Immunol Res DOI 10.1007/s12026-007-8014-9

Th17 cells: a new fate for diVerentiating helper T cells

Zhi Chen · John J. O’Shea

! Humana Press Inc. 2007

Abstract Classically naïve CD4+ have been thought to diVerentiate into two possible lin-eages, T helper 1 (Th1) or T helper 2 (Th2) cells. Within this paradigm the pathogenesis ofautoimmunity was suggested to predominantly relate to Th1 cells and the production ofIFN-!. However, there were many aspects of this model that did not seem to Wt, not theleast of which was that IFN-! was protective in some models of autoimmunity. During thepast 2 years, remarkable progress has been made to characterize a new lineage of helper Tcells. Designated Th17 cells, this lineage selectively produces proinXammatory cytokinesincluding IL-17, IL-21, and IL-22. In the mouse, the diVerentiation of this new lineage isinitiated by TGF"-1 and IL-6 and IL-21, which activate Stat3 and induce the expression ofthe transcription factor retinoic acid-related orphan receptor (ROR!t). IL-23, which alsoactivates Stat3, apparently serves to maintain Th17 cells in vivo. In human cells, IL-1, IL-6,and IL-23 promote human Th17 diVerentiation, but TGF"-1 is reportedly not needed.Emerging data have suggested that Th17 plays an essential role in the host defense againstextracellular bacteria and fungi and in pathogenesis of autoimmune diseases. Selectivelytargeting the Th17 lineage may be beneWcial for the treatment of inXammatory and autoim-mune diseases.

Keywords T cells · Cytokines · Interleukins · Immunoregulation · Th1 · Th2 · Th17 · Regulatory T cells

Z. Chen · J. J. O’SheaMolecular Immunology and InXammation Branch, National Institutes of Arthritis, Musculoskeletal and Skin Diseases, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

J. J. O’Sheae-mail: osheajo@mail.nih.gov

Z. Chen (&) · J. J. O’SheaFaculty of Medicine, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finlande-mail: zchen@utu.W

Immunol Res

Introduction

For about two decades, helper T cells have been considered to diVerentiate into twolineages, Th1 or Th2 [1–3]. The identiWcation of Th1/Th2 cells as two distinct subsets byCoVman and Mosman was a remarkable contribution to the Weld of immunology in that itallowed an understanding of how CD4+ T cells could shape appropriate response to diVer-ent pathogens. However, more recent studies have suggested the existence of other subpop-ulations of helper T cells that are also important in immunoregulation and host defense. Forinstance, regulatory T cells (Treg) are a separate subpopulation of immunosuppressive Tcells that express the transcription factor Foxp3. Tregs can develop in the thymus (naturalor nTregs) or can be induced in the periphery (inducible or iTregs). We now know thatTregs are essential for preserving peripheral tolerance. In addition, the existence of othersubsets of T cells has included cells that preferentially produce TGF"-1 and IL-10,although data are less clear as to whether these cells are truly discrete subsets marked byexpression of a distinct “master” regulatory transcription factor.

The Th1/Th2 paradigm was further challenged by the identiWcation of another lineage,which selectively produces IL-17. During the past 2 years, information on this newly iden-tiWed T helper subset, designated Th17 cells, has accumulated at a dizzying pace [4–14]. Inthis review, we discuss these recent advances in understanding the regulation and functionof Th17 cells (Fig. 1).

Fig. 1 New complexities in CD4+ T cell diVerentiation. Once activated by occupancy of the T cell receptor(TCR), naïve CD4+ T cells in the presence of IL-12 diVerentiate to become Th1 cells, which produce IFN-!.This process is regulated by Stat4 and T-bet. IFN-! has important functions in host defense against intracel-lular pathogens. In response to IL-4, naïve T cells diVerentiate to Th2 cells through the activation of Stat6 andGATA3. The signature cytokine made by Th2 cells is IL-4, which plays a key role in host defense againsthelminth infections. In conjunction with TCR stimulation, naïve T cells cultured with TGF"-1 can becomeCD4+CD25+Foxp3+ cells with suppressive eVects. Although TGF"-1 can upregulate Foxp3 expression, inthe presence of IL-6, mouse naïve T cells develop to become IL-17-producing Th17 cells. In human T cells,IL-1 synergizes with IL-6 and IL-23 inducing Th17 diVerentiation. Stat3 and ROR!t are the key transcriptionfactors regulating this process. Th17 diVerentiation is inhibited by IFN-!, IL-4, IL-2, retinoic acid, andSOCS3

Helper T cell subsets

Th17

Th2

Th1

IL-12

IL-4

IL-6,IL-21,IL-1, IL-23

TGFIL-2, RA

IFN

IL-4

Intracellular pathogens

Helminth infections

Extracellular pathogens

Immune suppression

Stat3/ROR t

Stat6/Gata3

Stat4/T-bet

SOCS3

Treg TGF

Stat5/Foxp3

TGF -1 (mouse)

