[Show abstract][Hide abstract] ABSTRACT: Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-β for early differentiation. Several Smad-independent pathways including the JNK and the RhoA-ROCK pathways have been implicated in the induction of RORγt, the master regulator of Th17, however, molecular mechanisms underlying Smad-independent pathway remain largely unknown. To identify novel pathways involved in Th17 differentiation, we screened 285 chemical inhibitors for known signaling pathways. Among them, we found that Kenpaullone, a GSK3-β and CDK inhibitor, efficiently suppressed TGF-β-mediated RORγt induction and enhanced Foxp3 induction in primary T cells. Another CDK inhibitor, Roscovitine, but not other GSK3-β inhibitors, suppressed Th17 differentiation and enhanced iTreg development. Kenpaullone and Roscovitine suppressed experimental autoimmune encephalomyelitis (EAE), a typical Th17-mediated autoimmune disease model. These two compounds enhanced STAT5 phosphorylation and restored IL-2 production in the presence of TGF-β. These data suggest that CDK inhibitors modulate TGF-β-signaling pathways, which restore TGF-β-mediated suppression of IL-2 production, thereby modifying the Th17/iTreg balance.
Biochemical and Biophysical Research Communications 05/2013; 435(3). DOI:10.1016/j.bbrc.2013.04.096 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mushroom known as Reishi (Ganoderma lucidum) has been used as an herbal medicine for tumor treatment and immune system activation. Because its effects on the differentiation of effector T helper cells have not yet been fully understood, we investigated the effects of Reishi and those of its principal ingredient, β-glucan, on the activation of dendritic cells and the differentiation of Th17 cells. Reishi extracts as well as purified β-glucan (Curdran) activated DCs and caused them to produce large amounts of IL-23. β-glucan also enhanced and sustained the transcription of IL-23p19. The MEK-ERK signaling pathway positively regulates IL-23p19 transcription in β-glucan-stimulated DCs. In a mixed leukocyte reaction, Reishi-stimulated DCs preferentially induced Th17 cells. Furthermore, orally-administrated Reishi increased the percentages of Th17 cells and the transcription levels of antimicrobial peptides. Our results show that Reishi and β-glucan activate DCs to produce large amounts of IL-23, which induces Th17 differentiation both in vitro and in vivo.
Biochemical and Biophysical Research Communications 05/2012; 422(1):174-80. DOI:10.1016/j.bbrc.2012.04.135 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Th17 cells, which have been implicated in autoimmune diseases, require STAT3 signaling activated by IL-6 or IL-23 for their development. Other Th1 and Th2 cytokines such as IL-2, IFN-γ and IL-4 strongly suppress Th17 development. Recently, CP-690,550 (tofacitinib), originally developed as a JAK3 inhibitor, has been shown to be effective in phase III clinical trials of rheumatoid arthritis and collagen-induced arthritis (CIA) models, but the precise mechanism of the effect, especially with respect to Th17 cells, is poorly understood. To our surprise, a low dose CP-690,550 was found to accelerate the onset of experimental autoimmune encephalomyelitis (EAE) at a concentration that suppressed CIA. At an early stage after immunization, more IL-17 production was observed in 15mg/kg body weight CP-690,550-treated mice than in untreated mice. In vitro, CP-690,550 inhibited both Th1 and Th2 development, while promoting Th17 differentiation at 10-50nM concentrations. Enhancement of Th17 by CP-690,550 is probably due to suppression of IL-2 signaling, because anti-IL-2 antibodies cancel the Th17-promoting effect of CP-690,550. CP-690,550 selectively inhibited IFN--induced STAT1, IL-4-induced STAT6 and IL-2-induced STAT5 at 3-30nM, while suppression of IL-6-induced STAT3 phosphorylation required a concentration greater than 100nM. In HEK293T cells, CP-690,550 less effectively suppressed JAK1-mediated STAT3 phosphorylation compared with JAK3. These results suggest that CP-690,550 has a different effects among JAKs and STATs, thereby affecting helper T cell differentiation, and murine autoimmune disease models.
