The role of IL-17-producing Foxp3⁺ CD4⁺ T cells in inflammatory bowel disease and colon cancer

Abstract

The intestinal epithelium and underlying lamina propria contain T cells that play important roles in maintaining colonic homeostasis. These T cells mediate substantial and specific regulation to ensure that pathogenic microorganisms are eliminated while commensal bacteria are tolerated. There is considerable evidence supporting the notion that the altered ratio between Foxp3⁺CD4⁺ T regulatory cells and T effector cells in the colonic microenvironment might contribute to the initiation and progression of inflammation and eventually development of colon cancer. Recent findings on the heterogeneity and plasticity of T regulatory cells, such as the identification of IL-17⁺Foxp3⁺CD4⁺ and the RORγt⁺Foxp3⁺CD4⁺ subsets, in patients with colorectal inflammation and cancer have provided a new twist in our understanding of the pathogenesis of colonic diseases. Phenotypic and functional properties of IL-17-producing Foxp3⁺CD4⁺ T cells as well as the significant implications of these cells in the initiation and progression of colorectal diseases are discussed in this review.

1. Introduction

Regulatory T (T_Reg) cells expressing the transcription factor forkhead box P3 (Foxp3) are naturally present in the immune system. T_Reg cells suppress the activation, proliferation and effector functions of a wide range of immune cells, including CD4⁺ and CD8⁺ T cells, natural killer (NK) and NKT cells, B cells and antigen presenting cells in vitro and in vivo [1]. T_Reg cells have key roles in the prevention of autoimmune responses and the development of immunopathology as well as in the maintenance of homeostasis. As a double-edged sword, T_Reg cells can also suppress antitumor immune responses and can favor tumor progression. The unique functional properties of T_Reg cells make these cells a primary target in the search for new cell-based immunotherapeutic approaches. However, recent studies showed that T_Reg cells are not a homogeneous and terminally differentiated cell population. Instead, these cells are heterogeneous in gene expression, phenotype and function. In addition, T_Reg cells are strongly affected by other immune components including effector cells and innate immune cells during initiation and progression of disease. Thus, to achieve the translational goal of applying T_Reg cells in the treatment of diseases, understanding of the T_Reg cell immunobiology in the context of various tissues and pathogenesis of various diseases is required. Lymphocytes of mucosal tissues form a relatively autonomous immune subsystem, with specialized adaptations appropriate for this particular microenvironment [2-4]. The intestinal mucosa is normally maintained in a state of controlled inflammation in which equilibrium exists between protective immunity and tolerance to self-antigen and commensal bacteria [5]. Protective immunity against different classes of pathogens depends on the generation of distinct types of immune responses mediated and coordinated by effector cells specifically T_H1, T_H2 and T_H17. On the other hand, T_Reg cells are involved in the maintenance of tolerance. A series of observations suggest that T_Reg cells correlate with poor prognosis in many cancer types, including breast, lung, melanoma, and ovarian carcinoma [6] due to their suppressive effects on anti-tumor immunity. However, several studies have shown that T_Reg cells are protective in cancer by virtue of their ability to control inflammation in an IL-10 dependent manner [7]. In fact, Foxp3 expression has been indicative of better prognosis in gastric cancer, head and neck, and breast cancer [8-10]. Recent identification of IL-17-producing Foxp3⁺CD4⁺ T cells in IBD, colon cancer and polyposis might provide a potential explanation for the conflicting observations regarding the role of T_Reg cells in cancer. Furthermore, the advancements in understanding T_Reg immunobiology locally and systemically and the influence of inflammatory microenvironments on the differentiation of effector cells and on the stability of T_Reg cells would improve the use of T_Reg as cell-based immunotherapy.

