Inflammatory Bowel Diseases:
Basic Science Review Article
Keeping the Bowel Regular: The Emerging Role of Treg as a Therapeutic Target in Inflammatory Bowel Disease
Gibson, David J. MbChB, BAO*,†; Ryan, Elizabeth J. PhD*,†; Doherty, Glen A. Mb, PhD*,†
*Centre for Colorectal Disease, St. Vincent’s University Hospital, Dublin, Ireland; and
†School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland.
Reprints: David J Gibson, MbChB, BAO, Centre for Colorectal Disease, St. Vincent’s University Hospital, Elm Park, Dublin, Ireland (e-mail: firstname.lastname@example.org).
The authors have no conflicts of interest to disclose.
Received May 15, 2013
Accepted June 06, 2013
Abstract: The understanding of the intricate mechanisms by which gut immune cells interact with each other and the intestinal flora is constantly developing. The mucosal immune system must retain the ability to mount a prompt response to intestinal pathogens while maintaining tolerance for commensal organisms. Effector T lymphocytes drive inflammation, whereas their actions are counteracted by populations of regulatory T cells (Treg), which act as an endogenous suppressor of mucosal inflammation. There is growing evidence that a loss of this delicate counterbalance is important in the etiology of inflammatory bowel disease (IBD). Here, we review studies highlighting alterations in Treg in the pathogenesis of IBD. Observations of dynamic changes in Treg activity with successful IBD treatment have highlighted their functional importance and potential to also serve as a biomarker of disease activity and to predict response to therapy. Furthermore, we explore the potential for adoptive transfer of Treg as part of IBD treatment.
Inflammatory bowel diseases (IBDs) are a group of chronic, relapsing inflammatory disorders of the intestine with 2 major subgroups: Crohn’s disease (CD) and ulcerative colitis (UC). Although the pathogenesis of IBD is not fully understood, there is an emerging consensus that IBD results from a disrupted balance between the mucosal immune system and the gut microflora. The intestinal flora is altered in IBD patients, and this so-called “dysbiosis” may represent both cause and effect. Mucosal barrier disruption facilitates recruitment and activation of a potent proinflammatory immune response, which fails to resolve and results in persistent mucosal inflammation.1,2 The failure of resolution points to potential defects in endogenous mechanisms in the intestinal immune system, which regulate and suppress intestinal inflammation.
In this review, we aim to clarify recent advances in this field, specifically in relation to human regulatory T cells (Treg), a vital player in the maintenance of immune homeostasis in the gut. We will examine the evidence suggesting that Treg are altered in patients with IBD. In addition, we will highlight the observations of numerical and functional alterations in Treg associated with changes in disease activity and treatment response, notably response to antitumor necrosis factor (TNF) α therapy. Finally, we will discuss the potential use of Treg as part of IBD therapeutics, in particular highlighting recent human studies where Treg transfer successfully suppressed inflammation in IBD, representing proof of concept.
ROLE OF Treg IN THE HUMAN INTESTINE
Treg constitute 1% to 5% of peripheral blood T cells in healthy adults3 and play a key role in suppression of inappropriate immune responses. In the lamina propria (LP), a range of luminal antigens can generate proinflammatory responses and Treg play an important role in preventing development of pathological immune-mediated responses.4,5 They exert their homeostatic influence by applying a dominant negative regulation on other immune cells, such as T effectors. Treg can mediate contact-dependent suppression, i.e., via molecules such as cytotoxic T-lymphocyte antigen 46 or by production of cytokines such as interleukin (IL)-10 and transforming growth factor (TGF) β. Treg both produce and respond to TGF-β, an anti-inflammatory cytokine that plays an important role in the maintenance of immune homeostasis.7 In addition, Treg can also generate anti-inflammatory signals via other mechanisms, such as through the action of cell surface ecto-enzymes such as CD39, which neutralize the proinflammatory effects of extracellular nucleotides such as ATP.8 Infusion of Treg is capable of curing experimental colitis in animal models,9,10 and this has prompted a rapid expansion in research in this area. However, much remains to be discovered about the phenotype, function, and plasticity of human Treg before we can translate much of this knowledge to the clinic.