Biochemical and Biophysical Research Communications 02/2012; 418(2):234-40. DOI:10.1016/j.bbrc.2011.12.156 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atopic dermatitis (AD) is a common pruritic inflammatory disease triggered by a defective skin barrier and immunodysregulation. AD has been considered a typical example of a Th2 response associated with allergic disease. In the early phases of the disease, symptoms include IgE hyperproduction, eosinophil accumulation, and mast cell activation; in the chronic phase, a Th1-dominant immune response is also observed at the sites of AD skin lesions. The role of IL-17-producing Th (Th17) cells in AD has not been established. In the current study, we found that pyridone 6 (P6), a pan-JAK inhibitor, delayed the onset and reduced the magnitude of skin disease in an AD-like skin-disease model of NC/Nga mice. P6 reduced IFN-γ and IL-13, whereas it enhanced IL-17 and IL-22 expression. In vitro, P6 also inhibited both Th1 and Th2 development, whereas it promoted Th17 differentiation from naive T cells when present within a certain range of concentrations. This was probably because P6 strongly inhibited STAT1, STAT5, and STAT6 phosphorylation, whereas STAT3 phosphorylation was less efficiently suppressed by P6 at the same concentration. Furthermore, IL-22 protects keratinocytes from apoptosis induced by IFN-γ, and administration of IL-17 and IL-22 partially ameliorated skin diseases in NC/Nga mice. These results suggested that the JAK inhibitor P6 is therapeutic for AD by modulating the balance of Th2 and Th17.
The Journal of Immunology 09/2011; 187(9):4611-20. DOI:10.4049/jimmunol.1100649 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes irreversible joint damage and significant disability. However, the fundamental mechanisms underlying how inflammation and joint destruction in RA develop and are sustained chronically remain largely unknown. Here, we show that signal transducer and activator of transcription 3 (STAT3) is the key mediator of both chronic inflammation and joint destruction in RA. We found that inflammatory cytokines highly expressed in RA patients, such as IL-1β, tumor necrosis factor alpha and IL-6, activated STAT3 either directly or indirectly and in turn induced expression of IL-6 family cytokines, further activating STAT3 in murine osteoblastic and fibroblastic cells. STAT3 activation also induced expression of receptor activator of nuclear factor kappa B ligand (RANKL), a cytokine essential for osteoclastogenesis, and STAT3 deficiency or pharmacological inhibition promoted significant reduction in expression of both IL-6 family cytokines and RANKL in vitro. STAT3 inhibition was also effective in treating an RA model, collagen-induced arthritis, in vivo through significant reduction in expression of IL-6 family cytokines and RANKL, inhibiting both inflammation and joint destruction. Leukemia inhibitory factor expression and STAT3 activation by IL-1β were mainly promoted by IL-6 but still induced in IL-6-deficient cells. Thus, our data provide new insight into RA pathogenesis and provide evidence that inflammatory cytokines trigger a cytokine amplification loop via IL-6-STAT3 that promotes sustained inflammation and joint destruction.
International Immunology 09/2011; 23(11):701-12. DOI:10.1093/intimm/dxr077 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin 10 (IL-10) and regulatory T cells (Tregs) maintain tolerance to intestinal microorganisms. However, Il10(-/-)Rag2(-/-) mice, which lack IL-10 and Tregs, remain healthy, suggesting the existence of other mechanisms of tolerance. Here, we identify suppressor of cytokine signalling 1 (SOCS1) as an essential mediator of immune tolerance in the intestine. Socs1(-/-)Rag2(-/-) mice develop severe colitis, which can be prevented by the reduction of microbiota and the transfer of IL-10-sufficient Tregs. Additionally, we find an essential role for prostaglandin E2 (PGE2) in the maintenance of tolerance within the intestine in the absence of Tregs. Socs1(-/-) dendritic cells are resistant to PGE2-mediated immunosuppression because of dysregulated cytokine signalling. Thus, we propose that SOCS1 and PGE2, potentially interacting together, act as an alternative intestinal tolerance mechanism distinct from IL-10 and Tregs.
