The CCR4-NOT deadenylase complex safeguards thymic positive selection by down-regulating aberrant pro-apoptotic gene expression.

A repertoire of T cells with diverse antigen receptors is selected in the thymus. However, detailed mechanisms underlying this thymic positive selection are not clear. Here we show that the CCR4-NOT complex limits expression of specific genes through deadenylation of mRNA poly(A) tails, enabling positive selection. Specifically, the CCR4-NOT complex is up-regulated in thymocytes before initiation of positive selection, where in turn, it inhibits up-regulation of pro-apoptotic Bbc3 and Dab2ip. Elimination of the CCR4-NOT complex permits up-regulation of Bbc3 during a later stage of positive selection, inducing thymocyte apoptosis. In addition, CCR4-NOT elimination up-regulates Dab2ip at an early stage of positive selection. Thus, CCR4-NOT might control thymocyte survival during two-distinct stages of positive selection by suppressing expression levels of pro-apoptotic molecules. Taken together, we propose a link between CCR4-NOT-mediated mRNA decay and T cell selection in the thymus.

Prostaglandin E Receptor 4 Antagonist in Cancer Immunotherapy: Mechanisms of Action.

A highly expressed prostaglandin E2 (PGE2) in tumor tissues suppresses antitumor immunity in the tumor microenvironment (TME) and causes tumor immune evasion leading to disease progression. In animal studies, selective inhibition of the prostaglandin E receptor 4 (EP4), one of four PGE2 receptors, suppresses tumor growth, restoring the tumor immune response toward an antitumorigenic condition. This review summarizes PGE2/EP4 signal inhibition in relation to the cancer-immunity cycle (C-IC), which describes fundamental tumor-immune interactions in cancer immunotherapy. PGE2 is suggested to slow down C-IC by inhibiting natural killer cell functions, suppressing the supply of conventional dendritic cell precursors to the TME. This is critical for the tumor-associated antigen priming of CD8+ T cells and their translocation to the tumor tissue from the tumor-draining lymph node. Furthermore, PGE2 activates several key immune-suppressive cells present in tumors and counteracts tumoricidal properties of the effector CD8+ T cells. These effects of PGE2 drive the tumors to non-T-cell-inflamed tumors and cause refractory conditions to cancer immunotherapies, e.g., immune checkpoint inhibitor (ICI) treatment. EP4 antagonist therapy is suggested to inhibit the immune-suppressive and tumorigenic roles of PGE2 in tumors, and it may sensitize the therapeutic effects of ICIs in patients with non-inflamed and C-IC-deficient tumors. This review provides insight into the mechanism of action of EP4 antagonists in cancer immunotherapy and suggests a C-IC modulating opportunity for EP4 antagonist therapy in combination with ICIs and/or other cancer therapies.

Transcriptional Regulation of Differentiation and Functions of Effector T Regulatory Cells.

Foxp3-expressing regulatory T (Treg) cells can suppress the activity of various types of immune cells and play key roles in the maintenance of self-tolerance and in the regulation of immune responses against pathogens and tumor cells. Treg cells consist of heterogeneous subsets that have distinct phenotypes and functions. Upon antigen stimulation, naïve-like thymus-derived Treg cells, which circulate in secondary lymphoid organs, can differentiate into effector Treg (eTreg) cells and migrate to and control immune homeostasis of peripheral tissues. eTreg cells are heterogeneous in terms of their ability to localize to specific tissues and suppress particular types of immune responses. Differentiation and function of diverse eTreg subsets are regulated by a variety of transcription factors that are activated by antigens and cytokines. In this article, we review the current understanding of the transcriptional regulation of differentiation and function of eTreg cells.

JunB regulates homeostasis and suppressive functions of effector regulatory T cells.

Foxp3-expressing CD4+ regulatory T (Treg) cells need to differentiate into effector Treg (eTreg) cells to maintain immune homeostasis. T-cell receptor (TCR)-dependent induction of the transcription factor IRF4 is essential for eTreg differentiation, but how IRF4 activity is regulated in Treg cells is still unclear. Here we show that the AP-1 transcription factor, JunB, is expressed in eTreg cells and promotes an IRF4-dependent transcription program. Mice lacking JunB in Treg cells develop multi-organ autoimmunity, concomitant with aberrant activation of T helper cells. JunB promotes expression of Treg effector molecules, such as ICOS and CTLA4, in BATF-dependent and BATF-independent manners, and is also required for homeostasis and suppressive functions of eTreg. Mechanistically, JunB facilitates the accumulation of IRF4 at a subset of IRF4 target sites, including those located near Icos and Ctla4. Thus, JunB is a critical regulator of IRF4-dependent Treg effector programs, highlighting important functions for AP-1 in Treg-mediated immune homeostasis.

