Early target genes of IL-12 and STAT4 signaling in th cells

IL-12 signaling through STAT4 is essential for induction of optimal levels of IFN-gamma production and commitment of Th1 cells. The molecular mechanism that controls how IL-12 and STAT4 signaling induces Th1 differentiation is poorly described. To identify the early target genes of IL-12 and STAT4 signaling, oligonucleotide arrays were used to compare the gene expression profiles of wild-type and STAT4-knockout murine Th cells during the early Th1 differentiation. According to the results, 20 genes were regulated in an IL-12- and STAT4-dependent manner. Importantly, Ifngamma was clearly the first gene induced by IL-12 in a STAT4-dependent manner. Most of the other defects in gene expression in STAT4-knockout cells were seen after 48 h of Th1 polarization. In addition to IL-12 signaling mediated by STAT4, STAT4-independent induction of a number of genes was observed immediately in response to Th1 induction. This induction was at least in part driven by IFN-gamma independently of STAT4. Importantly, addition of exogenous IFN-gamma into Th1 cell cultures of STAT4-knockout cells restored the defect in IFN-gamma production further demonstrating the critical role of IFN-gamma in early Th1 differentiation.     

Distinct characteristics of murine STAT4 activation in response to IL-12 and IFN-alpha

The role of type I IFN in Th1 development, STAT4 activation, and IFN-gamma production in murine T cells has remained unresolved despite extensive examination. Initial studies indicated that IFN-alpha induced Th1 development and IFN-gamma production in human, but not murine, T cells, suggesting species-specific differences in signaling. Later studies suggested that IFN-alpha also induced Th1 development in mice, similar to IL-12. More recent studies have questioned whether IFN-alpha actually induces Th1 development even in the human system. In the present study, we compared the capacity of IL-12 and IFN-alpha to induce Th1 differentiation, STAT4 phosphorylation, and IFN-gamma production in murine T cells. First, we show that IFN-alpha, in contrast to IL-12, cannot induce Th1 development. However, in differentiated Th1 cells, IFN-alpha can induce transient, but not sustained, STAT4 phosphorylation and, in synergy with IL-18, can induce transient, but not sustained, IFN-gamma production in Th1 cells, in contrast to the sustained actions of IL-12. Furthermore, loss of STAT1 increases IFN-alpha-induced STAT4 phosphorylation, but does not generate levels of STAT4 activation or IFN-gamma production achieved by IL-12 or convert transient STAT4 activation into a sustained response. Our findings agree with recent observations in human T cells that IFN-alpha-induced STAT4 activation is transient and unable to induce Th1 development, and indicate that IFN-alpha may act similarly in human and murine T cells.     

Activated STAT4 has an essential role in Th1 differentiation and proliferation that is independent of its role in the maintenance of IL-12R beta 2 chain expression and signaling

In this study we demonstrated that CD4(+) T cells from STAT4(-/-) mice exhibit reduced IL-12R expression and poor IL-12R signaling function. This raised the question of whether activated STAT4 participates in Th1 cell development mainly through its effects on IL-12 signaling. In a first approach to this question we determined the capacity of CD4(+) T cells from STAT4(-/-) bearing an IL-12Rbeta2 chain transgene (and thus capable of normal IL-12R expression and signaling) to undergo Th1 differentiation when stimulated by Con A and APCs. We found that such cells were still unable to exhibit IL-12-mediated IFN-gamma production. In a second approach to this question, we created Th2 cell lines (D10 cells) transfected with STAT4-expressing plasmids with various tyrosine-->phenylalanine mutations and CD4(+) T cell lines from IL-12beta2(-/-) mice infected with retroviruses expressing similarly STAT4 mutations that nevertheless express surface IL-12Rbeta2 chains. We then showed that constructs that were unable to support STAT4 tyrosine phosphorylation (in D10 cells) as a result of mutation were also incapable of supporting IL-12-induced IFN-gamma production (in IL-12Rbeta2(-/-) cells). Thus, by two complementary approaches we demonstrated that activated STAT4 has an essential downstream role in Th1 cell differentiation that is independent of its role in the support of IL-12Rbeta2 chain signaling. This implies that STAT4 is an essential element in the early events of Th1 differentiation.     

