TGF-β is necessary for induction of IL-23R and Th17 differentiation by IL-6 and IL-23

TGF-β and IL-6 induce Th17 differentiation, and IL-23 is required for expansion and maintenance of Th17 cells. Recently, it was shown that IL-6 up-regulates IL-23R mRNA in naive CD4⁺ T cells and therefore IL-6 and IL-23 synergistically promote Th17 differentiation. However, the molecular mechanism whereby IL-6 and IL-23 induce Th17 differentiation and the relevance to TGF-β remain unknown. Here, we found that IL-6 up-regulated IL-23R mRNA expression, and IL-6 and IL-23 synergistically augmented its protein expression. The combination induced Th17 differentiation, and TGF-β1 further enhanced it. IL-6 augmented endogenous TGF-β1 mRNA expression, whereas the amount of TGF-β produced was not enough to induce Th17 differentiation by IL-6 alone. However, unexpectedly, the up-regulation of IL-23R and induction of Th17 differentiation by IL-6 and IL-23 were almost completely inhibited by anti-TGF-β. These results suggest that the induction of IL-23R and Th17 differentiation by IL-6 and IL-23 is mediated through endogenously produced TGF-β.     

IDO upregulates regulatory T cells via tryptophan catabolite and suppresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a Th1 and Th17 cell-mediated autoimmune disease of the CNS. IDO and tryptophan metabolites have inhibitory effects on Th1 cells in EAE. For Th17 cells, IDO-mediated tryptophan deprivation and small molecule halofuginone-induced amino acid starvation response were shown to activate general control nonrepressed 2 (GCN2) kinase that directly or indirectly inhibits Th17 cell differentiation. However, it remains unclear whether IDO and tryptophan metabolites impact the Th17 cell response by mechanisms other than the GCN2 kinase pathway. In this article, we show that IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 cell responses and reduced regulatory T cell (Treg) responses. Administration of the downstream tryptophan metabolite 3-hydroxyanthranillic acid (3-HAA) enhanced the percentage of Tregs, inhibited Th1 and Th17 cells, and ameliorated EAE. We further demonstrate that Th17 cells are less sensitive to direct suppression by 3-HAA than are Th1 cells. 3-HAA treatment in vitro reduced IL-6 production by activated spleen cells and increased expression of TGF-β in dendritic cells (DCs), which correlated with enhanced levels of Tregs, suggesting that 3-HAA-induced Tregs contribute to inhibition of Th17 cells. By using a DC-T cell coculture, we found that 3-HAA-treated DCs expressed higher levels of TGF-β and had properties to induce generation of Tregs from anti-CD3/anti-CD28-stimulated naive CD4(+) T cells. Thus, our data support the hypothesis that IDO induces the generation of Tregs via tryptophan metabolites, such as 3-HAA, which enhances TGF-β expression from DCs and promotes Treg differentiation.     

Role of TGF-β and IL-6 in dendritic cells,Treg and Th17 mediated immune response during experimental cerebral malaria

The role of cytokines in Plasmodium infection have been extensively investigated, but pro and anti inflammatory cytokines mediated imbalance during malaria immune-pathogenesis is still unrevealed. Malaria is associated with the circulating levels of Interleukin-6 (IL-6) and transforming growth factor β (TGF-β), but association between these two cytokines in immune response remains largely obscured. Using mouse model, we proposed that IL-6 and TGF-β are involved in immune regulation of dendritic cells (DC), regulatory T cells (Treg), T-helper cells (Th17) during P. berghei ANKA (PbA) infection. Association between the cytokines and the severity of malaria was established with anti-TGF-β treatment resulting in increased parasitemia and increased immunopathology, whereas; anti-IL-6 treatment delays immunopathology during PbA infection. Further, splenocytes revealed differential alteration of myeloid DC (mDC), plasmocytoid DC (pDC), Treg, Th17 cells following TGF-β and IL-6 neutralization. Interestingly anti-TGF-β reduces CD11c⁺CD8⁺ DC expression, whereas anti-IL-6 administration causes a profound increase during PbA infection in Swiss mice. We observed down regulation of TGF-β, IL-10, NFAT, Foxp3, STAT-5 SMAD-3 and upregulation of IL-6, IL-23, IL-17 and STAT-3 in splenocytes during PbA infection. The STAT activity probably plays differential role in induction of Th17 and Treg cells. Interestingly we found increase in STAT-3 and decrease in STAT-5 expression during PbA infection. This pattern of STAT indicates that possibly TGF-β and IL-6 play a crucial role in differentiation of DCs subsets and Treg/Th17 imbalance during experimental cerebral malaria (ECM).     

