論文

Foxp3+ regulatory T (Treg) cells maintain immune homeostasis by limiting different types of inflammatory responses. Here, we report that miR-146a, one of the miRNAs prevalently expressed in Treg cells, is critical for their suppressor function. The deficiency of miR-146a in Treg cells resulted in a breakdown of immunological tolerance manifested in fatal IFNγ-dependent immune-mediated lesions in a variety of organs. This was likely due to augmented expression and activation of signal transducer and activator transcription 1 (Stat1), a direct target of miR-146a. Likewise, heightened Stat1 activation in Treg cells subjected to a selective ablation of SOCS1, a key negative regulator of Stat1 phosphorylation downstream of the IFNγ receptor, was associated with analogous Th1-mediated pathology. Our results suggest that specific aspects of Treg suppressor function are controlled by a single miRNA and that an optimal range of Stat1 activation is important for Treg-mediated control of Th1 responses and associated autoimmunity. © 2010 Elsevier Inc.

Although it has been well established that TGF-β plays a pivotal role in immune regulation, the roles of its downstream transcription factors, Smad2 and Smad3, have not been fully clarified. Specifically, the function of Smad2 in the immune system has not been investigated because of the embryonic lethality of Smad2-deficient mice. In this study, we generated T cell-specific Smad2 conditional knockout (KO) mice and unexpectedly found that Smad2 and Smad3 were redundantly essential for TGF-β-mediated induction of Foxp3-expressing regulatory T cells and suppression of IFN-γ production in CD4+ T cells. Consistent with these observations, Smad2/Smad3-double KO mice, but not single KO mice, developed fatal inflammatory diseases with higher IFN-γ production and reduced Foxp3 expression in CD4+ T cells at the periphery. Although it has been suggested that Foxp3 induction might underlie TGF-β-mediated immunosuppression, TGF-β still can suppress Th1 cell development in Foxp3-deficient T cells, suggesting that the Smad2/3 pathway inhibits Th1 cell development with Foxp3-independent mechanisms. We also found that Th17 cell development was reduced in Smad-deficient CD4+ T cells because of higher production of Th17-inhibotory cytokines from these T cells. However, TGF-β-mediated induction of RORγt, a master regulator of Th17 cell, was independent of both Smad2 and Smad3, suggesting that TGF-β regulates Th17 development through Smad2/3-dependent and -independent mechanisms. Copyright © 2010 by The American Association of Immunologists, Inc.

Although it has been well established that TGF-β plays a pivotal role in immune regulation, the roles of its downstream transcription factors, Smad2 and Smad3, have not been fully clarified. Specifically, the function of Smad2 in the immune system has not been investigated because of the embryonic lethality of Smad2-deficient mice. In this study, we generated T cell-specific Smad2 conditional knockout (KO) mice and unexpectedly found that Smad2 and Smad3 were redundantly essential for TGF-β-mediated induction of Foxp3-expressing regulatory T cells and suppression of IFN-γ production in CD4+ T cells. Consistent with these observations, Smad2/Smad3-double KO mice, but not single KO mice, developed fatal inflammatory diseases with higher IFN-γ production and reduced Foxp3 expression in CD4+ T cells at the periphery. Although it has been suggested that Foxp3 induction might underlie TGF-β-mediated immunosuppression, TGF-β still can suppress Th1 cell development in Foxp3-deficient T cells, suggesting that the Smad2/3 pathway inhibits Th1 cell development with Foxp3-independent mechanisms. We also found that Th17 cell development was reduced in Smad-deficient CD4+ T cells because of higher production of Th17-inhibotory cytokines from these T cells. However, TGF-β-mediated induction of RORγt, a master regulator of Th17 cell, was independent of both Smad2 and Smad3, suggesting that TGF-β regulates Th17 development through Smad2/3-dependent and -independent mechanisms. Copyright © 2010 by The American Association of Immunologists, Inc.