Naive CD4+IL-17A/FIL-21IL-22TNF

Helper T cell subsets

Th17

Th2

Th1

IL-12

IL-4

IL-6,IL-21,IL-1, IL-23

TGFIL-2, RA

IFN

IL-4

Intracellular pathogens

Helminth infections

Extracellular pathogens

Immune suppression

Stat3/ROR t

Stat6/Gata3

Stat4/T-bet

SOCS3

Treg TGF

Stat5/Foxp3

TGF -1 (mouse)

Naive CD4+IL-17A/FIL-21IL-22TNF

Immunol Res

The Th1/Th2 paradigm

For years, we considered only two possible fates for naïve CD4+ T cells—Th1 or Th2 cells.Th1 cells produce the signature cytokine, IFN-!, a critical factor that promotes cellularimmunity and host defense against a variety of pathogens, especially intracellular organ-isms. Interleukin-12 acting via the transcription factor STAT4, in concert with another tran-scription factor, T-BET, are critical for Th1 diVerentiation. In contrast, Th2 development isinitiated by IL-4 signaling with the participation of the transcription factors STAT6 andGATA3. The hallmark cytokine secreted by Th2 cells is IL-4, which is crucial for hostdefense against helminths and the pathogenesis of asthma and allergy. We have learned agreat deal about the regulation of Ifng gene and the Th2 cluster of genes (Il4, Il5, and Il13)[15]. Th1 and Th2 lineage decisions appear to be made at a very early stage of T helperdiVerentiation with respective Th1/Th2 cytokines enforcing their own expression andinhibiting alternative commitment. The counter regulation of Th1 and Th2 cells occurs atdiVerent levels including regulation of receptor levels, expression of transcription factors,epigenetic changes, and even intra- and interchromosomal interactions [1–3, 16]. We havebegun to understand how transcription factors might link intrachromosomal interactionsand epigenetic modiWcations, but clearly there is much to be learned about these processes.Nonetheless, as a result of these mechanisms, Th1 and Th2 cells develop into mature eVec-tors with stable phenotypes and important roles in host defense.

Autoimmunity revisited

While the dichotomous Th1/Th2 model has led to important advances in the understandingof immunoregulation and host defense against model pathogens, it became increasinglyclear that the model failed to reXect the true complexity of fate decision of helper T cellsand did not do a particularly good job of explaining autoimmune disease.

Traditionally autoimmune diseases were assumed to be associated with dysregulatedTh1 responses. However, a puzzling point was that IFN-! deWciency did not attenuatesome models of autoimmune diseases like experimental autoimmune encephalomyelitis(EAE); on the contrary, IFN-! deWciency worsened the disease [17]. Subsequently, a newcytokine subunit, designated p19, was identiWed by Oppmann et al. [18]. This subunitwas recognized to form a heterodimer with a subunit of the p40 subunit, previously notedto be a component of IL-12. This new dimeric cytokine comprising p19 and p40 wasnamed IL-23. IL-23 is produced by dendritic cells and induces IFN-! production and pro-liferation of both mouse and human memory T cells. Human p19 (IL23A) gene resideson chromosome 12.

Interestingly, IL-23 was found not to bind the IL-12R"2 subunit of the IL-12 receptor;rather, it utilizes the IL-12R"1 subunit in conjunction with its own receptor termed IL-23R,which is a member of the hematopoietin receptor superfamily [19]. The receptor is predom-inantly expressed on T and NK cells, with lower expression being detected in monocytes,dendritic cells, or B-cell lines [19, 20]. Human IL23R is located on chromosome 1, within150 kb of IL12RB2. Binding of IL-23 to its receptor leads to the activation of Jak2 andTyk2, which in turn leads to the activation of Stat1, Stat3, Stat4, and Stat5. However, stim-ulation through IL-23R gives stronger Stat3 phosphorylation and weaker Stat4 phosphory-lation compared to the strong, sustained tyrosine phosphorylation of Stat4 by IL-12 [18,19].

The complex biology of IL-12 and IL-23 is relevant to the pathogenesis of autoimmu-nity in that gene targeting of IL-12p40 attenuates the development of disease in many mod-

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els of autoimmunity. Similarly, anti-p40 antibody is eYcacious in the treatment of Crohn’sdisease and psoriasis [21, 22]. These eVects were initially misattributed to interference withIL-12 actions—contributing to the notion that IFN-! and Th1 responses were the bad actorsin autoimmunity. However the generation of IL23 (p19¡/¡)—deWcient mice shed newlight on this conundrum. In two diVerent animal models of autoimmunity, EAE and colla-gen-induced arthritis, it was found that mice deWcient in the IL-23 p19 subunit or the IL-12/23 p40 subunit were resistant to the diseases. In contrast, deWciency of IL-12 p35 hadincreased susceptibility to diseases [23, 24]. These two landmark studies directly chal-lenged the Th1/Th2 paradigm, particularly the pathogenic role of Th1/IFN-! axis in auto-immunity. However, through speciWc deletion of IL-23 (p19¡/¡ mice), it is nowrecognized that IL-23 and not IL-12 is the culprit, at least in many of the animal models[25–30]. Of note was that the proportion of IFN-! producing cells was not reduced in IL-23-deWcient mice, again arguing against a role for Th1 cells in the pathogenesis of thesemodels of autoimmune disease.

