Recent reports have provided convincing evidence that IL-17-producing T cells play a key role in the pathogenesis of organ-specific autoimmune diseases, a function previously attributed exclusively to IFN-γ-secreting Th1 cells. Furthermore, it appears that IL-17-producing T cells can also function with Th1 cells to mediate protective immunity to pathogens. Although much of the focus has been on IL-17-secreting CD4+ T cells, termed Th17 cells, CD8+ T cells, γδ T cells and NKT cells are also capable of secreting IL-17. The differentiation of Th17 cells from naïve T cells appears to involve signals from TGF-β, IL-6, IL-21, IL-1β and IL-23. Furthermore, IL-1α or IL-1β in synergy with IL-23 can promote IL-17 secretion from memory T cells. The induction or function of Th17 cells is regulated by cytokines secreted by the other major subtypes of T cells, including IFN-γ, IL-4, IL-10 and at high concentrations, TGF-β. The main function of IL-17-secreting T cells is to mediate inflammation, by stimulating production of inflammatory cytokines, such as TNF-α, IL-1β and IL-6, and inflammatory chemokines that promote the recruitment of neutrophils and macrophages.
More than 20 years ago Mosmann and Coffman reported that distinct CD4+ T-cell subtypes, termed Th1 and Th2 cells, could be distinguished on the basis of cytokine secretion and function 1. The Th1–Th2 model provided a useful but simplistic basis for our understanding of the mechanisms of immunity to infection and also attempted to explain the role of T cells in the pathogenesis of autoimmunity and allergy/asthma. The original hypothesis was that IFN-γ-secreting CD4+ cells mediated protective immunity to intracellular pathogens by promoting macrophage activation and stimulating production of complement fixing and virus neutralizing antibodies, but also promoted inflammatory responses and mediated the pathology in certain autoimmune diseases. In contrast, CD4+ T cells that secreted IL-4, IL-5, IL-10 and IL-13 were considered to be the main helper T cells providing help for B-cell production of antibody, especially IgG1 in mice, and mediated protective immunity to extracellular pathogens. These Th2 cells were also anti-inflammatory, controlling Th1 responses, but at the same time mediated allergic reactions and had an inflammatory and pathological role in asthma.
The identification of additional T-cell subtypes has led to a shift in the Th1–Th2 paradigm and has helped to explain some anomalies in the original model. In the mid 1990s suppressor T cells were re-discovered as Treg cells and were shown to have a major role in controlling immune responses to self-antigens, thereby preventing autoimmunity. Furthermore, these cells rather than the reciprocal Th1 or Th2 populations were shown to be the major regulator of effector T cells, functioning to maintain self-tolerance, prevent autoimmunity and limit collateral damage during immune responses to pathogens 2, 3.
There were a number of other observations which could not be explained on the basis of the two Th1 and Th2 subtypes. Despite the assumption that Th1 cells mediated autoimmunity, mice deficient in IFN-γ or IFN-γ receptors were found to have increased susceptibility to EAE and collagen-induced arthritis (CIA) 4, 5. Furthermore, EAE was exacerbated in mice deficient in the Th1 polarizing cytokine, IL-12 6, 7. It was then discovered that mice deficient in the novel IL-12 family member, IL-23 were resistant to EAE 6. IL-23 had previously been shown to play a role in promoting differentiation and/or survival of a population of CD4+ T cells that secreted IL-17, 8, 9, later termed Th17 cells. In addition, it was demonstrated that adoptive transfer of IL-23-polarized autoantigen-specific Th17 effector cells were able to transfer EAE more effectively than Th1 cells 9. IL-17 and IL-23 have also been shown to have a role in the pathogenesis of CIA 10 and colitis 11, 12. These studies established a pathogenic role for Th17 cells in organ-specific autoimmune and chronic inflammatory diseases. Th17 cells have also been shown to play a protective role in immunity to infection, where they help to promote pathogen clearance by enhancing neutrophil recruitment to sites of infection and activating macrophages 13, 14.
This review addresses the contribution of different T-cell subtypes to IL-17 production, the role of innate cytokines in promoting the differentiation and expansion of IL-17-producing T cells, the stimuli and signalling pathways for induction of these innate cytokines, the regulation of IL-17 production and the function of IL-17 in the pathogenesis of autoimmunity and in protection against infection.
T-cell subtypes that secrete IL-17
It was initially reported that IL-17 could be secreted by human CD4+T cells 15 and that IL-17 mRNA was elevated in the CSF and blood of patients with MS 16. It was later demonstrated that IL-17-producing T cells were a distinct linage from Th1 or Th2 cells 17–19. Furthermore, it was demonstrated that the transcriptional factor RORγt directed development of IL-17-secreting T cells 20, whereas T-bet and GATA3, which are involved in the development of Th1 and Th2 cells, respectively, inhibited differentiation of Th17 cells 18, 19 (Fig. 1). In addition, the induction of Th17 cells and severity of EAE were reduced in RORγt−/− mice 20.