Naive T cells can differentiate into either Treg or T cells of the effector lineage,11 as depicted in Figure 1. The balance of differentiation is critical in maintenance of intestinal homeostasis. Tipping the balance may allow effector T cells to proliferate in an unregulated fashion, promoting the development of chronic intestinal inflammation. In the setting of IBD, activated mucosal T lymphocytes are increased in number.12 Therefore, continuous Treg differentiation and trafficking in the gut is required to dampen immune reactions to commensal bacteria and dietary antigens.13
Broadly speaking, Treg can be divided into subtypes: natural or adaptive. Natural regulatory T cell (nTreg) are CD4+CD25+ and originate in the thymus. Adaptive regulatory T cells (iTreg) are CD4+ and acquire CD25 positivity outside the thymus, typically in the setting of inflammation.13 A third, less well-defined population of Treg also exists, which are termed type 1 regulatory (Tr1) cells. Tr1 cells do not express FoxP3. They are induced peripherally in the presence of IL-10 both in vitro and in vivo,14 but there are little data on their role in intestinal immunity and so our discussion will focus on the first 2 categories of cells.
Treg were initially defined by surface expression of CD4+CD25+.15 However, as CD25, the alpha-chain of the IL-2 receptor, is commonly induced on T-cell activation,16 additional cell markers are clearly needed to define this population. In most studies, Treg are now identified as CD4+ T cells with a combination of high levels of CD25 and low levels of CD127 expression (CD4+CD25brightCD127low). Other surface markers such as CD39 have also been recognized as being associated with the Treg phenotype. However, it is the expression of the transcription factor, Fox P3, which is crucial to the regulatory activity of Treg17,18 and in defining the Treg populations.
CD4+FoxP3+ Treg are crucial to intestinal homeostasis,19 so much so that they have been described as “master regulators” of immune homeostasis.20,21
The functional importance of FoxP3 is underlined by the observation that in humans, mutations in FoxP3 causes development of immune deregulation polyendocrinopathy enteropathy and X-linked syndrome.22 Immune deregulation polyendocrinopathy enteropathy and X-linked syndrome is characterized by severe inflammation. The most frequently affected organ in this syndrome is the intestine, which exemplifies the important of this transcription factor in gut homeostasis.
Natural Tregs are generated in the thymus under the control of MHC class II-dependent T-cell receptors.23 Differentiation of adaptive Treg occurs in more varied circumstances than that of natural Treg,24 and the different microenvironments in which adaptive Treg develop in vivo is still not fully understood. The distinct signaling pathways controlling thymic (nTreg) versus peripheral (iTreg) induction of Treg has been investigated. Although thymic differentiation can occur in the absence of TGF β, TGFβ seems crucial for the activation of the suppressive capacity of Treg.25 Induction of FoxP3 seems dependent on TGFβ signaling, both in vivo and in vitro. The intestine is known to be rich in TGF β, thus facilitating local Treg differentiation.26 Gut-associated lymphoid tissue provides an environment for de novo generation of adaptive Treg, independent of thymic control, in response to activation by microbial antigens.27 Indeed, the intestinal microbiota promotes intestinal Treg-cell development, as Treg cell accumulation in the colon is reduced in germ-free mice.28
Activation of the innate immune system by microbiota occurs via a myriad of pattern-recognition receptors such as the Toll-like receptors (TLR) and NOD-like receptors.29
In mice, a deficiency in TLR-9 results in increased frequency of FoxP3+Treg,30 whereas deficiencies in TLR-2 result in a reduction of FoxP3+ Tregs.31
Both nTreg and iTreg secrete the anti-inflammatory cytokine IL-10. Although under steady-state conditions in the spleen and mesenteric lymph nodes Treg produce very little IL-10, they are the main CD4+ T-cell population producing IL-10 in the colonic LP.32 Recent studies have highlighted the importance of the anti-inflammatory properties of IL-10 in the human intestine. Loss of function in IL-10 and the IL-10 receptor are associated with early onset IBD with an aggressive phenotype.33 Furthermore, allogeneic hematopoietic stem cell tranplants has been used to good effect in IL-10- and IL-10 receptor-deficient patients.34,35 However, therapy based on recombinant IL-10 does not seem to provide any benefit for the treatment of active CD.36 Understanding the role of IL-10 in the regulation of inflammation in the human intestine may help us to better exploit its anti-inflammatory properties clinically.
