Induction of regulatory Tr1 cells and inhibition of T_H17 cells by IL-27

Abstract

Accumulating evidence indicates that IL-27, a member of the IL-12 family of cytokines, alleviates the severity of autoimmune diseases in both mice and men. The IL-27-induced activation of Signal transducer and activator of transcription (Stat)1 and Stat3 promotes the generation of IL-10-producing type 1 regulatory T (Tr1) cells that inhibit effector T cells. In addition, IL-27 also suppresses the development of pathogenic IL-17-producing CD4⁺ T cells (T_H17) cells, suggesting that pharmacological manipulations of IL-27 signaling pathway could be exploited therapeutically in regulating tissue inflammation. Here, we review how IL-27 controls inflammation through the regulation of Tr1 and T_H17 responses.

1. Introduction

Since the original classification by Mosmann and Coffman of CD4⁺ helper T (T_H) lymphocytes into T_H1 and T_H2 subsets[1], the repertoire of T_H subsets has expanded to include additional effector and regulatory T cell subsets such as T_H17 cells and regulatory T cells (Foxp3⁺ Tregs and Tr1 cells). T_H1 cells, which predominantly produce interferon (IFN)-γ and lymphotoxin, are essential for eliminating intracellular pathogens, but were also regarded as the major effector T cells in inducing tissue inflammation in organ-specific autoimmunity. However, mice lacking the component of T_H1-IFN-γ pathway (Il12^−/−, Ifng^−/−, Ifngr1^−/−, Il12rb2^−/−) were not protected but overly susceptible to autoimmune diseases including Experimental Autoimmune Encephalomyelitis (EAE)[2], Experimental Autoimmune Uveitis (EAU)[3] and collagen-induced arthritis (CIA)[4]. Subsequent studies revealed that T_H17 cells, instead of T_H1 cells, induce tissue inflammation in autoimmune diseases. Although T_H17 cells are essential for eliminating extracellular pathogens [5, 6], exaggerated T_H17 response promotes autoimmunity. Elevated amounts of IL-17A and IL-17F are detected in several autoimmune diseases including multiple sclerosis (MS) [7], rheumatoid arthritis (RA) [8] and psoriasis[9]. The involvement of T_H17 cells in tissue inflammation was confirmed in mouse models such as EAE where IL-17-neutralizing antibodies ameliorate clinical scores [10] or CIA where IL-17-deficient animals develop attenuated disease[11]. The differentiation factors for both mouse and human T_H17 cells were found to be a combination of TGF-β1 and IL-6 or TGF-β1 and IL-21[12]. The activation of Signal transducer and activator of transcription (Stat)3 by IL-6 or IL-21 is critical for inducing the expression of the T_H17 cell master transcription factors retinoid-related orphan receptor (ROR)γt, encoded by the gene Rorc, and RORα (Rora) [13] [14, 15]. Rorc^−/− and Rora^−/− mice show defective T_H17 cell generation [15]. In addition, Chip-Sequencing analysis revealed Stat3 binding sites in the promoters regions of il17a and il17f gene[12]. Furthermore RORγt drives the expression of GM-CSF that is essential for inducing pathogenic T_H17 cells, and mice deficient in making GM-CSF are resistant to develop EAE[16]. These observations indicate that RORγt is essential for the development of T_H17 cells. Indeed T_H17 cell generation can be inhibited by directly targeting RORγt using small chemical compounds such as digoxin and SR1001[17]. While IL-23 is not required for the induction of T_H17 cell differentiation, IL-23 has a prominent role in expansion and stabilization of pathogenic T_H17 cells [18–20]. Both IL-12p19^−/− and IL-23R^−/− mice are resistant to EAE, and few T_H17 cells are found in the central nervous system (CNS) of those mice[21–23]. The IL-23-T_H17 pathway has been shown to be critical in many autoimmune diseases, which is consistent with the fact that IL-23R polymorphisms has been genetically associated with a number of human autoimmune diseases including psoriasis, inflammatory bowel diseases (IBD) and ankylosing spondylitis[24]. More recent studies suggested that T_H17 cells could also be induced with the combination of IL-1β, IL-6 and IL-23 in the absence of TGF-β1, suggesting that T_H17 cells might actually represent a heterogeneous population of proinflammatory cells that are highly pathogenic and can be induced by multiple different ways.