The importance of IL-23 was also exempliWed by transgenic overexpression of p19,which resulted in runting, infertile, premature death, systemic inXammation, anemia, ele-vated serum levels of IL1 and TNF-#, and chronic expression of acute phase proteins. Thiswas associated with neutrophilia and the neutrophilic inWltration in multiple organs. A sim-ilar phenotype was reproduced by bone marrow reconstitution of wild-type recipient micewith cells that transgenically expressed p19. Thus overexpression of p19 was reminiscentof the actions of IL-6 and granulocyte colony stimulating factor (G-CSF) [31]. Studies haveshown the increased mRNA levels of both IL-23p19, mainly produced by keratinocytes,and p40 in psoriatic skin lesions suggesting the dominant player for psoriasis pathogenesisis IL-23 than IL-12 [32, 33]. The role of IL-23 on human autoimmune diseases is high-lighted by recent Wndings showing the association of IL-23R polymorphisms and the prev-alence of several human autoimmune diseases [34, 35].

The connection between IL-23 and IL-17

If IL-23 and not IL-12 was the major player in autoimmune disease, then how was IL-23working to mediate immune pathology? In 2003, Aggarwal et al. reported that puriWed Tcells activated with anti-CD3 cultured with supernatant from LPS-treated dendritic cellsproduced IL-17. They went on to identify that the cytokine that promoted IL-17 expres-sion in memory T cells was in fact IL-23, and this eVect was inhibited by anti-p40 [36].Ironically, IL-17A is a proinXammatory cytokine initially identiWed in mouse cytotoxicT cells more than 10 years ago. This family now includes 6 family members: IL-17 (IL-17A), IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F [37–40] that share 16–50% aminoacid identity and have diVerent tissue expression patterns. IL-17 acts on a wide varietyof cells including epithelial cells, endothelial cells, Wbroblasts, synoviocytes, and mye-loid cells. IL-17 induces the secretion of a variety of mediators including chemokines,such as IL-8, CXC ligand (CXCL) 1, and CXCL6, as well as cytokines like IL-6, granu-locyte macrophage colony-stimulating factor, G-CSF, TNF-#, and IL-1". The net eVectis that IL-17 itself can induce neutrophil inWltration and production of inXammatorycytokines.

While links between IL-23 and IL-17 were Wrst noted in in vitro studies, it quicklybecame clear that there was a connection between IL-23 and IL-17 in models of autoim-mune disease including EAE, arthritis, and inXammatory bowel disease (IBD). The patho-genic eVect of IL-17 on EAE was documented using IL-17¡/¡ mice, which showeddelayed onset, reduced severity scores, and histological changes, with early recovery of

Immunol Res

EAE [41]. Accordingly, neutralization of IL-17 also attenuated the disease [42]. Moreover,loss of IL-23 was associated with reduced proportion of IL-17-producing cells. Finally,adoptive transfer of T cells showed that Th17 cells, but not Th1 cells caused EAE [43]. Inhumans, microarray analysis of MS plaques isolated at autopsy demonstrated increasedmRNA of IL-17 mRNA compared with the controls [44]. Vaknin-Dembinsky et al. showedthat dendritic cells from MS patients secrete more IL-23 compared with healthy controls aswell as more IL-17 from T cells [45].

The roles of IL-23 and IL-17 were also assessed in models of arthritis. Mice lacking p19have reduced incidence of collagen-induced arthritis associated with fewer IL-17-produc-ing CD4+ T cells [24]. Conversely, overexpression of IL-17 in murine knee joint usingadenoviral vector resulted in joint inXammation, cartilage proteoglycan depletion, and boneerosion [46]. Sato et al. have compared the eVects of diVerent helper T cell subsets onosteoclastogenesis and provide data that bone resorption is mediated by IL-17 and not Th1and Th2 cells [47]. In humans, elevated serum level of IL-17 has been detected in RApatients [48, 49]. Furthermore, elevated levels of IL-17 were detected in synovial Xuidfrom patients with rheumatoid arthritis, and osteoclast formation was inhibited by anti-IL-17 antibody, suggesting an eVect on bone resorption [47, 50–53].