JunB is essential for IL-23-dependent pathogenicity of Th17 cells.

CD4+ T-helper cells producing interleukin-17 (IL-17), known as T-helper 17 (TH17) cells, comprise heterogeneous subsets that exhibit distinct pathogenicity. Although pathogenic and non-pathogenic TH17 subsets share a common RORγt-dependent TH17 transcriptional programme, transcriptional regulatory mechanisms specific to each of these subsets are mostly unknown. Here we show that the AP-1 transcription factor JunB is critical for TH17 pathogenicity. JunB, which is induced by IL-6, is essential for expression of RORγt and IL-23 receptor by facilitating DNA binding of BATF at the Rorc locus in IL-23-dependent pathogenic TH17 cells, but not in TGF-ß1-dependent non-pathogenic TH17 cells. Junb-deficient T cells fail to induce TH17-mediated autoimmune encephalomyelitis and colitis. However, JunB deficiency does not affect the abundance of gut-resident non-pathogenic TH17 cells. The selective requirement of JunB for IL-23-dependent TH17 pathogenicity suggests that the JunB-dependent pathway may be a therapeutic target for autoimmune diseases.

[Transcription factors in the development of myeloid cells].

Hematopoietic stem cells give rise to various blood cell types with diverse functions, although these different cell types harbor essentially identical genome sequences. The basis for this cell type diversity is the establishment of specific gene expression patterns through transcription factor regulation. Transcription factors recognize and bind to specific nucleotide sequences in target genes and recruit chromatin modifiers to alter the epigenetic status of these genes, thereby controlling their expression. Dysregulation of these processes can cause diseases such as leukemia. Due to rapid advances in high-throughput experimental techniques including chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA-seq, the study of transcription factors is now entering a new era. In this review, we update the current knowledge of developmental pathways in myeloid cells, particularly mononuclear phagocytes (i.e., monocytes/macrophages and dendritic cells), and the transcription factors known to be required for their development. We subsequently provide an overview of the cooperative and antagonistic mechanisms by which the myeloid transcription factors regulate their target genes, with an emphasis on chromatin biology.

Regulation of myelopoiesis by the transcription factor IRF8.

Interferon regulatory factor-8 (IRF8) is a transcription factor expressed in hematopoietic cells, particularly in mononuclear phagocytes [monocytes/macrophages and dendritic cells (DCs)] and their progenitors. Various studies have demonstrated that IRF8 is essential for the development of monocytes, DCs, eosinophils, and basophils. Conversely, IRF8 suppresses the generation of neutrophils. Accordingly, Irf8 (-/-) mice develop immunodeficiency and a chronic myeloid leukemia (CML)-like disease. Mutations and loss of expression of the human IRF8 gene are also associated with immunodeficiency and CML, respectively. Recent findings have begun to reveal the transcription factor network and epigenetic changes governed by IRF8. For example, in mononuclear phagocyte progenitors, IRF8 cooperates with PU.1 to promote the formation of promoter-distal enhancers to induce monocyte-related genes including the critical downstream transcription factor gene Klf4. On the other hand, IRF8 blocks C/EBPα activity to suppress the neutrophil differentiation program. Indeed, Irf8 (-/-) mononuclear phagocyte progenitors fail to efficiently generate monocytes and DCs and, instead, aberrantly give rise to neutrophils. This article provides an overview of recent advances in our understanding of the role of IRF8 in myelopoiesis and related diseases.

Transcription factor IRF8 plays a critical role in the development of murine basophils and mast cells.

Basophils and mast cells play critical roles in host defense against pathogens and allergic disorders. However, the molecular mechanism by which these cells are generated is not completely understood. Here we demonstrate that interferon regulatory factor-8 (IRF8), a transcription factor essential for the development of several myeloid lineages, also regulates basophil and mast cell development. Irf8(-/-) mice displayed a severe reduction in basophil counts, which was accounted for by the absence of pre-basophil and mast cell progenitors (pre-BMPs). Although Irf8(-/-) mice retained peripheral tissue mast cells, remaining progenitors from Irf8(-/-) mice including granulocyte progenitors (GPs) were unable to efficiently generate either basophils or mast cells, indicating that IRF8 also contributes to the development of mast cells. IRF8 appeared to function at the GP stage, because IRF8 was expressed in GPs, but not in basophils, mast cells, and basophil/mast cell-restricted progenitor cells. Furthermore, we demonstrate that GATA2, a transcription factor known to promote basophil and mast cell differentiation, acts downstream of IRF8. These results shed light on the pathways and mechanism underlying the development of basophils and mast cells.