2. Heterogeneity of T_Reg cells

Foxp3⁺CD4⁺ T_Reg cells are not homogeneous in gene expression, phenotype and suppressive function. The transcription factor Foxp3, a master control gene for the development and function of both mouse and human T_Reg cells [11-14], is currently the definitive marker of T_Reg cells. However, Foxp3 can be transiently expressed in activated human T cells. This transient Foxp3 expression in T cells does not enable suppression but, instead, makes separation of T_Reg cells from activated T effector cells difficult to perform. Therefore, studies to determine the frequency of various T cell subsets in diseases require further scrutiny and mandate the use of multiple parameters instead of Foxp3 alone to define T_Reg cells. In spite of multiple attempts for the identification of the proper marker combination including CD25, CD127 and CD62L to delineate human T_Reg cells, the identification and purification of human T_Reg remains a challenge. Recent studies have shown that CD45RA or CD45RO, which are mutually exclusive, are particularly useful markers when combined with CD25 and/or Foxp3 [15]. CD45RA⁺Foxp3⁺ naïve T_Reg cells, are present in peripheral blood and prevalent in cord blood [16, 17] and are considered the human counterparts of mouse natural T_Reg cells that develop in the thymus. Naïve T_Reg cells proliferate after in vitro stimulation via their TCR and are highly resistant to apoptosis [18], in contrast to CD45RO⁺Foxp3⁺ T_Reg cells, which tend to be hyporesponsive and apoptotic after activation in vitro. Effector CD45RO⁺Foxp3⁺ T_Reg cells, which derive mainly from naïve T_Reg cells, as well as CD45RA⁺Foxp3⁺ naïve T_Reg cells have potent in vitro suppressive activity [15]. In contrast to the human system, naïve mouse CD4⁺ T cells lack transient and promiscuous expression of Foxp3 after activation. But naive mouse T cells readily convert to Foxp3⁺ T_Reg (iT_Reg) cells following in vitro stimulation with TGF-β or retinoic acid [19]. These induced T_Reg cells share many phenotypic markers and regulatory functions natural T_Reg cells, but do not exhibit the signature gene transcription profile of natural T_Reg cells [20]. Instead, the gene profile of induced T_Reg cells is Foxp3 independent [15].

3 Characterization of in vitro induced IL-17-producing Foxp3⁺CD4⁺ T cells

T_Reg cells are well known to produce IL-10 and TGF-β during activation and these cytokines are considered to be part of the mechanisms via which T_Reg cells mediate their suppressive function [21]. Recent findings reveal that T_Reg cells are capable of producing IL-17. There is an intimate link between T_H17 cells and T_Reg cells. Exposure of antigen-activated naïve T cells to TGF-β in vitro results in transcriptional up-regulation of both Foxp3 and RORγt [22]. Furthermore, T cells co-expressing Foxp3 and RORγt have been identified in vivo in both mice and humans [23]. Foxp3 is able to physically bind to RORγt and to inhibit the transcriptional activity of RORγt thereby blocking IL-17 production. Therefore, in steady state conditions, T_Reg cells are unable to produce IL-17 [22]. A number of research groups have reported that during activation in the presence of appropriate inflammatory stimuli in vitro, both human and murine T_Reg cells display a IL17⁺Foxp3⁺CD4⁺ phenotype and can produce IL-17 [24-27]. Recent studies including our own work have identified that IL-1β is a critical mediator in the conversion of T_Reg cells into IL-17-producing cells in vitro. In the presence of IL-1β, T_Reg cells express increased levels of RORγt and are able to secrete IL-17 in response to T cell receptor (TCR)-mediated stimulation. The activation of MAPK pathways via IL-1β/IL-1R might have an active role in this differentiation process [24]. We have determined that Runx1, a transcription factor required for the expression of Foxp3 and RORγt, is highly expressed in IL-17-prducing Foxp3⁺CD4⁺ cells compared to Foxp3⁺CD4⁺ T cells, and silencing of Runx1 with small interfering RNA significantly reduced the numbers of IL-17-producing cells induced by IL-1β [28]. Thus, sustained expression of Runx1 in T_Reg cells by IL-1β might be responsible for the conversion of T_Reg cells into IL-17-producing RORyt⁺Foxp3⁺CD4⁺ cells. Although these in vitro generated IL-17-producing Foxp3⁺CD4⁺ T cells retain phenotypic properties of T_Reg cells, including expression of CD25, CTLA-4 and GITR, these cells represent a distinct subset of T_Reg cells. IL-17-producing Foxp3⁺CD4⁺ T cells express increased levels of ICOS compared to T_Reg cells, and more importantly, display potent suppressive activity and are capable of inducing apoptosis of responder T cells [28].

