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Reasons for rarity of Th17 cells in inflammatory sites of human disorders
Seminars in Immunology 25 (2013) 299–304
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Review
Reasons for rarity of Th17 cells in inflammatory sites of human disorders Francesco Annunziato a,b,∗ , Veronica Santarlasci a , Laura Maggi a , Lorenzo Cosmi a,b , Francesco Liotta a,b , Sergio Romagnani a a b
Department of Experimental and Clinical Medicine and DENOTHE Center, University of Florence, Florence 50134, Italy Regenerative Medicine Unit and Immunology and Cellular Therapy Unit of Azienda Ospedaliera Careggi, Florence 50134, Italy
a r t i c l e Keywords: Th17 Th1 RORC CD161 IL-4I1 Tob1
i n f o
a b s t r a c t T helper 17 (Th17) cells have been reported to be responsible for several chronic inflammatory diseases. However, a peculiar feature of human Th17 cells is that they are very rare in the inflammatory sites in comparison with Th1 cells. The first reason for this rarity is the existence of some self-regulatory mechanisms that limit their expansion. The limited expansion of human Th17 cells is related to the retinoic acid orphan (ROR)C-dependent up-regulation of the interleukin (IL)-4 induced gene 1 (IL4I1), which encodes for a l-phenylalanine oxidase, that has been shown to down-regulate CD3 expression in T cells. This results in abnormalities of the molecular pathway which is responsible for the impairment of IL-2 production and therefore for the lack of cell proliferation in response to T-cell receptor (TCR) signalling. IL4I1 up-regulation also associates with the increased expression of Tob1, a member of the Tob/BTG anti-proliferative protein family, which is involved in cell cycle arrest. A second reason for the rarity of human Th17 cells in the inflammatory sites is their rapid shifting into the Th1 phenotype, which is mainly related to the activity of IL-12 and TNF-␣. We have named these Th17-derived Th1 cells as non-classic because they differ from classic Th1 cells for the expression of molecules specific for Th17 cells, such as RORC, CD161, CCR6, IL4I1, and IL-17 receptor E. This distinction may be important for defining the respective pathogenic role of Th17, non-classic Th1 and classic Th1 cells in many human inflammatory disorders. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Activated CD4+ Th cells can be subdivided into lineages based on their cytokine secretion, transcription factor expression and immunological function. Initially, CD4+ Th cells were thought of as having one of two possible fates: type 1 (Th1) or type 2 (Th2) cells. Th1 cells express the transcription factor T-bet, secrete IFN␥ and protect the host mainly against intracellular infections. Th2 cells express GATA-3, secrete interleukin (IL)-4, IL-5, IL-9 and IL-13 and mediate host defense against helminthes [1,2]. Recently, additional Th subsets have been identified that preferentially produce other cytokines. Of these, the most intensively studied is the Th17 cell subset that selectively produces IL-17A. Th17 cells are not only critical for host protection against extracellular pathogens, but they have also been found to be highly pathogenic, inasmuch as they
∗ Corresponding author at: Department of Experimental and Clinical Medicine, University of Florence, Viale Pieraccini 6, Firenze 50134, Italy. Tel.: +39 055 4271351; fax: +39 055 4271500. E-mail address: francesco.annunziato@unifi.it (F. Annunziato). 1044-5323/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.smim.2013.10.011
are responsible for several chronic inflammatory and autoimmune disorders [3,4]. Although there are many similarities between murine and human Th17 cells, these cells in the two species seem also to exhibit some phenotypic and functional differences. In particular, all human memory Th17 cells express CD161 [5], the human equivalent of NK.1.1, that has not been found in murine Th17 cells. Moreover, human Th17 cells have been reported to recruite neutrophil granulocytes not only by inducing the production of CXCL8 by several non-immune cell types, but also via the direct production of this chemokine [6]. A cross-talk between human Th17 cells and neutrophils seems to exist, since neutrophils can in turn recruite Th17 cells [6]. A third still controversial difference concerns the origin of murine and human Th17 cells. In mouse, Th17 have been reported to develop only following the in vitro stimulation of naïve Th cells with the mixture of IL-6 and transforming growth factor (TGF)- [7], whereas in human studies the in vitro addition of TGF- has not been found to be critical [8–10]. However, more recent studies in mice have also denied the essential role of TGF- [11,12]. In our studies, we found that human Th17 cells originate from retinoic acid orphan receptor (ROR)C-expressing precursors, which are all contained within a small subset of CD4+ CD161+ T cells present
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Fig. 1. Some examples of Th17 rarity in human diseases. Cytoflurimetric analysis of IL-17A and IFN-␥ production by CD4+ T cells, isolated from the inflamed intestine of one patient with Crohn’s disease, the skin of one patient with psoriasis, the SF of one patient with JIA, the CSF of one patient with MS, the thyroid of one patient with Hashimoto’s thyroiditis following their stimulation with PMA plus ionomycin.