TGF-beta-exposed plasmacytoid dendritic cells participate in Th17 commitment

TGF-β is required for both Foxp3(+) regulatory T cell (Treg) and Th17 commitment. Plasmacytoid DCs (pDC) have been shown to participate to both Treg and Th17 commitment as well. However, few studies have evaluated the direct effect of TGF-β on pDC, and to our knowledge, no study has assessed the capacity of TGF-β-exposed pDC to polarize naive CD4(+) T cells. In this paper, we show that TGF-β-treated pDC favor Th17 but not Treg commitment. This process involves a TGF-β/Smad signal, because TGF-β treatment induced Smad2 phosphorylation in pDC and blockade of TGF-β signaling with the SD208 TGF-βRI kinase inhibitor abrogated Th17 commitment induced by TGF-β-treated pDC. Moreover, TGF-β mRNA synthesis and active TGF-β release were induced in TGF-β-treated pDC and anti-TGF-β Ab blocked Th17 commitment. Unexpectedly, TGF-β treatment also induced increased IL-6 production by pDC, which serves as the other arm for Th17 commitment driven by TGF-β-exposed pDC, because elimination of IL-6-mediated signal with either IL-6- or IL-6Rα-specific Abs prevented Th17 commitment. The in vivo pathogenic role of TGF-β-treated pDC was further confirmed in the Th17-dependent collagen-induced arthritis model in which TGF-β-treated pDC injection significantly increased arthritis severity and pathogenic Th17 cell accumulation in the draining lymph nodes. Thus, our data reveal a previously unrecognized effect of TGF-β-rich environment on pDC ability to trigger Th17 commitment. Such findings have implications in the pathogenesis of autoimmune diseases or immune responses against mucosal extracellular pathogens.     

Ex vivo IL-1 receptor type I expression in human CD4+ T cells identifies an early intermediate in the differentiation of Th17 from FOXP3+ naive regulatory T cells

IL-17-producing CD4(+) Th (Th17) cells are a unique subset of proinflammatory cells expressing the retinoic acid-related orphan receptor γt and associated with different forms of inflammatory autoimmune pathologies. The development of Th17 cells, mediated by TGF-β and IL-1, is closely related to that of FOXP3(+) suppressor/regulatory T cells (Treg). In this study, we report that ex vivo expression of IL-1RI in human circulating CD4(+) T cells identifies a subpopulation of FOXP3(+) Treg that coexpress retinoic acid-related orphan receptor γt, secrete IL-17, and are highly enriched among CCR7(+) central memory cells. Consistent with the concept that IL-1RI expression in Treg identifies a subpopulation at an early stage of differentiation, we show that, in Th17 populations differentiated in vitro from natural naive FOXP3(+) Treg, IL-1RI(+) IL-17-secreting cells are central memory cells, whereas IL-1RI(-) cells secreting IL-17 are effector memory cells. Together with the absence of detectable IL-1RI and IL-17 expression in resting naive CD4(+) T cells, these data identify circulating CCR7(+) Treg expressing IL-1RI ex vivo as early intermediates along an IL-1-controlled differentiation pathway leading from naive FOXP3(+) Treg to Th17 effectors. We further show that, whereas IL-1RI(+) central memory Treg respond to stimulation in the presence of IL-1 by generating IL-17-secreting effectors, a significant fraction of them maintain FOXP3 expression, consistent with an important role of this population in maintaining the Treg/Th17 memory pool in vivo.     