As mentioned, a fine balance exists between Treg and effector T cells in the healthy state. One arm of the effector response is the Th17 pathway, which has an antagonistic relationship with Treg. The same naive T cell pool that generates Treg is also capable of producing Th17 effector T cells,37 and this is coordinated by TGF-β. Reduced frequency of Treg in IBD is associated with high levels of Th17.38 In the presence of infection or inflammation, IL-6 suppresses Treg production with the activation of a predominantly Th17 mediated proinflammatory response.39 IL-1β is also required for the differentiation of Th17 induction.40 In a manner similar to TGF-β, IL-6 is an important signaling protein, and in its presence, as found in active inflammation, Treg induction is blocked.41
TReg FUNCTION: LESSONS FROM MOUSE MODELS OF IBD
The role of Treg has been well studied in a range of murine models of IBD, and the results of these are summarized in Table 1. From initial studies, it became apparent that FoxP3 is integral to intestinal homeostasis.42,43 Subsequent studies have highlighted the mechanism by which naive T cells differentiate into Treg,20,26 the key role of Treg production of IL-10,10,32 and the mechanisms by which Treg suppress effector T cells44,45 and exert their anti-inflammatory role. The Wiskott–Aldrich syndrome protein is required for Treg function, and Wiskott–Aldrich syndrome protein knockout (WKO) nTreg lack the suppressive ability.46 Numbers of Treg were found to be reduced in colitic mice compared with controls,47 and further research provided an exciting potential avenue for IBD treatment, with the cure of colitis by the transfer of Treg.9 In the majority of these studies, Treg were defined by CD4+CD25+FoxP3+.
Treg LEVELS AND FUNCTION IN HUMAN IBD
The evidence in relation to the precise role of Tregs in IBD in humans is somewhat conflicting.
The key clinical studies that have evaluated the phenotype/function of Treg in both peripheral blood and the intestine of IBD patients are summarized in Table 2 and highlight the somewhat inconsistent findings. Although numerous trials have supported the hypothesis of reduced Treg in the active state,48–51 contradictory findings have also been found.52–55 In a comparison of patients with both UCand CD, versus control patients, Maul et al found that, in peripheral blood, increased Treg activity is found in remission, whereas, at mucosal level, higher levels of FoxP3 were found in inflamed mucosa. Contrasting to this, Saruta et al found increased Treg activity in active CD versus controls, both peripherally and in intestinal mucosa.