[Show abstract][Hide abstract] ABSTRACT: Th2 cytokines and their downstream Janus kinase (JAK)-signal transducer and activation of transcription (STAT) pathways play a critical role in allergic asthma. We studied the effects of a pan-JAK inhibitor, pyridone 6 (P6), on asthmatic responses in a mouse model and investigated the mechanism for its biological effects. Mice were sensitized and challenged by ovalbumin (OVA). P6 treatment during the challenge phase suppressed eosinophilia in bronchoalveolar lavage (BAL) fluids but did not affect airway hyperresponsiveness (AHR). To improve the efficacy of the JAK inhibitor, P6 was encapsulated in polylactic-coglycolic acid nanoparticles (P6-PLGA). P6-PLGA treatment just before OVA challenge suppressed both airway eosinophilia and AHR. Although the IL-13 levels in BAL fluids and the OVA-specific IgE levels in serum after the challenge phase treatment with P6-PLGA were similar to those after a sham treatment, the eotaxin levels in BAL fluids and lung mCLCA3/Gob-5 expression were decreased in P6-PLGA-treated mice. Interestingly, the local IL-13 levels and serum OVA-specific IgE were decreased, while IL-17-producing T cells were increased by P6-PLGA treatment during the sensitization plus challenge phases. In vitro, P6 strongly suppressed the differentiation of Th2 from naive CD4 T cells, but it partly enhanced Th17 differentiation. P6 potently suppressed IL-13-mediated STAT6 activation and mCLCA3/Gob-5 expression in mouse tracheal epithelial cells. These findings suggest that the JAK inhibitor P6 suppresses asthmatic responses by inhibiting Th2 inflammation and that application of PLGA nanoparticles improves the therapeutic potency of P6.
Biochemical and Biophysical Research Communications 01/2011; 404(1):261-7. DOI:10.1016/j.bbrc.2010.11.104 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, T cell cytokines such as IL-17 and IFN-γ have been shown to play important roles in the progression of brain injury induced by ischemia. We have shown that IL-23 from infiltrated macrophages activates γδT cells, thereby inducing IL-17 from these cells. However, deletion of the IL-23 gene in mice showed a more dramatic protective effect against brain ischemia reperfusion (I/R) model than γδT cell depletion did, suggesting that IL-23 plays some other pivotal role in brain injury in addition to its role in IL-17 induction. To develop therapeutic methods based on these findings, we examined the effect of the JAK kinase inhibitor CP-690550 and an anti-IL12/23 monoclonal antibody on an I/R model. CP-690550 efficiently inhibited IL-17 production from memory T cells in vitro and partly suppressed infarct volume increase after I/R. Anti-p40 antibody, which blocks both IL-12 and IL-23, efficiently suppressed I/R injury and improved recovery of neurological deficits. The number of IL-17-producing cells was decreased by anti-p40 antibody treatment. Thus the JAK inhibitor and anti-p40 antibody, both of which have already been under trial for the treatment of several human inflammatory diseases, appear to be promising therapeutic agents for the amelioration of stroke.
