Nitric oxide modulates TGF-beta-directive signals to suppress Foxp3+ regulatory T cell differentiation and potentiate Th1 development

TGF-β can induce Foxp3(+) inducible regulatory T cells (Treg) and also synergize with IL-6 and IL-4 to induce Th17 and Th9 cells. We now report that NO modulates TGF-β activity away from Treg but toward the Th1 lineage. NO potentiated Th1 differentiation in the presence of TGF-β in both IL-12-independent and -dependent fashions by augmenting IFN-γ-activated STAT-1 and T-bet. Differentiation into Treg, Th1, and Th17 lineages could be modulated by NO competing with other cofactors, such as IL-6 and retinoic acid. NO antagonized IL-6 to block TGF-β-directed Th17 differentiation, and together with IL-6, NO suppressed Treg development induced by TGF-β and retinoic acid. Furthermore, we show that physiologically produced NO from TNF and inducible NO synthase-producing dendritic cells can contribute to Th1 development predominating over Treg development through a synergistic activity induced when these cells cocluster with conventional dendritic cells presenting Ag to naive Th cells. This illustrates that NO is another cofactor allowing TGF-β to participate in development of multiple Th lineages and suggests a new mechanism by which NO, which is associated with protection against intracellular pathogens, might maintain effective Th1 immunity.     

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).     

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.     

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.     

Zinc deficiency drives Th17 polarization and promotes loss of Treg cell function

A high number of illnesses and disorders are connected to zinc deficiency. Equally, T cell polarization and a balance between different T helper (Th) cell subsets are essential. Therefore, in this study, the influence of zinc deficiency on T cell polarization and on respective signaling pathways was investigated. We uncovered a significantly increased number of regulatory T cells (Treg) and Th17 cells in expanded T cells during zinc deficiency after 3 days of combined treatment with IL-2 and TGF-β1 (Treg) or IL-6 and TGF-β1 (Th17). No difference in Th1 and Th2 cell polarization between zinc-deficient and zinc-adequate status was prominent. On the molecular level, Smad signaling was significantly enhanced by stimulation with TGF-β1/IL-6 during zinc deficiency compared to adequate zinc condition. This represents an explanation for the elevated Th17 cell numbers associated with autoimmune disease especially during zinc deficiency. Moreover, Treg cell numbers are increased during zinc deficiency as well. However, those cells might be nonfunctional since a lower expression of miR-146a was uncovered compared to normal zinc concentrations. In summary, an adequate zinc homeostasis is fundamental to slow down or probably stop the progression of autoimmune diseases and infections. Therefore, supplementing zinc might be a therapeutic approach to dampen autoimmune diseases connected to Th17 cells.     

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.     

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.     

IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer

Foxp3(+)CD4(+) regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17(+)CD4(+) T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17(+)Foxp3(+)CD4(+) T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17(+)Foxp3(+)CD4(+) T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17(+)Foxp3(+)CD4(+) T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17(+)Foxp3(+)CD4(+) T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6(+) T cells or Treg cells. IL-17(+)Foxp3(+)CD4(+) T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17(+)Foxp3(+) cells may be "inflammatory" Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.     

Helminth protection against autoimmune diabetes in nonobese diabetic mice is independent of a type 2 immune shift and requires TGF-β

Leading hypotheses to explain helminth-mediated protection against autoimmunity postulate that type 2 or regulatory immune responses induced by helminth infections in the host limit pathogenic Th1-driven autoimmune responses. We tested these hypotheses by investigating whether infection with the filarial nematode Litomosoides sigmodontis prevents diabetes onset in IL-4-deficient NOD mice and whether depletion or absence of regulatory T cells, IL-10, or TGF-β alters helminth-mediated protection. In contrast to IL-4-competent NOD mice, IL-4-deficient NOD mice failed to develop a type 2 shift in either cytokine or Ab production during L. sigmodontis infection. Despite the absence of a type 2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4(+)CD25(+)Foxp3(+) regulatory T cell frequencies and numbers, respectively, and helminth infection increased the proliferation of CD4(+)Foxp3(+) cells. However, depletion of CD25(+) cells in NOD mice or Foxp3(+) T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGF-β, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. Changes in Th17 responses did not seem to play an important role in helminth-mediated protection against autoimmunity, because helminth infection was not associated with a decreased Th17 immune response. This study demonstrates that L. sigmodontis-mediated protection against diabetes in NOD mice is not dependent on the induction of a type 2 immune shift but does require TGF-β.     