Although Th17 cells have derived their name from IL-17, they secrete a range of cytokines, including IL-17A, IL-17F, IL-21, IL-22, TNF-α and IL-6, which have both overlapping and distinct roles in host defence and inflammation 21, 22. Furthermore, although T cells are a major source of IL-17, they are not the only source of this cytokine. Neutrophils, as well as T cells, have been shown to produce IL-17 in response to IL-15 23. In addition, the precise subpopulation(s) of T cells that secrete IL-17 remain unclear.
Most studies have focused on CD4+ T cells and have shown that IL-17-secreting T cells are expanded in, and can transfer symptoms of, autoimmunity in mouse models, such as EAE, and indeed the terms ‘Th17 cell’ or ‘ThIL-17 cell’ arose from the observations that CD4+ Th cells are a major source of this cytokine and can produce it independently of IFN-γ or IL-4 18, 19. However, human memory CD8+ T cells have been shown to secrete IL-17 in vitro following stimulation of PBMC with PMA and ionomycin 24 and murine CD8+ T cells produce IL-17 in response to IL-1 and IL-23 25 or conditioned medium from DC activated with Klebseilla pneumoniae26.
Unconventional T cells can also secrete IL-17 when activated with appropriate stimuli in vitro or in vivo during infection or inflammation 27–30. Invariant NKT cells that lack the NK1.1 marker have been found to secrete high concentrations of IL-17, but not IFN-γ, and to mediate neutrophil influx into the lungs during airway inflammation 27. It has recently been demonstrated that NKT cells, which express IL-23R, secrete IL-17 following stimulation with anti-CD3 and IL-23, independently of IL-6 30. Furthermore, NKT cells can secrete IL-17 in response to the synthetic ligand α-galactosylceramide 27, 30.
γδ T cells and other non-CD4+CD8+ T cells are the primary source of IL-17 during Mycobacterium tuberculosis infection 28. Vδ1+ γδ T cells are also a major source of IL-17 in Escherichia coli infection, where they promote neutrophil recruitment 29. Studies in the CIA model have suggested that IL-17-secreting Vγ4/Vδ4+ γδ T cells may have a pathogenic role in autoimmunity 31. Therefore, T cells other than CD4+ Th cells secrete IL-17 and function in autoimmunity and protective immunity to pathogens and the term ‘Th17’ should only be used when it is shown that IL-17 is produced by CD4+ T cells. Furthermore, while Th17 can be clearly identified as a distinct population of CD4+ T cells from Th1 cells and to employ distinct transcriptional factors for their development, a population of cells that co-express IL-17 and IFN-γ have been identified 32–34. IL-17+IFN-γ+CD4+and IL-17+IFN-γ−CD4+ T cells express receptors for both IL-12 and IL-23, and stimulation with IL-12 enhances IFN-γ production and T-bet expression 35. However, the function of these IL-17 and IFN-γ double-positive T cells and their lineage relationship to Th1 and Th17 cells remain to be defined.
Differentiation and expansion of IL-17-secreting CD4+ T cells
The differentiation of naïve T cells into effector, pathogenic or Treg is controlled by a number of factors, including the activation status of the APC and the cytokine milieu at the site of induction. Following the discovery of Th1 and Th2 cells, it was demonstrated that the differentiation of Th2 cells is enhanced by IL-4, while IL-12 and IFN-γ are responsible for the differentiation of Th1 cells. More recently it has been shown that TGF-β and IL-10 have a role in the induction or peripheral conversion of Treg cells. It was initially reported that the IL-12 family member, IL-23, was the major stimulus for promoting the differentiation of Th17 cells 18, 19, 22, 36, 37. The study by Cua and colleagues demonstrated that it was possible to induce EAE in IL-12p35−/− but not IL-23p19−/− or IL-12p40−/− mice 36. However, naïve murine T cells do not express IL-23R and do not differentiate into Th17 cells in response to IL-23 stimulation in vitro22, 38. Conversely, it was demonstrated that IL-23 could expand a population of IL-17-producing T cells from memory CD4+ T cells in vitro37 and IL-23-driven IL-17 secreting cells were pathogenic in EAE 22. These studies also suggested that IL-23 induced differentiation of IL-17-secreting CD4+ T cells (Th17 cells), a distinct lineage from the IFN-γ-secreting Th1 cells, driven by IL-12 18, 19. It was then demonstrated that IL-6 and TGF-β could promote differentiation of murine Th17 cells from naïve CD4+ T cells co-stimulated with anti-CD3 and CD28 or APC in vitro39–41. Furthermore, development of Th17 cells was impaired in vivo in TGF-β mice 40 and in mice with defective TGF-β signalling in T cells 42. It was later reported that IL-21 was a key cytokine in the differentiation of murine Th17 cells and that it may act in an autocrine loop involving IL-6 and TGF-β 43–45 (Fig. 2).