in the umbilical cord blood (UCB) and newborn thymus and fully develop into Th17 cells in vitro only in the presence of a combination of IL-1 and IL-23. Accordingly, IL-23 receptor (IL-23R)- and CCR6expressing CD4+ T cells are also all contained within the CD161+ fraction [5]. More recently, we showed by using single cell PCR on sorted UCB CD4+CD161+ T cells that RORC+ precursors are only a minority of this subset and only part of them express RORC and IL-23R, suggesting that Th17 cell precursors are present in UCB and possibly in newborn thymus in different stages of differentiation (Mazzoni et al., unpublished data). Finally, it has been reported that human Th17 cells may exhibit a different cytokine profile according to their antigen-specificity. Candida albicans-specific Th17 cells could produce IL-17 and IFN-␥, but no IL-10, whereas Staphylococcus aureus-specific Th17 cells produced IL-17 and could produce IL-10 upon restimulation [13]. An interesting feature of both murine and human Th17 cells is their rarity in the inflammatory sites. This aspect has been underestimated in mice, but it has been widely investigated in humans. Here, therefore, we discuss the mechanisms which have been found to be responsible for the rarity of Th17 cells in the inflamed tissues of human diseases. 2. Reasons for rarity of Th17 cells in inflammatory sites Despite their suggested highly pathogenic role, Th17 cells are certainly a minority in the inflammatory sites of some murine experimental models in comparison to high numbers of infiltrating Th1 cells [14–16]. A very few Th17 cells have also been found by us in the inflamed intestine of patients with Crohn’s disease [5,17], the skin of patients with psoriasis (unpublished results), the synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA) [18], the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) (unpublished results) and the thyroid of patients with Hashimoto’s thyroiditis (unpublished results) (Fig. 1). Moreover, a very few Th17 cells were observed in the peripheral blood (PB) of patients with
severe chronic asthma [19], despite the numbers of these circulating Th17 cells were increased in comparison with those found in healthy subjects. There are different reasons that account for the rarity of human Th17 cells in chronic inflammatory disorders, which can be distinguished into two major categories, the first being the existence of different self-regulatory mechanisms that limit their expansion and the second the high transience of their phenotype, inasmuch as Th17 cells tend to rapidly shift towards the Th1 profile. 2.1. Mechanisms that limited Th17 cell expansion In the last few years we have identified at least two different mechanisms that limit human Th17 cell expansion: the poor ability to produce IL-2 in response to T-cell receptor (TCR) signaling, and the reduced capacity to enter into cell cycle. 2.1.1. Impaired IL-2 production in response to TCR signalling Our initial studies derived from the observation that while in vitro stimulation with anti-CD3–CD28 induced strong proliferation of Th1 cells, it was quite ineffective on the proliferation of Th17 cells [20]. IL-2 expression was therefore measured in the supernatants of the above mentioned cell cultures and found to be produced by Th1, but not by Th17, cells upon anti-CD3–CD28 stimulation. The impairment of IL-2 production by Th17 cells was apparently related to abnormalities of the molecular pathway that allows the activation of IL-2 gene promoter [20]. Gene expression profile of Th1 versus Th17 cell clones by a microarray analysis showed that IL4I1 mRNA was substantially up-regulated in Th17 compared to Th1 cells. IL4I1 encodes a secreted l-amino-acid oxidase expressed by B lymphocytes [21] and by dendritic cells [22], which inhibits human T lymphocyte proliferation in vitro by inducing a temporary decrease in CD3 chain expression resulting from the enzymatic production of H2 O2 [22]. Interestingly, IL4I1 silencing in Th17 cell clones by using IL4I1-specific siRNA substantially increased cell
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Fig. 2. Scheme of self-regulatory mechanisms of Th17 expansion. Scheme illustrating the self-regulatory mechanisms responsible for impaired Th17 proliferation in response to TCR triggering. RORC-dependent IL4I1 up-regulation results in reduced expression of CD3 chains and therefore in abnormalities of the molecular pathways that allow the activation of the IL-2 gene promoter. IL4I1 up-regulation in human Th17 cells also associates with increased expression of Tob1 that impairs the progression into the cell cycle. In human Th17 cells there is also a reduction of Skp2, which not only can act as a Tob inhibitor, but also interacts with, ubiquitinates, and promotes the degradation of the forkhead transcription factor FOXO1, able to promote Th17 cell survival and to inhibit IL-2 production.
proliferation, IL2 mRNA expression, and IL-2 production by Th17 cells. Taken together, these results demonstrate that IL4I1 mRNA is highly expressed in Th17 cells and that its activity is responsible, at least in part, for the impairment of cell proliferation and of IL-2 production by Th17 cells [20]. Additional experiments showed that IL4I1 mRNA expression was regulated by the Th17 cell master gene RORC, inasmuch as its product directly bound to the IL4I1 promoter [20].
2.1.2. Arrest of cell cycle progression In a subsequent study, we have shown that in human Th17 cells, IL4I1 also maintains high levels of Tob1 (Santarlasci et al., unpublished results), a member of the Tob/BTG anti-proliferative protein (APRO) family, which prevents the cell cycle progression mediated by TCR stimulation [23]. Of note, the results of this study also demonstrated that human Th17 cells exhibit reduced levels of Skp2, that interacts with Tob1 and promotes its ubiquitin-dependent degradation [24]. Moreover, it is known that Skp2 interacts with, ubiquitinates, and promotes the degradation of, the forkhead transcription factor FOXO1 [25], which has been shown to promote Th17 cell survival and to inhibit IL-2 production [26]. Although the mechanisms responsible for the Skp2 reduced expression in Th17 cells remain presently unclear, it may also contribute to the maintenance of high Tob1 and FOXO1 levels in human Th17 cells. [25]. Tob1 expression in human Th17 cells was related to IL4I1, inasmuch as IL4I1 silencing induced a substantial decrease of Tob1 expression (Santarlasci et al., unpublished results). These data suggest that IL4I1 up-regulation in human Th17 cells limits their TCR-mediated expansion not only by blocking the molecular pathway involved in the activation of the IL-2 promoter, but also by maintaining high levels of Tob1 which impairs both the cell cycle entry and the IL-2 production. These findings are consistent with recent observations showing that a low expression of Tob1 in CD4+ T cells of individuals presenting with an initial central nervous system (CNS) demyelinating event, correlated with high risk for progression to multiple
sclerosis [27]. Very recently, by investigating the role of Tob1 in experimental autoimmune encephalomyelitis (EAE), it was found that immunization of Tob1−/− mice with myelin oligodendrocyte glycoprotein MOG peptide 35–55 resulted in an earlier disease onset, an increase in the maximum clinical score, and a greater and sustained disease severity in comparison with wild type mice [28]. The exacerbated EAE phenotype in Tob1−/− mice associated with augmented CNS inflammation, increased infiltration of CD4+ and CD8+ T cells, and higher numbers of myelin-reactive Th1 and Th17 cells, whereas the numbers of regulatory T cells were reduced [28]. Thus, our data provide the explanation for the reason why Tob1 can play an important regulatory role in the development and/or severity of this important disorder of CNS and perhaps of other Th17-related chronic inflammatory processes in both humans and mice. The self-regulatory mechanisms responsible for the limited expansion of human Th17 cells are illustrated in Fig. 2.
2.2. Plasticity towards the Th1 phenotype Another important reason for explaining the rarity of Th17 cells in the inflammatory sites is their high plasticity, which allows these cells to produce IFN-␥ and then rapidly shift to the Th1 phenotype. The first demonstration of the Th17 cell shifting towards the Th1 phenotype was provided in our initial study on these cells performed in 2007 [17]. When we examined the cytokine profile of T cells derived from the inflamed mucosa of patients with Crohn’s diseases, we found that they were mainly characterized by the production of IFN-␥ alone (prevalence of Th1 cells, as already found years before) [29], but there were also a few T cells producing IL-17, but not IFN-␥ (Th17 cells), and other cells producing both IL-17A and IFN-␥, that we named as Th17/Th1 cells [17]. To investigate the origin of these Th17/Th1 cells, we cultured Th17 cell clones in vitro in the presence of IL-12. After one week of culture, a proportion of Th17 cells started to produce IFN-␥ and after two weeks all of them shifted towards the Th1
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Fig. 4. Mechanisms of Th17 cell shifting into non-classic Th1 cells. Human Th17 cells can be shifted in vitro and in vivo by IL-12 and/or TNF-␣ to produce IFN-␥ in addition to IL-17A and then to become non-classic Th1 cells.
