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Th17 cells were identified as an independent lineage of CD4+ T cells that secrete a distinctive set of immunoregulatory cytokines, including IL-17A, IL-17F, IL-22, and IL-21. These cytokines collectively play roles in inflammation and autoimmunity and in the response to extracellular pathogens. The expression of the lineage-specific transcription factor RORγt leads to Th17 lineage commitment; however, it has become increasingly clear that the population of cells designated as Th17 cells is not homogeneous. Although these cells collectively produce characteristic Th17 cytokines, not all are produced by each individual cell in the population. The cytokines produced by individual cells are presumably affected in part by the specific local cytokine milieu. In this review, we discuss the current understanding of the specific functional characteristics and regulation of Th17 cytokines and clarify how they mediate the actions of Th17 cells.
In the past few years, much attention has focused on the characterization of the Th17 lineage and elucidating the mechanism(s) underlying its role in inflammatory diseases. A range of inflammatory cytokines, including TGFβ, IL-6, IL-21, IL-23, and IL-1β, have been shown to participate in the generation of Th17 cells, with some species-specific differences being noted [1-3]. In turn, IL-17A, IL-17F, IL-22, and IL-21 are secreted by and involved in the in vivo function of Th17 cells . Interestingly, it has become clear that there are probably multiple subsets of effector cells within the collective population of cells known as Th17 cells, whose differentiation is controlled by the RORγt transcription factor . As such, Th17 cells represent a mixture of cells rather than a homogeneous population, whose specific secreted cytokine profile presumably depends on the cytokine milieu in which the cells develop. While much remains to be discovered about the mechanisms that regulate Th17 function, studies of these cells should help in the identification of therapeutic targets in inflammation, autoimmune disease, and pathogen responses.
IL-17, now denoted IL-17A, is the hallmark cytokine of Th17 cells and has been shown to function as a proinflammatory cytokine that upregulates a number of chemokines and matrix metalloproteases, leading to the recruitment of neutrophils into sites of inflammation . Of IL-17 family cytokines, IL-17A is most similar to IL-17F, sharing approximately 60% amino acid identity, and these are the IL-17 family members that are expressed mainly by T cells, whereas IL-17B, IL-17C, and IL-17E (also known as IL-25) are more broadly expressed. IL-17 family cytokines are a distinctive group and do not fit in either as type I or type II structural cytokines but instead form cysteine knot structures analogous to NGF and PDGF . IL-17A and IL-17F both are produced by Th17 cells [4, 7]. These two cytokines are co-expressed and can be secreted as either homodimers or as heterodimers , and they signal through receptors containing IL-17RA and IL-I7RC receptor components, but the formal stoichiometry of the receptor chains is still unknown .
When naive T cells undergo Th17 differentiation in vitro, three dimeric forms of IL-17 are secreted, with highest amounts of IL-17F/F followed by IL-17F/A and finally IL-17A/A . Each of these three forms of IL-17 is biologically active in vitro when assayed for its ability to induce the chemokines CXCL1 and CXCL5 in lung epithelial cells, but IL-17A/A is more potent than IL-17F/F in the induction of neutrophil recruitment to the lung, with IL-17A/F having an intermediate effect. IL-17A/A is the least abundant form in early differentiation but the most abundant in fully differentiated Th17 cells .
The signaling pathways used by IL-17A and IL-17F in target cells are similar, with Act1 (an adapter protein involved in activation of NF-κB) and TRAF6 being downstream of receptor binding . IL-17R cytoplasmic domains share homology with the TlR domain of Toll-like receptors . Despite their activation of similar pathways, however, the biological actions of IL-17A and IL-17F differ. For example, in an experimental model of allergic encephalitis, IL-17A KO mice exhibit a reduction in disease development whereas IL-17F KO mice show nearly identical early kinetics of disease onset as WT mice. Conversely, in an allergic airway model, IL-17F KO but not IL-17A KO mice exhibit a significant reduction in expression of CXCL5, a chemokine involved in neutrophil recruitment . In the DSS-induced colitis model, IL-17A KO mice had more severe weight loss and colonic epithelial damage, whereas IL-17F KO mice have less severe responses than WT mice . Thus, although these two cytokines share cell surface receptors and signaling components, the effect of the selective deletion of each cytokine differs, indicating differences in their biological actions and/or in their expression levels during disease development.