The transcription factor IRF8 counteracts BCR-ABL to rescue dendritic cell development in chronic myelogenous leukemia.

BCR-ABL tyrosine kinase inhibitors (TKI) have dramatically improved therapy for chronic myelogenous leukemia (CML). However, several problems leading to TKI resistance still impede a complete cure of this disease. IFN regulatory factor-8 (IRF8) is a transcription factor essential for the development and functions of immune cells, including dendritic cells. Irf8(-/-) mice develop a CML-like disease and IRF8 expression is downregulated in patients with CML, suggesting that IRF8 is involved in the pathogenesis of CML. In this study, by using a murine CML model, we show that BCR-ABL strongly inhibits a generation of dendritic cells from an early stage of their differentiation in vivo, concomitant with suppression of Irf8 expression. Forced expression of IRF8 overrode BCR-ABL (both wild-type and T315I-mutated) to rescue dendritic cell development in vitro, indicating that the suppression of Irf8 causes dendritic cell deficiency. Gene expression profiling revealed that IRF8 restored the expression of a significant portion of BCR-ABL-dysregulated genes and predicted that BCR-ABL has immune-stimulatory potential. Indeed, IRF8-rescued BCR-ABL-expressing dendritic cells were capable of inducing CTLs more efficiently than control dendritic cells. Altogether, our findings suggest that IRF8 is an attractive target in next-generation therapies for CML.

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner.

Type-1 immunity plays a crucial role in host defense against various tumors and infectious diseases. Here, we first demonstrated that extract of Larix Leptolepis (ELL), one of the most popular timbers at Hokkaido area in Japan, strongly activated Type-1 immunity. ELL induced production of Type-1 cytokines such as IL-12 and TNF-α from bone marrow-derived dendritic cells (BMDCs) in TLR2- and TLR4-dependent manner and remarkably up-regulated the expression of MHC and co-stimulatory molecules. In addition, antigen-specific CTLs were significantly augmented by the combined administration of ELL, antigen and BMDCs. Finally, we revealed that combination therapy using ELL, antigen and BMDCs significantly inhibited the growth of established tumor in mouse model. Thus, these findings suggested that ELL would be a novel adjuvant for inducing an activation of Type-1-dependent immunity including activation of BMDCs and induction of tumor-specific CTLs, which is applicable to the therapy of cancer and infectious diseases.

Effects of AhR ligands on the production of immunoglobulins in purified mouse B cells.

The aryl hydrocarbon receptor (AhR) has been shown to play important roles in the immune system, and contributions of AhR ligands to the differentiation and functions of Th17/Treg cells have recently been established. However, it has not been fully clarified whether AhR plays roles in B cell differentiation and functions. The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly potent AhR agonist, was reported to suppress the production of immunoglobulin M (IgM) in a transformed mouse B cell line. However, TCDD exhibits high toxicity toward cells and has unknown activities except for its action as an AhR agonist. In the present study, we tried to clarify how an endogenously generated AhR agonist affects mouse B cell differentiation and functions in terms of the direct effects on the expression of Ig subclasses in purified mouse B cells stimulated with an anti-CD40 antibody and interleukin-4. The AhR agonist 2-(1'H-indole-3'- carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), which is derived via tryptophan metabolism, suppressed the expression of not only IgM, but also IgG1 and IgE. ITE was also found to suppress the expression of secreted-type Ig mRNAs and plasma cell-specific genes. These findings indicate that the endogenous AhR agonist suppresses B cell differentiation into Ig-secreting plasma cells.

Toll-like receptor-dependent IL-12 production by dendritic cells is required for activation of natural killer cell-mediated Type-1 immunity induced by Chrysanthemum coronarium L.

Type-1 immunity has an essential role for our host defenses against cancer and outer pathogens such as bacteria and virus. We demonstrated here that the edible plant extract of Chrysanthemum coronarium L. (C. coronarium) remarkably activates Type-1 immunity in a Toll-like receptor (TLR)2-, TLR4-, and TLR9-dependent manner. In the present experiments, the extract of C. coronarium significantly induces interferon (IFN)-γ production by mouse spleen cells. In addition, the IFN-γ production by spleen cells was completely blocked by the addition of anti-Interleukin (IL)-12 monoclonal antibodies. We confirmed that NK1.1(+) natural killer (NK) cells, NKT cells, and CD11c(+) dendritic cells (DC) were immediately activated after the stimulation with the extract of C. coronarium and the IFN-γ production was abolished in NK1.1(+) cell-depleted spleen cells. The stimulation with the extract of C. coronarium caused DC maturation involving with up-regulations of surface expression levels of MHC class I, MHC class II, CD40, and CD86 as well as induction of IL-12 production. The IFN-γ production induced by the extract was significantly reduced in the spleen cells depleted CD11c(+) cells. Furthermore, the IFN-γ production after the stimulation was strongly reduced in TLR4- and partially in TLR2- and TLR9-deficient spleen cells. Thus, we demonstrated the cellular mechanism for the activation of Type-1 immunity via NK cells, NKT cells, and DC by the extract of C. coronarium. These findings strongly suggest that C. coronarium would be a promising immuno-improving adjuvant, which might be useful for prevention of infectious, cancer, and allergic diseases through the activation of Type-1 immunity.