Exaggerated inflammatory responses are prevented by regulatory T cell subsets that suppress activation of effector T cells. CD4⁺ regulatory T cells comprise Foxp3⁺ regulatory T-cells (Tregs) and IL-10-producing regulatory type I cells (Tr1) cells [25]. Foxp3⁺Tregs are important to maintain self-tolerance as illustrated by the severe autoimmune inflammation observed in mice deficient in Foxp3[26] or in patients with dysfunctional FOXP3 protein[27]. Although Foxp3⁺ Tregs inhibit effector T cell responses, they lose their suppressive functions in inflammatory conditions[28]. Therefore, IL-10-producing Tr1 cells might be crucial in controlling tissue inflammation. In humans, Tr1 cells were first described in severe combined immunodeficient (SCID) patients who had developed long-term tolerance to stem cell allografts, supporting the existence of these cells in humans and suggesting that they may play a role in mediating T cell tolerance [29]. Tr1 cells mediate immune suppression by secreting the suppressive cytokine IL-10 and by killing effector cells via Granzyme-B and Perforin [30, 31]. While IL-10 was initially described to be the differentiation factor for Tr1 cells, these T cells could not expand in the presence of IL-10. Therefore there was an emphasis on identifying growth/differentiation factors for Tr1 cells. Recent identification of IL-27 as a differentiation/growth factor for Tr1 cells has revived the interest in examining their role in tissue inflammation [32–34].

2. IL-27 dampens autoimmune inflammation

IL-27, an heterodimeric cytokine composed by the subunit p28 (IL-27p28) and the Epstein-Barr virus-induced gene 3 (EBI3), is mainly produced by activated antigen-presenting cells APCs[35]. IL-27 signals through a receptor complex consisting of the common IL-6 receptor chain, gp130, and the unique IL-27 receptor alpha chain (IL-27Ra or WSX-1) that is homologous to IL-12Rβ2 of IL-12 receptor[35, 36]. Based on the structural homology between IL-12 and IL-27 and their receptors, IL-27 was initially described as a proinflammatory cytokine that could induce T_H1 differentiation, which was consistent with the ability of IL-27 to induce T-bet (Tbx21), the master transcription factor for the generation of T_H1 cells. Subsequent work, using both T_H1 and T_H2 associated pathogens, established that IL-27 suppresses T_H cells (T_H1, T_H2 and T_H17 cells) functions in vivo, as Il27ra^−/− mice showed enhanced T cell functions (reviewed in [37]). However, the mechanism by which IL-27-induced inhibition of T cell functions was not understood until the discovery that IL-27 can induce IL-10 production from CD4⁺ T cells.

3. IL-27 controls T cell responses

3.1 Regulation of T_H1 and T_H2 differentiation

While IL-27 induces T-bet and expression of IL-12Rβ2 in naïve CD4⁺ T cells, IL-27 signaling is not mandatory for T_H1 differentiation as illustrated by mice lacking the IL-27R subunit (Il27ra^−/−) that can mount adequate T_H1 responses to eliminate intracellular pathogens [38–40]. Moreover, Il27ra^−/− mice die due to uncontrolled immunopathology and severe tissue inflammation associated with exaggerated T cell responses and enhanced production of IFN-γ and TNF-α [38–40]. IL-27 was also reported to control the generation of T_H2 cells. IL-27 treatment during Strongyloides venezuelensis infection decreases T_H2 responses against the parasite and treated mice failed to develop intestinal mastocytosis and exhibited a marked delay in parasite expulsion [41]. Furthermore, intranasal administration of IL-27 inhibits OVA-induced airway hyperresponsiveness and inflammation in OVA-sensitized animals[41]. At the transcriptional level, IL-27 has been shown to suppress the master T_H2 transcription factor GATA-3[41]. Recently, genome-wide association study (GWAS) has shown that a single nucleotide polymorphism (SNP) in the IL-27p28 gene was associated with an increased susceptibility to asthma[42] or COPD[43] and IL-27 has been proposed as a potential treatment for bronchial asthma.