The studies on IL-6 and TGF-β could not find a role for IL-23 in the differentiation of IL-17-secreting T cells in vitro or in vivo, but suggested that IL-23 may be required for expansion or survival of Th17 cells 39–41. However, in parallel with these findings, we reported that IL-1α or IL-1β synergized with IL-23 to promote IL-17 production by murine T cells in the presence or absence of TCR engagement, whereas T cells from IL-1 receptor type I-deficient (IL-1RI−/−) mice failed to induce IL-17 production in response to IL-23 25. In addition, the induction of antigen-specific Th17 cells (but not Th1 or Th2 cells) by immunization with antigens and adjuvants was abrogated and the severity of EAE was significantly lower in IL-1RI−/− compared with wild-type mice 25. An independent study demonstrated that IL-1α/β double knockout mice were also resistant to EAE, whereas IL-1ra−/− mice were more susceptible 46. It was also demonstrated that IL-1β is a critical mediator of chronic destructive murine arthritis, probably through the induction of Th17 cells 47. In a murine model of arthritis, where disease is induced by commensal bacteria in IL-1Ra−/−, a defect in TLR4 controlled the production of IL-17 by inducing IL-23 and IL-1 production by APC 48. In Leishmania amazonensis infection, IL-1β enhanced IL-17 production in vitro and in vivo, and also enhanced IFN-γ and IL-10 production 49.
Collectively, these studies suggested that IL-1 can act directly on murine T cells to enhance IL-17 production, either alone or in the presence of IL-23. These findings were confirmed and extended by a study which reported that both mouse and human memory T cells, but not naïve T cells, secrete IL-17 in vitro in response to stimulation with IL-1 and IL-23 in the presence of anti-CD3 38. However, Wilson et al. demonstrated that either IL-1β or IL-23 could promote the differentiation of human Th17 cells from naïve T cells in vitro following co-stimulation with anti-CD2, anti-CD3 and anti-CD28 50. An independent study reported that IL-1β and IL-6 induced differentiation of naïve human T cells into Th17 cells following co-stimulation with anti-CD3 and anti-CD28 in vitro51. These studies found that, unlike murine T cells, human naïve T cells did not differentiate into IL-17-secreting T cells in response to TGF-β and IL-6 in vitro38, 50, 52, 53. Furthermore, in a study of patients with genetic traits affecting TGF-β, IL-1, IL-6 and IL-23 production, enhanced TGF-β responses, due to mutations in TGFB1 and TGFBR2, did not alter expression of Th17 cells, whereas mutations in STAT3 and IL-12RB1 impaired dedifferentiation or expansion of Th17 cells in vivo54.
Three recent reports have demonstrated that TGF-β does have a role in promoting differentiation of human Th17 cells in vitro and that previous failures to identify a role for TGF-β reflected the high content of this cytokine in fetal bovine or human serum used to culture the T cells 53, 55, 56. It appears that high concentrations of TGF-β, such as that present in FCS can inhibit Th17 differentiation, but that lower concentrations may promote it. Yang et al. reported that TGF-β and IL-21 can induce differentiation of naïve human T cells cultured in serum-free medium in the presence of anti-CD3 and anti-CD28, whereas IL-1, IL-23 or IL-1 + IL-6 induced IL-17A production by central memory CD4+ T cells 53. Manel et al. showed that TGF-β with IL-1β, IL-23 and IL-2 promoted development of human Th17 cells in vitro in serum-free conditions; IL-6 or IL-21 also combined with IL-1β and IL-2 to drive Th17 differentiation, but not as effectively as with IL-23 55. Finally, in a systematic examination of the factors involved in promoting different cytokines production by Th17, Volpe et al. reported that TGF-β, IL-23, IL-1β and IL-6 all contributed to human Th17 cell differentiation in vitro in serum-free medium, but that these cytokines differentially modulated IL-17, IL-21, IL-22 and IL-6 production by the Th17 cells 56.
These new findings suggest that there is less difference between mouse and human Th17 than originally thought. Furthermore, the evidence of definitive roles for IL-6/IL-21 with TGF-β versus IL-1 and IL-23 are not necessarily incompatible, since IL-23 and IL-1 may act on a distinct cell type to that activated by TGF-β and IL-6 or IL-21. IL-1 and IL-23 may have greater function in promoting the activation or expansion of memory Th17 cells. However, experiments with IL-6−/− mice demonstrated that IL-6 is not essential for production of the Th17 cytokine, IL-22, and its protective role against liver inflammation associated with hepatitis 57. Furthermore, conditioned medium form LPS-stimulated DC induced IL-17 production by naïve T cells in the presence of anti-gp130 antibody or with LPS-stimulated DC from IL-6−/− mice 58, suggesting IL-6 is not always essential for differentiation of murine, as well as human, Th17 cells. However, IL-6-defective mice are resistant to induction of EAE 59 and other reports have demonstrated that IL-6 is important for the development of Th17 cells 39, 41.