Optimization of human Th17 cell differentiation in vitro<Emphasis Type="Italic">:</Emphasis> evaluating different polarizing factors

Regarding discrepancies that exist among different studies which have tried to clarify critical factors in human Th17 cell differentiation, the aim of this study was to identify the best condition for human Th17 differentiation and to clarify the possible role of TGF-β in differentiation of these cells. Naïve CD4⁺ T cells were isolated from cord blood samples and cultured either in X-VIVO 15 serum-free medium or RPMI 1640 containing 10% FBS. Purified cells were treated with different combinations of polarizing cytokines (TGF-β, IL-1β, IL-6, IL-23 and IL-21) followed by analysis of the expression of characteristic genes and their relevant cytokines by real-time quantitative RT-PCR and ELISA method, respectively. Our data indicate that a combination of TGF-β plus IL-6 and IL-23 cytokines in X-VIVO 15 serum-free medium could be applied as the best condition for developing human Th17 cells in compare with other studied cytokine treatments. It is shown that TGF-β could be considered as a positive regulator for human Th17 cell differentiation only if applied in average concentrations. Interestingly, polarizing treatments in absence of TGF-β, induced double-secreting Th17 cells which co-express IL-17 and IFN-γ whereas polarization in presence of TGF-β-induced single-secreting (only IL-17 expressing) Th17 cells.     

IL-1β and TGF-β act antagonistically in induction and differentially in propagation of human proinflammatory precursor CD4+ T cells

Cytokines are critical messengers that control the differentiation of Th cells. To evaluate their impact on the fate of human naive CD4(+) T cells from cord and adult blood, early T cell differentiation was monitored after T cell activation in the presence of pro- and anti-inflammatory cytokines. Interestingly, the analysis of Th cell lineage-specific molecules revealed that IL-1β on its own mediates differentiation of Th cells that secrete a wide range of proinflammatory cytokines and stably express CD69, STAT1, IFN-γ, and IL-17. Notably, our data suggest that IL-1β induces Th17 cells independent of RORC upregulation. In contrast, TGF-β that triggers RORC prevents Th17 cell development. This suppressive function of TGF-β is characterized by inhibition of STAT1, STAT3, and CD69. However, after repeated anti-CD3 and anti-CD28 stimulation, we observe that TGF-β provokes an increase in Th17 cells that presumably relies on reactivation of a default pathway by preferential inhibition of IFN-γ. Hence, our data extend the view that the principal cytokines for determining Th cell fate are IL-12 for the Th1 lineage, IL-4 for the Th2 lineage, and TGF-β in conjunction with IL-6 for the Th17 lineage. We propose that IL-1β induces a general proinflammatory Th cell precursor that, in the presence of the lineage-specifying cytokines, further differentiates into one of the specific Th cell subpopulations.     

Loss of Sprouty4 in T cells ameliorates experimental autoimmune encephalomyelitis in mice by negatively regulating IL-1β receptor expression

Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-β for early differentiation. To gain pathogenicity, however, Th17 cells require IL-1β and IL-23. The underlying mechanism by which these confer pathogenicity is not well understood. Here we show that Sprouty4, an inhibitor of the PLCγ-ERK pathway, critically regulates inflammatory Th17 (iTh17) cell differentiation. Sprouty4-deficient mice, as well as mice adoptively transferred with Sprouty4-deficient T cells, were resistant to experimental autoimmune encephalitis (EAE) and showed decreased Th17 cell generation in vivo. In vitro, Sprouty4 deficiency did not severely affect TGF-β/IL-6-induced Th17 cell generation but strongly impaired Th17 differentiation induced by IL-1/IL-6/IL-23. Analysis of Th17-related gene expression revealed that Sprouty4-deficient Th17 cells expressed lower levels of IL-1R1 and IL-23R, while RORγt levels were similar. Consistently, overexpression of Sprouty4 or pharmacological inhibition of ERK upregulated IL-1R1 expression in primary T cells. Thus, Sprouty4 and ERK play a critical role in developing iTh17 cells in Th17 cell-driven autoimmune diseases.     