Fig. 3. First demonstration of Th17 plasticity. First demonstration of the plasticity of Th17 cells, based on: (A) the observation that there are human CD4+ T cells able to produce both IL-17A and IFN-␥; (B) the possibility to induce the production of IFN-␥ by a human Th17 clone cultured for one week in the presence of IL-12 (from Ref. [17]).
phenotype [17]. The transient nature of the Th17 phenotype is now considered as an established fact even in mice [30] (Fig. 3). 2.2.1. Mechanisms responsible for Th17 shifting to Th1 In humans, Th17-derived Th1 cells can be easily recognized and separated from classic Th1 cells because they express CD161. Based on this finding, we examined the CD4+ T cell populations present in the PB and SF of patients with JIA in comparison with CD4+ T cell populations of the PB from healthy children. There was a significant enrichment for CD4+CD161+ cells in the SF in comparison with PB of JIA children. CD4+ T cells able to produce IL-17A were found virtually only in the CD161+ fraction and appeared to be significantly higher in the SF than in PB of JIA children. By contrast, IFN-␥-producing cells could be detected within either the CD161+ or the CD161− fraction of PB and SF from both healthy and JIA children, respectively, their proportions being significantly higher in the SF than in PB of JIA children [18]. Levels of RORC mRNA were also significantly higher in CD4+CD161+ T-cell fractions, irrespective of whether they produced IL-17A or IFN-␥ alone, in comparison with CD161− Th1 cells from both healthy and JIA children [18]. Th17
cells were then purified from the PB of healthy subjects and cultured in presence or absence of the pooled SF from JIA children, or IL-12 or the same SF plus an anti-IL-12 neutralizing mAb. Culturing in either the presence of IL-12 or the pooled SF from JIA resulted in reduced proportions of IL-17+IFN-␥− (Th17) and increased proportions of both IL-17+IFN-␥+ (Th17/Th1) and IL-17−IFN-␥+ (Th1), cells. The addition of an anti-IL-12 mAb to cultures containing the SF of JIA completely reversed their modulatory effects, suggesting they were at least partially due the Th1-polarizing activity of IL-12 [18]. The demonstration that Th17 cells from the SF of JIA children could be shifted in vitro to Th1 cells allowed us to hypothesize that a similar Th17 to Th1 shifting could also occur in vivo in the SF of JIA children, explaining the high frequency of CD161+ Th1 cells. To provide support to this hypothesis, we compared the TCR-BV repertoire of highly purified CD161+IL-17−IFN-␥+ cells with that of CD161+IL-17+IFN-␥− cells derived from SF of a JIA patient and we found that these two types of cells had a common origin [18]. Finally, the possibility that the presence of CD4+CD161+ T cells in the SF correlated with disease activity was investigated. The proportions of CD4+CD161+ in SF directly correlated with levels of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) of JIA children. Of note, a positive correlation between the proportions of CD161+ T cells producing both IL-17A and IFN-␥ in the SF and levels of ESR and CRP was found [18], thus supporting the hypothesis that these cells may play a role in disease activity. Based on all these finding we concluded that Th17 cell-derived Th1 cells were clearly distinct from traditionally known Th1 cells and we, therefore, decided to name these cells as “non-classic” in order to distinguish them from classic Th1 cells [18]. In a subsequent study, we found that etanercept in vitro increased the numbers of CD4+CD161+ Th17/Th1 and Th17 cells, while it decreased the proportions of non-classic, but not of classic Th1, cells. We also observed that TNF-␣ was able to induce in vitro the transition of Th17 lymphocytes towards the nonclassicTh1 phenotype, probably thanks to the high expression of TNFRII which was observed in Th17 cells. Accordingly, the proportions of CD4+CD161+ Th1 lymphocytes in PB of patients treated with etanercept were lower than in untreated ones (Maggi et al., unpublished results). These findings suggest that not only IL-12, but also TNF-␣ is able to favour the shifting of Th17 cells into nonclassic Th1 cells. A scheme illustrating the mechanisms of shifting of human Th17 into non-classic Th1 cells is shown in Fig. 4. 2.2.2. The distinction between non-classic from classic Th1 cells Based on all these findings, we asked whether non-classic Th1 cells could be distinguished from classic Th1 cells on the basis of other markers besides the expression of CD161. To this end, we assessed a panel of T-cell clones, as well as CD161+ or CD161−CD4+ T cells derived ex vivo from the circulation of healthy subjects or the SF of patients with JIA. The results showed that non-classic Th1 cells
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could be identified not only of the basis of CD161 expression, but also by the consistent expression of RORC, IL-17 receptor E, CCR6 and IL4I1, which were all virtually absent from classic Th1 cells [31]. More recently, we also looked at possible differences in the epigenetic regulation of non-classic and classic Th1 cells. As expected, non-classic Th1 cells exhibited complete demethylation of the analyzed regions of interest (ROI) of RORC2 gene promoter, as happens in Th17 cells, as well as partial methylation of the analyzed ROI in IL-17A gene promoter, whereas classic Th1 cells did not (Mazzoni et al., unpublished results). Taken all together, these findings suggest the possibility to distinguish these two cell subsets by using such a panel of markers even in inflamed tissues and therefore provide the opportunity to better establish the respective pathogenic roles of Th17, as well as non-classic and classic Th1 cells in different chronic inflammatory disorders [32].