Transforming growth factor-β (TGF-β) has been shown to play an essential role in the suppression of inflammation, yet recent studies have revealed the positive roles of TGF-β in inflammatory responses. For example, TGF-β induces Foxp3-positive regulatory T cells (iTregs) in the presence of interleukin-2 (IL-2), while in the presence of IL-6, it induces pathogenic IL-17 producing Th17 cells. TGF-β inhibits the proliferation of immune cells as well as cytokine production via Foxp3-dependent and -independent mechanisms. Little is known about molecular mechanisms involved in immune suppression via TGF-β; however, Smad2/3 have been shown to play essential roles in Foxp3 induction as well as in IL-2 and IFN-γ suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Interaction between TGF-β and other cytokine signaling is important in establishing the balance of immunity and tolerance. © The Authors 2010. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Transforming growth factor-β (TGF-β) has been shown to play an essential role in the suppression of inflammation, yet recent studies have revealed the positive roles of TGF-β in inflammatory responses. For example, TGF-β induces Foxp3-positive regulatory T cells (iTregs) in the presence of interleukin-2 (IL-2), while in the presence of IL-6, it induces pathogenic IL-17 producing Th17 cells. TGF-β inhibits the proliferation of immune cells as well as cytokine production via Foxp3-dependent and -independent mechanisms. Little is known about molecular mechanisms involved in immune suppression via TGF-β; however, Smad2/3 have been shown to play essential roles in Foxp3 induction as well as in IL-2 and IFN-γ suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Interaction between TGF-β and other cytokine signaling is important in establishing the balance of immunity and tolerance. © The Authors 2010. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Ebola virus initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal Ebola virus infections. Here we report that host Toll-like receptor 4 (TLR4) is a sensor for Ebola virus glycoprotein (GP) on virus-like particles (VLPs) and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines and suppressor of cytokine signaling 1 (SOCS1) in a human monocytic cell line and in HEK293-TLR4/MD2 cells stably expressing the TLR4/MD2 complex. Ebola virus GP was found to interact with TLR4 by immunoprecipitation/Western blot analyses, and Ebola virus GP on VLPs was able to stimulate expression of NF-κB in a TLR4-dependent manner. Interestingly, we found that budding of Ebola virus VLPs was more pronounced in TLR4-stimulated cells than in unstimulated control cells. In sum, these findings identify the host innate immune protein TLR4 as a sensor for Ebola virus GP which may play an important role in the immunopathogenesis of Ebola virus infection. Copyright © 2010, American Society for Microbiology.

Ebola virus initially targets monocytes and macrophages, which can lead to the release of proinflammatory cytokines and chemokines. These inflammatory cytokines are thought to contribute to the development of circulatory shock seen in fatal Ebola virus infections. Here we report that host Toll-like receptor 4 (TLR4) is a sensor for Ebola virus glycoprotein (GP) on virus-like particles (VLPs) and that resultant TLR4 signaling pathways lead to the production of proinflammatory cytokines and suppressor of cytokine signaling 1 (SOCS1) in a human monocytic cell line and in HEK293-TLR4/MD2 cells stably expressing the TLR4/MD2 complex. Ebola virus GP was found to interact with TLR4 by immunoprecipitation/Western blot analyses, and Ebola virus GP on VLPs was able to stimulate expression of NF-κB in a TLR4-dependent manner. Interestingly, we found that budding of Ebola virus VLPs was more pronounced in TLR4-stimulated cells than in unstimulated control cells. In sum, these findings identify the host innate immune protein TLR4 as a sensor for Ebola virus GP which may play an important role in the immunopathogenesis of Ebola virus infection. Copyright © 2010, American Society for Microbiology.