As mentioned above, Foxp3⁺T_reg cells represent a heterogeneous population consisting of committed T_Reg cells and of a minor subpopulation that retains developmental plasticity [29]. It is tempting to speculate that this plasticity-retaining subset gives rise to T_Reg cells capable of producing IL-17. Several attempts have been made to identify the origin of IL-17-producing Foxp3⁺CD4⁺ T cells. Most of the studies have demonstrated that effector CD45RO⁺ T_Reg cells but not CD45RA⁺ naïve T_Reg cells are capable of producing IL-17 upon TCR stimulation in the presence of the combination of IL-1β, IL-2, IL-23 and IL-21 [23]. CD45RA⁺ naïve T_Reg cells retain stable CpG methylation across the RORC locus even upon prolonged ex vivo expansion and as a consequence, they display only a marginal tendency to express RORγτ and to develop into IL-17-producing cells. In contrast, stimulation-induced DNA demethylation of RORC occurs selectively in effector CD45RA- T_Reg cells [30]. IL-17-producing Foxp3⁺CD4⁺ T cells like T_H17 cells express CCR6, a trait of tissue-homing effector T cells [31, 32], suggesting that CCR6 combined with other markers such as CD45RA, CD45RO and Foxp3 could be used to identify IL-17-producing T_Reg cells in vitro and in vivo. In addition, studies by Raffin et al. have determined that IL-1 receptor type 1 (IL-1R1) positive effector T_Reg cells can become IL-17-secreting effectors in response to TCR-mediated stimulation in the presence of IL- 1β, suggesting that ex vivo expression of IL-1R1 in human T_Reg cells identifies an early intermediate in the conversion of IL-17-producing cells from T_Reg cells [33].

4. Roles of T_Reg cells and T_H17 cells in inflammation and colon cancer

The intestinal tract contains large numbers of immune cells involved in the encounter with microbial antigens. Under normal conditions, the lumen of the intestine is covered by a layer of intestinal epithelial cells (IECs), which are joined together by tight junctions forming a protective layer impeding bacterial invasion [34]. Immune responses in the colon take place in three distinct compartments: the lamina propria, the isolated lymphoid follicles (ILFs) scattered throughout the colon, and the mesenteric lymph nodes. The latter two sites may play important roles in initiating immune responses and in dictating quantitative and qualitative features of the responses. Cells in the lamina propria are involved in antigen sampling, and this is the site in which effector cells accumulate during inflammation [35].

T_Reg cells actively suppress enteroantigen-reactive cells and contribute to the maintenance of intestinal immune homeostasis. Distinct T_Reg cell subsets coexist in the intestinal mucosa and mesenteric lymph nodes. Studies have shown that commensal bacteria can direct the development of Foxp3⁺ T_Reg cells with a unique ‘inducible” genetic signature, suggesting the existence of a mechanism via which microbiota can actively promote mucosal tolerance by inducing the differentiation of conventional T cells into Foxp3⁺ T_Reg cells [36]. In fact, it is widely accepted that ‘inducible’ rather than ‘natural’ T_Reg cells are particularly effective in maintaining immune homeostasis of the intestine (Figure 1). The use of murine models of inflammatory bowel disease (IBD) has indicated that these IL-10-producing inducible T_Reg cells play an important role in preventing or limiting colitis [37]. The precise origin of these inducible T_Reg cells remains unclear.

Figure 1. Induction of T_Reg cell mediated gut immune tolerance.

