IL-6 is a pleiotropic cytokine involved in the physiology of virtually every organ system. Recent studies have demonstrated that IL-6 has a very important role in regulating the balance between IL-17-producing Th17 cells and regulatory T cells (Treg). The two T-cell subsets play prominent roles in immune functions: Th17 cell is a key player in the pathogenesis of autoimmune diseases and protection against bacterial infections, while Treg functions to restrain excessive effector T-cell responses. IL-6 induces the development of Th17 cells from naïve T cells together with TGF-β; in contrast, IL-6 inhibits TGF-β-induced Treg differentiation. Dysregulation or overproduction of IL-6 leads to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA), in which Th17 cells are considered to be the primary cause of pathology. Given the critical role of IL-6 in altering the balance between Treg and Th17 cells, controlling IL-6 activities is potentially an effective approach in the treatment of various autoimmune and inflammatory diseases. Here, we review the role of IL-6 in regulating Th17/Treg balance and describe the critical functions of IL-6 and Th17 in immunity and immune-pathology.
CD4+ Th cells are essential regulators of immune responses and inflammatory diseases. CD4+ Th cells can be divided into various subsets, such as Th1, Th2 and Treg. The development of Th1 cells, which activate macrophages and are highly effective in clearing intracellular pathogens, is coupled to the sequential actions of IL-12 and IFN-γ 1, 2. Th2 cells, the differentiation of which is driven by IL-4, are important for the production of immunoglobulin E and the clearance of extracellular organisms 3–5. While the Th1-Th2 paradigm provided a reasonable basis for further exploration of the mechanisms of immunity to infection and autoimmune diseases, several lines of evidence suggest that Th1 cells are not the true culprits in the induction and progression of many autoimmune diseases, as originally proposed. For example, IFN-γ-deficient (KO) mice do not show resistance to autoimmunity. On the contrary, these mice are even more susceptible to autoimmunity 6, which led us and others to hypothesize that there may be an additional Th subset that is distinct from Th1 cells. Indeed a “new” subset of Th cells that produces IL-17, which would eventually come to be known as the Th17 cells, were shown to play a crucial role in the induction of autoimmune diseases such as rheumatoid arthritis (RA) and experimental autoimmune encephalomyelitis (EAE), and allergen-specific responses 7–9. Furthermore, Th17 cells are crucial for defense against fungi and extracellular bacteria 10. In addition to these effector subsets, there are distinct regulatory subsets, including the CD4+ Treg, which is characterized by the forkhead/winged helix transcription factor forkhead box P3 (Foxp3). TGF-β1 promotes Treg differentiation, which in turn suppresses adaptive T-cell responses and prevent autoimmunity 11, 12.
IL-6 is a pleiotropic cytokine with various biological activities in immune regulation, hematopoiesis, inflammation, and oncogenesis. IL-6 induces the generation of Th17 cells from naïve T cells together with TGF-β and inhibits TGF-β-induced Treg (iTreg) differentiation 13–15. These observations support the notion that IL-6 blockade may be an innovative treatment strategy for autoimmune diseases. Indeed, it has been demonstrated that targeting IL-6 activities can be an effective approach in the treatment of several autoimmune diseases 16, 17. In this review, we discuss the novel function of the pleiotropic cytokine IL-6 in Th17/Treg development, their relationships in autoimmune and inflammatory diseases, and the clinical applications of this knowledge.
IL-6 was cloned as B-cell stimulatory factor-2 in 1986 18. IL-6 has various biological activities, including a stimulatory effect on the growth of murine plasmacytoma and human myeloma and induction of acute phase reaction; it also functions as a hepatocyte stimulating factor 19–23. In addition to these stimulatory effects, IL-6 also has inhibitory function, e.g. an inhibitory effect on the antiviral antibody response after immunization with a vesicular stomatitis virus 23.
