellular signaling, seems to have a protective role against inflammation-related CRC induced in mice by AOM + DSS treatment [41]. However, it is worth noting that MyD88 signaling has been implicated in cancer promotion in different mouse models of CRC [42,43]. Important players in colitis-associated CRC growth are tumor associated macrophages (TAM), which can be divided into M1 and M2 types [44]. M1 macrophages, activated by IFN-γ and microbial products, are capable of killing pathogens and priming antitumor immune responses through the up-regulation of major histocompatibility complex (MHC) molecules and the production of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-12). Contrary, M2 or “alternatively activated macrophages”, which are induced in vitro by IL-4, IL-10, and IL-13, are known to support angiogenesis and inhibit anti-cancer immunity through the production of TGF-β and IL-10. However, although most TAM are considered to have an M2 phenotype and promote inflammation-related CRC, most tumor-promoting cytokines are “M1 cytokines” [45]. In addition to TAM, MDSC are suggested to promote colitis-associated CRC by dampening anti-tumor immune responses [45]. A possible role for innate immune cells in the pathogenesis of this cancer type was highlighted by Hayakawa and coworkers, who showed that ASK1(−/−) mice, in which macrophages are impaired in their ability to kill bacteria, develop a more severe colitis and more numerous and larger tumors than wild-type mice following AOM + DSS treatment [46].

TNF-α, mainly produced by activated M1 macrophages and T lymphocytes, plays a pivotal role in the inflammatory process by binding TNF receptors (TNF-R) p55 and p75 on target cells. Reports on the role of TNF-α in cancer suggest that the cytokine may either promote or inhibit CRC cell growth [48]. Inflammatory cells may use a TNF-α outburst to stimulate ROS and kill transformed cells. On the other hand, chronic production of low doses TNF-α could sustain tumor growth [48]. TNF-α is crucial in the pathogenesis of colitis and the use of anti-TNF-α monoclonal antibodies is beneficial in IBD patients [49]. Similarly, TNF-α signaling seems to be involved in colitis-associated CRC. Popivanova and coworkers showed that TNF-Rp55−/− mice treated with AOM + DSS developed a milder colitis and fewer tumors than wild-type mice [50]. It was also shown that wild-type mice transplanted with bone marrow of TNF-Rp55-deficient mice developed significantly fewer tumors after AOM + DSS treatment than either wild-type mice or TNF-Rp55−/− mice transplanted with wild-type bone marrow. Moreover, mice given anti-TNF-α were less susceptible to both colitis and colitis-associated CRC [50]. As TNF-α is a powerful inducer of NF-kB, a pleiotropic transcription factor supporting both inflammation and carcinogenesis [51] and blockade of NF-kB activation in the intestinal epithelium significantly reduces the incidence of colitis-associated tumors [52], the pro-inflammatory and tumor-promoting effects of TNF-α in this model could be mediated by NF-kB.

IL-6 is a multifunctional cytokine produced by T cells, B cells, macrophages and fibroblasts with a crucial role in the generation of Th17 cells [53]. IL-6 also regulates epithelial cell proliferation and survival either by binding to the membrane-bound IL-6 receptor (IL-6Rα/gp130) on target cells or to soluble IL-6Rα (sIL-6Rα) with subsequent trans-signaling [54]. These receptors initiate a signal cascade leading to activation of signal transducer and activator of transcription (STAT)3, with the downstream effect of inducing anti-apoptotic and proliferative genes [55,56]. However, it is worth noting that two recent studies, performed in ApcMin/+ mice, showed STAT3 to exert opposite roles on colon carcinogenesis depending on the tumor stage [57,58]. IL-6 has been linked to IBD pathogenesis [59,60] and been suggested to play a pivotal role in colitis-associated CRC. Accordingly, in the AOM + DSS mouse model, Becker and colleagues demonstrate that IL-6 is highly produced by CD4+ T cells and promotes tumor cell proliferation via STAT3 activation [61]. In a similar model, Grivennikov and coworkers show that IL-6 expressed by lamina propria myeloid cells protects normal and malignant epithelial cells from apoptosis in a STAT3-dependent fashion [56]. An essential role of IL-6 trans-signaling in the development of colitis-associated CRC is also reported by Matsumoto and colleagues [62]. Recently, Li et al. found higher expressions of IL-6 and STAT3 in both patients with active UC and those who had progressed to CRC, compared with patients with inactive disease or control patients [63]. In the same study, the expression of the suppressor of cytokine signaling (SOCS)3, a protein that hampers the transcription of IL-6-target genes, is found to be decreased in UC patients who had developed CRC, thus suggesting an important role for SOCS3 in UC-related CRC [63]. Consistently, in the AOM + DSS model, mice with deletion of SOCS3 in intestinal epithelial cells show increased STAT3 and NF-κB activation and enhanced susceptibility to developing colonic tumors following AOM + DSS treatment [64]. Finally, Gerlach and colleagues demonstrate a critical role for the transcription factor NFATc2 in IL-6 signaling and colitis-associated CRC [65]. In this study, NFATc2-knockout mice produced reduced IL-6 and were almost completely protected from the AOM + DSS-induced tumors, in contrast to wild-type mice, which bore multiple colonic lesions. Notably, administration of hyper-IL-6 abrogated protection from tumor progression in NFATc2-deficient mice and restored tumor incidence to levels observed in wild-type mice [65].