Although some of these conflicting observations may be explained by the use of limited panels of Treg markers in some studies, this alone is not sufficient to explain the somewhat discordant findings. Transient FoxP3 expression may be observed on activation of effector T cells, making reliance on FoxP3 expression alone unreliable.56 In addition, it is now clear that the concept of strict reciprocity between Treg and Th17 cell phenotypes is not valid. T-cell populations not only positive for FoxP3 but also producing IL-17 have recently been observed in the intestinal LP of IBD patients. Interestingly, despite producing IL-17, these cells seem to retain a suppressive phenotype.57
A correlation between disease activity and Treg concentration has been described. In the setting of UC, Holmen et al58 found a positive correlation between Treg frequency and disease activity. This further confuses matters but may suggest that, although increased in the active state, the function of Treg is impaired, and as such, T-effector cells are inadequately suppressed. In other autoimmune conditions such as rheumatoid arthritis (RA), there is evidence that Treg function is compromised in active disease,59 and similarly, in the setting of systemic lupus erythematous, Treg function is deficient.60
No definite evidence of a functional defect in human Treg has been identified to date in IBD studies. It has been suggested that, rather than a primary defect in Treg activity, IBD may be more associated with an effector cell phenotype that is resistant to Treg suppression. Colitogenic T cells obtained from patients with IBD have been described, which seem resistant to the anti-inflammatory effect of TGF-β.13 Augmented activity of the signaling molecule Smad7, a negative regulator of TGF-β signaling has been proposed as a mechanism, and antisense oligonucleotides, which would inhibit Smad7 activity, are in early clinical trials in IBD.61 However, the absence of spontaneous colitis in Smad7 transgenic mice, which overexpress this molecule, has caused some to question the importance of this pathway.62 It is also clear that effective treatment of IBD is associated with dynamic changes in human Treg and that Treg resistance has not proved a barrier to the effective use of adoptive transfer of Treg as an IBD therapy.
ROLE OF TNF IN IBD/EFFECTS OF ANTI-TNFα THERAPY ON Treg
Anti-TNFα therapy, administered either intravenously or subcutaneously, is now well established as effective therapy for IBD.
Initially, animal models showed a role for TNFα in chronic intestinal inflammation,63 and subsequently, TNFα was found to have an important role in pathogenesis of CD64 and UC.65 Neutralization of TNFα results in the downregulation of proinflammatory cytokines, including IL-17, IL-23, and IL-666 with potential effects on T-cell differentiation in inflamed tissue.
However, approximately one-third of patients with IBD do not respond to anti-TNF treatment, whereas others lose responsiveness or become intolerant.67 The precise mechanisms underlying the action of anti-TNFα treatment, and the reasons for nonresponse have not been elucidated in full. Paradoxically, new onset IBD has been reported in some patients treated with anti-TNF agents for other inflammatory conditions.68,69 Given the fact that some anti-TNF therapies, such as etanercept, are ineffective in CD,70 this further illustrates the complex mechanism by which anti-TNF treatments exert their effect.
It is possible that the inflammation in patients who do not respond to anti-TNFα treatment is driven independently of TNFα. As can be seen in Figure 1, other proinflammatory cytokines, independent of TNFα pathways, such as IL-6 and IL-17, play an important role in the inflammatory cascade, further supported by numerous clinical trials in these areas.71–73
In other autoimmune conditions such as RA, anti-TNF treatment has been shown to have a restorative effect on Treg59 and further studies in RA have shown that anti-TNF treatment increases FoxP3 expression, thereby augmenting the Treg suppressive function.74,75 The effects of anti-TNF therapy on Treg also provide contrasting results when assessed in different IBD patient cohorts; the results of studies to date are summarized in Table 3.
One emerging theory is that there is increased apoptosis of Treg in patients with active IBD. In patients who respond to treatment, anti-TNF therapies seem to reduce this apoptosis, thereby increasing the ratio of Treg:T effectors,48 which may be important in the therapeutic efficacy of these agents.
Anti-TNF antibodies also seem to induce apoptosis of mucosal CD4+ cells in inflamed mucosa.76 In contrast, minimal apoptosis takes place in peripheral blood. This suggests that these agents are acting selectively on membrane-bound TNF-bearing pathogenic intestinal lymphocytes.77 However, other authors have produced data to contradict this theory, showing that anti-TNF therapy caused increased levels of Treg in peripheral blood, with a reciprocal decrease in intestinal mucosa.78
There does appear to be stronger evidence to support the theory of anti-TNF treatment increasing Treg function.49,50,79 However, given the fact that numerous articles have contradicted this evidence,51,80–83 we are left with more questions than answers.