3. Conclusions In this review, we have discussed the mechanisms possibly responsible for the rarity of Th17 cells in inflammatory sites of human diseases. This rarity has also been reported in experimental murine models of chronic inflammatory disorders where they are thought to play an important pathogenic role. However, the mechanisms responsible for this rarity have been extensively investigated only in humans. These studies have shown that the rarity of Th17 cells in the inflammatory sites of human diseases may be due at least in part to self-regulatory mechanisms that limit their expansion in response to TCR triggering, i.e. to antigen stimulation. One of these mechanisms consists of an impairment of IL-2 production, due to the RORC-dependent hyper-expression of IL4I1, which encodes for an enzyme produced by dendritic cells and capable of reducing the CD3 chains signalling, and therefore of inducing abnormalities in the molecular pathway that allows IL-2 gene promoter activation [20]. IL4I1 hyper-expression in Th17 cells also associates with the high expression of Tob1, a member of the Tob/BTG APRO family, which prevents the cell cycle progression mediated by TCR stimulation. Moreover, human Th17 cells exhibit reduced expression of Skp2, that promotes the ubiquitin-dependent Tob1 degradation [Santarlasci et al., unpublished results]. Although the mechanisms responsible for Skp2 reduction in human Th17 cells still remain unknown, one cannot exclude that, in addition to the maintenance of high Tob1 levels, it also favours the persistence of the forkhead transcription factor FOXO1, which also plays a pivotal role in growth arrest and apoptosis [25,26]. Thus, several self-regulatory mechanisms concur in limiting the expansion of human Th17 cells in response to antigen stimulation. These mechanisms are probably related to the peculiar function of Th17 cells, which may be devoted to reside in a quiescent state in peripheral tissues, where they provide a first line of protection against pathogens. Indeed, it has recently been reported that Th17 cells express a signature closely resembling the pattern observed in stem cell-like memory cells, i.e. the enhanced capacity to survive, self renew and generation of effector progeny [33]. Another explanation for the rarity of Th17 cells in inflamed tissues is their high plasticity, that allows these cells to rapidly shift in presence of pro-inflammatory cytokines, such as IL-12 and TNF-␣, to the production of IFN-␥ in addition to IL-17A, and subsequently even acquire a pure Th1 profile. The transient nature of Th17 cells, which was first discovered in our initial studies in humans [17], when mouse immunologists still considered the Th17 as a fixed phenotype, is now considered as a generally established fact [30]. Our subsequent studies have clearly demonstrated that Th17-derived Th1 cells are different from the already known, classic, Th1 cells and have therefore been named by us as non-classic
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Th1 cells [18]. The difference between non-classic and classic Th1 cells not only consists of the expression of different markers, such as RORC, CD161, CCR6, IL-17RE and IL4I1 [31,32], but also rests on the different degree of demethylation of the ROI of RORC2 and IL-17A gene promoter (Mazzoni et al., unpublished results). The reason for the rapid shifting of Th17 cells into non-classic Th1 cells is unclear, because the respective role of Th17, Th17/Th1, non-classic and classic Th1 cells in the pathogenesis of different chronic inflammatory disorders has not yet been clarified. Indeed, after the initial conceptual substitution of Th1 cells with Th17 cells as responsible for pathogenicity, mainly based on the results obtained in a murine model of autoimmunity in gene-deficient animals [14], a series of well-performed studies has strongly challenged this oversimplification. Th17 can be pathogenic in certain forms of experimental autoimmune encephalomyelitis (EAE) (those characterized by granulocyte infiltration), whereas Th1 cells are mainly responsible for the EAE models characterized by prevalent mononuclear cell infiltration, where Th17 cells do not seem to play any pathogenic role [34]. In the murine model of autoimmune disorder known as proteoglycan-induced arthritis, Th1 cells, but not Th17 cells, are pathogenic [35]. Either Th1 or Th17 cells have been found to be pathogenic in experimental autoimmune uveitis [36]. Neither IL-17A nor IL-17F contributes vitally to autoimmune neuroinflammation in mice [37]. Th17 cells can promote pancreatic inflammation but only induce type 1 (insulin-dependent) diabetes mellitus efficiently in lymphopenic mice after conversion into Th1 cells [38]. Accordingly, highly purified Th17 cells from BDC2.5NOD mice shift into Th1-like cells in non-obese diabetic/severe combined immunodeficiency recipient mice. The transferred Th17 cells, completely devoid of IFN-␥ at the time of transfer, rapidly converted to secrete IFN-␥ in the non-obese diabetic/severe combined immunodeficiency recipients. More importantly, the development of insulin-dependent diabetes mellitus was prevented by the treatment with anti-IFN-␥, but not with anti-IL17A, specific antibody [39]. More recently, two types of EAE were described, one being Tbet dependent and the other one mediated by Th17 and Th17/Th1 cells and T-bet-independent, suggesting that both classic and nonclassic Th1 (these latter together with Th17 and Th17/Th1) cells may be pathogenic according to the different experimental model [40,41]. On the other hand, in T-bet-knockouts in which Th17 cells cannot shift to Th1 cells, they are still capable of mediating EAE, albeit with a milder clinical course [42]. Thus, it seems that Th17, Th17/Th1, non-classic and classic Th1 cells may all be involved in the tissue damage, with a variable importance in the different types and/or phases of the inflammatory disorders. Therefore, based on all these findings, the reason why Th17 cells rapidly shift to the Th1 phenotype in the inflammatory sites remains presently unclear.