Like other autoimmune diseases, increased levels of IL-17 have been found in inXamedmucosa and sera from patients with IBD, especially in patients with active disease [54].Accordingly, in a T cell transfer model of colitis, IL-23 was found to be essential, acting toinduce IL-17 and IL-6 [25, 28, 55]. In a Helicobacter hepaticus model, it was also con-cluded that IL-23 acting through IL-17 but not IL-12 was essential for the development ofmaximal intestinal disease. However, in this study it was shown that severe intestinalinXammation was triggered by the synergistic eVect of both IFN-! and IL-17 responsesdriven by IL-23 [27].

Th17 diVerentiation

The emerging data therefore pointed to the role of IL-23 as an inducer of IL-17. So the nextissue was how the production of IL-17 Wts into our understanding of T cell diVerentiation.In fact, it was found that the development of IL-17 producing T cells was independent ofthe cytokines and the transcription factors required for Th1 or Th2 diVerentiation; indeed,IFN-! and IL-4 actually inhibited IL-23-dependent IL-17 production. Thus it was arguedthat IL-17 producing cells represented a new lineage CD4+ T cells—“Th17” cells andthat this subset of T cells is crucial in mediating inXammatory [42, 43, 56]. Compared withIL-12-stimulated Th1 cells, IL-23-stimulated CD4+ T cells express distinct pattern ofgenes, including IL-17A and IL-17F and these IL-23-stimulated T cells are important forthe pathogenesis of EAE [43].

Although the IL-23-IL-17 connection now seems quite straightforward, what was lessclear from these initial studies was how Th17 cells arose from naïve CD4+ T cells, as wenow know that naïve CD4+ T cells express a very low level of IL-23R. This issue wasaddressed by three independent groups that concluded that IL-23 was not the initiator ofthis process. Initially it was noted that co-culture of LPS-activated DC with naïve T cellsupregulated IL-17 production. This led to the idea that IL-6, IL-1, and TGF"-1 were criticalcytokines for Th17 diVerentiation, independent of IL-23 [4]. The criticality of IL-6 andTGF"-1 was noted in additional studies [5, 6]. TGF"-1-deWcient mice showed impairedTh17 development; conversely addition of TGF"-1 increased the number of IL-17-produc-ing cells. The studies were also of interest in that they linked generation of Th17 with Tregcells. That is, it had been known for sometime that in vitro culture of naïve CD4+ T cells

Immunol Res

with IL-2 and TGF"-1 induced expression of the transcription factor Foxp3 and conferredimmunosuppressive properties. In contrast, it was noted that IL-6 along with TGF"-1 notonly promoted Th17 diVerentiation but inhibited conversion to Foxp3-expressing Tregcells (discussed in detail below).

More recently it has become clear that IL-21 is selectively produced by Th17 cells. It isinduced by IL-6 in a Stat3-dependent manner and Stat3 may directly regulate the Il21 gene[8–10, 57]. Interestingly, IL-21 also promotes IL-17 production and inhibits IFN-! produc-tion [7–10]. In fact, IL-21 can replace IL-6 in vitro to potently induce Th17 diVerentiation.Furthermore, the deWciency of IL-21 or its receptor attenuates Th17 diVerentiation [8–10,57]. Therefore, analogous to Th1 and Th2 cells, IL-21 promotes Th17 diVerentiation in anautocrine manner.

Taken together the current view is that Th17 diVerentiation proceeds in three steps: IL-6acting in conjunction with TGF"-1 initiates Th17 diVerentiation, which is associated withIL-21 production. IL-6 also antagonizes iTreg diVerentiation. IL-21 acts as an autocrinefactor that enhances the commitment to this lineage choice and antagonizes Th1 diVerentia-tion. Finally, IL-23 serves to expand and maintain Th17 fate determination. Despite thestrong in vivo data pointing to a link between IL-23 and IL-17, it should be clear that this isnot a very precise deWnition of IL-23’s actions and its exact roles vis-à-vis inXammationwill need to be more carefully deWned in the future.1 InXammatory cytokines like IL-1 alsopromote IL-17 production.

The role of Th17 cells in autoimmunity is made more complicated but more interestingby the Wndings that Th17 cells also produce other cytokines including tumor necrosis factor(TNF) and IL-22 [13, 14, 29, 30, 58]. IL-22 is a member of IL-10 family, the human IL-22gene being located on chromosome 12q15 between IFNG and Il26 loci. The mouse Il22gene is located on chromosome 10, also in proximity to the Ifng locus [59, 60]. The IL22Ris expressed predominantly in skin, respiratory and digestive tissues, and occupancy of thereceptor induces expression of a variety of genes involved in host defense, including psori-asin (S100A7), calgranulin A (S100A8), and calgranulin B (S100A9) [61]. Expression ofIL-22 and beta-defensins is higher in skin from patients with psoriasis or atopic dermatitisthan skin from healthy individuals, and anti-psoriatic therapy reduces IL-22 and IL-22-reg-ulated gene expression [62]. Interestingly, blood brain barrier endothelial cells express IL-17 and IL-22 receptors, and IL-17 or IL-22 treatment disrupts the gap junction of thesecells thereby increasing their permeability. This allows Th17 cells to migrate across theblood brain barrier [63]. Although IL-22 is expressed by Th17 cells, previous studies havealso reported that IL-22 is produced by Th1 cells [64].