Biochemical and Biophysical Research Communications 10/2010; 402(3):500-6. DOI:10.1016/j.bbrc.2010.10.058 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammation has been shown to contribute to both tumor development and antitumor immunity. However, conditions determining these opposing effects are not well understood. Suppressor of cytokine signaling 1 (SOCS1) has been shown to play an important role in regulating inflammation and tumor development. It has been reported that silencing of SOCS1 gene in dendritic cells potentiates antitumor immunity, while SOCS1-deficiency in whole organs except for T and B cells enhances inflammation-mediated colon tumor development. To determine which types of cells are important for the suppression of tumor development by SOCS1-deficiency, we employed the conditional knockout strategy. SOCS1 gene was deleted in macrophages and neutrophils by crossing SOCS1-flox/flox mice with LysM-cre mice. Resulting conditional knockout (cKO) mice showed enhanced sensitivity to endotoxin shock. SOCS1-cKO mice survived much longer than wild-type mice after B16 melanoma transplantation. Colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) plus dextran sulfate sodium (DSS) was also reduced in SOCS1-cKO mice. SOCS1-deficiency in monocytic cells enhanced tumor-killing activity of macrophages and tumor-specific cytotoxic T cell activity. These results suggest that inflammation induced by SOCS1-deficiency in monocytes potentiates antitumor immune responses rather than tumor-promoting inflammation.
Cancer Science 05/2009; 100(4):730-6. DOI:10.1111/j.1349-7006.2009.01098.x · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Type I interferons (IFN-alpha/beta) are essential for immune defense against viruses and induced through the actions of the cytoplasmic helicases, RIG-I and MDA5, and their downstream adaptor molecule IPS-1. TRAF6 and the downstream kinase TAK1 have been shown to be essential for the production of proinflammatory cytokines through the TLR/MyD88/TRIF pathway. Although binding of TRAF6 with IPS-1 has been demonstrated, the role of the TRAF6 pathway in IFN-alpha/beta production has not been fully understood. Here, we demonstrate that TRAF6 is critical for IFN-alpha/beta induction in response to viral infection and intracellular double-stranded RNA, poly(I:C). Activation of NF-kappaB, JNK, and p38, but not IRF3, was impaired in TRAF6-deficient mouse embryo fibroblasts in response to vesicular stomatitis virus and poly(I:C). However, TAK1 was not required for IFN-beta induction in this process, since normal IFN-alpha/beta production was observed in TAK1-deficient mouse embryo fibroblasts. Instead, another MAP3K, MEKK1, was important for the activation of the IFN-beta promoter in response to poly(I:C). Forced expression of MEKK1 in combination with IRF3 was sufficient for the induction of IFN-beta, whereas suppression of MEKK1 expression by small interfering RNA inhibited the induction of IFN-beta by poly(I:C). These data suggest that IPS-1 requires TRAF6 and MEKK1 to activate NF-kappaB and mitogen-activated protein kinases that are critical for the optimal induction of type I interferons.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DCs) are specialized antigen-presenting cells that play pivotal roles in initiating immune responses. However, DC maturation is usually strongly restricted by the stromal microenvironment, especially in non-lymphoid tissues, such as skin and mucosa. Although suppression of DC maturation by stromal cells has been well documented, the molecular basis of this suppression has not been established. In this study, we examined the role of fibroblasts for DC maturation in vitro. The mouse embryonic fibroblasts (MEFs) strongly suppressed LPS-induced DC maturation. Although suppression of class II MHC and CD40 required DC-MEF contact, soluble factors in the culture supernatant of MEFs were sufficient for the suppression of IL-12 and tumor necrosis factor-alpha production. Using molecular-size selection and HPLC, we determined that prostaglandin E2 (PGE2) is a major soluble inhibitory factor secreted by MEFs. This was confirmed by the fact that cyclooxygenase inhibitors inhibited the production of the suppressive factor by MEFs. These results suggest that PGE2 is a major soluble factor produced by MEFs for the suppression of inflammatory cytokine production from DCs, while a contact mechanism between MEFs and DCs is required for the suppression to induce T cell-stimulating molecules.
International Immunology 09/2008; 20(9):1219-29. DOI:10.1093/intimm/dxn078 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity.