Context: Autosomal dominant inactivating sprouty-related EVH1 domain-containing protein 1 (SPRED1) mutations have recently been described in individuals presenting mainly with café au lait macules (CALMs), axillary freckling, and macrocephaly. The extent of the clinical spectrum of this new disorder needs further delineation. Objective: To determine the frequency, mutational spectrum, and phenotype of neurofibromatosis type 1-like syndrome (NFLS) in a large cohort of patients. Design, Setting, and Participants: In a cross-sectional study, 23 unrelated probands carrying a SPRED1 mutation identified through clinical testing participated with their families in a genotype-phenotype study (2007-2008). In a second cross-sectional study, 1318 unrelated anonymous samples collected in 2003-2007 from patients with a broad range of signs typically found in neurofibromatosis type 1 (NF1) but no detectable NF1 germline mutation underwent SPRED1 mutation analysis. Main Outcome Measures: Comparison of aggregated clinical features in patients with or without a SPRED1 or NF1 mutation. Functional assays were used to evaluate the pathogenicity of missense mutations. Results: Among 42 SPRED1-positive individuals from the clinical cohort, 20 (48%; 95% confidence interval [CI], 32%-64%) fulfilled National Institutes of Health (NIH) NF1 diagnostic criteria based on the presence of more than 5 CALMs with or without freckling or an NF1-compatible family history. None of the 42 SPRED1-positive individuals (0%; 95% CI, 0%-7%) had discrete cutaneous or plexiform neurofibromas, typical NF1 osseous lesions, or symptomatic optic pathway gliomas. In the anonymous cohort of 1318 individuals, 34 different SPRED1 mutations in 43 probands were identified: 27 pathogenic mutations in 34 probands and 7 probable nonpathogenic missense mutations in 9 probands. Of 94 probands with familial CALMs with or without freckling and no other NF1 features, 69 (73%; 95% CI, 63%-80%) had an NF1 mutation and 18 (19%; 95% CI, 12%-29%) had a pathogenic SPRED1 mutation. In the anonymous cohort, 1.9% (95% CI, 1.2%-2.9%) of individuals with the clinical diagnosis of NF1 according to the NIH criteria had NFLS. Conclusions: A high SPRED1 mutation detection rate was found in NF1 mutation-negative families with an autosomal dominant phenotype of CALMs with or without freckling and no other NF1 features. Among individuals in this study, NFLS was not associated with the peripheral and central nervous system tumors seen in NF1. ©2009 American Medical Association. All rights reserved.

Th cells have long been divided into two subsets, Th1 and Th2; however, recently, Th17 and inducible regulatory T (iTreg) cells were identified as new Thcell subsets. Although Th1- and Th 2-polarizing cytokines have been shown to suppress Th17 and iTreg development, transcriptional regulation of Th17 and iTreg differentiation by cytokines remains to be clarified. In this study, we found that expression of the growth factor independent 1 (Gfi1) gene, which has been implicated in Th2 development, was repressed in Th17 and iTreg cells compared with Th 1 and Th2 lineages. Gfi1 expression was enhanced by the IFN-γ/STAT1 and IL-4/STAT6 pathways, whereas it was repressed by the transforming growth factor-β1 stimulation at the promoter level. Over-expression of Gfi1 strongly reduced IL-17A transcription in the EL4 T cell line, as well as in primary T cells. This was due to the blockade of recruitment of retinoid-related orphan receptor γt to the IL-17A promoter. In contrast, IL-17A expression was significantly enhanced in Gfi1-deficient T cells under Th17-promoting differentiation conditions as compared with wild-type T cells. In contrast, the impacts of Gfi1 in iTregs were not as strong as in Th17 cells. Taken together, these data strongly suggest that Gfi1 is a negative regulator of Th17 differentiation, which represents a novel mechanism for the regulation of Th17 development by cytokines. © The Japanese Society for Immunology. 2009. All rights reserved.

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. © 2009 Japanese Cancer Association.

Infection with pathogens containing superantigens (Sags) can result in massive excessive CD4+ T cell activation and death in such conditions as toxic shock, food poisoning and autoimmune diseases. We here showed how enhancement of IL-6 signaling suppresses Sag-mediated activated CD4+ T cell death. Sag-induced CD4+ T cell death increased in IL-6 knockout (KO) mice, whereas it decreased in mice characterized by enhanced IL-6-gp130-STAT3 signaling. The serum concentration of IFN-γ was inversely correlated with the magnitude of IL-6 signaling, and IFN-γ deficiency inhibited Sag-induced activated CD4+ T cell death, suggesting that IL-6 suppresses CD4+ T cell death via IFN-γ expression. Interestingly, depletion of activated CD8+ T cells inhibited Sag-mediated increases in IFN-γ expression in IL-6 KO mice as well as the augmented CD4+ T cell death. The results demonstrate that IL-6-gp130-STAT3 signaling in activated CD8+ T cells contributes to Sag-induced CD4+ T cell death via IFN-γ expression, highlighting this signaling axis in CD8+ T cells as a potential therapeutic target for Sag-related syndromes. © The Japanese Society for Immunology. 2008. All rights reserved.