A recent report has demonstrated that expansion of autoantigen-specific T cells in the presence of IL-23 generates a population of IL-17-secreting T cells that can induce EAE, whereas T cells expanded in the presence of TGF-β and IL-6 are not pathogenic, because they co-induce IL-17- and IL-10-secreting T cells 60. However, studies in an experimental autoimmune uveitis (EAU) model demonstrated that IL-23p19−/− mice produce less IL-17 and were more resistant to induction of EAU 61. Treatment with anti-IL-23 reduced IL-17 production and attenuated EAU when administered immediately before and after induction of disease, but not at the effector stage of the disease, suggesting that IL-23 promotes the induction of murine Th17 cells in vivo61. This conclusion is consistent with a study in the EAE model involving adoptive transfer of T cells into IL-23p19−/− mice, which concluded that IL-23 played a critical role in the development, but not effector function, of encephalitogenic T cells 62. These studies, together with findings from my own group, showing that IL-23 can function with IL-1 to promote ‘innate’ IL-17 production from unconventional T cells in the absence of TCR engagement 25 (and Sutton and Mills, unpublished observations), suggest that the function of IL-23 is not to be confined to expansion of memory T cells.
Role of TLR and NOD-like receptors (NLR) ligands in promoting innate cytokines that drive Th17 cells
TLR, as well as NLR, act as sensors of pathogen infection and are located primarily on cells of the innate immune system, such as DC. Binding of ligands to TLR and NLR leads to the activation of NF-κB, IRF and MAP kinase signalling pathways 63. Activation of these signalling pathways is associated with enhancement of co-stimulatory molecule expression on DC and the production of inflammatory cytokines, which allows the DC to direct the induction of T-cell responses. TLR signalling has been implicated in precipitating, or exacerbating autoimmune diseases. One mechanism may involve induction of innate cytokines, such as IL-6, TGF-β, IL-1 and IL-23, and the subsequent induction of Th17 cells. Experimental autoimmune disease in many animal models such as EAE, EAU and CIA is induced by immunization with autoantigen in the presence of CFA; this is likely to involve the activation of innate inflammatory cytokines that drive Th1 and Th17 cells. Indeed there is already evidence that pathogens or PAMPs can promote the production of inflammatory cytokines from monocytes and DC, which activate IL-17 production by T cells 14, 38, 64. Stimulation of DC with TLR3, TLR4 and TLR9 ligands was found to induce MyD88-dependent production of cytokines that promoted differentiation of IL-17 producing CD4+ T cells 41. We have reported that the Gram-negative bacteria Bordetella pertussis or LPS purified from this bacteria induce TLR4-dependent IL-1 and IL-23 production from DC, which in turn leads to the induction of IL-17 production by murine antigen-specific memory T cells 14. Similarly, bacterial-derived ligands for the intracellular PRRs, NOD-2, such as MDP, have also been shown to drive IL-17 production by human memory T cells by stimulating IL-1 and IL-23 production by DC 38. Furthermore, hyphae of Candida albicans induce IL-23, but not IL-12 production from human monocytes and DC and thereby promote Th17, but not Th1 cells 32. It has also been demonstrated that Th17 cells can be induced independent of TLR and NLR activation. Engagement of the C-type lectin dectin-1 with curdlan, a β-glucan, activated IL-6, TNF and IL-23 production by DC, which promoted the differentiation of Th17 and Th1 cells 65.
Intracellular signalling molecules and transcription factors leading to IL-17 production in T cells
The transcriptional factor RORγt appears to be essential for the development of Th17 cells 20 and is also expressed by IL-17-secreting NKT cells 30 and IL-17-secreting γδ T cells (Sutton and Mills, unpublished data). STAT-3 has been shown to regulate IL-6 and IL-23 induced expression of RORγt and IL-17 production by CD4+ T cells 66, 67. Furthermore STAT3-defective mice have reduced IL-17 production and are resistant to the development of experimental autoimmune uveoretinitis 33. It has also been suggested that the NFAT and MAP kinase pathways are involved TCR-induced IL-17 production by human T cells 68. My group has demonstrated essential roles for phosphatidylinositol 3-kinase (PI3K), NF-κB and novel PKC isoforms in IL-1 and IL-23 induced IL-17 production by murine T cells 25. The latter is consistent with the observation that PKCθ-deficient mice are resistant to EAE and have reduced IL-17 in the CNS 69. Furthermore, studies in patients with rheumatoid arthritis have suggested that overproduction of IL-17 is dependent on signal transduction pathways involving PI3K/Akt and NF-κB 70.It has also been reported that IRF-4 is required for Th17 differentiation and IRF4-deficient are resistant to the development of EAE 71.
The signalling pathways that promote innate cytokines that drive induction or expansion of Th17 cell have also received some attention 72. Although a great deal is known about TLR-induced signalling in macrophages or DC that mediate IL-1, TNF-α and IL-12 production, less is known about the pathways that promote IL-23 production, except that they appear to be distinct from those that mediated IL-12-driven Th1 responses 73. We have preliminary evidence that distinct signalling pathways are involved in TLR/NLR agonist-induced IL-23 and IL-12p70 production by murine DC (Brereton and Mills, unpublished data). It has been reported that MyD88-mediated activation of NF-κB, IRF-1, IRF-5 and IRF-8 plays important roles in the expression of IL-12p35 and IL-12p40 genes and in driving Th1 in responses, whereas NF-κB, IRF3, IRF5 may be involved in IL-23 and IL-17 production 72, 73. In addition, it has been demonstrated that signalling through the C-type lectin dectin-1 in DC for IL-6, TNF-α and IL-23 production and consequent Th17 development involves Syk and CARD9 65.
