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Summary Helper T (Th) and regulatory T (Treg) cell differentiation programs promote the eradication of pathogens, while minimizing adverse immune reactions. Here, we found that Nr4a family of nuclear receptors supports Treg cell induction and represses Th1 and Th2 cell differentiation from naive CD4+ T cells. Nr4a factors are transiently induced in CD4+ T cells immediately after antigen stimulation, thereby mediating epigenetic changes. In differentiating Treg cells, Nr4a factors mainly upregulated the early responsive genes in the Treg cell-specifying gene set, either directly or in cooperation with Ets family transcription factors. In contrast, Nr4a factors repressed AP-1 activity by interrupting a positive feedback loop for Batf factor expression, thus suppressing Th2 cell-associated genes. In an allergic airway inflammation model, Nr4a factors suppressed the pathogenesis, mediating oral tolerance. Lastly, pharmacological activation of an engineered Nr4a molecule prevented allergic airway inflammation, indicating that Nr4a factors may be novel therapeutic targets for inflammatory diseases., Graphical abstract, Highlights • Among “Treg signature genes”, Nr4a factors mainly induce early responsive ones • Nr4a activate target genes directly or by supporting Ets factors' function • Nr4a factors repress Th2-driving positive feedback loop for Batf factor expression • Pharmacological activation of Nr4a factors' activity prevented airway inflammation, Biological Sciences; Immunology; Cell Biology

T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies1,2. CAR T cells have been less effective against solid tumours3–5, in part because they enter a hyporesponsive (‘exhausted’ or ‘dysfunctional’) state6–9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR T cells in solid tumours, we transferred hCD19-reactive CAR T cells into hCD19+ tumour-bearing mice. CD8+CAR+ tumour-infiltrating lymphocytes and CD8+ endogenous tumour-infiltrating lymphocytes expressing the inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1) by the initiating transcription factor NFAT (nuclear factor of activated T cells)10–12. CD8+ T cells from humans with cancer or chronic viral infections13–15 expressed high levels of NR4A transcription factors and displayed enrichment of NR4A-binding motifs in accessible chromatin regions. CAR T cells lacking all three NR4A transcription factors (Nr4a triple knockout) promoted tumour regression and prolonged the survival of tumour-bearing mice. Nr4a triple knockout CAR tumour-infiltrating lymphocytes displayed phenotypes and gene expression profiles characteristic of CD8+ effector T cells, and chromatin regions uniquely accessible in Nr4a triple knockout CAR tumour-infiltrating lymphocytes compared to wild type were enriched for binding motifs for NF-κB and AP-1, transcription factors involved in activation of T cells. We identify NR4A transcription factors as having an important role in the cell-intrinsic program of T cell hyporesponsiveness and point to NR4A inhibition as a promising strategy for cancer immunotherapy. Transfer of NR4A-deficient T cells expressing chimeric antigen receptors is shown to reduce tumour burden and increase survival by shifting T cell transcriptional programs away from exhaustion and towards increased effector function.

Summary: Regulatory T (Treg) cells develop from a self-reactive, CD4-single positive (CD4SP) precursor cell pool. Thus, Treg-fated developing thymocytes are expected to possess the potential to generate pathogenic self-reactive cells. However, no such pathogenic conversion has been observed, indicating mechanisms of defense to prevent such a deleterious event. Here, we show that, after the initial developmental phase, the Nr4a family of nuclear receptors promotes the development of Treg cells by cooperating with other Treg cell developmental machineries, as well as by forming a reinforcing loop with Foxp3. Nr4a-deficient Treg-fated thymocytes survive and can elicit autoimmunity, highlighting their roles in elimination of developing Treg precursors that fail to complete their development. Our findings reveal that the defective development of Treg-fated thymocytes is a potential route for the generation of pathogenic self-reactive cells, which is normally suppressed by Nr4a factors at both developmental and cell death levels. : Sekiya et al. find that developing precursors of Treg cells are a labile population compared to mature Treg cells and can convert to pathogenic self-reactive cells. They show that Nr4a family nuclear receptors prevent this pathogenic conversion of Treg precursors at the level of both development and cell death. Keywords: Treg, Nr4a, Foxp3, negative selection, immune tolerance, inflammatory cytokine