IL-17A, mainly secreted by Th17 cells and, to a lesser extent, by NKT cells, innate lymphoid cells and γδ T cells, exerts pro-inflammatory properties essential for the host protection against extracellular pathogens [66]. On the other hand, IL-17A has been implicated in the pathogenesis of many chronic inflammatory disorders and clinical trials with IL-17A-neutralizing antibodies have reported benefit in patients with psoriasis and rheumatoid arthritis [67]. However, IL-17A blockade was not beneficial in CD patient populations [68]. Consistent with this observation is also the demonstration that transfer of IL-17A-deficient T cells to immunocompromized mice promotes a colitis which is indistinguishable from that induced by wild-type T cells [69]. Similarly, studies in cancer models have shown a dual role of IL-17A in controlling neoplastic cell growth. Indeed, IL-17A inhibits tumor growth in a murine model of melanoma [70] and in implanted tumor models [71,72], while promotes malignant cell growth in mouse models of spontaneous intestinal cancer [73,74]. Using the AOM + DSS model, Hyun and colleagues showed that IL-17A-deficient mice developed a less severe colitis than wild-type mice, as demonstrated by the decreased cell infiltrate and the diminished expression of pro-inflammatory factors (e.g., TNF-α, IL-6) [75]. Consistently, in IL-17A-knockout mice STAT3 activation was markedly reduced compared with wild-type mice, and this associated with the development of fewer and smaller colonic tumors [75].

IL-21 is a pleiotropic cytokine produced by activated Th and NKT cells and T follicular helper cells [76]. Both pre-clinical and clinical studies have shown that IL-21 has potent anti-tumor effects due to its ability to expand the cytotoxic immune response [77]. However, IL-21 has been involved in the pathogenesis of many immune-mediated diseases including IBD [76]. Indeed, IL-21 is over-produced in the colonic mucosa of UC and CD patients [78] where it positively regulates Th17 cell responses [79]. Notably, two independent and recent studies revealed a key role for this cytokine in promoting colitis-associated CRC [80,81]. High levels of IL-21 have been observed in the gut of patients with UC-associated colon cancer and in mice with colitis-associated CRC induced by AOM + DSS [80]. In this model, IL-21-deficient mice developed a less severe colitis than wild-type mice, characterized by reduced mucosal damage, reduced infiltration of T cells, and diminished production of IL-6 and IL-17A. IL-21KO mice also developed fewer and smaller tumors compared with control mice [80]. Analysis of mechanisms underlying this effect revealed that IL-21 sustains CD4+ T cell infiltration in the tumor and peritumor areas and enhances the production of IL-6 and IL-17A as well as STAT3 activation. Moreover, colonic tumors developing in wild-type mice exhibited an increased infiltrate of both M2 macrophages and MDSC than those grown in IL-21-deficient mice [80]. In a very similar model, Fichtner-Feigl’s group showed that IL-21 promotes colitis-related CRC and associated this effect with increased tumor cell proliferation and impaired anti-tumor response of CD103+ CD8+ cytotoxic T cells specific for cancer cells [81].