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[25] Huang H, Regan KM, Wang F, Wang D, Smith DI, van Deursen JM, et al. Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation. Proc Natl Acad Sci U S A 2005;102:1649–54. [26] Oh HM, Yu CR, Golestaneh N, Amadi-Obi A, Lee YS, Eseonu A, et al. STAT3 protein promotes T-cell survival and inhibits interleukin-2 production through up-regulation of Class O forkhead transcription factors. J Biol Chem 2011;286:30888–97. [27] Corvol JC, Pelletier D, Henry RG, Caillier SJ, Wang J, Pappas D, et al. Abrogation of T cell quiescence characterizes patients at high risk for multiple sclerosis after the initial neurological event. Proc Natl Acad Sci U S A 2008;105: 11839–44. [28] Schulze-Topphoff U, Casazza S, Varrin-Doyer M, Michel K, Sobel RA, Hauser SL, et al. Tob1 plays a critical role in the activation of encephalitogenic T cells in CNS autoimmunity. J Exp Med 2013;210:1301–9. [29] Parronchi P, Romagnani P, Annunziato F, Sampognaro S, Becchio A, Giannarini L, et al. Type 1 T-helper cell predominance and interleukin-12 expression in the gut of patients with Crohn’s disease. Am J Pathol 1997;150:823–32. [30] Murphy KM, Stockinger B. Effector T cell plasticity: flexibility in the face of changing circumstances. Nat Immunol 2010;11:674–80. [31] Maggi L, Santarlasci V, Capone M, Rossi MC, Querci V, Mazzoni A, et al. Distinctive features of classic and nonclassic (Th17 derived) human Th1 cells. Eur J Immunol 2012;42:3180–8. [32] Annunziato F, Cosmi L, Liotta F, Maggi E, Romagnani S. Defining the human T helper 17 cell phenotype. Trends Immunol 2012;33:505–12. [33] Muranski P, Borman ZA, Kerkar SP, Klebanoff CA, Ji Y, Sanchez-Perez L, et al. Th17 cells are long lived and retain a stem cell-like molecular signature. Immunity 2011;35:972–85. [34] Kroenke MA, Carlson TJ, Andjelkovic AV, Segal BM. IL-12- and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition. J Exp Med 2008;205:1535–41. [35] Doodes PD, Cao Y, Hamel KM, Wang Y, Farkas B, Iwakura Y, et al. Development of proteoglycan-induced arthritis is independent of IL-17. J Immunol 2008;181:329–37. [36] Luger D, Silver PB, Tang J, Cua D, Chen Z, Iwakura Y, et al. Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J Exp Med 2008;205:799–810. [37] Haak S, Croxford AL, Kreymborg K, Heppner FL, Pouly S, Becher B, et al. IL-17A and IL-17F do not contribute vitally to autoimmune neuro-inflammation in mice. J Clin Invest 2009;119:61–9. [38] Martin-Orozco N, Chung Y, Chang SH, Wang YH, Dong C. Th17 cells promote pancreatic inflammation but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells. Eur J Immunol 2009;39:216–24. [39] Bending D, et al. Highly purified Th17 cells from BDC2.5NOD mice convert into Th1-like cells in NOD/SCID recipient mice. J Clin Invest 2009;119:565–72. [40] Duhen R, Glatigny S, Arbelaez CA, Blair TC, Oukka M, Bettelli E. Cutting Edge: the pathogenicity of IFN-␥-producing Th17 cells is independent of T-bet. J Immunol 2013;190:4478–82. [41] O’Connor RA, Cambrook H, Huettner K, Anderton SM. T-bet is essential for Th1mediated, but not Th17-mediated, CNS autoimmune disease. Eur J Immunol 2013;July, http://dx.doi.org/10.1002/eji.201343689. [42] Grifka-Walk HM, Lalor SJ, Segal BM. Highly polarized Th17 cells induce EAE via a T-bet independent mechanism. Eur J Immunol 2013;(July), http://dx.doi.org/10.1002/eji.201343723.
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Review
Reasons for rarity of Th17 cells in inflammatory sites of human disorders Francesco Annunziato a,b,∗ , Veronica Santarlasci a , Laura Maggi a , Lorenzo Cosmi a,b , Francesco Liotta a,b , Sergio Romagnani a a b
Department of Experimental and Clinical Medicine and DENOTHE Center, University of Florence, Florence 50134, Italy Regenerative Medicine Unit and Immunology and Cellular Therapy Unit of Azienda Ospedaliera Careggi, Florence 50134, Italy
a r t i c l e Keywords: Th17 Th1 RORC CD161 IL-4I1 Tob1
i n f o
a b s t r a c t T helper 17 (Th17) cells have been reported to be responsible for several chronic inflammatory diseases. However, a peculiar feature of human Th17 cells is that they are very rare in the inflammatory sites in comparison with Th1 cells. The first reason for this rarity is the existence of some self-regulatory mechanisms that limit their expansion. The limited expansion of human Th17 cells is related to the retinoic acid orphan (ROR)C-dependent up-regulation of the interleukin (IL)-4 induced gene 1 (IL4I1), which encodes for a l-phenylalanine oxidase, that has been shown to down-regulate CD3 expression in T cells. This results in abnormalities of the molecular pathway which is responsible for the impairment of IL-2 production and therefore for the lack of cell proliferation in response to T-cell receptor (TCR) signalling. IL4I1 up-regulation also associates with the increased expression of Tob1, a member of the Tob/BTG anti-proliferative protein family, which is involved in cell cycle arrest. A second reason for the rarity of human Th17 cells in the inflammatory sites is their rapid shifting into the Th1 phenotype, which is mainly related to the activity of IL-12 and TNF-␣. We have named these Th17-derived Th1 cells as non-classic because they differ from classic Th1 cells for the expression of molecules specific for Th17 cells, such as RORC, CD161, CCR6, IL4I1, and IL-17 receptor E. This distinction may be important for defining the respective pathogenic role of Th17, non-classic Th1 and classic Th1 cells in many human inflammatory disorders. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Activated CD4+ Th cells can be subdivided into lineages based on their cytokine secretion, transcription factor expression and immunological function. Initially, CD4+ Th cells were thought of as having one of two possible fates: type 1 (Th1) or type 2 (Th2) cells. Th1 cells express the transcription factor T-bet, secrete IFN␥ and protect the host mainly against intracellular infections. Th2 cells express GATA-3, secrete interleukin (IL)-4, IL-5, IL-9 and IL-13 and mediate host defense against helminthes [1,2]. Recently, additional Th subsets have been identified that preferentially produce other cytokines. Of these, the most intensively studied is the Th17 cell subset that selectively produces IL-17A. Th17 cells are not only critical for host protection against extracellular pathogens, but they have also been found to be highly pathogenic, inasmuch as they
∗ Corresponding author at: Department of Experimental and Clinical Medicine, University of Florence, Viale Pieraccini 6, Firenze 50134, Italy. Tel.: +39 055 4271351; fax: +39 055 4271500. E-mail address: francesco.annunziato@unifi.it (F. Annunziato). 1044-5323/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.smim.2013.10.011
are responsible for several chronic inflammatory and autoimmune disorders [3,4]. Although there are many similarities between murine and human Th17 cells, these cells in the two species seem also to exhibit some phenotypic and functional differences. In particular, all human memory Th17 cells express CD161 [5], the human equivalent of NK.1.1, that has not been found in murine Th17 cells. Moreover, human Th17 cells have been reported to recruite neutrophil granulocytes not only by inducing the production of CXCL8 by several non-immune cell types, but also via the direct production of this chemokine [6]. A cross-talk between human Th17 cells and neutrophils seems to exist, since neutrophils can in turn recruite Th17 cells [6]. A third still controversial difference concerns the origin of murine and human Th17 cells. In mouse, Th17 have been reported to develop only following the in vitro stimulation of naïve Th cells with the mixture of IL-6 and transforming growth factor (TGF)- [7], whereas in human studies the in vitro addition of TGF- has not been found to be critical [8–10]. However, more recent studies in mice have also denied the essential role of TGF- [11,12]. In our studies, we found that human Th17 cells originate from retinoic acid orphan receptor (ROR)C-expressing precursors, which are all contained within a small subset of CD4+ CD161+ T cells present
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Fig. 1. Some examples of Th17 rarity in human diseases. Cytoflurimetric analysis of IL-17A and IFN-␥ production by CD4+ T cells, isolated from the inflamed intestine of one patient with Crohn’s disease, the skin of one patient with psoriasis, the SF of one patient with JIA, the CSF of one patient with MS, the thyroid of one patient with Hashimoto’s thyroiditis following their stimulation with PMA plus ionomycin.