3.2 Inhibition of T_H17 cell differentiation

In addition to inhibiting both T_H1 and T_H2 development, IL-27 prevents the development of T_H17 cells in vitro and in vivo. Il27ra^−/− mice are overly susceptible to EAE compared to wild-type mice and present an increased accumulation of T_H17 cells in the draining lymph nodes and in the CNS [44]. In this model, neutralization of IL-17 in Il27ra^−/− mice during EAE disease course attenuated their disease phenotype [44]. Accordingly, recombinant IL-27 treatment decreases the disease incidence and severity in EAE with the inhibition of development of T_H17 cells [45]. Similarly, Il27ra^−/− mice chronically infected with T. gondii developed severe neuropathology mediated by CD4⁺ T cells, associated with increased T_H17 cell development. IL-27 inhibits the production of IL-17 by BMNCs from chronically infected mice stimulated with IL-23[46]. Finally in the absence of IL-27 during murine flu infection, flu-specific T cell responses are skewed towards T_H17[47].

Above observations clearly indicated that IL-27 is negative regulator of development of T_H17 cells. However, the mechanism by which IL-27 inhibits the development of T_H17 cells is not clearly understood. Accumulating data suggest that IL-27 utilizes multiple mechanisms to inhibit the development of T_H17 cells (Fig. 1 and 2). During T_H17 cell differentiation, IL-27 directly suppresses the expression of both RORγt, the master transcription factor of T_H17 cells [48] and RORα[49] (Fig. 1). IL-27 inhibits expression of RORγt in T_H17 cells both in mouse and man [48]. Interestingly, IL-27 decreases the expression of GM-CSF and thereby dampens the pathogenicity of T_H17 cells[16]. By blocking GM-CSF secretion and by inhibiting both RORα and RORγt expression, IL-27 interferes with T_H17 cell differentiation at several levels, explaining its potent ability to suppress the induction of T_H17 cells.

Figure 1. IL-27 inhibition of differentiating T_H17 cells.

On differentiating T_H17 cells, IL-27 inhibits the expression of transcription factors Rorγt and Rorα, thereby impairing the secretion the T_H17-related cytokines, IL-17A, IL-17F, IL-22 and GM-CSF.

Figure 2. IL-27 inhibition of committed T_H17 cells.

IL-27 induces the differentiation of Tr1 cells that inhibit T_H17 cells in an IL-10-dependant manner. IL27p28 monomers interfere with IL-6 cytokine signaling through gp130 and thereby inhibit the maintenance of T_H17 cells and their IL-17 secretion. IL-27 further induces t-bet expression that drives IFN-γ production and promotes the conversion of T_H17 cells into T_H1 cells.

Whether IL-27 can directly suppress effector/memory T_H17 cells or fully differentiated T_H17 cells is still debated. Indeed, T_H17 maintained in culture for at least two rounds become unresponsive to IL-27 as IL-27 fails to inhibit the expression of RORα and RORγt in these cells[49]. However, IL-27 could modulate effector/memory T_H17 cells using different strategies. Among the two IL-27 cytokine subunits, EBI3 is constitutively expressed but IL-27p28 secretion is transcriptionally regulated. IL-27p28 monomers can interfere with the IL-6-mediated production of IL-17 by preventing IL-6 signaling through gp130, suggesting that IL-27p28 monomers could also be exploited in regulating T cell responses [50]. IL-27p28 thus limits the generation and maintenance of T_H17 cells in vivo without directly interfering with T_H17 transcriptional program (Fig. 2). Furthermore, it has been proposed that T_H17 could be converted into T_H1 cells that are presumably less pathogenic [51, 52]. One putative mechanism by which IL-27 could converts T_H17 into T_H1 cells may be by inducing the expression of T-bet that drives IFN-γ expression and reduces the expression of IL-17 (Fig. 2). However, this hypothesis by which IL-27 may increase T_H17 plasticity has not been proven experimentally.