Role of IL-17-secreting T cells in immune-mediated diseases
Much of the focus on IL-17-secreting T cells has been on their role in promoting organ-specific autoimmunity and chronic inflammatory conditions. Indeed, the demonstration that IL-23- or IL-17-defective mice had reduced susceptibility to autoimmune and chronic inflammatory disease, such as EAE, CIA and colitis, has helped to explain previous anomalies on the role of IL-12 and IFN-γ. It was initially demonstrated that IL-23p19−/− mice are resistant to the development of EAE and CIA, whereas IL-12p35−/− mice had exacerbated disease 36, 74. It was later reported that the role of IL-23 involved induction or expansion of Th17 cells; autoantigen-specific Th17 cells transferred EAE to naïve mice 22. Furthermore, mice treated with anti-IL-17 neutralizing antibody 75 or IL-17−/− mice 76, 77 had reduced susceptibility to experimentally induced autoimmunity, though the attenuation was not as pronounced as that observed in IL-23−/− mice. This was complemented by studies in humans, which showed that CD4+ T cells from MS patients secrete higher levels of IL-17 than healthy donors following in vitro stimulation with plate-bound anti-CD3 78. Furthermore, DC from MS patients secreted more IL-23 in response to LPS than DC from healthy donors 78.
The precise role of IL-17 and Th17 cells in mediating autoimmunity is still not clear. IL-22, which is preferentially produced by Th17 cells, has been shown to mediate dermal inflammation and acanthosis, suggestive of a possible role for this cytokine in the pathogenesis of psoriasis 79. However, IL-22 has also been shown to have a protective role, independent of IL-17, in acute liver inflammation 57. It has been demonstrated that IL-17 promotes TNF-α and IL-1β 80, as well as chemokine production 13, and thereby promotes inflammation and tissue damage (Fig. 3). Since TNF-α and IL-1β have established roles in the pathogenesis of many autoimmune and chronic inflammatory disorders, it seems logical that IL-17 might mediate its effect by enhancing production of these pro-inflammatory cytokines. However, we have demonstrated that IL-1 is essential for the induction of IL-17-producing T cells in the development of EAE, but is dispensable for its effector function; IL-1RI−/− mice were resistant to EAE, but transfer of myelin oligodendrocyte glycoprotein-specific T cells cultured with antigen in the presence of IL-1 and IL-23 induced EAE in IL-1RI−/− mice 25.
Much of the recent reports on Th17 cells have suggested that IL-12-induced Th1 cells are not essential for induction of autoimmunity and that IFN-γ may actually suppress pathogenic Th17 cells, while Th1 cells may attenuate autoimmunity by inhibiting IL-17 production 18, 19 However, the relative roles of IFN-γ and IL-17 have not been fully elucidated and some recent reports have suggested that IFN-γ may promote inflammation, perhaps at different stages of disease or by stimulating distinct inflammatory responses. Administration of T-bet siRNA i.v. after the onset of clinical signs of EAE, and again 2 wk later, reduced the symptoms of EAE and this was associated with reduced IFN-γ, IL-17 and IL-23R expression; it was concluded that suppression of T-bet attenuated EAE by inhibiting IFN-γ and IL-17, the latter by reducing IL-23R expression 81. This study, as well as a report by Ivanov 20, demonstrated that IL-17 and IFN-γ are co-expressed in the CNS during EAE. In addition, CD4+ T cells producing both IL-17 and IFN-γ have been found in the gut of patients with Crohn's disease 35. It has also been demonstrated that T-bet knockout mice that lack Th1 cells do not develop EAE, despite having elevated IL-17-producing T cells 82. Furthermore, T cells from TCR Tg mice that recognized MBP Ac1-11 expanded in vitro with antigen and IL-12, secreted IFN-γ, but not IL-17 or IL-4, and were capable of inducing EAE in naïve mice 81. In addition, it has recently been reported that either Th1 or Th17 cells can mediate EAU 61. Transfer of a T-cell line specific for interphotoreceptor retinoid-binding protein peptide 161–180, which secreted IFN-γ but not IL-17, induced EAU in naïve recipients and treatment of recipient mice with anti-IL-17 did not attenuate disease 61. The same study also demonstrated that neutralization of IL-17 prevented disease induced by immunization with interphotoreceptor retionoid-binding peptide 161–180 with CFA. Further evidence that either Th1 or Th17 cells can mediated pathology in autoimmune disease was provided by the recent report demonstration that transfer of mylein-specific T cells modulated in vitro with either IL-12 or IL-23 could induce EAE in naïve mice 83.