IL-6 activates a receptor complex consisting of the IL-6 receptor (IL-6R) and the signal-transducing receptor subunit gp130 24, 25. IL-6R exists in both a transmembrane form and a soluble form. IL-6 binds to both of these forms, which can then interact with gp130 to trigger downstream signal transduction and gene expression. Although gp130 has no intrinsic kinase domain, members of the Janus Kinase (Jak) family, such as Jak1, Jak2, and tyrosine kinase 2 (Tyk2), are constitutively associated with gp130 26. Complexes of IL-6, IL-6R, and gp130 phosphorylate these kinases and then activate the cytoplasmic transcriptional factors, STAT1 and STAT3 27. Thus, IL-6 activates these kinases and transcriptional factors through IL-6R/gp130 complexes, which in turn leads to IL-6's downstream effects.
Th17 cells secrete IL-17A (IL-17), IL-17F, IL-22, IL-6, and TNF-α 8. The IL-17 family is composed of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F 28. IL-17 is a pleiotropic cytokine, which mediates tissue inflammation by inducing many pro-inflammatory cytokines (such as IL-6 and TNF-α) and chemokines 28, 29. IL-17 is also involved in immunity to bacteria through the recruitment and activation of neutrophils and macrophages 30. Importantly for the treatment of autoimmune diseases is the recent recognition of Th17 cells, as opposed to Th1 cells, as the primary effector cell type involved in both autoimmune diseases in human as well as mouse models of these diseases 31–34. Indeed, expression of IL-17 has been detected in the serum and target tissues of patients with various autoimmune diseases such as RA, multiple sclerosis (MS), and systemic lupus erythematous (SLE) 34, 35. Furthermore, IL-17 KO mice are resistant to the development of collagen-induced arthritis (CIA) and EAE, and IL-17 blockade prevents the development of EAE 32, 33, 36. These evidence indicate that the regulation of Th17 differentiation and its function is potentially an effective treatment for autoimmune diseases.
Differentiation of Th17 cells
Although initial reports claimed that IL-23 is required for the generation of Th17 cells from naïve T cells 37, 38, it was subsequently demonstrated that IL-23R is not expressed on naïve T cells. Instead, IL-23, as well as TNF-α, acts as a survival signals for Th17 cells 15, 39. The current consensus is that IL-6 induces Th17 differentiation together with TGF-β 13, 14. It has been reported that IL-21, similar to IL-6, can also initiate Th17 differentiation combined with TGF-β 40. On the other hand, it has been suggested that additional signals may be required to induce fully functional Th17 cells in vivo41. The combination of IL-6 and TGF-β induces the orphan nuclear receptors, retinoid-related orphan receptor (ROR) γt and RORα, which are the key transcription factors in determining the differentiation of the Th17 lineage 42, 43. STAT3 regulates IL-6-induced expression of RORγt and RORα and IL-17 production 44, 45. In contrast to STAT3 activation, STAT1 activation inhibits the development of Th17 cells. Although IL-6 activates both STAT3 and STAT1, it has been demonstrated that STAT3 activation is maintained while STAT1 activation is suppressed in Th17 cells 46.
The negative regulatory system for Th17 differentiation involves some STAT family members mediated through various cytokines. IL-27 and IFN-γ are responsible for the inhibition of Th17 development in a STAT1-dependent manner 47–49. It has been reported that IL-2 also inhibits Th17 development, an effect that is dependent on STAT5 50. Thus, STAT family members activated by various cytokines provide both positive and negative regulation of Th17 development.
In addition to the aforementioned transcription factors, interferon-regulatory factor 4 and T-bet are two relative new-comers to the scene, and these act as positive and negative regulators of Th17 commitment, respectively 51, 52. Furthermore, while retinoic acid inhibits Th17 development, dioxin, a ligand of aryl hydrocarbon receptor (Ahr), promotes the generation of Th17 cells 53–56. Ahr is induced under Th17-polarizing conditions, such as in the presence of TGF-β plus IL-6, and promotes Th17 development through inhibiting STAT1 and STAT5 activation 53. Ahr is one of the important factors in Th17 differentiation. Given that such nuclear receptors play an important role in Th17 differentiation, the control of nuclear receptor signaling may lead to novel approaches in the treatment of autoimmune diseases caused by Th17 cells.