IL-21 inhibits IFN-gamma production in developing Th1 cells through the repression of Eomesodermin expression

Exposure of naive Th cell precursors (Thp) to IL-21 inhibits IFN-gamma production from developing Th1 cells. The inhibition of IFN-gamma seen in IL-21-treated Thp cells is specific as the expression of other Th1 cytokines is unaffected. Recently, it has been reported that Eomesodermin (Eomes), a member of the T-box gene family, is expressed in developing CD8+ T cells and plays an important role in regulating IFN-gamma production and cytolytic effector function. In this study, we show that Eomes mRNA and protein are also expressed in developing Th1 cells, and exposure of naive Thp cells to IL-21 results in a decrease in Eomes expression. Moreover, the repression of Eomes expression by IL-21 is not due to an indirect effect of IL-21 on the expression of IFN-gamma or STAT4 and is independent of STAT1 and T-bet expression. Finally, we show that ectopic expression of Eomes prevents the inhibition of IFN-gamma production from IL-21-treated Thp cells. Taken together, these results demonstrate that Eomes plays a role in regulating IFN-gamma production in CD4+ T cells and IL-21 inhibits IFN-gamma production in developing Th1 cells through the repression of Eomes expression.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.     

Cutting edge: ectopic expression of the IL-12 receptor-beta 2 in developing and committed Th2 cells does not affect the production of IL-4 or induce the production of IFN-gamma

The IL-12 receptor-beta 2 (IL-12R beta 2) chain is expressed on Th1 cells and lost upon differentiation to the Th2 phenotype. This has been suggested as the basis for commitment of Th1 cells, because early differentiated Th2 cells do not reverse their phenotype and do not produce IFN-gamma on restimulation in the presence of IL-12. In this study, we ectopically expressed the IL-12 receptor-beta 2 (IL-12R beta 2) bicistronically with enhanced green fluorescent protein by retroviral infection in developing and committed Th2 cells. Restimulation of Th2 cells expressing this ectopic IL-12R beta 2 in the presence of IL-12 led to levels of IL-4 production similar to those in control Th2 cells. The expression of IL-12R beta 2 in Th2 cells did not lead to significant levels of IFN-gamma production, although IL-12-mediated STAT signaling and proliferation were restored. Thus, although the IL-12R beta 2 and IL-12-dependent STAT4 activation are required for Th1 responses, activation of this pathway is not sufficient to restore a Th1 phenotype in developing or committed Th2 cells.     

IL-13 regulates Th17 secretion of IL-17A in an IL-10-dependent manner

IL-13 is a central mediator of airway hyperresponsiveness and mucus expression, both hallmarks of asthma. IL-13 is found in the sputum of patients with asthma; therefore, IL-13 is an attractive drug target for treating asthma. We have shown previously that IL-13 inhibits Th17 cell production of IL-17A and IL-21 in vitro. Th17 cells are associated with autoimmune diseases, host immune responses, and severe asthma. In this study, we extend our in vitro findings and determine that IL-13 increases IL-10 production from Th17-polarized cells and that IL-13-induced IL-10 production negatively regulates the secretion of IL-17A and IL-21. To determine if IL-13 negatively regulates lung IL-17A expression via an IL-10-dependent mechanism in vivo, we used a model of respiratory syncytial virus (RSV) strain A2 infection in STAT1 knockout (KO) mice that increases lung IL-17A and IL-13 expression, cytokines not produced during RSV infection in wild-type mice. To test the hypothesis that IL-13 negatively regulates lung IL-17A expression, we created STAT1/IL-13 double KO (DKO) mice. We found that RSV-infected STAT1/IL-13 DKO mice had significantly greater lung IL-17A expression compared with that of STAT1 KO mice and that increased IL-17A expression was abrogated by anti-IL-10 Ab treatment. RSV-infected STAT1/IL-13 DKO mice also had increased neutrophil infiltration compared with that of RSV-infected STAT1 KO mice. Neutralizing IL-10 increased the infiltration of inflammatory cells into the lungs of STAT1 KO mice but not STAT1/IL-13 DKO mice. These findings are vital to understanding the potential side effects of therapeutics targeting IL-13. Inhibiting IL-13 may decrease IL-10 production and increase IL-17A production, thus potentiating IL-17A-associated diseases.     