in the umbilical cord blood (UCB) and newborn thymus and fully develop into Th17 cells in vitro only in the presence of a combination of IL-1 and IL-23. Accordingly, IL-23 receptor (IL-23R)- and CCR6expressing CD4+ T cells are also all contained within the CD161+ fraction [5]. More recently, we showed by using single cell PCR on sorted UCB CD4+CD161+ T cells that RORC+ precursors are only a minority of this subset and only part of them express RORC and IL-23R, suggesting that Th17 cell precursors are present in UCB and possibly in newborn thymus in different stages of differentiation (Mazzoni et al., unpublished data). Finally, it has been reported that human Th17 cells may exhibit a different cytokine profile according to their antigen-specificity. Candida albicans-specific Th17 cells could produce IL-17 and IFN-␥, but no IL-10, whereas Staphylococcus aureus-specific Th17 cells produced IL-17 and could produce IL-10 upon restimulation [13]. An interesting feature of both murine and human Th17 cells is their rarity in the inflammatory sites. This aspect has been underestimated in mice, but it has been widely investigated in humans. Here, therefore, we discuss the mechanisms which have been found to be responsible for the rarity of Th17 cells in the inflamed tissues of human diseases. 2. Reasons for rarity of Th17 cells in inflammatory sites Despite their suggested highly pathogenic role, Th17 cells are certainly a minority in the inflammatory sites of some murine experimental models in comparison to high numbers of infiltrating Th1 cells [14–16]. A very few Th17 cells have also been found by us in the inflamed intestine of patients with Crohn’s disease [5,17], the skin of patients with psoriasis (unpublished results), the synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA) [18], the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) (unpublished results) and the thyroid of patients with Hashimoto’s thyroiditis (unpublished results) (Fig. 1). Moreover, a very few Th17 cells were observed in the peripheral blood (PB) of patients with
severe chronic asthma [19], despite the numbers of these circulating Th17 cells were increased in comparison with those found in healthy subjects. There are different reasons that account for the rarity of human Th17 cells in chronic inflammatory disorders, which can be distinguished into two major categories, the first being the existence of different self-regulatory mechanisms that limit their expansion and the second the high transience of their phenotype, inasmuch as Th17 cells tend to rapidly shift towards the Th1 profile. 2.1. Mechanisms that limited Th17 cell expansion In the last few years we have identified at least two different mechanisms that limit human Th17 cell expansion: the poor ability to produce IL-2 in response to T-cell receptor (TCR) signaling, and the reduced capacity to enter into cell cycle. 2.1.1. Impaired IL-2 production in response to TCR signalling Our initial studies derived from the observation that while in vitro stimulation with anti-CD3–CD28 induced strong proliferation of Th1 cells, it was quite ineffective on the proliferation of Th17 cells [20]. IL-2 expression was therefore measured in the supernatants of the above mentioned cell cultures and found to be produced by Th1, but not by Th17, cells upon anti-CD3–CD28 stimulation. The impairment of IL-2 production by Th17 cells was apparently related to abnormalities of the molecular pathway that allows the activation of IL-2 gene promoter [20]. Gene expression profile of Th1 versus Th17 cell clones by a microarray analysis showed that IL4I1 mRNA was substantially up-regulated in Th17 compared to Th1 cells. IL4I1 encodes a secreted l-amino-acid oxidase expressed by B lymphocytes [21] and by dendritic cells [22], which inhibits human T lymphocyte proliferation in vitro by inducing a temporary decrease in CD3 chain expression resulting from the enzymatic production of H2 O2 [22]. Interestingly, IL4I1 silencing in Th17 cell clones by using IL4I1-specific siRNA substantially increased cell
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Fig. 2. Scheme of self-regulatory mechanisms of Th17 expansion. Scheme illustrating the self-regulatory mechanisms responsible for impaired Th17 proliferation in response to TCR triggering. RORC-dependent IL4I1 up-regulation results in reduced expression of CD3 chains and therefore in abnormalities of the molecular pathways that allow the activation of the IL-2 gene promoter. IL4I1 up-regulation in human Th17 cells also associates with increased expression of Tob1 that impairs the progression into the cell cycle. In human Th17 cells there is also a reduction of Skp2, which not only can act as a Tob inhibitor, but also interacts with, ubiquitinates, and promotes the degradation of the forkhead transcription factor FOXO1, able to promote Th17 cell survival and to inhibit IL-2 production.
proliferation, IL2 mRNA expression, and IL-2 production by Th17 cells. Taken together, these results demonstrate that IL4I1 mRNA is highly expressed in Th17 cells and that its activity is responsible, at least in part, for the impairment of cell proliferation and of IL-2 production by Th17 cells [20]. Additional experiments showed that IL4I1 mRNA expression was regulated by the Th17 cell master gene RORC, inasmuch as its product directly bound to the IL4I1 promoter [20].
2.1.2. Arrest of cell cycle progression In a subsequent study, we have shown that in human Th17 cells, IL4I1 also maintains high levels of Tob1 (Santarlasci et al., unpublished results), a member of the Tob/BTG anti-proliferative protein (APRO) family, which prevents the cell cycle progression mediated by TCR stimulation [23]. Of note, the results of this study also demonstrated that human Th17 cells exhibit reduced levels of Skp2, that interacts with Tob1 and promotes its ubiquitin-dependent degradation [24]. Moreover, it is known that Skp2 interacts with, ubiquitinates, and promotes the degradation of, the forkhead transcription factor FOXO1 [25], which has been shown to promote Th17 cell survival and to inhibit IL-2 production [26]. Although the mechanisms responsible for the Skp2 reduced expression in Th17 cells remain presently unclear, it may also contribute to the maintenance of high Tob1 and FOXO1 levels in human Th17 cells. [25]. Tob1 expression in human Th17 cells was related to IL4I1, inasmuch as IL4I1 silencing induced a substantial decrease of Tob1 expression (Santarlasci et al., unpublished results). These data suggest that IL4I1 up-regulation in human Th17 cells limits their TCR-mediated expansion not only by blocking the molecular pathway involved in the activation of the IL-2 promoter, but also by maintaining high levels of Tob1 which impairs both the cell cycle entry and the IL-2 production. These findings are consistent with recent observations showing that a low expression of Tob1 in CD4+ T cells of individuals presenting with an initial central nervous system (CNS) demyelinating event, correlated with high risk for progression to multiple
sclerosis [27]. Very recently, by investigating the role of Tob1 in experimental autoimmune encephalomyelitis (EAE), it was found that immunization of Tob1−/− mice with myelin oligodendrocyte glycoprotein MOG peptide 35–55 resulted in an earlier disease onset, an increase in the maximum clinical score, and a greater and sustained disease severity in comparison with wild type mice [28]. The exacerbated EAE phenotype in Tob1−/− mice associated with augmented CNS inflammation, increased infiltration of CD4+ and CD8+ T cells, and higher numbers of myelin-reactive Th1 and Th17 cells, whereas the numbers of regulatory T cells were reduced [28]. Thus, our data provide the explanation for the reason why Tob1 can play an important regulatory role in the development and/or severity of this important disorder of CNS and perhaps of other Th17-related chronic inflammatory processes in both humans and mice. The self-regulatory mechanisms responsible for the limited expansion of human Th17 cells are illustrated in Fig. 2.