3.3 Induction of Tr1 cells

IL-27, while inhibiting TGF-β-induced Foxp-3⁺ Tregs, induces IL-10⁺, IFNγ⁺ T cells that are immunosuppressive, a phenotype in line with the previously described Tr1 cells [32–34, 53, 54]. The role of IL-27 in generation of IL-10-producing Tr1 cells was further emphasized in vivo. IL-27 treated MOG-specific splenocytes lose their ability to transfer EAE in an IL-10 dependent manner [33]. Furthermore, during flu infection, IL-27 generates regulatory T cells that inhibit T_H17 cells by secreting IL-10 and IFN-γ. In the absence of IL-10, flu-specific T cell responses developed a stronger T_H17 component [47]. Furthermore, it has been shown that Tr1 cells can inhibit T_H17 cells in vivo in an IL-10 dependent manner during murine colitis [55] (Fig. 2). Akin to what has been observed in murine T cells, activation of naïve human T cells in the presence of IL-27 similarly induces Tr1 cells that produce both IFN-γ and IL-10 [56].

4. Molecular pathways involved in IL-27 biology

Similar to other type 1 cytokine receptors, IL-27 also induces the activation of Janus kinase/Stat pathway. IL-27 predominantly induces the phosphorylation of Stat1 and Stat3. Here we will discuss the IL-27-induced signaling events following the activation of the Stats and analyze their roles in inhibiting T_H17 cell and in inducing Tr1 cell differentiation.

4.1 IL-27 and Stat1 activation

4.1.1 Stat1 activation by IL-27 represses T_H17 differentiation and induces Tr1 cells

The activation of the IL-27 specific subunit WSX-1 drives the tyrosine phosphorylation of JAK1 that further activates Stat1. Indeed, JAK1, but not other JAKs, coprecipitates with the WSX1 subunit[57].

The Stat1 signaling pathway is necessary for IL-27-induced T-bet expression [58]. T-bet not only drives the expression of IFN-γ but also plays an important role in the inhibition of T_H17 cytokines, independently of IFN-γ. T-bet can reprogram committed T_H17 cells by repressing T_H17 gene program, which results in fewer transcripts of Rorc, il17a, il17f, il23r [59]. These finding were supported by studies showing that T-bet utilizes Runt-related transcription factor 1 (Runx1), a transcriptional activator that sequesters Rorc away from the regulatory regions on Rorc promoter[59]. Indeed Runx1 binding site is located upstream of T-bet binding site on Rorc promoter. By sequestering Runx1, T-bet inhibits the expression of RORγt, resulting impaired development of T_H17 cell[59] (Fig. 3).

Figure 3. Reciprocal regulation of T_H17 and Tr1 cells by IL-27.

The molecular mechanisms by which IL-27 promotes Foxp3⁻IL-10⁺Tr1 cell differentiation and represses T_H17 cell development through activation of Stat1 and Stat3 activation are shown. IL-27 activates Stat1 through the subunit WSX1 that inhibits RoRγt expression through t-bet-dependent as well as t-bet-independent pathways. Alternatively, IL-27 can promote t-bet expression in a Stat1 independent pathway via GADD45γ. In addition, IL-27 activates Stat3 signaling through gp130. Stat3 induction then drives Maf transcription. Maf together with Ahr transactivates il21 and il10 promoters. On the other hand, IL-27 inhibits Foxp3 transcription in a Stat3/Smad3 dependent manner.