Further complicating this story are the data documenting that IL-22 not only serves as aproinXammatory cytokine in some settings but also has anti-inXammatory properties. In avery recent study, in a hepatitis model, more extensive liver damage was observed in IL-22-deWcient mice compared to wild-type mice. Surprisingly, adoptive transfer of Th17 cellsto IL-22-deWcient mice actually protects liver damage. Furthermore, this eVect is IL-22-dependent but not IL-17-dependent [65]. This Wnding further supports the complexity of“Th17” cells which involve multiple cytokines and their divergent functions during inXam-mation. Although this may seem rather paradoxical, readers should recall that many classicproinXammatory cytokines (TNF, IL-2 etc.) have complicated, dualistic eVects [66].

1 Recent evidence indicates that Th17 cells can also make IL-10 and IL-23 is an important factor that regulatesthe pathogenicity of Th17 cells.

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Distinct regulation of human Th17 cell diVerentiation

Because of their pathological importance, it is obviously important to deWne factors that arerelevant in the regulation of human Th17 cells. However, several groups have found thatthe regulation of IL-17 expression in human CD4+ T cells is distinct from that in murinecells [12–14]. Indeed, optimal conditions for the development of IL-17-producing T cellsfrom murine naïve precursors are reportedly ineVective in human T cells [12–14]. SpeciW-cally TGF"-1 inhibits human Th17 diVerentiation in a dose-dependent manner [12].

In the recent studies by Acosta-Rodriguez et al. and Wilson et al., it is reported thatalthough IL-1 alone is not suYcient to induce sustained IL-17 expression, combining IL-1with IL-6 or IL-23 eVectively induced human Th17 development with sustained Ror!expression [12, 14]. By using various ligands of toll-like receptors with diVerent antigen-presenting cells, it was found that activation of monocytes with peptidoglycan (a ligand forTLR2) resulted in high-level IL-6 production with little IL-12 production. This provided aparticularly eVective stimulus for generating cells that selectively produce IL-17 [12].Naïve T cells express a low level of IL-23R; therefore IL-23 has small eVects on IL-17induction at early stages of Th17 diVerentiation [13]. However, IL-23R expression wasupregulated by TCR stimulation, IL-6, IL-21, and even IL-23 itself [9, 13, 67]. Accord-ingly, two groups have reported that IL-23 promotes IL-17 production in naïve T cells [13,14]. Furthermore, IL-1 was also found to act in concert with IL-23 to generate IL-17-pro-ducers [12, 14]. Of note is that cells stimulated with IL-23 alone or in combination with IL-1 generated a mixed population of IL-17 single producers and IL-17–IFN-! ‘double-pro-ducing’ cells. This is notable because IL-17- and IFN-!-double producers are consistentlydetected in memory CD4+ T cells [13]. Acosta-Rodriguez et al. have characterized the pat-tern of chemokine expression for Th17 and Th1 cells and have found that T cells thatexpress CCR6 and CCR4 only produce IL-17. In concert, T cells that express CCR6 andCXCR3 produce both IL-17 and IFN-! [68]. Whether these distinct patterns of chemokinereceptor expression mark subsets of mouse Th17 cells has not been examined.

A puzzling question is why are mouse and human Th17 cell diVerentiation so diVerent?An obvious explanation is that the source of cells and the extent to which they are truly“naïve” matters. In other words, studies of mouse T cells start with thymocytes, spleno-cytes, or lymph node cells, whereas the reported human studies all rely on peripheral bloodcells—an apples and oranges issue. This is an area in which we are likely hear a good dealmore. It is worth bearing in mind that IL-23 not only induces IL-17 production but is also apotent inducer of another inXammatory cytokine, IL-22 [13, 14].

Transcription factors that control Th17 cells

Unquestionably, we have a better idea of how cytokines drive Th17 diVerentiation, but theobvious next question was what are the molecular mechanisms that govern IL-17 expres-sion? What IL-6, IL-21, and IL-23 have in common is that they all activate the transcriptionfactor Stat3. Several groups have reported that conditional deletion of Stat3 in T cells abro-gates Th17 diVerentiation [67, 69–71]. Conversely, overexpression of a constitutivelyactive Stat3 allele promoted Th17 diVerentiation [67]. Importantly, deletion of Stat3 in Tcells also abrogates the development of autoimmunity in several models of disease [71].Conversely, SOCS3, which is an important negulative regulator of Stat3, also appears to bean important regulator of IL-17 production; in fact, deWciency of SOCS3 is associated withwidespread autoimmune diseases which features IL-17-dependent pathology. Furthermore,

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SOCS3-deWcient T cells were found to enhance phosphorylation of Stat3 in response toIL-23 and increased production of IL-17 [72].