Pertussis toxin by inducing IL-6 promotes the generation of IL-17-producing CD4 cells

Compelling evidence has now demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to autoimmune pathogenesis, whereas CD4+CD25+ T regulatory cells (Treg) play a major role in suppression of autoimmunity. Differentiation of proinflammatory Th17 and immunosuppressive Treg from naive CD4 cells is reciprocally related and contingent upon the cytokine environment. We and others have reported that in vivo administration of pertussis toxin (PTx) reduces the number and function of mouse Treg. In this study, we have shown that supernatants from PTx-treated mouse splenic cells, which contained IL-6 and other proinflammatory cytokines, but not PTx itself, overcame the inhibition of proliferation seen in cocultures of Treg and CD4+CD25- T effector cells. This stimulatory effect could be mimicked by individual inflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha. The combination of these cytokines synergistically stimulated the proliferation of CD4+CD25- T effector cells despite the presence of Treg with a concomitant reduction in the percentage of FoxP3+ cells and generation of IL-17-expressing cells. PTx generated Th17 cells, while inhibiting the differentiation of FoxP+ cells, from naive CD4 cells when cocultured with bone marrow-derived dendritic cells from wild-type mice, but not from IL-6-/- mice. In vivo treatment with PTx induced IL-17-secreting cells in wild-type mice, but not in IL-6-/- mice. Thus, in addition to inhibiting the development of Treg, the immunoadjuvant activity of PTx can be attributable to the generation of IL-6-dependent IL-17-producing CD4 cells.     

Retinoic acid increases Foxp3+ regulatory T cells and inhibits development of Th17 cells by enhancing TGF-beta-driven Smad3 signaling and inhibiting IL-6 and IL-23 receptor expression

The de novo generation of Foxp3+ regulatory T (Treg) cells in the peripheral immune compartment and the differentiation of Th17 cells both require TGF-beta, and IL-6 and IL-21 are switch factors that drive the development of Th17 cells at the expense of Treg cell generation. The major vitamin A metabolite all-trans retinoic acid (RA) not only enforces the generation of Treg cells but also inhibits the differentiation of Th17 cells. Herein we show that RA enhances TGF-beta signaling by increasing the expression and phosphorylation of Smad3, and this results in increased Foxp3 expression even in the presence of IL-6 or IL-21. RA also inhibits the expression of IL-6Ralpha, IRF-4, and IL-23R and thus inhibits Th17 development. In vitro, RA significantly promotes Treg cell conversion, but in vivo during the development of experimental autoimmune encephalomyelitis it does not increase the frequency of Treg cells in the face of an ongoing inflammation. However, RA suppresses the disease very efficiently by inhibiting proinflammatory T cell responses, especially pathogenic Th17 responses. These data not only identify the signaling mechanisms by which RA can affect both Treg cell and Th17 differentiation, but they also highlight that in vivo during an autoimmune reaction, RA suppresses autoimmunity mainly by inhibiting the generation of effector Th17 cells.     

T helper cells subtypes and their cytokine gene expression affected by carvacrol in sensitized mice administered during sensitization period

Th1, Th2, Th17, and Treg cells and their cytokine gene expressions in splenocytes of control mice, ovalbumin sensitized (S), and S treated with dexamethasone and carvacrol during a sensitization period were examined. Th2 and Th17 population as well as the gene expression of IL-4, IL-17, and TGF-β were increased, but Th1, Th1/Th2 ratio, the gene expression of IFN-γ and FOXP3 as well as the IFN-γ/IL-4 ratio were decreased in S compared with control group ( P < 0.001 for all cases). Carvacrol treatment caused significant reduction of Th2 and Th17 population as well as gene expression of IL-4, IL-17, and TGF-β but increase in Treg cells, Th1/Th2 ratio, gene expressions of FOXP3, IFN-γ, and IFN-γ/IL-4 ratio ( P < 0.05 to P < 0.001). The population of Th1, Th2, Th17 cells as well as the gene expression of IL-4, IL-17, and TGF-β were significantly decreased, but only Treg was increased in the dexamethasone treatment group ( P < 0.05 to P < 0.001). Carvacrol treatment during the sensitization period showed a more specific effect on Th1/Th2 imbalance in sensitized mice than dexamethasone, which may indicate the therapeutic potentials of carvacrol in disorders associated with Th1/Th2 imbalance such as asthma.     

Foxp3 inhibits RORgammat-mediated IL-17A mRNA transcription through direct interaction with RORgammat

The cytokine, transforming growth factor-beta1 (TGF-beta1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-beta1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-beta1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor gammat (RORgammat), was rapidly induced by TGF-beta1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORgammat binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORgammat-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORgammat through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORgammat-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg.     