Objectives. Human endogenous uveitis is one of the sight-threatening diseases associated with variety of systemic disorders, such as Behcet's disease and sarcoidosis. Recently, biosynthesized antibodies against inflammatory cytokines have been recognized to be useful to control the regional inflammation. In this study, we focused on the possibility of IL-6-based biological therapies for endogenous uveitis. We initially confirmed the significant increase of several inflammatory soluble factors including IL-6 in the vitreous fluids from refractory/chronic engogenous uveitis patients. Methods. To investigate the role of IL-6 in the formation of refractory ocular inflammation, we used the mouse experimental autoimmune uveitis (EAU) model. Both IL-6 and IL-23 are required for the development of IL-17-producing helper T subset (Th17) from naïve CD4+T cells. Results. In the EAU model, neither IL-6-deficient mice nor IL-23-deficient mice could induce Th17 cells and the EAU score was decreased in these mice in the entire time course. We also confirmed that systemic administration of anti-IL-6 receptor antibody ameliorates EAU by suppressing both systemic and regional Th17 responses. Conclusions. IL-6 is responsible for causing ocular inflammation, and it is, at least partially, due to IL-6-dependent Th17 differentiation. IL-6 may be a target for therapy of refractory endogenous uveitis in humans. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 regulates integrin-mediated T cell adhesion. In the present study, we find that STAP-2 expression affects Jurkat T cell migration after stromal cell-derived factor-1α (SDF-1α)-treatment. Furthermore, STAP-2-deficient T cells exhibit reduced cell migration after SDF-1α-treatment. Importantly, overexpression of STAP-2 in Jurkat T cells induces activation of small guanine triphosphatases, such as Rac1 and Cdc42. Regarding the mechanism for this effect, we found that STAP-2 associates with Vav1, the guanine-nucleotide exchanging factor for Rac1, and enhances downstream Vav1/Rac1 signaling. These results reveal a novel STAP-2-mediated mechanism for the regulation of SDF-1α-induced chemotaxis of T cells via activation of Vav1/Rac1 signaling. Copyright © 2009 by The American Association of Immunologists, Inc.

Suppressor of cytokine signaling 3 (SOCS3), a negative-feedback molecule for cytokine signaling, has been implicated in protection against liver injury. Previous studies have shown that overexpression of SOCS3 in the liver by adenovirus or membrane permeable recombinant protein protected the liver from various injuries. However it remained uncertain in which type of cells SOCS3 suppresses liver injury. In this study, we demonstrated that forced expression of SOCS3 in T and NKT cells suppressed ConA-induced hepatitis using T and NKT cell-specific SOCS3 transgenic (Lck-SOCS3 Tg) mice. IFN-γ and IL-4 production was reduced in Lck-SOCS3 Tg mice as well as splenocytes treated with ConA. IFN-γ and IL-4 levels were also reduced in Lck-SOCS3 Tg mice administrated with α-galactosylceramide, suggesting that SOCS3 in NKT cells has suppressive function. Sustained expression of SOCS3 in an NKT cell line also resulted in reduced expression of various cytokines and transcription factors. In contrast, T and NKT cell-specific SOCS3 conditional knockout (Lck-SOCS3 cKO) mice were hypersensitive to ConA-mediated hepatitis. Isolated SOCS3-deficient NKT cells produced higher levels of IFN-γ and IL-4. These data indicate that SOCS3 plays a negative regulatory role in NKT cell activation and that forced expression of SOCS3 in NKT cells is effective in preventing hepatitis. Copyright © 2009 by The American Association of Immunologists, Inc.