The extract of Japanese soybean, Kurosengoku activates the production of IL-12 and IFN-γ by DC or NK1.1(+) cells in a TLR4- and TLR2-dependent manner.

During the search for immuno-improving foods, we found that a variety of the Japanese soybean, Glycine max cv. Kurosengoku (Kurosengoku), which activated Type-1 immunity in a Toll-like receptor (TLR)4- and TLR2-dependent manner. Namely, the extract of Kurosengoku first caused production of IL-12 from DC and sequentially induced IFN-γ production by NK1.1(+) NK cells and NKT cells. The IFN-γ production was significantly blocked by neutralizing mAb against IL-12 or TLR4- and TLR2-deficient condition, indicating that TLR4- and TLR2-dependent activation of DC to produce IL-12 was essential for the production of IFN-γ from spleen cells by Kurosengoku. Moreover, the extract of Kurosengoku also enhanced production of IFN-γ from human PBMC by co-stimulation with anti-CD3 mAb in a TLR2- and TLR4-dependent manner. Thus, our findings strongly suggest that Kurosengoku might a novel immuno-improving food, which would be a useful tool for preventing the tip of immune balance in developed countries.

Mutation is the Main Driving Force in the Diversification of the Vibrio splendidus Clade.

The Vibrio splendidus clade is the biggest in Vibrionales composed of 11 described species (25). Diversification of these species may have occurred 260 million years ago. The main driving forces of speciation in this clade have never been studied. Population biological parameters (population base recombination rate (ρ), population base mutation rate (θ), and index of association (Ia)) were determined among 16 strains of 9 defined species in the Splendidus cluster. A comparison of individual gene phylogeny indicated significant incongruence in tree topology, which suggests the occurrence of recombination between species. Homologous recombination between species was detected at four loci. However, the mutation rate θ was higher than the recombination rate ρ, suggesting that mutation is the main driving force in the diversification of V. splendidus-related species.

Discovery of orally bioavailable cathepsin S inhibitors for the reversal of neuropathic pain.

Cathepsin S inhibitors are well-known to be an attractive target as immunological therapeutic agents. Recently, our gene expression analysis identified that cathepsin S inhibitors could also be effective for neuropathic pain. Herein, we describe the efficacy of selective cathepsin S inhibitors as antihyperalgesics in a model of neuropathic pain in rats after oral administration.

Essential role of Toll-like receptors for dendritic cell and NK1.1(+) cell-dependent activation of type 1 immunity by Lactobacillus pentosus strain S-PT84.

Activation of type 1 immunity plays a critical role in host defense mechanisms against infectious disease and tumor. Lactic acid bacteria, existing in the gastrointestinal tract, are one of the powerful tools to induce a type-1-dominant immunity, which may improve Th2-dependent allergic diseases. In the present work, we found that an oral intake of Lactobacillus pentosus strain, S-PT84 into mice significantly enhanced NK activity of spleen cells in vivo. We further revealed that NK1.1 positive NK cells and NKT cells are responsible cells for producing IFN-gamma after stimulation with S-PT84 in vitro. S-PT84 induced IFN-gamma-producing cells through activation of IL-12 production by CD11c(+)DCs in Toll-like receptor (TLR) 2- and/or TLR4-dependent manner. Interestingly, direct interaction between DCs and NK1.1(+) cells was also essential for the IFN-gamma production in response to the S-PT84 stimulation. Therefore, we concluded that S-PT84 effectively promoted type 1 immunity through IL-12 and IFN-gamma which were produced by DCs and NK1.1(+) cells, respectively. Thus, S-PT84 would be a nice immune modulator for improving immunobalance, which plays a pivotal role for controlling allergy, infectious diseases and tumor.

Unisized two-dimensional platinum clusters on silicon(111)-7x7 surface observed with scanning tunneling microscope.