IL-22 is an IL-10 family type II cytokine that is preferentially expressed by differentiated Th17 cells  and thus considered as a Th17 cytokine, but it also is expressed by other cells, including for example, both CD8+ T cells and CD11c+ dendritic cells . IL-22 acts via a receptor consisting of IL-22R and IL-10R2 (also known as IL-10Rβ), and activates the JAK-STAT pathway to primarily activate STAT3 . Although IL-22 is induced by the same factors, TGFβ and IL-6, that promote expression of IL-17A and IL-17F, when Th17-differentiated cells were examined at a single cell level, less than 10% of these cells also expressed IL-22. Interestingly, IL-23 was shown to drive preferential expansion of cells that co-express IL-22 and IL-17 .
In addition to their partial co-expression, IL-22 and IL-17 synergistically act to augment the expression of genes involved in the defense against microbial pathogens such as the β-defensin 2 gene in keratinocytes . Nevertheless, both in vivo and in vitro experiments also indicate distinctive roles for IL-17 and IL-22. For example, an analysis of mycobacteria-specific Th17 cells in exposed humans revealed that most of these cells expressed either IL-22 or IL-17 but not both cytokines . Among the IL-22-expressing cells, most did not produce IFNγ, indicating that although IL-22 can be expressed in Th1 cells , this is not always the case.
Mouse infectious and autoimmune models also indicate independent roles for IL-17 and IL-22. In a mouse model of autoimmune encephalitis, although both cytokines were co-expressed in Th17 cells infiltrating the central nervous system, IL-22 deficient mice retained maximal susceptibility to the disease, indicating that IL-22 was not responsible for the autoimmune pathology in this disease . Infection with Citrobacter rodentium results in different kinetics of IL-17 and IL-22 responses in the colon, with IL-22 being produced within the first several days and IL-17 expression delayed, becoming maximal only at day 12 after infection . In this model system, the ablation of IL-17 had no effect on disease progression, whereas IL-22 deficient mice had enhanced intestinal pathology and increased mortality , indicative of a protective effect for IL-22 but not for IL-17. In a mouse model for psoriasis, in which a large percentage of Th17 cells were found to co-express IL-22 and IL-17A, ablation or deficiency of IL-22 alone reversed the skin pathology [14, 18]. IL-22 also confers a strong protective effect in a mouse model for inflammatory hepatitis. In this disease, IL-22-expressing Th17 cells played a protective role in that mice deficient for IL-22 were highly susceptible to liver injury induced by concanavalin A, whereas IL-17 deficient mice displayed no difference in susceptibility . In this model, IL-22 upregulated anti-apoptotic BCL2 and BCL-XL in hepatocytes. In another study focused on the role of Th17 cells in the host defense to bacterial pneumonia, although both IL-17 and IL-22 were important mediators of host resistance to infection, only IL-22 played a protective role through the induction of lung epithelial cell proliferation and could facilitate bronchial epithelial cell wound repair in an in vitro system . A mechanism by which IL-22 mediated these protective responses was through the induction in epithelial cells of several members of a family of anti-microbial proteins (RegIIIβ and RegIIIγ), which can directly bind and kill Gram+ bacteria and which also can function as autocrine growth factors that play a role in epithelial layer repair processes .
IL-21 is a type I four α-helical bundle cytokine that was first noted to be produced by CD4+ T cells following antigen activation . IL-21 acts via a receptor containing IL-21R and the common cytokine receptor γ chain, γc, which is mutated in X-linked SCID . The inactivation of IL-21 together with IL-4 contributes to the non-functional B cells in this disease , and IL-21 was shown to drive terminal differentiation of B lymphocytes . Additionally, IL-21 can cooperate to drive CD8+ T cell proliferation, with additional actions on NK cells and dendritic cells . IL-21, like the other γc family cytokines, activates JAK1 and JAK3, but is distinctive from them in most potently activating STAT3 , which is the same STAT protein that is primarily activated by IL-22.
In addition to its known pleiotropic range of actions, IL-21 was recently reported to play an important role in the generation of Th17 cells: IL-21 was shown to be potently induced by IL-6 , Th17 in vitro differentiated cells expressed much higher levels of IL-21 mRNA and protein [27, 28], and in the absence of IL-6, IL-21 in combination with TGFβ could function as an alternative signal for the induction of Th17 cells . IL-21R and IL-21 deficient mice were used to show that IL-21 was critical for the induction of IL-23R on Th17 cells, allowing their further expansion by IL-23 [26, 28]. These studies collectively led to the conclusion that IL-21 could function as an autocrine growth factor for Th17 cells. Nevertheless, an analysis of Th17 cells in the IL-21R-deficient small intestine lamina propria revealed normal Th17 development even in the absence of IL-21 signaling , and in two independent studies, EAE disease development and cytokine production during the course of disease was similar in either IL-21 or IL-21R deficient mice to that observed in WT mice [30, 31]. Although IL-21 plus TGF-β can induce Th17 differentiation, IL-6 appears to be more potent than IL-21 .