In the steady state, gut immune tolerance is induced and maintained by a variety of mechanisms, which regulate the differentiation and activation of T_Reg cells. Some commensal bacteria can induce the differentiation of CD4⁺ T cells into T_Reg cells in a TLRs dependent manner. Clostridium spp. can induce generation of IL-10 producing T_Reg cells via epithelial cell-derived TGF-β, whereas Bacteroides fraglilis can induce IL-10 producing T_Reg cells via polysaccharide A (PSA). CD103⁺DC can also induce T_Reg cells through their ability to produce retinoic acid and TGF-β. T_Reg cells act on macrophage and myeloid cells to inhibit their ability to produce colitogenic cytokines including IL-12p40, Il-6 and IL-23, which are required for the differentiation of CD4⁺ T cells into Th1 and Th17 cells. Both Th1 and Th17 cells are associated with initiation and progression of IBD and colon cancer. T_Reg cells suppress inflammatory responses mainly through production of anti-inflammatory cytokines TGF-β and more importantly IL-10.

In mucosal surfaces, T_H17 cells are thought to protect the host from infections, whereas several types of T_Reg cells keep effector T cell populations in check and prevent T cell-mediated destruction of intestinal tissue [38-41]. There is compelling evidence that Crohn's disease (CD) is driven by T_H17 cells [42]. CD, one type of IBD, is believed to be the result of an aberrant response of the gut-associated lymphoid tissue to bacterial and/or dietary antigens. Increased serum IL-17 levels have been observed in patients with CD compared to healthy individual. In addition, IL-17 mRNA expression and the number of IL-17⁺ T cells were also increased in the inflamed mucosa from patients with active CD [43]. However, blocking IL-17 is ineffective for the treatment of CD, suggesting that although IL-17 is involved in the pathogenesis of CD, IL-17 itself is unlikely to serve as a therapeutic target for CD [44]. Increased numbers of T_H17 cells were also observed in the intestinal mucosa in patients with ulcerative colitis (UC), another well-characterized type of IBD [45]. It is unclear whether T_H17 cells play the same role in the pathogenesis of these two types of IBD or whether these two forms of IBD share a common mechanism underlying the accumulation of T_H17 cells as a consequence of the inflammation process in the intestinal mucosa. Of note, concomitantly with the increased frequency of T_H17 cells, there is also an increased number of T_Reg cells in the lamina propria, mesenteric lymph nodes, and inflamed intestinal mucosa of IBD patients [46]. Although the presence of IL-17, T_H17 cells and T_Reg cells in the inflamed intestine is well established, their dynamics and contribution to the IBD disease remain elusive.

T_Reg cells are an obstacle for immune surveillance and immune therapy of cancer due to suppressing anti-tumor immune responses. However, studies using Apc^Min/+ mice revealed a protective role of IL-10-producing T_Reg cells in bacterial-induced chronic inflammation and cancer [47] and even hereditary colon cancer [48], suggesting that T_Reg cells might play a protective role in cancer by suppressing inflammation (Figure 2). Mast cells are essential hematopoietic components promoting the development of adenomatous polyps [49]. Adoptive transfer of T_Reg cells protect against colon cancer by suppressing focal mastocytosis in adenomatous polyps due to IL-10 production [7]. Conversely, in polyp-ridden mice, T_Reg cells are aberrant, fail to produce IL-10 and instead produce IL-17 thereby promoting rather than suppressing mastocytosis [7, 50]. These observations suggest that the role of T_Reg cells in the development of cancer depends on their unique functional properties as identified by their ability to produce specific cytokines.

Figure 2. Opposing effects of Foxp3⁺T_Reg and RORγt⁺Foxp3⁺T_Reg cells in the development of IBD and colon cancer.