Generation and immunologic functions of Th17 cells in malignant gliomas

Th17 cells, a recently discovered inflammatory T cell subtype, have been implicated with autoimmune disorders. However, mechanism of generation or functions of intratumoral Th17 cells are still unclear. We have been investigating the mechanism of induction and role of Th17 cells in malignant gliomas using primary tumor as well as cell lines. We report here that: (1) a higher frequency of Th17 cells in gliomas were associated with higher number of myeloid (CD11b) cells as well as the expression of TGF-β1 or IL-6; (2) conditioned medium from glioma cells (Gl CM) induced Th17 cell differentiation, which was inhibited by anti-TGF-β1 and anti-IL-6; (3) glioma-associated monocytes secreted Th17-promoting cytokines IL-1β and IL-23; (4) CM from glioma and monocyte co-culture (Gl+Mo CM) induced high frequency of Th17 cells in naïve T cell culture, which was abrogated by anti-IL-1β and anti-IL-23 antibodies; (5) In vitro Gl+Mo CM-mediated Th17 generation was associated with a decrease in IFN-γ and a concomitant increase in IL-10 secretion. Anti-TGF-β1, but not anti-IL-6, significantly reversed this cytokine profile. These results demonstrate prevalence of Th17 cells in gliomas and implicate the cytokines derived from the tumor as well as infiltrating myeloid cells in the induction of Th17 cells in glioma microenvironment. Moreover, the data also suggest that glioma-associated Th17 cells may contribute to immune-suppression via TGF-β1-induced IL-10 secretion. Further studies on the mechanism of tumor-infiltration, developmental pathways, and pro-/anti-tumor functions of Th17 cells will provide rationale for developing novel adjuvant immunotherapeutic strategies for malignant gliomas.     

Dendritic Cells Induce a Subpopulation of IL-12Rβ2-Expressing Treg that Specifically Consumes IL-12 to Control Th1 Responses

Cytokines secreted from dendritic cells (DCs) play an important role in the regulation of T helper (Th) cell differentiation and activation into effector cells. Therefore, controlling cytokine secretion from DCs may potentially regulate Th differentiation/activation. DCs also induce de-novo generation of regulatory T cells (Treg) that modulate the immune response. In the current study we used the mixed leukocyte reaction (MLR) to investigate the effect of allospecific Treg on IL-12, TNFα and IL-6 secretion by DCs. Treg cells were found to markedly down-regulate IL-12 secretion from DCs following stimulation with TLR7/8 agonist. This down-regulation of IL-12 was neither due to a direct suppression of its production by the DCs nor a result of marked DC death. We found that IL-12 was rather actively consumed by Treg cells. IL-12 consumption was mediated by a subpopulation of IL-12Rβ2-expressing Treg cells and was dependent on MHC class-II expressed on dendritic cells. Furthermore, IL-12 consumption by Tregs increased their suppressive effect on T cell proliferation and Th1 activation. These results provide a new pathway of Th1 response regulation where IL-12 secreted by DCs is consumed by a sub-population of IL-12Rβ2-expressing Treg cells. Consumption of IL-12 by Tregs not only reduces the availability of IL-12 to Th effector cells but also enhances the Treg immunosuppressive effect. This DC-induced IL-12Rβ2-expressing Treg subpopulation may have a therapeutic advantage in suppressing Th1 mediated autoimmunity.     

Transforming growth factor-β protein inversely regulates in vivo differentiation of interleukin-17 (IL-17)-producing CD4+ and CD8+ T cells

TGF-β is a pleiotropic cytokine that predominantly exerts inhibitory functions in the immune system. Unexpectedly, the in vitro differentiation of both Th17 and Tc17 cells requires TGF-β. However, animals that are impaired in TGF-β signaling (TGF-βRIIDN mice) display multiorgan autoimmune disorders. Here we show that CD4(+) T cells from TGF-βRIIDN mice are resistant to Th17 cell differentiation and, paradoxically, that CD8(+) T cells from these animals spontaneously acquire an IL-17-producing phenotype. Neutralization of IL-17 or depletion of CD8(+) T cells dramatically inhibited inflammation in TGF-βRIIDN mice. Therefore, the absence of TGF-β triggers spontaneous differentiation of IL-17-producing CD8(+) T cells, suggesting that the in vivo and in vitro conditions that promote the differentiation of IL-17-producing CD8(+) T cells are distinct.