In an infection model, IL-23 was found to be dispensable for the generation of IL-17-producing T cells, but was essential for schistosome egg-induced immunopathology 84. This is consistent with a recent study on colitis, which demonstrated that IL-17-producing T cells are not required for the development of intestinal inflammation, but that IL-23 does have a pathogenic role by inhibiting Treg cells 85, and thereby enhancing Th1 responses. It has also been demonstrated that IL-23, as well as promoting IL-17 producing T cells, can enhance IFN-γ-producing Th1 cells 62. Thus, despite the initial assumptions that Th17, but not Th1, cells were the critical T cells that mediate the pathology in many autoimmune diseases, it now appears that Th1 cells may have both pathogenic and regulatory roles in certain autoimmune diseases, possibly at the induction and effector stages of disease.
Role of IL-17-secreting T cells in infection
There is increasing evidence of a role for IL-17 in protection against bacterial infections. The major function of IL-17 in immunity to bacteria appears to be to promote chemokine and pro-inflammatory cytokine production and consequent recruitment and activation of neutrophils and macrophages (Fig. 4). It has also been suggested that IL-17 may have a regulatory function limiting the accumulation and or activity of neutrophils during inflammation, by attenuating the anti-apoptotic effect of inflammatory cytokines 86. IL-17R-defective mice had reduced CXC chemokine production, neutrophil recruitment and increased bacterial load and mortality following challenge with K. pneumoniae13. Conversely, over-expression of IL-17 in the lungs enhanced local production of TNF-α, IL-1β and MIP-2 and enhanced neutrophil recruitment and clearance of K. pneumoniae87. Furthermore, DC stimulated with K. pneumoniae induced TLR4-dependent production of IL-23 from DC, which promotes IL-17 secretion from CD4 and CD8 T cells 26. Studies in IL-12p35, IL-12p40, IL-23p19 and IL-17R defective mice demonstrated that both IL-23-driven IL-17 and IL-12-driven Th1 responses were required for optimal protective immunity to K. pneumonia88. A dual role for IFN-γ and IL-17 has also been proposed for protective pulmonary immunity to M. tuberculosis89, 90. Here vaccination with M. tuberculosis antigens in adjuvants induces IL-17-producing CD4+ T cells, which after bacterial challenge promote chemokine production and recruit protective Th1 cells 89. IL-17-producing T cells, activated by innate IL-1 and IL-23 production, also function with Th1 cells to mediate natural and vaccine-induced immunity to B. pertussis14, 91.
Studies on the role of IL-17-producing T cells in immunity to fungal infections have generated more conflicting findings. Clearance of the fungal pathogen Crypotococcus neoformans is delayed and survival reduced in IL-23p19−/− mice 92. Similarly, IL-17AR−/− mice have decreased resistance to systemic challenge with C. albicans; higher fungal burden in the kidney and reduced survival in knockout mice were associated with reduced mobilization of peripheral neutrophils and their recruitment to the kidney 93. However, there is also evidence that IL-23 and IL-17 may have a negative role in immunity to fungal infection. IL-23p19−/− mice were less susceptible to intragastric infection with C. albicans and intranasal infection with Aspergillus fumigatus and this was associated with enhanced IL-12 and IFN-γ production 94. This study concluded that IL-23 and IL-17 impaired the anti-fungal immunity by suppressing Th1 responses and the fungicidal activity of neutrophils.
There is more limited information on the role of IL-17 in immunity to viruses and parasites. IL-17 has been detected in the cornea of mice infected with HSV-1 and mice lacking IL-17R had a transient decrease in neutrophil infiltration, but corneal pathology and viral clearance were not significantly different than in wild-type mice 95. We have recently found that antigen-specific Th17 cells are induced during infection with hepatitis C virus and that these are regulated by innate IL-10 and TGF-β induced in monocytes by the NS4 protein of the virus 34.
There is an increased parasite burden in the brain, spleen gut and liver of IL-17R−/− mice infected with Toxoplasma gondii and this was associated with reduced MIP-2 production and reduced infiltration of neutrophils to the infected tissue 96. Mice infected with Schistosoma mansoni and then immunized with soluble schistosome egg antigens (SEA) in CFA develop severe egg-induced immunopathology, with granulomatous inflammation mediated by IL-17 97. Furthermore S. mansoni-infected IL-23p19−/− mice with or mice treated with anti-IL-17 neutralizing antibody had significantly reduced hepatic granulomatous inflammation, suggesting that IL-17-producing T cells contribute to immunopathology 84.