Foxp3⁺ T_Reg cells suppress both T-cell mediated anti-tumor immune response and inflammatory response at least in part by secretion of IL-10 and TGF-β in response to TCR-mediated stimulation. Given their ability to suppress inflammatory processes, Foxp3⁺ T_Reg cells play an important role in controlling and suppressing the pathogenesis of IBD and subsequently the development of colon cancer. However, in the context of an inflammatory milieu, Foxp3⁺ T_Reg cells are able to express RORγt and have the potential to express and secrete IL-17. The resultant RORγt⁺Foxp3⁺T_Reg cells display potent suppressive activity on T-cell mediated immune responses, whereas their antiinflammatory function is compromised. As a consequence, RORγt⁺Foxp3⁺ T_Reg cells are unable to control inflammation and rather promote instead of suppressing the development of colon cancer.

IL-17, produced locally in the tumor microenvironment, plays important roles in both angiogenesis and tumor immunity. Inhibition of IL-17A at tumor sites by intratumoral injection of an adenovirus vector expressing siRNA against the mouse IL-17A gene (Ad-si-IL-17) significantly inhibited tumor growth. It was found that inhibition of IL-17 at tumor sites significantly suppressed CD31, MMP9 and VEGF expression in tumor tissue. Furthermore, the cytotoxic activity of tumor infiltrating CD8⁺ lymphocytes in mice treated with ad-si-IL17A was significantly higher than in control mice [51]. Further experimental evidence supports the idea that IL-17 promotes tumor growth. Specifically, ablation of IL-17 significantly reduced tumor development in mice bearing a heterozygote mutation in the adenomatous polyposis coli (APC) gene (Apc^Min/+ mice). There was also a decrease in inflammatory cytokines and proinflammatory mediators and reduced lymphocytic infiltration, suggesting that IL-17 promotes spontaneous intestinal tumorigenesis [52]. Consistently with a positive role of IL-17 in promoting tumor development, tumor tissues have a higher frequency of IL-17⁺ cells T cells compared with untransformed bowel tissues [53]. The role of IL-17 in promoting tumor growth provides additional support for the already well-established connection between inflammation and tumorigenesis. However, this study did not identify whether Ad-si-IL-17 induced IL-17 ablation only in T effector cells or whether its effects were also extended to T_Reg cells, a population that is also capable of producing IL-17 and is intimately linked to the development of inflammation and cancer in the bowel.

T_Reg cells can affect tumor progression by mediating suppression of the effector T-cell responses and by inducing production of angiogenetic factors by T_H17 cells. Tumor infiltrating T_H17 cells themselves have also been postulated to offset the anti-tumor response of IFN-γ⁺ effector cells [54]. Thus, both T_Reg cells and T_H17 cells can affect cancer progression through independent and interconnected effects although the precise mechanisms of such counter-regulation remain poorly understood. Understanding the regulation of the T_Reg/T_H17 axis may result in novel approaches for the control of tumor progression. Studies by Baba et al. suggested that human CD45RA⁺ T_Reg cells promote the differentiation of human T_H17 cells, implying that CD45RA⁺ T_Reg cells positively govern T_H17 development. These findings might provide an explanation why T_H17 cells are often detected alongside T_Reg cells within tumor tissues or systemically in cancer patients [7, 55, 56] and further suggest that the T_Reg population might be an appropriate target for cancer immunotherapy.

5. Paradoxical roles of IL-17-producing Foxp3⁺CD4⁺ cells in anti-inflammatory response and anti-tumor immunity

Inflammatory and tumor tissues often have lower frequency of CD69⁺ T effector cells but a higher frequency of T_H17 cells and T_Reg cells compared to healthy or untransformed tissues [53]. The mechanisms by which T_H17 cells and T_Reg cells accumulate in such inflammatory and tumor tissues and their roles in the pathogenesis of these diseases remain unsolved. The identification of IL-17⁺Foxp3⁺ T cells in inflamed intestinal mucosa of patients with IBD and cancer but not in healthy control might provide some clues to the above questions.