The immune system is characterized by its dynamic responses to various infectious agents through different types and intensities of immune reactions. However, the immune system must also tolerate self and nonharmful environmental antigens. The immune system also exhibits memory for the primary infection, which enables efficient clearance of the same pathogen upon subsequent infections. One of the cellular machineries that deals with such complex demands on the immune system is epigenetics. Epigenetic modifications, which include different covalent modifications on histones and DNA, are characterized by their “multidimensional” regulation of gene activities. These epigenetic hallmarks not only mediate the switching on and off of transcription, but also regulate the kinetics and robustness, maintain the transcriptional status, and define the responsiveness or unresponsiveness of genes to external stimuli, thus elaborating the well-organized immune system. This chapter describes the epigenetic regulation of immune cell development, initially presenting the basic epigenetic regulatory mechanisms of gene activity and then presenting the epigenetic mechanisms that mediate the development of various immune cell types, with examples from both innate and adaptive arms of immunity.

Adoptive T-cell immunotherapy is a promising approach to cancer therapy. Stem cell memory T (TSCM) cells have been proposed as a class of long-lived and highly proliferative memory T cells. CD8+ TSCM cells can be generated in vitro from naive CD8+ T cells via Wnt signalling; however, methods do not yet exist for inducing TSCM cells from activated or memory T cells. Here, we show a strategy for generating TSCM-like cells in vitro (iTSCM cells) from activated CD4+ and CD8+ T cells in mice and humans by coculturing with stromal cells that express a Notch ligand. iTSCM cells lose PD-1 and CTLA-4 expression, and produce a large number of tumour-specific effector cells after restimulation. This method could therefore be used to generate antigen-specific effector T cells for adoptive immunotherapy., Tumour-specific T cells can be expanded in vitro and adoptively transferred for therapy, but this strategy is limited by induction of short-lived T cell populations. Here the authors activate Notch signalling in cultured mouse or human T cells, resulting in the production of a long-lived stem cell memory T cell population that can fight tumours in mice.

In cancer immunotherapy, CD19-targeted CAR T cells have exhibited impressive clinical efficacy against B cell leukemias and lymphomas; however, they have been less effective against solid tumors. This is in part because CAR T cells enter a hyporesponsive (“exhausted” or “dysfunctional”) state that is triggered by chronic antigen stimulation and characterized by upregulation of several inhibitory surface receptors and loss of effector function. To identify transcriptional regulators and other candidates contributing to the diminished CAR T cell function in solid tumors, we developed a CAR T cell model in which recipient mice bearing murine melanoma tumors expressing human CD19 were adoptively transferred with CD19-targeted CAR T cells. We demonstrate that in both CD8+ CAR T tumor-infiltrating lymphocytes (TILs) and endogenous CD8+ TILs expressing high levels of PD-1 and TIM3, the Nr4a proteins Nr4a1 (Nur77), Nr4a2 (Nurr1), and Nr4a3 (Nor1) are prominent effectors of the transcriptional program downstream of NFAT: they promote the expression of inhibitory receptors and genes associated with early hyporesponsive state, and limit effector function. Most importantly, treatment of tumor-bearing mice with CD8+ CAR T cells lacking all three Nr4a factors (Nr4aTKO) resulted in tumor regression and prolonged survival. Nr4aTKO CD8+ CAR T TILs displayed a transcriptomic profile characteristic of effector function, including upregulation of granzymes and cytokines, and many of these gene expression changes were associated with altered regulatory element accessibility near effector genes. Our data identify Nr4a transcription factors as major players in the cell-intrinsic program of T cell hyporesponsiveness and point to Nr4a inhibition as a promising strategy for cancer immunotherapy. Citation Format: Joyce Chen, Isaac F. Lopez-Moyado, Hyungseok Seo, Chan-Wang J. Lio, Laura J. Hempleman, Takashi Sekiya, Akihiko Yoshimura, James P. Scott-Browne, Anjana Rao. Nr4a transcription factors limit CAR T-cell function in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 937.

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1–deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte–associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function., Author summary T cells are pivotal in orchestrating an adaptive immune response. They are produced in the thymus and undergo selection processes resulting in elimination of nonfunctional and self-reactive cells in order to prevent autoimmune disease. One type of T cells, called regulatory T cells (Treg cells), is generated either in the thymus or in the periphery through a process termed agonist selection. Peripheral Treg cells are crucial for the suppression of unwanted immune responses. However, too much suppressive function of Treg cells can also impair immune responses directed against tumors. Here, we have analyzed the mechanisms that regulate this process and show that a microRNA, miR-181a/b-1, controls de novo generation of Treg cells by modulating agonist selection. We show that thymic and peripheral Treg cells deficient in miR-181a/b-1 contain higher levels of the key effector molecule CTLA-4, and as a consequence, such Treg cells display increased suppressive capacity. We observe that the expression of miR-181a/b-1 in peripheral Treg cells is much lower when compared to thymocytes; thus, our study implies that peripheral Treg-cell effector function is imprinted during intrathymic differentiation.