To deWne the mechanism by which Stat3 might be acting on IL-17 production, chroma-tin immunoprecipitation was employed and the study revealed direct binding of Stat3 to theIL-17 locus [72]. This argues for very direct actions of Stat3 vis-à-vis IL-17 regulation.Additionally, chromatin immunoprecipitation assays have documented that Stat3 directlybinds to the Il21 promoter, identifying another very direct means by which Stat3 regulatesdiVerentiation of Th17 cells [8–10, 57]. Finally, several studies have shown that IL-23Rexpression is upregulated by IL-6 and IL-23 and this is dependent upon Stat3 [9, 10, 13]. Incontrast to Stat3, it has been reported that Stat4 and Stat6 are not required for IL-23-depen-dent IL-17 induction [42, 56].

Another transcription factor that is critical for Th17 diVerentiation is the retinoid orphanreceptor, ROR!t [73]. ROR!t is predominantly expressed in a subset of lamina propria Tcells but is also preferentially expressed by diVerentiating Th17 cells. In vitro, ROR!¡/¡ Tcells show reduced Th17 diVerentiation, whereas overexpression of ROR!t in T cellspromotes IL-17 expression. Furthermore, ROR!-deWcient mice are less susceptible to EAEsuggesting the essential role of ROR!t in the diVerentiation of Th17 cells and in autoimmu-nity [11].

Recent studies have shown that levels of ROR!t are signiWcantly reduced in Stat3-deW-cient T cells, consistent with the signiWcant downregulation of IL-17 in these cells [69–71].This suggests that Stat3 can regulate the expression of ROR!t, but the mechanism underly-ing this regulation has not been clearly clariWed. How exactly ROR!t, Stat3, or possiblyother unknown factors cooperate in regulating IL-17 expression is deWnitely an importantarea to be explored.

Brustle and colleagues have recently reported that interferon regulatory factor (IRF)-4 isa positive regulator in Th17 diVerentiation [74]. Irf4¡/¡ or wild-type Th cells transfectedwith IRF4 siRNA have impaired IL-17 production despite stimulation of TGF"-1 and IL-6.Moreover, Irf4¡/¡ mice are resistant to EAE, which is overcome by the transfer of wild-type CD4+ T cells. However, the molecular mechanism of the induction of IL-17 by IRF-4has not been clariWed. Moreover, the expression of this transcription factor in human Th17has not been explored.

Despite the importance of TGF"-1 in promoting Th17 diVerentiation, we are entirelyignorant of how it works to induce diVerentiation of this subset. TGF"-1 has pleiotropicfunctions in immune responses. For T cells, it is an important regulator for T cell prolifera-tion, survival and subsets’ diVerentiation [75]. TGF"-1 is produced by Treg cells andinhibits IL-2 production, thereby inhibiting T cell proliferation [76]. TGF"-1 inhibits theexpression of T-bet and Gata3, two “master” transcription factors, for Th1/Th2 develop-ment. Consequently, TGF-" inhibits both Th1 and Th2 diVerentiation [77, 78]. WhetherTGF"-1 is predominantly acting to inhibit other fates or actively promoting IL-17 produc-tion is unknown at this time.

Negative regulation of Th17 cells

Given the intense inXammatory nature of IL-17, it is not a surprise that the production ofthis cytokine is tightly controlled. In the initial studies of Th17 cells, it was recognized thatIFN-!, the prototypic cytokine produced by Th1 cells, inhibited Th17 diVerentiation [42,56]. Th1 cells also express the transcription factor T-bet and it is of interest that T-bet deW-cient T cells produce more IL-17 [79]; exactly how T-bet is working with respect to IL-17

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regulation has not been established. IL-4, the signature cytokine of Th2 cells, is also a neg-ative regulator of Th17 cell diVerentiation.

Interestingly, an IL-12-related cytokine, IL-27, has been reported to inhibit IL-17expression [56, 80–83]. Studies have shown that IL-27 limits EAE, Toxoplasma gondii-induced immunopathology, and experimental autoimmune uveoretinitis by suppressing thedevelopment of IL-17-producing T cells. Although similar to IL-6 in that its receptor isalso composed of gp130, IL-27 predominantly activates Stat1 and the suppressive eVect ofIL-27 on Th17 development has been documented to be Stat1-dependent [81–83].