Treg play a critical role in maintaining immune homeostasis and preventing autoimmune diseases 12, 57. Although there are many types of Treg, naturally occurring Treg (nTreg) and iTreg are the best characterized. nTreg are generated in the thymus and acquire the expression of the transcription factor Foxp3. iTreg are generated from naïve T cells in the periphery or in vitro, after stimulation with antigens and TGF-β 58. In addition to nTreg and iTreg, other T cells with regulatory properties have been reported, including the CD8+ Treg, Tr1 cells, and Th3 cells 59–61. Although the activation of self-reactive T cells causes various autoimmune diseases, they are controlled by peripheral tolerance mechanisms such as Treg. Therefore, it is critical to maintain tolerance via Treg in order to prevent autoimmune diseases.
A balance between Th17 and Treg is crucial for immune homeostasis. TGF-β is required for Th17 and Treg differentiation and it can induce both Foxp3 and RORγt expression 62; however, as described, this treatment led exclusively to Treg differentiation, as Foxp3 is able to associate with RORγt and inhibit RORγt transcriptional activation 62. In the presence of IL-6 however, this inhibition was abrogated, and Th17 differentiation was initiated 13, 14. Thus, IL-6 acts as a potent pro-inflammatory cytokine in T cells through promotion of Th17 differentiation and inhibiting Treg differentiation (Fig. 1), indicating that the control of IL-6 normalizes the balance between Th17 and Treg and may alleviate autoimmune symptoms.
Anti-IL-6R therapy for autoimmune diseases in mice and human
The overproduction of IL-6 and abnormalities in IL-6 signal transduction are causative factors in autoimmune disorders including RA. At present, there is increasing clarification of the role of IL-6 in diseases such as RA in which Th17 cells are considered to be the primary cause of pathology. We first focus on anti-IL-6R therapy for the pathogenesis of CIA and of EAE – animal disease models that exhibit symptoms close to those of human RA and human MS, respectively. Although Th1 cells were once considered to be the major cause of pathogenesis in CIA and EAE, it has become clear that Th17 cells are actually the major cause 7–9. As discussed, given the critical role of IL-6 (and TGF-β) in inducing Th17 differentiation 13, 14, suppression of Th17 differentiation by anti-IL-6R antibodies is most likely the mechanism underlying resistance to CIA and EAE, as suggested by data from murine models 61, 62.
Closer examination of the CIA and RA model reveals the intricate details of IL-6's role in Th17 differentiation. In the CIA model, CIA is induced by administering type II collagen together with an adjuvant on day 0 and day 21 in mice. Interestingly, although anti-IL-6R antibodies administered on day 0 suppressed the induction of Th17 cells in the regional lymph nodes and the development of arthritis, antibodies administered on day 14 did not suppress Th17 cells or arthritic symptoms. These results indicate that the inhibition of Th17 differentiation caused by anti-IL-6R antibodies is necessary for CIA suppression and that for CIA to develop, IL-6 is required for the initial differentiation of Th17 cells from naïve T cells, but not for the maintenance of Th17 cells after differentiation 63. Moreover, we investigated whether a suppressing effect of Th17 development is observed with TNF inhibitor therapy in CIA. Intriguingly, when a TNF-soluble receptor (TNFR-Fc) was administered during the initial CIA induction period (days 0–14), arthritis and Th17 differentiation could not be suppressed; however, when TNFR-Fc was administered after day 21, arthritis is substantially suppressed although Th17 cells were increased to levels similar to those in control mice and mice treated with TNFR-Fc on days 0–14. 63. These results suggest that IL-6 inhibitor treatment in CIA acts primarily on initial CD4+ T-cell response including Th17 differentiation, rather than on the effector phase including angiogenesis and osteoclast differentiation. In contrast, it suggests that the main point of action in TNF inhibitor therapy is different from that of IL-6 inhibitor therapy, i.e. TNF inhibition does not play a role in initial Th17 differentiation but instead acts on the effector phase.