Aryl hydrocarbon receptor (AhR) is crucial for various immune responses. The relationship between AhR and infection with the intracellular bacteria Listeria monocytogenes (LM) is poorly understood. Here, we show that in response to LM infection, AhR is required for bacterial clearance by promoting macrophage survival and reactive oxygen species (ROS) production. AhR-deficient mice were more susceptible to listeriosis, and AhR deficiency enhances bacterial growth in vivo and in vitro. On the other hand, pro-inflammatory cytokines were increased in AhR-deficient macrophages infected with LM despite enhanced susceptibility to LM infection in AhR-deficient mice. Subsequent studies demonstrate that AhR protects against macrophage cell death induced by LM infection through the induction of the antiapoptotic factor, the apoptosis inhibitor of macrophages, which promotes macrophage survival in the setting of LM infection. Furthermore, AhR promotes ROS production for bacterial clearance. Our results demonstrate that AhR is essential to the resistance against LM infection as it promotes macrophage survival and ROS production. This suggests that the activation of AhR by its ligands may be an effective strategy against listeriosis.

The aryl hydrocarbon receptor (AhR), a ligand-activated nuclear transcription factor, is known to mediate the toxic and carcinogenic effects of various environmental pollutants, while AhR has been shown to protect animals from various types of tissue injury. ConA-induced hepatitis is known as a mouse model of acute liver injury. Here, we found a protective role of AhR in ConA-induced hepatitis. AhR is induced in the liver during ConA-induced hepatitis, and Ahr (-/-) mice were highly sensitive to this model. Bone marrow chimera experiments indicate that Ahr (-/-) hematopoietic cells are responsible for hypersensitivity to ConA-induced hepatitis. We found that IFN-γ from invariant NKT cells was up-regulated and IL-22 from innate lymphoid cells (ILCs) was abolished in Ahr (-/-) mice. In addition, IL-22 production was still observed in Rag2 (-/-) mice but it was severely reduced in Ahr (-/-) Rag2 (-/-) mice. ConA-induced IL-22 production was also dependent on retinoic acid-related orphan receptor γt. These results show that AhR has crucial protective roles in ConA-induced liver injury via promoting IL-22 production from ILCs and suppressing IFN-γ expression from NKT cells.

Suppressor of cytokine signaling-1 (SOCS1) has been shown to be an essential negative regulator of cytokine responses, including those of IFNγ, IL-2, IL-4 and IL-7. SOCS1 deficiency resulted in hyperactivation not only of T cells in general but also of NKT cells specifically. Consistent with previous reports, T- and NKT-cell-specific deletion of Socs1 in mice resulted in enhanced sensitivity to ConA-induced hepatitis. Compared with wild-type (WT) NKT cells, SOCS1-deficient NKT cells produced larger quantities of IFNγ in response to ConA and proliferated faster in response to IL-2 and IL-15. To our surprise, however, SOCS1-deficient NKT cells did not respond to the synthetic glycolipid ligand alpha-galactosylceramide (α-GalCer), though they did respond to sulfatide. α-GalCer-CD1d-tetramer-positive type I NKT [invariant NKT (iNKT)] cells were marginally detected in the periphery of SOCS1-conditional knockout (cKO) mice, suggesting that most of the SOCS1-deficient NKT cells at the periphery were type II NKT cells. Consistently, invariant Vα14 expression was much lower in SOCS1-deficient NKT cells than in WT NKT cells, indicating that iNKT cell homeostasis was abnormal in SOCS1-cKO mice. This reduction in iNKT cells was not observed in mice of an IFNγ-deficient background. These results suggest that SOCS1 is an important regulator of the balance between type I and type II NKT cells at the periphery.