However, in the presence of interleukin (IL)-6, TGF-β1 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-β1
and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor
γt (RORγt), was rapidly induced by TGF-β1 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 RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated
IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region
of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into
[Show abstract][Hide abstract] ABSTRACT: Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling-1 (SOCS1), which is a negative-feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver-specific SOCS1-conditional-knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)-induced hepatitis, which has been shown to be dependent on activated T and natural killer T (NKT) cells. Although serum cytokine level and NKT cell activation were similar in wild-type (WT) and SOCS1-deficient mice after ConA treatment, proapoptotic signals, including signal transducers and activators of transcription 1 (STAT1) and Jun-terminal kinase (JNK) activation, were enhanced in SOCS1-deficient livers compared with those in WT livers. SOCS1-deficient hepatocytes had higher expression of Fas antigen and were more sensitive to anti-Fas antibody-induced apoptosis than were WT hepatocytes. Furthermore, SOCS1-deficient hepatocytes were more sensitive to tumor necrosis factor (TNF)-alpha-induced JNK activation and apoptosis. These data indicate that SOCS1 is important to the prevention of hepatocyte apoptosis induced by Fas and TNF-alpha. In contrast, SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA-induced liver injury. Conclusion: These findings indicate that SOCS1 plays important negative roles in fulminant hepatitis and that forced expression of SOCS1 is therapeutic in preventing hepatitis.
[Show abstract][Hide abstract] ABSTRACT: Suppressor of cytokine signaling 1 (SOCS1) is an important negative regulator for cytokines; however, the role of SOCS1 in Th17 differentiation has not been clarified. We generated T cell-specific SOCS1-deficient mice and found that these mice were extremely resistant to a Th17-dependent autoimmune disease model, experimental autoimmune encephalomyelitis. SOCS1-deficient naive CD4(+) T cells were predominantly differentiated into Th1 and poorly into Th17 in vitro. These phenotypes were canceled in IFN-gamma(-/-) background, suggesting that a large amount of IFN-gamma in SOCS1-deficient T cells suppressed Th17 differentiation. IL-6 plus TGF-beta enhanced retinoic acid receptor-related orphan receptor (ROR)-gammat expression and suppressed IFN-gamma production in wild-type T cells, whereas these effects were severely impaired in SOCS1-deficient T cells. These phenotypes can be partly explained by STAT3 suppression by enhanced SOCS3 induction through hyper-STAT1 activation in SOCS1-deficient T cells. In addition, SOCS1-deficient T cells were much less sensitive to TGF-beta. Suppression of Th1 differentiation by TGF-beta was impaired in SOCS1-deficient T cells. TGF-beta-mediated Smad transcriptional activity was severely inhibited in SOCS1-deficient cells in the presence of IFN-gamma. Such impairment of TGF-beta functions were not observed in SOCS3-overexpressed cells, indicating that suppression of Smads was independent of SOCS3. Therefore, SOCS1 is necessary for Th17 differentiation by suppressing antagonistic effect of IFN-gamma on both STAT3 and Smads. Induction of SOCS3 can partly explain IFN-gamma-mediated STAT3 suppression, while other mechanism(s) will be involved in IFN-gamma-mediated Smad suppression. SOCS1-deficient T cells will be very useful to investigate the molecular mechanism for the STAT1-mediated suppression of Th17 development.
The Journal of Immunology 04/2008; 180(6):3746-56. DOI:10.4049/jimmunol.180.6.3746 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cgamma-2 (PLCgamma2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMphi) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCgamma2 knockout mice. Thus, PLCgamma2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCgamma2-knockdown cells, PGN-induced IkappaB-alpha phosphorylation and p38 activation were reduced. Moreover, PLCgamma2 was necessary for the full production of TNF-alpha and IL-6. These data indicate that the PLCgamma2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.
Genes to Cells 03/2008; 13(2):199-208. DOI:10.1111/j.1365-2443.2007.01159.x · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-kappaB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-kappaB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax.
Biochemical and Biophysical Research Communications 02/2008; 365(1):189-94. DOI:10.1016/j.bbrc.2007.10.172 · 2.30 Impact Factor