Stat1^−/− and T-bet^−/− mice exhibit an increased number of T_H17 cells both during systemic inflammation in vivo or during T_H17 cells differentiation in vitro. IL-17 production is greater in the absence of T-bet compared to the absence of Stat1[60]. This may be related to the fact that T-bet might also be induced in a Stat1 independent manner. In this vein, Owaki et al have shown that IL-27 induces a Stat1 independent T-bet expression[61]. Indeed IL-27 induces the expression of GADD45γ that further drives the phosphorylation of p38 MAPK leading to T-bet expression (Fig. 3).

It has been further proposed that Stat1 could inhibit RORα and RORγt expression in differentiating T_H17 cells in a T-bet independent manner (Fig. 3). While a direct inhibitory effect of Stat1 on RORα and RORγt expression has not been ruled out, Stats could also indirectly affect T_H17 responses by promoting the function of auxiliary inhibitory T_H17 factors. Different repressors of T_H17 cells differentiation have been identified, including Ets-1, which negatively regulates T_H17 cell differentiation[62]. Stat1 and Ets-1 have been shown to bind together[63] and might cooperate to inhibit T_H17 cell differentiation by directly or indirectly interfering with RORγt function in T_H17 cells.

IL-27 has been shown to induce IL-10 expression from CD4⁺ T cells using both Stat1 and Stat3 pathways (Fig. 3). Indeed, in the absence of Stat1 signaling, IL-27 driven IL-10 production is decreased. While it is clear that the Stat1 driven IL-10 secretion is independent of t-bet signaling, the underlying mechanisms still remain unclear [34].

4.2 IL-27 and Stat3 activation

4.2.1 Stat3 activation by IL-27 does not enhance T_H17 cell differentiation

IL-27 utilizes gp130 subunit of IL-6 receptor complex, which results in activation of Stat3 signaling. A genetic defect in Stat3 signaling in humans, in hyperIgE syndrome, results in defective T_H17 cells and in unrelenting fungal infections, supporting the critical role of Stat3 in the generation of T_H17 cells[64]. At the first glance, it is puzzling that IL-6 and IL-27, which both activate Stat3 pathways, have antagonistic properties. It has been proposed that IL-6 leads to a faster and more persistent pattern of Stat3 phosphorylation that is crucial to drive pro-inflammatory signals downstream Stat3. pStat-3 directly binds to il17a and il17f promoters and transactivate these genes by collaborating with other trancription factors like IRF-4 and RORγt. Furthermore, the formation of Stat1-Stat3 heterodimers in response to IL-27 rather than the formation of mainly Stat3 homodimers in response to IL-6 or IL-21 may play a role in the difference between IL-6 and IL-27 signaling. Indeed preliminary data from our laboratory supports this hypothesis. In addition, IL-6 activation rapidly induces Stat3 repressor SOCS3[65]. SOCS3 is an essential negative regulator of Stat3 phosphorylation and constrains T_H17 cell differentiation[66, 67]. While IL-27 induces expression of SOCS3, IL-27-mediated inhibition of IL-17 production is independent of SOCS3[46]. It therefore seems unlikely that IL-27-induced SOCS3 contributes to the inhibition of T_H17 cells. Instead, the inhibition of T_H17 differentiation might mainly be mediated through Stat1 and T-bet as discussed above.

4.2.2 Stat3 activation by IL-27 promotes Tr1 cell differentiation

IL-27-induced Stat3 phosphorylation is essential for the anti-inflammatory role of IL-27, as it triggers IL-10 secretion from CD4⁺ T cells[34] (Fig. 3). Sustained activation of Stat3 leads to the induction of the transcription factor Maf[68]. We and others have recently shown that Maf is essential for IL-10 production induced by IL-27[53]. Similarly to Stat3 deficient CD4⁺ T cells, Maf deficient CD4⁺ T cells cannot produce IL-10 in response to IL-27. It has been further shown that Maf directly transactivates il10 and il21 promoters[53]. In addition to Maf, IL-10 production by IL-27 is regulated by the ligand activated transcription factor Aryl hydrocarbon receptor (AhR) that binds to Maf resulting in a complex that induces both il10 and il21 transcription[69]. The finding of AhR involvement in IL-10 production is significant as it provides impetus to design AhR ligands that can modulate the anti-inflammatory properties of Tr1 cells both in vitro and in vivo (reviewed in [31]). The expression of the cytokine IL-21 is further essential for IL-27-induced-IL-10 production[53] (reviewed in [37]). In the absence of IL-21, IL-10 production is reduced in Tr1 cells. IL-21 secretion can be further amplified by AhR activation[69].