2.2. Plasticity towards the Th1 phenotype Another important reason for explaining the rarity of Th17 cells in the inflammatory sites is their high plasticity, which allows these cells to produce IFN-␥ and then rapidly shift to the Th1 phenotype. The first demonstration of the Th17 cell shifting towards the Th1 phenotype was provided in our initial study on these cells performed in 2007 [17]. When we examined the cytokine profile of T cells derived from the inflamed mucosa of patients with Crohn’s diseases, we found that they were mainly characterized by the production of IFN-␥ alone (prevalence of Th1 cells, as already found years before) [29], but there were also a few T cells producing IL-17, but not IFN-␥ (Th17 cells), and other cells producing both IL-17A and IFN-␥, that we named as Th17/Th1 cells [17]. To investigate the origin of these Th17/Th1 cells, we cultured Th17 cell clones in vitro in the presence of IL-12. After one week of culture, a proportion of Th17 cells started to produce IFN-␥ and after two weeks all of them shifted towards the Th1
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Fig. 4. Mechanisms of Th17 cell shifting into non-classic Th1 cells. Human Th17 cells can be shifted in vitro and in vivo by IL-12 and/or TNF-␣ to produce IFN-␥ in addition to IL-17A and then to become non-classic Th1 cells.
Fig. 3. First demonstration of Th17 plasticity. First demonstration of the plasticity of Th17 cells, based on: (A) the observation that there are human CD4+ T cells able to produce both IL-17A and IFN-␥; (B) the possibility to induce the production of IFN-␥ by a human Th17 clone cultured for one week in the presence of IL-12 (from Ref. [17]).
phenotype [17]. The transient nature of the Th17 phenotype is now considered as an established fact even in mice [30] (Fig. 3). 2.2.1. Mechanisms responsible for Th17 shifting to Th1 In humans, Th17-derived Th1 cells can be easily recognized and separated from classic Th1 cells because they express CD161. Based on this finding, we examined the CD4+ T cell populations present in the PB and SF of patients with JIA in comparison with CD4+ T cell populations of the PB from healthy children. There was a significant enrichment for CD4+CD161+ cells in the SF in comparison with PB of JIA children. CD4+ T cells able to produce IL-17A were found virtually only in the CD161+ fraction and appeared to be significantly higher in the SF than in PB of JIA children. By contrast, IFN-␥-producing cells could be detected within either the CD161+ or the CD161− fraction of PB and SF from both healthy and JIA children, respectively, their proportions being significantly higher in the SF than in PB of JIA children [18]. Levels of RORC mRNA were also significantly higher in CD4+CD161+ T-cell fractions, irrespective of whether they produced IL-17A or IFN-␥ alone, in comparison with CD161− Th1 cells from both healthy and JIA children [18]. Th17
cells were then purified from the PB of healthy subjects and cultured in presence or absence of the pooled SF from JIA children, or IL-12 or the same SF plus an anti-IL-12 neutralizing mAb. Culturing in either the presence of IL-12 or the pooled SF from JIA resulted in reduced proportions of IL-17+IFN-␥− (Th17) and increased proportions of both IL-17+IFN-␥+ (Th17/Th1) and IL-17−IFN-␥+ (Th1), cells. The addition of an anti-IL-12 mAb to cultures containing the SF of JIA completely reversed their modulatory effects, suggesting they were at least partially due the Th1-polarizing activity of IL-12 [18]. The demonstration that Th17 cells from the SF of JIA children could be shifted in vitro to Th1 cells allowed us to hypothesize that a similar Th17 to Th1 shifting could also occur in vivo in the SF of JIA children, explaining the high frequency of CD161+ Th1 cells. To provide support to this hypothesis, we compared the TCR-BV repertoire of highly purified CD161+IL-17−IFN-␥+ cells with that of CD161+IL-17+IFN-␥− cells derived from SF of a JIA patient and we found that these two types of cells had a common origin [18]. Finally, the possibility that the presence of CD4+CD161+ T cells in the SF correlated with disease activity was investigated. The proportions of CD4+CD161+ in SF directly correlated with levels of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) of JIA children. Of note, a positive correlation between the proportions of CD161+ T cells producing both IL-17A and IFN-␥ in the SF and levels of ESR and CRP was found [18], thus supporting the hypothesis that these cells may play a role in disease activity. Based on all these finding we concluded that Th17 cell-derived Th1 cells were clearly distinct from traditionally known Th1 cells and we, therefore, decided to name these cells as “non-classic” in order to distinguish them from classic Th1 cells [18]. In a subsequent study, we found that etanercept in vitro increased the numbers of CD4+CD161+ Th17/Th1 and Th17 cells, while it decreased the proportions of non-classic, but not of classic Th1, cells. We also observed that TNF-␣ was able to induce in vitro the transition of Th17 lymphocytes towards the nonclassicTh1 phenotype, probably thanks to the high expression of TNFRII which was observed in Th17 cells. Accordingly, the proportions of CD4+CD161+ Th1 lymphocytes in PB of patients treated with etanercept were lower than in untreated ones (Maggi et al., unpublished results). These findings suggest that not only IL-12, but also TNF-␣ is able to favour the shifting of Th17 cells into nonclassic Th1 cells. A scheme illustrating the mechanisms of shifting of human Th17 into non-classic Th1 cells is shown in Fig. 4. 2.2.2. The distinction between non-classic from classic Th1 cells Based on all these findings, we asked whether non-classic Th1 cells could be distinguished from classic Th1 cells on the basis of other markers besides the expression of CD161. To this end, we assessed a panel of T-cell clones, as well as CD161+ or CD161−CD4+ T cells derived ex vivo from the circulation of healthy subjects or the SF of patients with JIA. The results showed that non-classic Th1 cells
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could be identified not only of the basis of CD161 expression, but also by the consistent expression of RORC, IL-17 receptor E, CCR6 and IL4I1, which were all virtually absent from classic Th1 cells [31]. More recently, we also looked at possible differences in the epigenetic regulation of non-classic and classic Th1 cells. As expected, non-classic Th1 cells exhibited complete demethylation of the analyzed regions of interest (ROI) of RORC2 gene promoter, as happens in Th17 cells, as well as partial methylation of the analyzed ROI in IL-17A gene promoter, whereas classic Th1 cells did not (Mazzoni et al., unpublished results). Taken all together, these findings suggest the possibility to distinguish these two cell subsets by using such a panel of markers even in inflamed tissues and therefore provide the opportunity to better establish the respective pathogenic roles of Th17, as well as non-classic and classic Th1 cells in different chronic inflammatory disorders [32].