Emerging connections between Treg and Th17 cells

There are at least two types of regulatory T cells: naturally occurring Treg (nTreg) cells,which are generated in thymus; and induced Treg (iTreg) cells, which can be generatedduring in vitro culture of naïve T cells by cytokines. Presumably the latter represent amodel of peripheral conversion that occurs especially in the gut. In mouse, both these twotypes of Tregs are CD4+CD25+Foxp3+ and both have suppressive eVects that are essentialfor maintaining self-tolerance and for controlling pathological immune responses [6, 84,85]. CD4+CD25-naïve T cells can convert into CD4+CD25+Foxp3+ suppressor cells whenexposed to TGF"-1 and IL-2 [84, 86]. Thus it was of considerable interest that TGF"-1 inthe context of other cytokines (IL-6, IL-21) generated highly inXammatory Th17 cells. Asindicated above, it is still not clear how TGF"-1 can regulate fate determination in develop-ing helper T cells.

IL-2 is a well-known T cell growth factor in vitro; however, mice lacking IL-2, IL-2R#,or IL-2R" all exhibit reduction in Treg cells and severe autoimmune diseases [87–89]. Thishas been explained based on the role of IL-2 in promoting the diVerentiation of Treg cells.IL-2 activates the transcription factor Stat5. Stat5 in turn binds the Foxp3 gene and Stat5deWciency is associated with profound reduction in Tregs [88, 89]. However, we noted thatthe autoimmunity associated with IL-2 deWciency is also associated with increased IL-17production [69]. In vitro, Th17 cell diVerentiation is enhanced by blocking IL-2, whereasaddition of exogenous IL-2 interfered with Th17 diVerentiation. Since IL-2 in combinationwith TGF"-1 promotes the conversion of naïve CD4+ T cells to Foxp3-expressing Tregcells, while simultaneously inhibiting Th17 diVerentiation, it appears that T cells may havean endogenous means of limiting IL-17 production. In addition, Stat5 deWciency is alsoassociated with expansion of Th17 cells [69]. Stat5 also appears to bind the Il17 locus andmay serve as a transcriptional repressor in this context.

The importance of Treg in maintaining peripheral tolerance is nicely illustrated in vari-ous models of IBD. Recent studies documenting the prevalence of Th17 cells in gut pro-vide yet another rationale for why it is so important to attenuate immune responses in thegut [25, 27, 28, 90]. In this regard, it is notable that a subset of dendritic cells (CD103+)exist in that gut that have immunosuppressive properties. The subpopulation of DCs in gutis specially equipped for converting antigen-speciWc T cells into Foxp3+ cells. This conver-sion process leads to the upregulation of #4"7 integrin and CCR9 permitting the newlyformed Treg cells to home to the gut. A series of recent studies show that these mucosalDCs produce retinoic acid, which inhibits ROR!t expression and Th17 diVerentiation[91–96]. Retinoic acid has previously been known to inhibit Th1 and Th2 diVerentiation.Of interest was that not only did retinoic acid inhibit Th17 diVerentiation, but it also upreg-ulated Foxp3 expression and conferred immunosuppressive activities. Through the use ofselective retinoic acid receptor (RAR) inhibitors, it was shown that maintenance of Foxp3expression appears to be dependent upon the function of RARs.

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A very recent paper shows that in gut lamina propria macrophages also produce retinoicacid and IL-10, and they too promote the generation of Treg cells. In contrast, lamina pro-pria DCs produce IL-6 and IL-23 and induce Th17 cells. Of note is that the diVerentiationof both cells depends on TGF"-1 [97].

Th17 cells and infection

The role of Th17 cells in the pathogenesis of autoimmunity has been emphasized, primarilybecause the apparent limitations of the Th1/Th2 paradigm forced a reconsideration of themechanisms of these disorders and thereby led to the understanding of the role of this newsubset. However, it should be obvious that the evolutionary pressure for this specializedsubset likely came from its role in host defense. As discussed, the receptor for IL-17 iswidely expressed on many cell types where it induces the expression of chemokines, proin-Xammatory cytokines, and colony-stimulating factors. These cytokines and chemokines inturn induce the recruitment of neutrophils and other myeloid cells, which is a critical fea-ture of many infectious diseases [98].

Accordingly, it has been established that IL-17 is critical for protection against Gram-negative bacteria, including Klebsiella pneumoniae [99, 100]. IL-17R-deWcient miceshowed reduced survival rate in a K. pneumoniae pneumonia model, due to the failurerecruitment of neutrophils to the lung. This was associated with reduced expression ofG-CSF, SCF, MIP-1#, MIP-1", and MIP-2 [99]. Like IL-17R¡/¡ mice, IL-12/IL-23p40¡/¡, IL-23, and p19¡/¡ mice are more susceptible to Klebsiella pneumoniae infection—another piece of data pointing to an IL-23/IL-17 link. Of note was that IL-12 p35¡/¡ micealso had increased susceptibility to infection and IL-12 was required for IFN-! expression.Thus, in this circumstance both Th1 and Th17 responses are involved in optimal host defenseagainst K. pneumoniae [101]. Additionally, Bordetella pertussis-infected human monocyte-derived dendritic cells produce high level of IL-23 but not IL-12 which results in enhancedlevel of IL-17 production by T cells during the in vitro culture [102, 103].