Regulation of IL-17-secreting T cells
Since Th17 cells mediate inflammatory responses with the potential to induce immunopathology during infection, it is important that these cells are tightly regulated. Indeed, it appears that the induction or function of Th17 cells is suppressed by cytokines secreted by most other T-cell subtypes. The first reports demonstrating that Th17 cells were a distinct subtype from Th1 cells showed that IFN-γ was capable of suppressing the IL-17 production induced by stimulation with IL-6 and TGF-β or IL-1 and IL-23 18, 19, 25, 58. This conclusion was mostly based on the evidence of the enhancement or inhibition of IL-17 production following addition of anti-IFN-γ or recombinant IFN-γ, respectively, during T-cell activation in vitro. There is also evidence that IFN-γ can inhibit IL-17 production in vivo40. In a type I diabetes model, treatment with Ig-glutamic acid decarboxylase (GAD)2 protected against disease by inducing IFN-γ, which inhibited IL-17-producing T cells; neutralization of IFN-γ, but not IL-10, enhanced IL-17 production 98. Further evidence that IFN-γ may suppress IL-17 production was provided by the study in the experimental antigen-induced arthritis model, where it was demonstrated that arthritis was exacerbate in IFN-γ−/− mice due to increased Th17 responses 99. IL-17 production was also shown to be enhanced in IFN-γ−/− mice infected with BCG, whereas addition of recombinant IFN-γ inhibited in vitro activation of Th17 cells by DC infected with BCG 100. In an experimental autoimmune myocarditis model, T-bet-mediated IFN-γ production by CD8+ T cells was found to regulate IL-17 production and thereby control the severity of myocarditis 101. IFN-γ may also play a role in controlling pathogenic T cells in autoimmunity by inducing peripheral conversion of CD4+CD25− T cells into CD4+FoxP3+ Treg cells 102. While all these reports suggest that IFN-γ may suppress induction of Th17 cells, there is also evidence that IL-23 and IL-17 may negatively regulate IFN-γ production; anti-IL-23 and anti-IL-17 enhanced IFN-γ production in mice with fungal infections 94. It has also been demonstrated that IFN-γ can enhance IL-23p19 and IL-12p40 mRNA, whereas IL-4 suppresses IL-12p19 and IL-12p40 in response to Sendai virus infection 103. Furthermore, the Th2 cytokine IL-4 can suppress IL-6 and TGF-β-induced differentiation of Th17 cells in vitro18, 19.
Since the major role of Treg cells is to control inflammatory and autoimmune responses, it would appear logical that they should suppress Th17 cells; however, the role of different Treg cells or their secreted cytokines in controlling IL-17 is still not clear. Recent studies have demonstrated that Foxp3 can inhibit RORγt-mediated IL-17 production by murine T cells 104, 105. However in humans, although CD4+CD25+Foxp3+ Treg cells are effective regulators of Th1 responses, they do not appear to suppress Th17 cells 35, 64. Furthermore, anti-IL-10 enhanced IFN-γ but had little effect on IL-17 production by soluble SEA-stimulated lymph node cells from mice 84. Conversely, IL-10 was higher in granuloma cells from IL-23p19−/− compared with wild-type mice infected with S. mansoni and then immunized with SEA in CFA 84. Nevertheless, IL-10 and TGF-β have established roles in controlling chronic inflammatory and autoimmune diseases. IL-10 mRNA expression rises, whereas IFN-γ declines, during stabilization of the clinical symptoms of EAE 106. Transgenic mice expressing human IL-10 are highly resistant to EAE 107, whereas disease is exacerbated in IL-10−/− mice 108. Recent studies have demonstrated that IL-27 suppresses IL-17 production by naïve T cells stimulated with IL-6 and TGF-β in combination with anti-CD3 and anti-CD28 58. It has been reported that IL-27 and IL-6 can induce IL-10 production from Th1, Th2 and Th17 cells through activation of STAT-1 and STAT-6 109. Furthermore, it has been demonstrated that IL-27 inhibits IL-17 production and EAE by inducing IL-10 from IFN-γ+T-bet+Foxp3− T cells 110. Finally, it has recently been reported that IL-35, a heterodimer of Epstein–Barr-virus-induced gene 3 and IL-12p35, which is highly expressed by mouse Foxp3+ Treg cells 111 and induces IL-10 production by Treg cells, suppresses Th17 cell differentiation and attenuates CIA 112.