In cancer immunotherapy, CD19-targeted CAR T-cells have exhibited impressive clinical efficacy against B cell leukemias and lymphomas; however, they have been less effective against solid tumors. This is in part because CAR T-cells enter a hyporesponsive (“exhausted” or “dysfunctional”) state that is triggered by chronic antigen stimulation and characterized by upregulation of several inhibitory receptors and loss of effector function. To identify transcriptional regulators and other candidates contributing to the diminished function of CAR T-cells in solid tumors, we developed a CAR T-cell model in which recipient mice bearing murine melanoma tumors expressing the human CD19 antigen were adoptively transferred with CD19-targeted CAR T-cells. Genome-wide analyses of these mouse tumor-infiltrating lymphocytes (TILs) showed that endogenous CD8+ TILs and CAR T TILs selected for expression of high levels of PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of the Nr4a nuclear receptor family of transcription factors by the transcription factor NFAT. We demonstrate that in both CAR T TILs and endogenous CD8+ TILs, the Nr4a proteins Nr4a1 (Nur77), Nr4a2 (Nurr1), and Nr4a3 (Nor1) are prominent effectors of the transcriptional program downstream of NFAT: they promote the expression of inhibitory receptors and genes associated with early stages of the exhausted/ dysfunctional state, and limit effector function. Most importantly, treatment of tumor-bearing mice with CAR T-cells lacking all three Nr4a transcription factors (Nr4aTKO) resulted in tumor regression and prolonged survival. Nr4aTKO tumor-infiltrating CAR T-cells displayed a gene expression profile characteristic of effector function, including increased expression of granzymes, cytokines and the transcription factor T-bet. Many of these gene expression changes were associated with altered regulatory element accessibility near effector genes. Our data identify Nr4a transcription factors as major players in the cell-intrinsic program of T-cell hyporesponsiveness and point to Nr4a inhibition as a promising strategy for cancer immunotherapy. Citation Format: Joyce Chen, James P. Scott-Browne, Isaac F. Lopez-Moyado, Laura J. Hempleman, Hyungseok Seo, Takashi Sekiya, Akihiko Yoshimura, Anjana Rao. Nr4a transcription factors limit CAR T-cell function in solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A178.

SOCS1 is required to restrict IFN-γ and IL-17 expression and maintain Foxp3 expression in and function of regulatory T cells., Regulatory T cells (Treg cells) maintain immune homeostasis by limiting inflammatory responses. SOCS1 (suppressor of cytokine signaling 1), a negative regulator of cytokine signaling, is necessary for the suppressor functions of Treg cells in vivo, yet detailed mechanisms remain to be clarified. We found that Socs1−/− Treg cells produced high levels of IFN-γ and rapidly lost Foxp3 when transferred into Rag2−/− mice or cultured in vitro, even though the CNS2 (conserved noncoding DNA sequence 2) in the Foxp3 enhancer region was fully demethylated. Socs1−/− Treg cells showed hyperactivation of STAT1 and STAT3. Because Foxp3 expression was stable and STAT1 activation was at normal levels in Ifnγ−/−Socs1−/− Treg cells, the restriction of IFN-γ–STAT1 signaling by SOCS1 is suggested to be necessary for stable Foxp3 expression. However, Ifnγ−/−Socs1−/− Treg cells had hyperactivated STAT3 and higher IL-17A (IL-17) production compared with Ifnγ−/−Socs1+/+ Treg cells and could not suppress colitis induced by naive T cells in Rag2−/− mice. In vitro experiments suggested that cytokines produced by Socs1−/− Treg cells and Ifnγ−/−Socs1−/− Treg cells modulated antigen-presenting cells for preferential Th1 and Th17 induction, respectively. We propose that SOCS1 plays important roles in Treg cell integrity and function by maintaining Foxp3 expression and by suppressing IFN-γ and IL-17 production driven by STAT1 and STAT3, respectively.

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.

Cell suspension cultures of Phytolacca americana L. (pokeweed) are capable of glycosylating capsaicinoids that have several biomedical applications. To identify the capsaicinoid glucosyltransferase involved in this biotransformation, we isolated three full-length cDNAs (PaGTs) encoding homologs of plant secondary product glycosyltransferases from cultured cells of P. americana L. These glycosyltransferase cDNAs were heterologously expressed in Escherichia coli cells and the expressed products were functionally characterized. Although all of these glycosyltransferases displayed broad glucosyl-acceptor specificities toward phenolics, capsaicinoid glucosyltransferase activity was found only for one of the cloned enzymes, PaGT3. Phylogenetic analysis showed that PaGT3 is the most closely related to betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis, and in fact, it displayed a weak betanidin 5-O-glucosyltransferase activity. Transcription analyses showed that the expression of PaGT3 in P. americana L. was strongly induced by exposure of the cells to capsaicin (0.65 mM). These results show that PaGT3 should be, at least in part, responsible for the capsaicinoid glucosyltransferase activity of this plant.