IL-17⁺Foxp3⁺ cells accumulated in colorectal cancer (CRC) tissue express CCR6, TGF-β and IL-6, and significantly suppress CD8⁺ cell-mediated immune responses during in vitro culture [57, 58]. Interestingly, the suppressive activity of IL-17⁺Foxp3⁺ cells is attenuated by pretreatment with an anti-IL-17 antibody, suggesting that this subset of T cells may be a novel therapeutic target in the treatment of CRC [58]. In a similar study, abundant IL-17⁺Foxp3⁺ cells expressing high levels of TGF-β, CXCR3, CCR6, and RORγt were also detected in CRC. In addition, co-culture of IL-17⁺Foxp3⁺ cells isolated from transformed tissues with sphere cells generated from bone marrow derived mononuclear cells under hypoxia, could induce sphere cells to express CRC markers such as CD133, CD44s, CD166, EpCAM, and ALDH1 [59, 60]. Addition of neutralizing anti-IL-17 antibody in the co-culture abolished the expression of CRC markers in sphere cells, suggesting that IL-17⁺Foxp3⁺ T_Reg cells drive cancer-initiating mechanisms and that IL-17 itself plays a critical role in this process [60].

A different study showed that T_Reg cells become pathogenic in the context of CRC when they acquire expression of RORγt [61]. In this model, expression of RORγt marks a distinct subset of activated T_Reg cells, which have the potential to express IL-17 and have been identified in human CRC tumor. These RORγt-expressing T_Reg cells expand in CRC in a manner dependent on cancer-stage. Moreover, enriched RORγt⁺ T_Reg cells obtained from CRC display potent T-cell suppressive function. Conversely, RORγt deficiency restored the anti-inflammatory properties of T_Reg cells, protected against polyposis and improved anti-tumor immunity as determined by increased IFN-γ production [61]. These findings suggest that RORγt expressed in Foxp3⁺ T_Reg cells might serve as a primary therapeutic target for improvement of the anti-inflammatory effects of T_Reg cells, thereby inhibiting tumor development. Furthermore, these findings suggest that RORγt⁺ Treg rather than IL-17-producung cells might have a causative role in the pathogenesis of CRC development in the context of bowel inflammation (Figure 1).

6. Conclusions

The immunobiology of T_Reg cells has been intensively investigated since the discovery of T_Reg cells and their master transcription factor Foxp3, more than a decade ago. Such efforts have led to significant advances in our understanding of the development of T_Reg cells and the molecular mechanisms underlying their suppression function. Recent studies have focused on understanding the reciprocal relationship between T_Reg and other immune components surrounding T_Reg cells. Through these studies it has been recognized that T_Reg cells are subjected to regulation mediated by other immune cell populations, and as a result, the functional and phenotypic properties of T_Reg cells can be altered. Currently developing evidence supports the idea that the role of T_Reg cells in various disease settings varies considerably depending on disease stage, inflammatory conditions and activation of effector cells. Identification of the IL-17⁺Foxp3⁺T_Reg and RORγt⁺Foxp3⁺ T_Reg subsets in human IBD and CRC confirm that Foxp3⁺ T_Reg cells are capable of expressing transcription factors that control the differentiation of effector cells and can potentially produce proinflammatory cytokines. Although the exact mechanisms underlying the generation of IL-17⁺ T_Reg cells remain unclear, soluble mediators such as IL-1β, TGF-β and IL-23 might be involved in this process. The precise role of these T_Reg subsets in inflammation and tumor immunity is still debatable but most studies agree that IL-17⁺T_Reg and RORγt⁺T_Reg suppress anti-tumor immunity and promote tumor growth. Thus, targeting RORγt and IL-17 in T_Reg cells might be required in order to improve the properties of T_Reg cells employed for cell-based therapies in the context of inflammation and cancer.

Highlights.

• Foxp3⁺ T_Reg cells are able to express RORγt and produce IL-17

• IL-17⁺ T_Reg cells are accumulated in the inflamed intestinal mucosa.

• IL-17⁺ T_Reg cells have potent T-cell suppressive activity.

• RORγt ⁺ T_Reg cells are unable to suppress inflammation.

• IL-17⁺/RORγt⁺ T_Reg cells are pathogenic T_Reg cells with proinflammatory properties.