IL-22-induced regulatory CD11b+ APCs suppress experimental autoimmune uveitis

We have previously reported that IL-17(+) interphotoreceptor retinoid-binding protein (IRBP) 161-180-specific T cells have a strong pathogenic effect in experimental autoimmune uveitis (EAU) induced in B10RIII mice; however, this pathogenic activity is not solely attributable to the major cytokine, IL-17, produced by these cells. To determine whether other cytokines produced by Th17 cells show a stronger association with their pathogenic activity, we studied the role of IL-22 in EAU. IL-22 is one of the major cytokines produced by these cells. Our results showed that administration of small doses of IL-22 to EAU-susceptible mice significantly reduced the severity of EAU. In addition, mice treated with IL-22 generated decreased numbers of IFN-γ(+) and IL-17(+) uveitogenic T cells, but increased numbers of Foxp3(+) regulatory T cells. Mechanistic studies showed that the effect of the injected IL-22 was on CD11b(+) APCs, which expressed increased levels of IL-22R during induction of disease following immunization with uveitogenic Ag. In vitro IL-22 treatment of CD11b(+) APCs collected from Ag-primed mice resulted in increased expression of programmed death ligand-1 and the production of increased amounts of IL-10 and TGF-β. Moreover, IL-22-treated CD11b(+) APCs caused IRBP161-180-specific T cells to lose their uveitogenic activity and acquire immunosuppressive activity, which suppressed the induction of EAU by additional pathogenic IRBP161-180-specific effector T cells.     

Effect of short-term interferon-β treatment on cytokines in multiple sclerosis: significant modulation of IL-17 and IL-23

Therapeutic effect of interferon-β (IFN-β) treatment has been associated with modulation of the balance between Th1, Th17, Th2 and regulatory T (Treg) cells, whereas the impact of disease modifying drugs on Th9-immunity in multiple sclerosis (MS) has not been studied. To investigate the short-term effects of IFN-β treatment on cytokines in MS, we determined serum levels of IL-17, IL-23, IL-10, IL-4, IFN-γ, IL-9 and TGF-β in relapsing remitting MS patients before and 2 months after IFN-β treatment by ELISA. MS patients showed increased IL-17, IL-23 and IL-4 levels and decreased IL-9 levels as compared to healthy controls. IFN-β treatment only reduced IL-17 and IL-23 levels, whereas the levels of other cytokines remained unchanged. IFN-β treatment appears to exert its earliest therapeutic effect on Th17-immunity. The influence of IL-9 on MS pathogenesis needs to be further studied.     

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.     

Human CCR4+ CCR6+ Th17 cells suppress autologous CD8+ T cell responses

The role of Th17 cells in cancer patients remains unclear and controversial. In this study, we have analyzed the phenotype of in vitro primed Th17 cells and further characterized their function on the basis of CCR4 and CCR6 expression. We show a novel function for a subset of IL-17-secreting CD4(+) T cells, namely, CCR4(+)CCR6(+)Th17 cells. When cultured together, CCR4(+)CCR6(+)Th17 cells suppressed the lytic function, proliferation, and cytokine secretion of both Ag-specific and CD3/CD28/CD2-stimulated autologous CD8(+) T cells. In contrast, CCR4(-)CCR6(+) CD4(+) T cells, which also secrete IL-17, did not affect the CD8(+) T cells. Suppression of CD8(+) T cells by CCR4(+)CCR6(+)Th17 cells was partially dependent on TGF-β, because neutralization of TGF-β in cocultures reversed their suppressor function. In addition, we also found an increase in the frequency of CCR4(+)CCR6(+), but not CCR4(-)CCR6(+) Th17 cells in peripheral blood of hepatocellular carcinoma patients. Our study not only underlies the importance of analysis of subsets within Th17 cells to understand their function, but also suggests Th17 cells as yet another immune evasion mechanism in hepatocellular carcinoma. This has important implications when studying the mechanisms of carcinogenesis, as well as designing effective immunotherapy protocols for patients with cancer.     

Inducible adeno-associated virus-mediated IL-2 gene therapy prevents autoimmune diabetes

IL-2 and TGF-β1 play key roles in the immunobiology of Foxp3-expressing CD25(+)CD4(+) T cells (Foxp3(+)Treg). Administration of these cytokines offers an appealing approach to manipulate the Foxp3(+)Treg pool and treat T cell-mediated autoimmunity such as type 1 diabetes. However, efficacy of cytokine treatment is dependent on the mode of application, and the potent pleiotropic effects of cytokines like IL-2 may lead to severe side effects. In the current study, we used a gene therapy-based approach to assess the efficacy of recombinant adeno-associated virus vectors expressing inducible IL-2 or TGF-β1 transgenes to suppress ongoing β cell autoimmunity in NOD mice. Intramuscular vaccination of recombinant adeno-associated virus to 10-wk-old NOD female mice and a subsequent 3 wk induction of IL-2 was sufficient to prevent diabetes and block the progression of insulitis. Protection correlated with an increased frequency of Foxp3(+)Treg in the periphery as well as in the draining pancreatic lymph nodes and islets. IL-2 induced a shift in the ratio favoring Foxp3(+)Treg versus IFN-γ-expressing T cells infiltrating the islets. Induction of IL-2 had no systemic effect on the frequency or activational status of T cells and NK cells. Induction of TGF-β1 had no effect on the Foxp3(+)Treg pool or the progression of β cell autoimmunity despite induced systemic levels of activated TGF-β1 that were comparable to IL-2. These results demonstrate that inducible IL-2 gene therapy is an effective and safe approach to manipulate Foxp3(+)Treg and suppress T cell-mediated autoimmunity and that under the conditions employed, IL-2 is more potent than TGF-β1.