The role of TGF-β in regulating IL-17 production by T cells is more controversial. Although TGF-β is secreted by antigen-specific Treg cells, in particular Th3 cells in the gut, and induces peripheral conversion of CD4+CD25−Foxp3− T cells to CD4+CD25+Foxp3+ Treg cells 113, this cytokine has also been shown to function with IL-6 or IL-21 to promote the differentiation of murine and human Th17 cells from naïve CD4+ T cells in vitro39–41, 43–45. Initial studies in humans suggested that TGF-β did not activate IL-17 production, either alone or in the presence of IL-6 38, 50, 52. However, recent studies have shown that in association with IL-21 or IL-1 and IL-23, low concentrations of TGF-β do promote human Th17 differentiation 53, 55, 56. However, studies in mice showed that while TGF-β acted in synergy with IL-1, IL-23 or IL-6 to promote IL-17 production, it reduced the expression of IL-23R on naïve murine T cells stimulated with IL-6 or IL-21 44. Conversely, it has been demonstrated that TGF-β can inhibit IL-17 production by naïve human T cells stimulated with IL-1β and IL-6 in the presence of anti-CD3 and anti-CD28 51. Furthermore, IL-22, produced by human Th17 cells, in response to in vitro stimulation with IL-6 or IL-6 and IL-2, is inhibited by TGF-β 50, 79. In the study by Zheng et al., it was demonstrated that TGF-β enhanced IL-17 and inhibited IL-22 production by IL-6-stimulated murine T cells, suggesting that IL-22 and IL-17, as well as having distinct functions in vivo, may be differentially regulated by TGF-β 79. A recent report has demonstrated that TGF-β induces RORγt as well as Foxp3 expression in a T-cell line and that over-expression of Foxp3 inhibited IL-17 mRNA expression through direct interaction with RORγt 104. Another study showed that at low concentrations TFG-β synergizes with IL-6 and IL-21 to enhance IL-23R expression and Th17 differentiation, but that high concentrations of TGF-β suppressed IL-23R expression and favored Foxp3 expression, which inhibited the function of RORγt and Th17 differentiation 105. We have recently demonstrated that neutralization of TGF-β can enhance antigen-specific IL-17 production by CD4+ T cells from hepatitis C virus infected patients and that recombinant TGF-β can inhibit IL-1 and IL-23 production by human monocytes, suggesting that TGF-β can suppress development of human Th17 cells in vivo by inhibiting innate cytokines that promote their induction or expansion 34.
Conclusions and prospects for therapies that target Th17 cells
The discovery of IL-17-producing T cells and the realization that they play a critical role in the pathogenesis of autoimmune diseases has resulted in an explosion of publications on these cells over the past 2–3 years and has opened up the potential to develop new therapeutic approaches for autoimmune diseases in humans. Much of the focus in the literature has been on the cytokines that promote the differentiation of the so-called ‘Th17 cells’ from naïve CD4+ T cells. While the differences observed using different experimental approaches and between human and mouse have not been fully resolved, the picture that is emerging is that multiple cytokines, including IL-1, IL-6, IL-21, TNF-α, IL-23 and TGF-β, stimulated by inflammatory stimuli, including TLR and NLR agonists and during sterile inflammation, can promote the differentiation or expansion of IL-17-secreting T cells. Studies with knockout mice have identified essential roles for IL-1 25, IL-6 59, IL-23 62 and TGF-β 42 in the development of pathogenic T cells that mediated autoimmunity. These cytokines are obvious targets for the development of immunotherapeutics for autoimmune diseases. Indeed, antibodies, antagonists or receptor antagonists to IL-1 and TNF-α are already in use for a range of autoimmune and chronic inflammatory conditions and emerging evidence suggests that these may function to prevent induction of Th17 cells instead of, or as well as, mediating the downstream inflammatory responses and resulting tissue damage 25. Antibodies to IL-23 are in clinical development and pre-clinical studies have already shown efficacy in animal models of autoimmunity 114. Targeting the IL-17 or IL-17R families also has potential, but is complicated by the fact that Th17 cells secrete a range of cyokines, including IL-17A, IL-17F, IL-21, IL-22, IL-1, TNF-α and IL-6, which have overlapping and distinct functions in inflammatory processes. Targeting intracellular signalling molecules or transcriptional factors involved in the activation of IL-17 or IL-23 production is an alternative approach amenable to the development of low molecular weight drugs, but is also complicated by the fact that many of the signalling pathways may not be unique to the IL-23-IL-17 axis and may also inhibit responses of other cell types involved in protective immunity. However, there is evidence that IL-12-driven Th1 responses and the IL-23-IL-17 axis may involve distinct signalling pathways in DC 73 (Brereton and Mills, unpublished data). Therefore, it may be possible to specifically block IL-23 and IL-17 production, without affecting IL-12 and Th1 responses, thus limiting the immunosuppressive effects of the immunotherapy to attenuation of pathogenic Th17 cells. However, Th17 cells, as well as Th1 cells, have established roles in protective immunity to infection and may also contribute to immunity to tumors. Therefore prolonged inhibition of Th17 cells does have the risk of increasing susceptibility to infection and possibly to tumors. Nevertheless, inhibiting Th17 cells or the cytokines that promote their induction or expansion is a more targeted approach than inhibiting antigen presentation, co-stimulatory molecule expression or T-cell activation and therefore does have considerable potential to generate a new family of immunotherapeutic drugs against autoimmune and chronic inflammatory disorders.
Kingston Mills's research is funded by Science Foundation Ireland, The Health Research Board of Ireland, Enterprise Ireland and the EU 6th and 7th framework programmes. I am grateful to Jean Fletcher, Caroline Sutton, Sarah Higgins, Padraig Ross, Corrina Brereton Stephen Lalor and Cheryl Sweeney for their discussion and helpful comments and for allowing me to quote their unpublished work.
Conflict of interest: Kingston Mills is a co-founder and shareholder in Opsona therapeutics, a start-up company involved in the development of anti-inflammatory therapeutics.