4.2.3 Stat3 activation by IL-27 and inhibition of Foxp3

IL-27 inhibits the generation of Foxp3⁺Tregs[70]. The fact that Foxp3⁺Tregs express IL-27R strongly suggested that IL-27 might block the development of those regulatory cells in vitro[71]. IL-27 indeed leads to a decreased expression of Foxp3 through a mechanism that is at least partially dependent on Stat3[70]. Smad3 binding to Foxp3 promoter is implicated in Foxp3 transcription. It has been proposed that IL-27-induced pStat3 binds to a gene silencer region (enhancer II) in a conserved region of Foxp3 gene that reduces the acetylation in the region of Smad3 binding site and decreases the binding of pSmad 3 to Foxp3 promoter [72]. This results in a decreased accessibility and binding of Smad3 to Foxp3 promoter and thereby decreases Foxp3 transcription (Fig. 3). IL-27 impacts Foxp3⁺Treg development and function in vivo. Indeed mice that overexpress both IL-27 subunits, IL-27p28 and EBI3, have decreased number of Foxp3⁺Tregs and developed spontaneous inflammation similar to mice that lack Foxp3⁺Tregs such as the scurfy Foxp3 mutant mice or IL-2^−/− mice[73]. Interestingly, IL-27 transgenic mice are deficient in IL-2. Those results are in accordance with another recent study showing that IL-27 inhibits Foxp3⁺Treg in vivo in a murine T cell transfer colitis model. Il27ra^−/− deficient T cells transferred an attenuated disease due to a larger percentage of transferred cells expressing Foxp3 compared to wild-type T cells[74].

5. Therapeutic implications

5. 1. IL-27 confers protection against Multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system resulting in inflammation, demyelization and axonal loss. It is a common neurological disorder, which attacks young adults. T_H17 cells were shown to contribute to MS development[75]. By contrast, IL-27 protects against autoimmune inflammation in the mouse model EAE as exemplified by Il27ra^−/− mice which develop an accelerated EAE disease course compared to WT controls and show increased levels of T_H17 cells in the CNS [44]. Furthermore, daily intrathecal treatment with IL-27 during EAE alleviates the disease and decreases both the inflammation in the brain and the number of infiltrating T_H17 cells[45]. Similarly in a T cell adoptive transfer model, pre-treatment of autoreactive CD4⁺ T cells with IL-27 leads to a reduction of their pathogenicity in an IL-10 dependent manner[33]. Interestingly, IL-27 was also shown to mediate the protective effect of Bone marrow stromal cells (BMSCs) that prevent EAE in mice and suppress IL-17 production[76].

Support for IL-27 in regulating autoimmune tissue inflammation has also been provided in humans. The immunomodulatory drug IFN-β, used in the first line of treatment for MS, has been shown to induce IL-27 production from dendritic cells (DCs). Interferon (IFN)-β, a member of the type I interferon family, is an approved treatment for relapsing remitting MS (RRMS) that reduces the rate of relapses by 30%. While the therapeutic mechanisms of IFN-β remain poorly understood, recent studies indicate that IL-27 contributes to its regulatory properties both in mouse[77] and human[78] [79]. One limitation of IFN-β treatment is that 20–50% of patients fail to respond to therapy thus delaying a change in the treatment strategy of those patients. While the presence of neutralizing antibodies (Nabs) against IFN-β in the blood has been proposed to correlate with treatment failure[80], a proportion of non-responder patients do not develop Nabs, limiting the use of Nabs to predict the response to IFN-β therapy [81]. IL-27 secretion from PBMC from RRMS patients has been proposed as a predictive factor of clinical response to IFN-β treatment. Indeed, PBMC isolated from RRMS patients that respond to IFN-β treatment secrete more IL-27 when exposed in vitro to IFN-β than PBMC isolated from “non-responder” patients[78]. Finally, other therapies proposed for treating MS, such as Statins, which in addition to their cholesterol-lowering activity have anti-inflammatory properties, were shown to increase in vitro IL-27 secretion from human monocytes of MS patients [82]