3. Conclusions In this review, we have discussed the mechanisms possibly responsible for the rarity of Th17 cells in inflammatory sites of human diseases. This rarity has also been reported in experimental murine models of chronic inflammatory disorders where they are thought to play an important pathogenic role. However, the mechanisms responsible for this rarity have been extensively investigated only in humans. These studies have shown that the rarity of Th17 cells in the inflammatory sites of human diseases may be due at least in part to self-regulatory mechanisms that limit their expansion in response to TCR triggering, i.e. to antigen stimulation. One of these mechanisms consists of an impairment of IL-2 production, due to the RORC-dependent hyper-expression of IL4I1, which encodes for an enzyme produced by dendritic cells and capable of reducing the CD3 chains signalling, and therefore of inducing abnormalities in the molecular pathway that allows IL-2 gene promoter activation [20]. IL4I1 hyper-expression in Th17 cells also associates with the high expression of Tob1, a member of the Tob/BTG APRO family, which prevents the cell cycle progression mediated by TCR stimulation. Moreover, human Th17 cells exhibit reduced expression of Skp2, that promotes the ubiquitin-dependent Tob1 degradation [Santarlasci et al., unpublished results]. Although the mechanisms responsible for Skp2 reduction in human Th17 cells still remain unknown, one cannot exclude that, in addition to the maintenance of high Tob1 levels, it also favours the persistence of the forkhead transcription factor FOXO1, which also plays a pivotal role in growth arrest and apoptosis [25,26]. Thus, several self-regulatory mechanisms concur in limiting the expansion of human Th17 cells in response to antigen stimulation. These mechanisms are probably related to the peculiar function of Th17 cells, which may be devoted to reside in a quiescent state in peripheral tissues, where they provide a first line of protection against pathogens. Indeed, it has recently been reported that Th17 cells express a signature closely resembling the pattern observed in stem cell-like memory cells, i.e. the enhanced capacity to survive, self renew and generation of effector progeny [33]. Another explanation for the rarity of Th17 cells in inflamed tissues is their high plasticity, that allows these cells to rapidly shift in presence of pro-inflammatory cytokines, such as IL-12 and TNF-␣, to the production of IFN-␥ in addition to IL-17A, and subsequently even acquire a pure Th1 profile. The transient nature of Th17 cells, which was first discovered in our initial studies in humans [17], when mouse immunologists still considered the Th17 as a fixed phenotype, is now considered as a generally established fact [30]. Our subsequent studies have clearly demonstrated that Th17-derived Th1 cells are different from the already known, classic, Th1 cells and have therefore been named by us as non-classic
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Th1 cells [18]. The difference between non-classic and classic Th1 cells not only consists of the expression of different markers, such as RORC, CD161, CCR6, IL-17RE and IL4I1 [31,32], but also rests on the different degree of demethylation of the ROI of RORC2 and IL-17A gene promoter (Mazzoni et al., unpublished results). The reason for the rapid shifting of Th17 cells into non-classic Th1 cells is unclear, because the respective role of Th17, Th17/Th1, non-classic and classic Th1 cells in the pathogenesis of different chronic inflammatory disorders has not yet been clarified. Indeed, after the initial conceptual substitution of Th1 cells with Th17 cells as responsible for pathogenicity, mainly based on the results obtained in a murine model of autoimmunity in gene-deficient animals [14], a series of well-performed studies has strongly challenged this oversimplification. Th17 can be pathogenic in certain forms of experimental autoimmune encephalomyelitis (EAE) (those characterized by granulocyte infiltration), whereas Th1 cells are mainly responsible for the EAE models characterized by prevalent mononuclear cell infiltration, where Th17 cells do not seem to play any pathogenic role [34]. In the murine model of autoimmune disorder known as proteoglycan-induced arthritis, Th1 cells, but not Th17 cells, are pathogenic [35]. Either Th1 or Th17 cells have been found to be pathogenic in experimental autoimmune uveitis [36]. Neither IL-17A nor IL-17F contributes vitally to autoimmune neuroinflammation in mice [37]. Th17 cells can promote pancreatic inflammation but only induce type 1 (insulin-dependent) diabetes mellitus efficiently in lymphopenic mice after conversion into Th1 cells [38]. Accordingly, highly purified Th17 cells from BDC2.5NOD mice shift into Th1-like cells in non-obese diabetic/severe combined immunodeficiency recipient mice. The transferred Th17 cells, completely devoid of IFN-␥ at the time of transfer, rapidly converted to secrete IFN-␥ in the non-obese diabetic/severe combined immunodeficiency recipients. More importantly, the development of insulin-dependent diabetes mellitus was prevented by the treatment with anti-IFN-␥, but not with anti-IL17A, specific antibody [39]. More recently, two types of EAE were described, one being Tbet dependent and the other one mediated by Th17 and Th17/Th1 cells and T-bet-independent, suggesting that both classic and nonclassic Th1 (these latter together with Th17 and Th17/Th1) cells may be pathogenic according to the different experimental model [40,41]. On the other hand, in T-bet-knockouts in which Th17 cells cannot shift to Th1 cells, they are still capable of mediating EAE, albeit with a milder clinical course [42]. Thus, it seems that Th17, Th17/Th1, non-classic and classic Th1 cells may all be involved in the tissue damage, with a variable importance in the different types and/or phases of the inflammatory disorders. Therefore, based on all these findings, the reason why Th17 cells rapidly shift to the Th1 phenotype in the inflammatory sites remains presently unclear.
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