Sprouty proteins have been shown to negatively regulate a variety of receptor tyrosine kinase (RTK) signaling pathways and are considered to be tumor suppressor proteins. The pathophysiological functions of Sproutys in vivo remain to be investigated. In this study, we examined the physiological function of Sprouty4 as an angiogenic regulator, using Sprouty4 knockout (KO) mice and cells.We found that transplanted tumor cells grow much faster in Sprouty4 KO mice than in wild type (WT) mice, which we associate with enhanced neovascularization in the tumors transplanted into Sprouty4 KO mice. Moreover, vascular endothelial growth factor (VEGF)-A-induced angiogenesis and vascular permeability in vivo were enhanced in Sprouty4 KO mice compared with WT mice. Ex vivo angiogenesis, which we induced by VEGF-A, basic fibroblast growth factor (bFGF), and sphingosine-1-phosphate (S1P), was also enhanced in the aortas of Sprouty4 KO mice. We demonstrated that Sprouty4 suppresses Ras-independent VEGF-A and S1P signaling,while it does not affect Ras-dependent VEGF-C signaling. These data indicate that Sprouty4 selectively suppresses Ras-independent angiogenic factor signals and is an important negative regulator of pathophysiological angiogenesis. © 2009 Japanese Cancer Association.

Atherosclerosis is an inflammatory vascular disease responsible for the first cause of mortality worldwide. Recent studies have clearly highlighted the critical role of the immunoinflammatory balance in the modulation of disease development and progression. However, the immunoregulatory pathways that control atherosclerosis remain largely unknown. We show that loss of suppressor of cytokine signaling (SOCS) 3 in T cells increases both interleukin (IL)-17 and IL-10 production, induces an antiinflammatory macrophage phenotype, and leads to unexpected IL-17-dependent reduction in lesion development and vascular inflammation. In vivo administration of IL-17 reduces endothelial vascular cell adhesion molecule-1 expression and vascular T cell infiltration, and significantly limits atherosclerotic lesion development. In contrast, overexpression of SOCS3 in T cells reduces IL-17 and accelerates atherosclerosis. We also show that in human lesions, increased levels of signal transducer and activator of transcription (STAT) 3 phosphorylation and IL-17 are associated with a stable plaque phenotype. These results identify novel SOCS3-controlled IL-17 regulatory pathways in atherosclerosis and may have important implications for the understanding of the increased susceptibility to vascular inflammation in patients with dominant-negative STAT3 mutations and defective Th17 cell differentiation. © 2009 Taleb et al.

Lymphocyte recruitment and activation have been implicated in the progression of cerebral ischemia-reperfusion (I/R) injury, but the roles of specific lymphocyte subpopulations and cytokines during stroke remain to be clarified. Here we demonstrate that the infiltration of T cells into the brain, as well as the cytokines interleukin-23 (IL-23) and IL-17, have pivotal roles in the evolution of brain infarction and accompanying neurological deficits. Blockade of T cell infiltration into the brain by the immunosuppressant FTY720 reduced I/R-induced brain damage. The expression of IL-23, which was derived mostly from infiltrated macrophages, increased on day 1 after I/R, whereas IL-17 levels were elevated after day 3, and this induction of IL-17 was dependent on IL-23. These data, together with analysis of mice genetically disrupted for IL-17 and IL-23, suggest that IL-23 functions in the immediate stage of I/R brain injury, whereas IL-17 has an important role in the delayed phase of I/R injury during which apoptotic neuronal death occurs in the penumbra. Intracellular cytokine staining revealed that T lymphocytes, but not CD4 + helper T cells, were a major source of IL-17. Moreover, depletion of T lymphocytes ameliorated the I/R injury. We propose that T lymphocytes, including T lymphocytes, could be a therapeutic target for mitigating the inflammatory events that amplify the initial damage in cerebral ischemia.

Diabetic nephropathy (DN) associated with type 2 diabetes is the most common cause of end-stage renal disease (ESRD) and a serious health issue in the world. Currently, molecular basis for DN has not been established and only limited clinical treatments are effective in abating the progression to ESRD associated with DN. Here we found that diabetic db/db mice which lack the leptin receptor signaling can be used as a model of ESRD associated with DN. We demonstrated that p70S6-kinase was highly activated in mesangial cells in diabetic obese db/db mice. Furthermore, systemic administration of rapamycin, a specific and potent inhibitor of mTOR, markedly ameliorated pathological changes and renal dysfunctions. Moreover, rapamycin treatment shows a significant reduction in fat deposits and attenuates hyperinsulinemia with few side effects. These results indicate that mTOR activation plays a pivotal role in the development of ESRD and that rapamycin could be an effective therapeutic agent for DN. © 2009 Elsevier Inc. All rights reserved.