IL-17 also appears to play an important role in protection against Mycobacterium tuber-culosis [104, 105]. However, IL-17 does not seem to have any protective eVect during theprimary infection; rather, it is thought to play a more important role during the chronicinfection or secondary immune responses [106]. In contrast, IFN-! is required for the pri-mary immune response against Mycobacterium tuberculosis [107]. This is another situationin which IL-17 and IFN-! appear to work in concert in host defense.

IL-17 also appears to be important in Lyme disease. That is, enhanced IL-17 productionwas observed in response to Borrellia burgdorferi stimulation of both in mouse and humanT cells [108]. Furthermore, IL-17 production was detected in T cells from synovial Xuidfrom patients with Lyme arthritis.

Two recent studies have also indicated that Th17 cells are also involved in antifungaldefense both in mouse and human [68, 109]. Zymosan, a cell wall polysaccharide of yeast,and Curdlan, "-1,3-glucan from C. albicans, preferentially induce IL-23 production.Accordingly, this therefore favors Th17 diVerentiation; and in vivo, mice infected withC. albicans have large numbers of IL-17-producing CD4+ T cells, although IL-17/IFN-!double producers are prominent in this setting [68, 109].

Thus it would appear that in contrast to Th1 and Th2 cells, which protect against intra-cellular bacteria and helminthes, a major function of Th17 cells is to protect against extra-cellular bacteria and fungi. This being said, in some circumstances (e.g., Mycobacterialinfection) IFN-! and IL-17 appear to work in concert; clearly the role of IL-17 in hostdefense will be an exciting area of future investigation.

Immunol Res

Concluding remarks

The past 4 years have witnessed remarkable advances that have led to identiWcation of thenew lineage of helper T cells, Th17 cells. These studies have provided many surprises withrespect to our understanding of lineage commitment of helper T cells—certainly life ismuch more complicated than previously assumed. The simple Th1/Th2 dichotomy hasbeen replaced by a more Xexible multilineage model with reciprocal regulation. Despite thecomplexity, the demise of the Th1/Th2 paradigm allows immunologists to escape from theproblem of Wtting square pegs into round holes—this was the state of the art of conceptual-izing T cells and autoimmune disease a few years ago. It is very clear now that Th17 cellsoccupy the center-stage in the pathogenesis of autoimmunity, but additionally, their role inhost defense is also becoming more obvious. Despite the immense amount that we havelearned about the regulation of the cells, there are numerous unanswered questions.

Even seemingly simple questions like how TGF"-1 is working to regulate Th17 andTreg diVerentiation do not have a clear answer at this point. Similarly, how is Ror!regulated? Does Stat3 directly regulate its transcription? The Wnding that endogenousretinoids upregulate Foxp3 and downregulate Ror! causes one to speculate whether theremight be endogenous ligands that positively regulate Ror!. Might there be subsets ofdendritic cells and macrophages that produce such a ligand and in this manner promoteTh17 diVerentiation?

Although we learned much about Th17 cells, the extent to which they represent a fullycommitted, distinct lineage of helper T cells is somewhat unclear. Moreover, how theyrelate to Treg cells and Th1 cells is also unclear. In a recent study, it was reported thatFoxp3-expressing Tregs could diVerentiate into Th17 cells [110]. Additionally, IL-17+IFN!+ and IL-17¡IFN!+ cells are observed in disease models and in human memorycells [12–14, 68, 72, 73, 111]. Do these represent distinct subsets of helper cells or just atransitional stage between two subsets? Are they derived from the same precursor? Howplastic are these cells and what might control their ultimate fate determination?

Other cytokines, such as IL-17F, IL-22, TNF, have also been reported being producedby Th17 cells [29, 43, 58]. Unlike IL-17, relatively little is known about the regulation ofIL-17F and IL-22. Do the transcription factors (Stat3 and ROR!t) regulating IL-17A alsoupregulate these two cytokines? More importantly, although it is known that IL-22 plays animportant role in the pathogenesis of psoriasis, much more needs to be learned about therelative eVects of these cytokines in autoimmunity and host defense. Nonetheless, a wealthof studies now support the critical eVects of Th17 in autoimmunity. As such, IL-17 andTh17 cells certainly represent an attractive therapeutic target in the treatment of autoim-mune and inXammatory diseases.

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