The endoderm is a multipotent progenitor cell population in the embryo that gives rise to the liver, pancreas, and other cell types and provides paradigms for understanding cell-type specification. Studies of isolated embryo tissue cells and genetic approaches in vivo have defined fibroblast growth factor/mitogen-activated protein kinase (FGF/MAPK) and bone morphogenetic protein (BMP) signaling pathways that induce liver and pancreatic fates in the endoderm. In undifferentiated endoderm cells, the FoxA and GATA transcription factors are among the first to engage silent genes, helping to endow competence for cell-type specification. FoxA proteins can bind their target sites in highly compacted chromatin and open up the local region for other factors to bind; hence, they have been termed "pioneer factors." We recently found that FoxA proteins remain bound to chromatin in mitosis, as an epigenetic mark. In embryonic stem cells, which lack FoxA, FoxA target sites can be occupied by FoxD3, which in turn helps to maintain a local demethylation of chromatin. By these means, a cascade of Fox factors helps to endow progenitor cells with the competence to activate genes in response to tissue-inductive signals. Understanding such epigenetic mechanisms for transcriptional competence coupled with knowledge of the relevant signals for cell-type specification should greatly facilitate efforts to predictably differentiate stem cells to liver and pancreatic fates.

CD4(+) T cells play central roles in adaptive immunity, driving appropriate immune responses to invading pathogens of diverse types. Four major CD4(+) T cell subsets, Th1, Th2, Th17, and Treg cells are differentiated from naive CD4(+) T cells upon ligation of their T cell receptors with antigens, depending on the cytokines they receive. Th1 cells, which are induced by IL-12 and IFN-γ, mediate host defense against intracellular pathogens by exclusively expressing IFN-γ. Th2 cells, which are induced by IL4, secrete IL-4, IL-5, and IL-13, and protect hosts from helminths. IL-6 plus TGF-β induces Th17 cells, another Th subset identified relatively recently, express IL-17 and play important roles in the eradication of extracellular bacteria and fungi. Treg cells, which play central roles in immune suppression, are composed of either thymus-derived Treg cells (tTreg cells), which are directly developed from CD4-single positive (CD4-SP) cells in the thymus, or peripherally derived Treg cells (pTreg cells), which are induced by TGF-β plus IL-2 from naive CD4(+) T cells. Although the regulated induction of Th cells results in proper eradication of pathogens, their excess activation results in various immune-associated diseases. For example, aberrant activation of Th1 and Th17 has been implicated in autoimmune diseases, excess Th2 activity causes atopic diseases, and impaired function of Treg cells due to abrogation of Foxp3 has been shown to cause fatal inflammatory disorders both in human and in mouse. The methods for in vitro differentiation of each Th subset described above are presented here. We hope these methods will facilitate understanding of differentiation and function of CD4(+) T cells and pathogenesis of various inflammatory diseases.

We investigated the expression and function of a panel of Toll-like receptors (TLRs) in human basophils. Basophil preparations constitutively expressed TLR2, TLR4, TLR9 and TLR10 mRNAs (TLR4 > TLR2 >> TLR9, TLR10). Although TLR mRNA expression in basophils was generally less prominent compared with those in neutrophils and monocytes, basophils expressed significantly higher levels of TLR2 and TLR4 mRNA than eosinophils. Various TLR ligands (Pam3Cys-Ser-Lys4, poly I:C, lipopolysaccharide, R-848, CpG DNA) were tested, but none affected the expression level of adhesion molecule CD11b or the viability of freshly purified basophils. On the other hand, when basophils were pretreated with interferon-γ before stimulation with TLR ligands, only the TLR4 ligand, lipopolysaccharide, upregulated CD11b expression. However, the surface levels of TLR2 and TLR4 on the interferon-γ-treated basophils showed no obvious changes. These results suggest that TLR4 on basophils may be involved in the pathogenesis of infection-induced exacerbation of allergic inflammation by modulating basophil functions.