5.2 IL-27 protects against rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory disorder that principally attacks synovial joints. T_H17 cells and IL-17 expression is elevated in RA synovial tissue and fluid macrophages compared to controls[83, 84]. Elevated levels of IL-17 have been reported in the animal model of RA, collagen-induced arthritis (CIA), and IL-17 neutralization prevents bone destruction suggesting a pathological role of T_H17 cells in the development of RA [85]. Administration of IL-27 in mice suffering from CIA reduces the severity of the disease, as shown by reduced cellular infiltration in the joints, synovial hyperplasia, and joint erosion[84]. IL-27 treatment further decreases serum levels of IL-6. In addition, lymphocytes isolated from spleen and lymph node of IL-27-treated mice produce significantly reduced amounts of IFN-γ and IL-17 when cultured with type II collagen in vitro compared with lymphocytes from control mice. Similar results were obtained when IL-27 was ectopically expressed in the joints [86]. These studies highlight in the therapeutic potential of IL-27 in RA, especially with the feasibility of local, intra-articular, administration of recombinant IL-27.

5.3 Controversial role of IL-27 in inflammatory Bowel Disease

IL-27 is implicated in the pathogenesis of IBD, Crohn’s disease and ulcerative colitis. Genome wide studies have identified SNPs in the gene encoding p28 subunit associated with a lower expression of IL-27 and early onset inflammatory bowel disease, which would be consistent with a protective role of IL-27 in IBD[87]. Two other studies have found transcripts for IL-27p28[88] and Ebi3[89] to be overexpressed in biopsy samples from IBD patients. The function of IL-27 has been assessed using different murine models of IBD. In the mouse IBD model of acute inflammation, which relies on the presence of dextran Sulfate Sodium (DSS) to induce inflammation, Il27ra^−/− mice receiving 5–10% DSS in drinking water were more susceptible to disease[90]. Il27ra^−/− deficient mice showed a reduction in T_H1 IFNγ-producing cells and an increase in T_H17 cells in gut-associated lymphoid tissue pointing toward an important regulatory role of IL-27 in dampening T_H17 cell function [90]. In the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 (EBI3 and p28 subunits generated as a single-chain human IL-27) treatment significantly improved in a dose-dependent manner the extent of the lesions as well as necrosis, ulceration and thickening of mucosal epithelium. scIL-27 suppressed several inflammatory cytokines in inflamed colon, including IL-17 [91]. However, in a T cell transfer colitis model, IL-27 was shown to exert proinflammatory effects as it suppressed induced Treg development in vivo[74]. In contrast, in the DSS model, Il27ra^−/− mice treated with lower doses of DSS (0.5% in drinking water), were protected compared to WT controls[92]. The implication of different pathogenic or regulatory subsets and the heterogenicity of the models may explain the different responses to IL-27 treatment in murine models of colitis. However, in models where T_H17 cells are implicated in the development of the disease, the anti-inflammatory role of IL-27 appears to be dominant. Indeed, T_H17 cells have been shown to be crucial for the development of TNBS-induced colitis as IL-17 receptor A (IL-17RA) knockout mice do not develop TNBS colitis[93] and IL-17F-deficient mice develop more severe DSS colitis than controls[94]. A better understanding of the pathogenesis of IBD should provide additional insight into the role of IL-27 in colitis.