In addition to its ability to promote Th17 differentiation, IL-21 is produced by in vitro differentiated Th17 cells. Interestingly, however, one study used intracellular staining to show that the majority of IL-21-producing “Th17” cells did not produce either IL-17A or IL-17F . In this study, IL-21 expression was inhibited by TGFβ , a cytokine known to be critical for the induction of IL-17 production, indicating that within the Th17 differentiated population, IL-17 and IL-21 are not always made by the same individual cells. The distinctive production of these cytokines is further underscored by the induction of IL-21 in TCR-stimulated CD4+ T cells  and by the more recent demonstration that T follicular helper (Tfh) cells [33, 34] produce high levels of IL-21 yet do not express either IL-17 or RORγt. In fact, mice deficient in RORγt expressed normal levels of IL-21, demonstrating Th17-independent IL-21 production. Interestingly, Th17 cells can also be generated by using vasoactive intestinal peptide, a neuroendocrine mediator, plus TGFβ in the absence of IL-6, and these cells also produce abundant IL-17 and IL-22 but no detectable IL-21 or IL-6 .
As noted above, IL-21 has pleiotropic effects on multiple lineages, including CD8+ T cells, B cells, NK cells, and dendritic cells . Thus, its contribution to Th17 function is only one action of this cytokine, and likely is context dependent. The recent finding that IL-17A induces a positive feedback loop for enhanced IL-6 expression  suggests that IL-17 production, in the proper cytokine milieu, may be able to amplify IL-21 production. Whereas IL-21 can contribute to Th17 lineage development and may mediate some actions of these cells, there is clearly IL-21-independent Th17 differentiation as well.
One of the early observations about Th17 development was that the differentiation of Th17 cells could be inhibited by Th1 cytokine IFN-γ [37, 38], but in the inflammatory lesions that develop in a number of autoimmune diseases, Th17 and Th1 cells can be co-localized , suggesting the possibility of redundant functional relationships between these lineages in disease pathology . Differentiated Th17 cells are known to express IFN-γ and Th1 cells can express IL-22, which is consistent with functional overlap of these lineages. A systematic computational cluster analysis of the expression of a panel of lineage-specific cytokines as influenced by removal of single inducing factors showed that the removal of TGFβ induced a shift in the Th17 expression pattern that clustered more closely with the Th1 pattern . Several recent studies have demonstrated that Th17 cells can undergo phenotype switching to either IFNγ/IL-17 co-expressing or IFNγ expressing cells in the context of either an in vivo inflammatory response  or during in vitro induction with Th1-inducing cytokines . These studies thus suggest that slight changes in the cytokine priming environment can mediate significant phenotypic shifts related to cytokines expressed by Th17 cells.
In summary, we have reviewed Th17 cells as a population of cells whose differentiation is driven by IL-6 + TGF-β (Figure 1). This process interestingly requires IL-23 for optimal differentiation, and IL-21 also can contribute to Th17 differentiation, depending on the biological context, although Th17 differentiation in the gut in vivo appears to be largely independent of IL-21. IL-17A and IL-17F are the hallmark cytokines produced by Th17 cells, with IL-21 and IL-22 being produced by a fraction of the cells. IL-17A and IL-17F are pro-inflammatory, whereas IL-22 exerts protective actions. The importance of Th17 cells as a source of IL-21 remains unclear given its production by other populations of cells including CD4+ T cells after antigen encounter and by Tfh cells. It is possible that the production of IL-21 by Th17 cells contributes to a a number of the broad range of actions mediated by this cytokine. Interestingly, IL-6, IL-22, and IL-21 all use STAT3 as a major signaling molecule, underscoring the role of STAT3 in both the differentiation as well as some of the actions mediated by these cells. Additionally, however, NF-κB-related pathways are important given the activation of NF-κB by IL-17A and IL-17F. Th17 cells, at least in part via these cytokines and signaling pathways, play important roles in multiple inflammatory/pathological processes. The manipulation of these cytokines can thus have impact on the regulation of these processes.
The authors would like to thank Drs. Yrina Rochman, Jian-Xin Lin, and Hyok-Joon Kwon for critical reading of the manuscript. R.S. and W.J.L. are supported by the Division of Intramural Research, NHLBI.