Background 5-Lipoxygenase (5-LO) products have been strongly implicated in the pathogenesis of allergic diseases. In addition to their physiologic effects on residential cells, 5-LO products are capable of stimulating various eosinophil functions. However, little is known regarding the effects of 5-LO products on basophil functions. Objective This study was designed to elucidate the effects of the main 5-LO products (ie, leukotriene [LT] B 4 , LTD 4 , and 5-oxo-6,8,11,14-eicosatetraenoic acid [5-oxo-ETE]), as well as their receptor expression on human basophils. Methods We studied the effects of 5-LO products on Ca 2+ mobilization, migration, CD11b expression, and degranulation of human basophils. Expression of the receptors for LTC 4 /D 4 /E 4 (cysteinyl leukotriene 1 [CysLT 1 ] and CysLT 2 ), LTB4 (BLT 1 and BLT 2 ), and 5-oxo-ETE (oxoeicosanoid [OXE]) was assessed by means of real-time PCR and flow cytometry. Results At the mRNA level, basophils strongly expressed OXE and predominantly expressed CysLT 1 and BLT 2 . The expression level of OXE mRNA in basophils was approximately 20-fold higher than in neutrophils and similar to that in eosinophils. At the protein level, basophils expressed CysLT 1 , CysLT 2 , BLT 1 , and OXE, but not BLT 2 . All products elicited a transient increase of cytosolic calcium, with the order of magnitude being LTB 4 >5-oxo-ETE>LTD 4 . 5-Oxo-ETE induced a strong basophil migratory response that was almost equivalent to that of prostaglandin D 2 . LTB 4 elicited significant degranulation of IL-3–primed basophils. In contrast, no functional significance was observed for LTD 4 . Conclusion Among 5-LO products, 5-oxo-ETE induces a potent basophil migratory response, and LTB 4 elicits degranulation under certain conditions. Our results strongly suggest that 5-oxo-ETE might afford opportunities for therapeutic targeting in allergic inflammation.

Eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 bind specifically and exclusively to CC chemokine receptor (CCR) 3, which is a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Bronchial epithelial cells represent an important source of chemokines, and thus we investigated in vitro and in vivo expression of eotaxin-2 and eotaxin-3 in bronchial epithelial cells in comparison with that of eotaxin-1. Immunohistochemistry showed increased expression of both eotaxin-2 and eotaxin-3 in addition to eotaxin-1 in asthmatics. Considerable amounts of eotaxins were secreted by bronchial epithelial lineage. As with eotaxin-1 production, generation of eotaxin-2 and eotaxin-3 by bronchial epithelial cells was up-regulated by IL-4 and IL-13, and attenuated by IFN-gamma and glucocorticoids. In addition to eotaxin-1 expression, but also eotaxin-2 and eotaxin-3 expression in the bronchial epithelium should be taken into consideration when developing the therapeutic strategies to treat eosinophilic airway diseases.

Interleukin-12 (IL-12) is believed to play an important role in inducing Th1-type cytokine profiles. Atopic dermatitis (AD) and psoriasis vulgaris (PsV) are considered to be Th2 and Th1 type disease, respectively. The IL-12 p40 subunit gene (IL12B) is located at chromosome 5q31-33 and linkage findings of AD on 5q31 were reported. Recently single nucleotide polymorphism (SNP) (1188A/C) of IL12B has been reported. In function, it has been reported that this SNP is associated with IL12B mRNA expression levels. To learn whether this SNP is associated with susceptibility to AD or PsV, we investigated the genotype and allele frequencies of the SNP in AD patients, in PsV patients and in controls, examining 164 AD patients, 143 PsV patients and 100 healthy individuals in Japanese population. Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism method. The A allele was decreased in AD patients (40.9%, p=0.031) and increased in PsV patients (60.1%, p=0.035) compared with controls (50.5%). This suggests that IL12B SNP is associated with susceptibility to AD and PsV, presumably by affecting the Th1/Th2 balance.

Interleukin (IL)-13 plays an important role in the induction of immunoglobulin E (IgE) and in the pathogenesis of atopic dermatitis (AD). We investigated the allele and genotype frequencies of three IL-13 single nucleotide polymorphisms (SNPs) (A704C and C1103T in the promoter region and G4257A in exon 4) in Japanese patients with AD. For A704C and C1103T SNPs, there were no significant differences in allele or genotype frequencies between AD patients and controls. For G4257A SNP, A allele was significantly increased in AD patients (39.5%) compared with controls (29.4%) (P = 0.016). The same proportion of each genotype and allele was observed in the patient subgroup with and without asthma. Serum IgE levels and peripheral eosinophil counts were not significantly different among genotypes in G4257A SNP. There was also no significant difference in allele or genotype frequencies between AD patients with mild disease and those with severe disease, between those with family history of AD and those without it, or between those with family history of atopic disorders and those without it. This result suggests that 4257A allele is associated with susceptibility to AD and that it may function in the pathogenesis of AD itself, presumably by other mechanisms than inducing IgE production.