6. Open questions and concluding remarks

While IL-27 promotes Tr1 cells, it inhibits CD4⁺Foxp3⁺Tregs induced by TGF-β. These observation are reminiscent of the action of AhR ligands such as FICZ that promotes Tr1 cells but inhibits Foxp3⁺Tregs. This paradoxical effect on regulatory T cells might stem from different and/or complementary roles of regulatory T cells. Tr1 cells but not Foxp3⁺Tregs may develop in situ in the inflamed tissue as IL-27 can be secreted be resident cells in the target organ, such as in the brain during EAE and MS. Foxp3⁺ Tregs can not inhibit highly pathogenic effector T cells in the target organ[95] but they induce tolerogenic plasmacytoid dendritic cell (DC) that secrete IL-27 thus promoting Tr1 cell generation [32]. Under inflammatory settings, Foxp3⁺Tregs can produce cytokines that belong to other lineages [96, 97] and we propose that Tr1 cells could be more stable and thereby regulate tissue inflammation at the target site.

IL-27 controls inflammation by inhibiting T_H17 cells and by promoting the development of IL-10-producing regulatory Tr1 cells. Despite their opposite in vivo functions, Tr1 and T_H17 cells harbor striking similarities. First, they rely on the transcription factors Maf and AhR for their generation. Second, they require IL-21 for their growth. Third, they produce IL-10. In this regard, Ghoreschi et al showed that T_H17 differentiated with TGF-β and IL-6 (T_H17(β)) produced IL-10 and were poorly pathogenic in vivo in contrast to T_H17 cells induced by IL-6, IL-1β and IL-23 (T_H17)(23)) that did not produce IL-10 and were highly pathogenic. In addition, TGF-β induced T_H17 expressed higher levels of Maf and AhR compared to T_H17 induced with IL-1, IL-6 and IL-23 (23). This observation would thus be in line with a previous work suggesting that the Maf-driven induction of IL-10 in T_H17 cells reduced their pathogenicity[98]. Since we have shown that the expression of Maf and AhR is required for the production of IL-10 and IL-21 in Tr1 cells, it might be interesting to explore whether IL-27 could actually be converting T_H17 to Tr1 cells. We are currently conducting a functional transcriptional analysis of Tr1 (differentiated with IL-27) and T_H17 (IL-6 and TGF-β) cells using a computational approach and a whole genome microarray analysis to address this question.

In the same line, IL-21 has been ascribed a functional role in promoting both T_H17[99, 100] and Tr1 cells[53]. The role of IL-21 during autoimmune disease such as EAE is controversial. While initial studies have proposed that IL-21R^−/− mice presented a less severe EAE disease [100], longer observation of EAE disease course showed that IL-21R^−/− mice developed a more severe disease [101, 102]. Besides being a growth factor for T_H17 cells [103], IL-21 may behave as an anti-inflammatory effect by promoting IL-10 secretion from different T cell subtypes. It remains to be seen whether IL-27 and its downstream cytokine IL-21 can modulate the pathogenicity and stability of different subtypes of T_H17 cells that have been further treated with IL-23. In conclusion, IL-27 not only induces the generation of anti-inflammatory Tr1 cells but broadly controls autoimmune responses by inhibiting effector T cells in various target organs.

Highlights.

• IL-27 controls autoimmune responses by promoting Tr1 cells and inhibiting T_H17 cells.

• IL-27 triggers Stat1 and Stat3 signaling

• IL-27 induces Maf and AhR that control IL-10 secretion from Tr1 cells

• Stat1 activation represses T_H17 cells and induces Tr1 cells

• Stat3 activation enhances Tr1 cells

• IL-27 alleviates human autoimmune diseases.

Abbreviation

Tr1 cells

type 1 regulatory T cells

T_H17

T helper 17

Stat

Signal Transducer and Activator of Transcription

Maf

Transcription factor Maf

Ahr

Aryl hydrocarbon receptor