IFN-gamma suppresses STAT6 phosphorylation by inhibiting its recruitment to the IL-4 receptor

Polarized Th1 cells show a stable phenotype: they become insensitive to IL-4 stimulation and lose the potential to produce IL-4. Previously, we reported that IFN-gamma played a critical role in stabilizing Th1 phenotype. However, the mechanism by which IFN-gamma stabilizes Th1 phenotype is not clear. In this study, we compared STAT6 phosphorylation in wild-type (WT) and IFN-gamma receptor knockout (IFNGR(-/-)) Th1 cells. We found a striking diminution of STAT6 phosphorylation in differentiated WT Th1 cells, but not in differentiated IFNGR(-/-) Th1 cells. The impairment of STAT6 phosphorylation in differentiated WT Th1 cells was not due to a lack of IL-4R expression or phosphorylation. Jak1 and Jak3 expression and phosphorylation were comparable in both cell types. No differential expression of suppressor of cytokine signaling 1 (SOCS1), SOCS3, or SOCS5 was observed in the two cell types. In addition, Src homology 2-containing phosphatase mutation did not affect IL-4-induced STAT6 phosphorylation in differentiated Th1 cells derived from viable motheaten (me(v)/me(v)) mice. These results led us to focus on a novel mechanism. By using a pulldown assay, we observed that STAT6 in WT Th1 cells bound less effectively to the phosphorylated IL-4R/GST fusion protein than that in IFNGR(-/-) Th1 cells. Our results suggest that IFN-gamma may suppress phosphorylation of STAT6 by inhibiting its recruitment to the IL-4R.     

Synergistic effects of IL-4 and IL-18 on IL-12-dependent IFN-gamma production by dendritic cells

Mouse splenic dendritic cells (DCs) produce IFN-gamma in response to IL-12. In the present study, we analyzed effects of Th1 and Th2 cytokines on IFN-gamma production by DCs. IL-18 produced by DCs and macrophages acts in an autocrine manner and augments IL-12-induced IFN-gamma production by DCs as also observed in T and NK cells. Surprisingly, IL-4, a Th2 cytokine, also acts synergistically with IL-12 on IFN-gamma production by DCs. In addition, IL-4 markedly enhances IFN-gamma production when DCs are stimulated through CD40 or MHC class II. These results indicate that both Th1 and Th2 cytokines act on DCs during T cell-DC interaction upon Ag presentation. p38 mitogen-activated protein kinase is constitutively activated in mature DCs and is required for IFN-gamma production by DCs. IL-18 but not IL-4 or IL-12 further activates the p38 mitogen-activated protein kinase activity, suggesting that IL-4 and IL-18 enhance IFN-gamma production through distinct intracellular signal transduction pathways in DCs.     

Cellular basis of the role of diesel exhaust particles in inducing Th2-dominant response

There is growing evidence that diesel exhaust particles (DEP) can induce allergic diseases with increased IgE production and preferential activation of Th2 cells. To clarify the cellular basis of the role of DEP in the induction of Th2-dominant responses, we examined the effects of DEP on the cytokine production by T cells stimulated with anti-CD3/CD28 Ab and on that by monocyte-derived dendritic cells (MoDCs) stimulated with CD40L and/or IFN-gamma. We examined IFN-gamma, IL-4, IL-5, IL-8, and IL-10 produced by T cells and TNF-alpha, IL-1beta, IL-10, and IL-12 produced by MoDCs using real-time PCR analysis or by ELISA. To highlight the effects of DEP, we compared the effects of DEP with those of dexamethasone (DEX) and cyclosporin A (CyA). DEP significantly suppressed IFN-gamma mRNA expression and protein production, while it did not affect IL-4 or IL-5 mRNA expression or protein production. The suppressive effect on IFN-gamma mRNA expression was more potent than that of DEX and comparable at 30 mug/ml with 10(-7) M CyA. The suppressive effect on IFN-gamma production was also more potent than that of either DEX or CyA. DEP suppressed IL-12p40 and IL-12p35 mRNA expression and IL-12p40 and IL-12p70 production by MoDCs, while it augmented IL-1beta mRNA expression. Finally, by using a thiol antioxidant, N-acetyl cysteine, we found that the suppression of IFN-gamma production by DEP-treated T cells was mediated by oxidative stress. These data revealed a unique characteristic of DEP, namely that they induce a Th2 cytokine milieu in both T cells and dendritic cells.     

IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.     

An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+ T cells

IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-gamma production. It has been recently reported that IL-27 induces T-bet and IL-12Rbeta2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rbeta2 expression, and synergistic IFN-gamma production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rbeta2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation.     

IL-12 protects against coxsackievirus B3-induced myocarditis by increasing IFN-gamma and macrophage and neutrophil populations in the heart

Th1-type immune responses, mediated by IL-12-induced IFN-gamma, are believed to exacerbate certain autoimmune diseases. We recently found that signaling via IL-12Rbeta1 increases coxsackievirus B3 (CVB3)-induced myocarditis. In this study, we examined the role of IL-12 on the development of CVB3-induced myocarditis using mice deficient in IL-12p35 that lack IL-12p70. We found that IL-12 deficiency did not prevent myocarditis, but viral replication was significantly increased. Although there were no changes in the total percentage of inflammatory cells in IL-12-deficient hearts compared with wild-type BALB/c controls by FACS analysis, macrophage and neutrophil populations were decreased. This decrease corresponded to reduced TNF-alpha and IFN-gamma levels in the heart, suggesting that macrophage and/or neutrophil populations may be a primary source of TNF-alpha and IFN-gamma during acute CVB3 myocarditis. Increased viral replication in IL-12-deficient mice was not mediated by reduced TNFRp55 signaling, because viral replication was unaltered in TNFRp55-deficient mice. However, STAT4 or IFN-gamma deficiency resulted in significantly increased viral replication and significantly reduced TNF-alpha and IFN-gamma levels in the heart, similar to IL-12 deficiency, indicating that the IL-12/STAT4 pathway of IFN-gamma production is important in limiting CVB3 replication. Furthermore, STAT4 or IFN-gamma deficiency also increased chronic CVB3 myocarditis, indicating that therapeutic strategies aimed at reducing Th1-mediated autoimmune diseases may exacerbate common viral infections such as CVB3 and increase chronic inflammatory heart disease.     

IL-4 promotes airway eosinophilia by suppressing IFN-gamma production: defining a novel role for IFN-gamma in the regulation of allergic airway inflammation

Airway eosinophilia in asthma is dependent on cytokines secreted by Th2 cells, including IL-5 and IL-4. In these studies we investigated why the absence of IL-4 led to a reduction in airway, but not lung tissue, eosinophils. Using adoptively transferred, in vitro-generated TCR-transgenic Th2 cells deficient in IL-4, we show that this effect is independent of IL-5 and Th2 cell generation. Airway eosinophilia was no longer inhibited when IL-4(-/-) Th2 cells were transferred into IFN-gammaR(-/-) mice, indicating that IFN-gamma was responsible for reducing airway eosinophils in the absence of IL-4. Intranasal administration of IFN-gamma to mice after IL-4(+/+) Th2 cell transfer also caused a reduction in airway, but not lung parenchymal, eosinophils. These studies show that IL-4 indirectly promotes airway eosinophilia by suppressing the production of IFN-gamma. IFN-gamma reduces airway eosinophils by engaging its receptor on hemopoietic cells, possibly the eosinophil itself. These studies capitalize on the complex counterregulatory effects of Th1 and Th2 cytokines in vivo and clarify how IL-4 influences lung eosinophilia. We define a new regulatory role for IFN-gamma, demonstrating that eosinophilic inflammation is differentially regulated at distinct sites within the respiratory tract.