Antigen-specific regulatory T cells (Tregs) possess the potential to reduce excess of immune responses following allogeneic hematopoietic stem cell transplantation. Although antigen-specific in vitro- expanded Tregs (iTregs) have long been considered as a promising therapeutic agent against allo-immune reactions such as graft versus host disease (GVHD), accumulating evidences have suggested that iTregs easily lose their characteristics with quick cessation of Foxp3 expression , a lineage specifying transcription factor of Tregs, compared with in vivo -generated natural Tregs (nTregs). It has been revealed that insufficient demethylation of the CpG islands in conserved noncoding sequence 2 (CNS2) region of the Foxp3 locus mainly explains such instability of Foxp3 expression in iTregs. In order to overcome this drawback, we investigated the optimum way to generate stable iTregs for the prevention of a murine model of acute GVHD. We created a major-MHC mismatched GVHD model by transferring CD45.1 (H-2Kb) bone marrow (BM) cells together with effector T cells (CD4+25- and CD8 cells) into lethally irradiated (8Gy/body) 8-12-week old BALB/C (H-2Kd) recipients. In this model, recipient mice usually die within 7-10 days after bone marrow transplant (BMT) due to severe acute GVHD. Alloantigen-specific iTregs were generated by co-culturing naive T cells from human CD2 Foxp3 reporter mice (C57BL6/J background, H-2Kb) with BALB/C-derived antigen presenting cells in the presence of TGF-β, IL-2 and retinoic acid. Alloantigen-specific iTregs were then harvested 6-7 days after co-culture, by FACS-sorting the CD4+Foxp3+ population. Subsequently, sorted iTregs (106 cells) were transferred intravenously together with effector T cells (106 cells) and BM cells into recipients. We tested a panel of pharmacological agents and gene transduction during co-culture for their effects on iTreg stability, with FACS-based evaluation on day 7 after BMT. Among a panel of agents and gene modification tested, we found that only vitamin C-treated iTregs effectively improved Foxp3 maintenance compared with untreated iTregs (90%, vitamin C-treated iTregs vs 40%, untreated iTregs) in the GVHD model mice. As reported, vitamin C facilitated DNA demethylation of the Foxp3 CNS2 in iTregs in a Tet DNA demethylase-dependent manner. Bisulfite sequencing revealed a significant acceleration of CpG demethylation at the Foxp3 CNS2 by vitamin C, and the extent of demethylation achieved with vitamin C treatment reached to an equivalent level to those seen in nTregs. Furthermore, vitamin C mediated demethylation was extended to other Treg cell-specific regulatory lesions such as Tnfrsf18, Ikzf4, Ctla4 . On the other hand, untreated iTregs remain methylated at these loci to the same degree as naïve T cells (p In summary, vitamin C stabilized Foxp3 expression with specific demethylation of Foxp3 CNS2 as well as Treg-associated genes in antigen-specific iTregs, preventing them from conversion into inflammatory ex- Foxp3 iTregs, thus ameliorating the pathogenesis of a murine model of GVHD. Our findings strongly suggest the potential of clinical application of Treg therapy combined with vitamin C treatment, for GVHD and autoimmune diseases. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Transplantation of endothelial cells (ECs) is a promising therapeutic approach for ischemic disorders. In addition, the generation of ECs has become increasingly important for providing vascular plexus to regenerated organs, such as the liver. Although many attempts have been made to generate ECs from pluripotent stem cells and nonvascular cells, the minimum number of transcription factors that specialize in directly inducing vascular ECs remains undefined. Here, by screening 18 transcription factors that are important for both endothelial and hematopoietic development, we demonstrate that ets variant 2 (ETV2) alone directly converts primary human adult skin fibroblasts into functional vascular endothelial cells (ETVECs). In coordination with endogenous FOXC2 in fibroblasts, transduced ETV2 elicits expression of multiple key endothelial development factors, including FLI1, ERG, and TAL1, and induces expression of endothelial functional molecules, including EGFL7 and von Willebrand factor. Consequently, ETVECs exhibits EC characteristics in vitro and forms mature functional vasculature in Matrigel plugs transplanted in NOD SCID mice. Furthermore, ETVECs significantly improve blood flow recovery in a hind limb ischemic model using BALB/c-nu mice. Our study indicates that the creation of ETVECs provides further understanding of human EC development induced by ETV2.