EAE is induced by administering the myelin sheath framework protein myelin oligodendrocyte glycoprotein peptide together with an adjuvant and pertussis toxin. When anti-IL-6R antibodies were administered immediately after antigen stimulation, the occurrence of EAE could be suppressed in the same manner as for CIA. Under this treatment regimen, no Th17 cells were found in the draining lymph nodes or the spinal cord. Moreover, few immune cells such as T cells, B cells, and macrophages were observed in the lesion of spinal cord 64. On the other hand, the effect of anti-IL-6R antibodies on Th17 cells and the disease onset was abolished when anti-IL-6R antibody administration was delayed. Thus, IL-6 is required for the initial differentiation phase of Th17 also in the EAE model, and it appears that IL-6 also acts on cells other than Th17 cells because few immune cells were observed in the lesion of spinal cord after anti-IL-6R antibody administration. These results show that IL-6 inhibitor therapy is highly effective in suppressing the occurrence of EAE 64.
In humans, anti-IL-6R antibodies (Tocilizumab) has become a novel therapeutic strategy for some inflammatory and autoimmune diseases, including RA, systemic-onset juvenile idiopathic arthritis, Crohn's disease (CD), Castleman's disease, multiple myeloma, and systemic lupus erythematous 17, 65–67. Tocilizumab can block the IL-6 signals induced by the interaction of IL-6 and IL-6R as well as the neutralization of the soluble receptors by establishing the antibody against the receptors (Fig. 2). In RA patients, Tocilizumab significantly improved the symptoms and ACR (American College of Rheumatology) improvement scores 20, 50, and 70 (ACR 20, 50, and 70 indicate 20, 50, and 70% improvement in disease activity, respectively) were 89, 70, and 47%, respectively, and normalized C-reactive protein and serum amyloid A in the patients within 6 wk 68. Although a role of Th17 in RA is less clear, it has been reported that IL-17 is detected in the synovial fluid from RA patients and acts as a potent stimulator of osteoclastogenesis 69. Given that IL-6 is important for Th17 differentiation not only in mice but also in humans, Tocilizumab may improve the symptoms of RA through regulating the development of Th17 cells. Although it has been shown that IL-6 blockade by Tocilizumab is therapeutically effective for other inflammatory diseases such as Castleman's disease, juvenile idiopathic arthritis, and CD 17, 65, 66, it remains controversial whether Tocilitumab can inhibit Th17 differentiation in a manner that is clinically meaningful. Furthermore, it remains to be demonstrated how Tocilizumab contributes to the treatment of the above-mentioned autoimmune diseases and whether Tocilizumab can also exert therapeutic benefits on other autoimmune disorders.
IL-6 has a wide range of biological activity including immune responses, inflammation, and hematopoiesis. It has been reported that IL-6 levels are increased in various autoimmune diseases including RA, CIA, and CD, suggesting that IL-6 blockade may be an effective strategy for such autoimmune diseases. In fact, targeting IL-6 has been a promising novel approach for the treatment of inflammatory and autoimmune diseases. Inflammatory T cells are thought to be central to the pathology of autoimmune diseases. Especially, the recently identified T-cell subset, the Th17 cells, shows pro-inflammatory functions and plays a critical role in various autoimmune disorders. On the other hand, Treg are important in the maintenance of immune homeostasis. Both an excess in Th17 function or increased numbers of Th17, and defects in Treg function or reduced numbers of Treg, may trigger inflammatory disorders. IL-6 participates in the differentiation of both of these two T-cell subsets. Whereas IL-6 induces Th17 differentiation together with TGF-β, IL-6 inhibits TGF-β-induced Treg differentiation, indicating that IL-6 is a very important factor in determining Th17/Treg balance. Taken together, the dysregulation of IL-6 may lead to imbalances in Th17/Treg and subsequently inflammatory diseases. These evidences explain why IL-6 blockade is an effective treatment for several autoimmune diseases; however, it remains to be fully understood how Tocilizumab exerts its therapeutic effects. Future studies will be required to establish the safety and full benefit of Tocilitumab.
This work was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation and Chugai-Roche Pharmaceutical Co. Ltd, Tokyo, Japan.
Conflict of interest: The authors declare no financial or commercial conflict of interest.