The exact markers that exclusively define Treg in humans remain an area of active debate. CD4+CD25+FoxP3+ cells may define a significant majority, but, as already described, Tr1 cells do not express FoxP3.
The varying ways in which different studies have defined Treg populations, as illustrated in Table 2, may help explain some of the contrasting observations. Markers such as cytotoxic T-lymphocyte antigen 4,84 Ki67,85 and CD127 negativity confer further purity.86 Other cell surface markers such as CD39 and CD73 may be useful in identifying Treg, particularly those subpopulations with a propensity to Th17 differentiation.87,88 As described by Collison and Vignali,89 Treg functional suppression assays appear crucial to classify this T-cell population and future studies need to focus on isolation and characterization of human Treg populations in IBD patients.
FUTURE DIRECTIONS—Treg AS IBD THERAPY
Given the sustained interest in the role of Treg in IBD pathogenesis, efforts persist into how Treg can be incorporated into a viable form of therapy for humans with IBD.
The transition of knowledge from animal to human models is always challenging. What may be effective treatment in animal models may have little effect in humans, and the limitations of murine models of IBD compared with humans are clear.90
The challenges of Treg transfer therapy are manifold. If Treg are to be infused, it will be necessary to select a cell population, which is a pure pool of Treg. Avoidance of contamination with effector T cells is critical. In addition, it is not known whether in vitro T cells infused in vivo will retain their regulatory capacity or differentiate into effector cells. If the latter occurs, there could be an unwanted exacerbation of the autoimmune process. This is supported by studies showing differentiation of Treg at the site of inflammation into T effectors of the Th1 and Th17 lineage.91,92
Ongoing studies are identifying further cell surface molecules to help differentiate T-cell subpopulations and, therefore, offer the potential for a more pure collection.93 There is the potential that harvested Treg could undergo ex vivo reconditioning with agents such as rapomycin and retinoic acid, with a view to potentiating their suppressive function.94 Emerging biologic treatments in IBD, such as ustekinumab, have targeted ILs such as IL-23, involved in the promotion of the Th17 effector pathway,95,96 could be used in conjunction with Treg transfer to maximize clinical effectiveness.
One unanswered question relates to the patient-to-patient variability in the intestinal microbiota, which has a critical role in the evolution of IBD.
With each patient comes a different microenvironment. It is known that some commensals specifically promote FoxP3 Treg,28,97 whereas others have shown that commensal bacteria exploit the TLR pathway to actively suppress immunity.98
How in vitro T cells will adapt to contrasting gut microenvironments in vivo is unknown. Manipulation of the intestinal flora may provide an additional avenue to potentiating the effectiveness of adoptive transfer Treg therapy.
Recent clinical data offer significant grounds for optimism in this field as the CATS1 study, performed by Desreumaux et al,99 provides evidence of the efficacy of Treg treatment in the setting of refractory CD. In this open-labeled study, autologous antigen-specific Treg were collected, purified ex vivo, and then infused at varying doses. Results were promising, and with 1 concentration of infusion, there was a 75% response rate. Admittedly, study numbers were small, but this landmark study may pave the way for the introduction of Treg-based treatment in IBD.
Although many questions remain unanswered, it is clear that Treg play a critical role in gut homeostasis and that numerical and functional alterations in Treg are observed in humans with IBD, which seems important in disease pathogenesis. With improved characterization of the full panel of Treg markers in humans, a better understanding of the role of Treg is emerging. Changes in Treg that are observed in conjunction with successful use of anti-TNF agents may explain some of their beneficial effects on IBD disease activity. New molecular approaches to promote the regulatory pathway in T cells, whereas dampening the effector pathway, have considerable promise as a part of future anti-inflammatory strategies. Recent clinical data provide proof of concept that adoptive transfer of Treg has potential as an effective disease therapy for IBD. Further studies in this area should be prioritized.
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inflammatory bowel disease; anti-TNF treatment; immune system; Treg; FoxP3
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