Uni-sized platinum clusters (size range of 5-40) on a silicon(111)-7 x 7 surface were prepared by depositing size-selected platinum cluster ions on the silicon surface at the collision energy of 1.5 eV per atom at room temperature. The surface thus prepared was observed by means of a scanning tunneling microscope (STM) at the temperature of 77 K under an ambient pressure less than 5 x 10(-9) Pa. The STM images observed at different cluster sizes revealed that (1) the clusters are flattened and stuck to the surface with a chemical-bond akin to platinum silicide, (2) every platinum atom occupies preferentially the most reactive sites distributed within a diameter of approximately 2 nm on the silicon surface at a cluster size up to 20, and above this size, the diameter of the cluster increases with the size, and (3) the sticking probability of an incoming cluster ion on the surface increases with the cluster size and reaches nearly unity at a size larger than 20.

Lysophospholipase I identified as a ghrelin deacylation enzyme in rat stomach.

Ghrelin, discovered in rat stomach as an endogenous growth hormone secretagogue, is octanoylated at the Ser3 residue. Since this octanoylation is essential for the functions of ghrelin, the enzymes that catalyze acylation for ghrelin biosynthesis and deacylation (deactivation step) must be considered as important regulators. We found that rat stomach homogenate contained ghrelin deacylation activity, and we isolated the active fractions by column chromatography. After sequencing and expressing candidate proteins, the ghrelin deacylation enzyme in the stomach was identified as lysophospholipase I (LysoPLA I). The enzyme properties were examined using recombinant rat LysoPLA I expressed in Escherichia coli. K(m) and V(max) values were determined as 6.5 microM and 2.3 micromol/min/mg for ghrelin and 2.2 x 10(2) microM and 0.5 micromol/min/mg for lysophosphatidylcholine (LysoPC), respectively. The deacylation of both substrates was inhibited by methyl arachidonyl fluorophosphonate (MAFP), which is known as an irreversible inhibitor of LysoPLA I. These results reveal that LysoPLA I catalyzes the removal of n-octanoic acid from ghrelin to form des-acyl ghrelin. Identification of the ghrelin deacylation enzyme in the stomach and a deacylation inhibitor will be helpful in investigating ghrelin biosynthesis.

Low-energy impact of X-(H2O)n (X = Cl,I) onto solid surface.

We investigated dissociation of X-(H2O)n (X = Cl, I, n = 13-31) by the impact onto a (La0.7Ce0.3)B6(100) surface at a collision energy Ecol of 1-5 eV per water molecule in a tandem time-of-flight mass spectrometer equipped with a translation-energy analyzer. The mechanism of the dissociation was elucidated on the basis of the measurements of the mass spectrum and the translational energies of the product anions, X-(H2O)m (m = 0-4), scattered from the surface. It was concluded that (1) the parent cluster anion impacted on the surface undergoes dissociation on the surface under quasiequilibrium with its characteristic time varying with Ecol and n, and (2) the total collision energy introduced is partitioned preferentially to the translational motions of the products on the surface and to the rotational, the vibrational, and the lattice vibrational motions (surface) in this order. The quasiequilibrium model is applicable, even at the collision energy as low as 1 eV, because the translational modes are found to be statistically distributed while the other modes are not much populated by dynamical and energetics limitation.

Distinct usages of phospholipase C gamma and Shc in intracellular signaling stimulated by neurotrophins.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the neurotrophin family, bind to and activate TrkA, TrkB and TrkC, respectively, members of the Trk receptor tyrosine kinase family, to exert various effects including promotion of differentiation and survival, and regulation of synaptic plasticity in neuronal cells. Many reports have suggested that different neurotrophins show distinct biological functions, although molecular mechanisms by which neurotrophins exert their different functions remain unclear. In the present study, we found distinct usages of phospholipase Cgamma (PLCgamma) and Shc in intracellular signaling stimulated by neurotrophins. BDNF stimulated much stronger interactions of PLCgamma with Trk than NGF and NT-3 in PC12 cells stably expressing TrkB and cultured cerebral cortical neurons, respectively, although BDNF, NGF and NT-3 induced similar levels of tyrosine phosphorylation of Trk. Furthermore, the cultured cortical neurons showed large PLCgamma-dependent increases in intracellular Ca(2+) levels in response to BDNF compared with NT-3. In Shc signaling, NGF, but not BDNF, displayed interactions between Trk and Shc in a phenylarsine oxide (PAO; an inhibitor of tyrosine phosphatase)-dependent manner in TrkB-expressing PC12 cells. These results indicated that neurotrophins stimulate distinct kinds of interactions between Trk and PLCgamma and between Trk and Shc. These differences may lead to the distinct biological functions of neurotrophins.