Xenoestrogens (XEs) are a diverse group of chemicals that mimic estrogenic actions and may have adverse effects on human health. The influence of these compounds on cytokine production or immune system function remains unclear. In this study we have examined the effects of 17beta-estradiol (E2) and XEs on chemoattractant cytokine (chemokine) production and analyzed the molecular mechanism. Monocyte chemoattractant protein-1 (MCP-1), also termed monocyte chemotactic and activating factor, is a member of the chemokine family and attracts mainly blood monocytes. Human mammary tumor cell line MCF-7 cells produce a large quantity of MCP-1 in response to interleukin-1alpha (IL-1alpha). Addition of E2 to MCF-7 cells inhibited MCP-1 production in a dose-dependent manner. XEs, bisphenol A, and NP also inhibited MCP-1 production, although the potency was 3-4 orders of magnitude lower than that of E2. E2, bisphenol A, and NP inhibited MCP-1 messenger RNA expression in MCF-7 cells. Two closely located nuclear factor-kappaB sites, A1 and A2, have been identified in the promoter of the human MCP-1 gene. A luciferase construct containing this enhancer region (pGLM-ENH) was activated by IL-1alpha, and a mutation at either the A1 or A2 site resulted in a loss of IL-1alpha responsiveness. Treatment with E2 or XEs decreased the IL-1alpha-inducible pGLM-ENH luciferase activity significantly. In an electrophoretic mobility shift assay and supershift analysis, we found that treatment with E2 or XEs diminished the IL-1alpha-induced complex formation with both A1 and A2 probes, which was identified immunochemically to consist of nuclear factor-kappaB, p50, and p65. The IL-1alpha-induced p50/c-Rel complex to the A2 probe was also, to a lesser extent, decreased by E2 or XE treatment. The effects of E2 and XEs on the expression of MCP-1 seem to be much more dramatic than the effects of these agents on the promoters used in the luciferase assay, suggesting the involvement of an additional site(s) of the promoter region of the MCP-1 gene or posttranscriptional regulation of MCP-1 gene expression by E2 and XEs. This work represents the first report describing possible regulation of immune system function by XEs through inhibiting chemokine production.

IL-9 is a pleiotropic cytokine that can regulate autoimmune and allergic responses. Th9 cells can develop from naive T cells or Th2 cells through stimulation by TGF-β in vitro. In this study, we demonstrated that Smad2 and Smad3 are necessary for IL-9 production from T cells in an OVA-induced asthma model using T cell–specific Smad2- and Smad3-deficient mice. Smad2 and Smad3 were also redundantly essential for TGF-β signaling to induce histone modifications for Il9 transcription. Although Smad2/3 was recruited to the Il9 promoter by TGF-β stimulation, they are not sufficient to activate the Il9 promoter. By the screening the transcription factors, we found that IFN regulatory factor 4 (IRF4) was essential for the Smad2/3-mediated Il9 promoter activation. In addition, Smad2/3 physically interacted with IRF4, and Smad2/3 did not bind to the Il9 promoter and could not induce Th9 in IRF4-deficient T cells. Similarly, IRF4 could not stimulate Il9 transcription in the absence of Smad2/3, and TGF-β enhanced IRF4 recruitment to the Il9 promoter in a Smad2/3-dependent manner. We propose that Smad2/3 and IRF4 cooperatively transactivate the Il9 promoter and play an important role in regulating allergic immune responses by inducing Th9 cells.

Choroidal neovascularization (CNV) is a characteristic of age-related macular degeneration. Genome-wide association studies have provided evidence that the immune system is involved in the pathogenesis of age-related macular degeneration; however, the role of inflammatory cytokines in CNV has not been established. In this study, we demonstrated that IL-17 had a strong potential for promoting neovascularization in a vascular endothelial growth factor–independent manner in laser-induced experimental CNV in mice. Infiltrated γδT cells and Thy-1+ innate lymphoid cells, but not Th17 cells, were the main sources of IL-17 in injured eyes. IL-23 was dispensable for IL-17 induction in the eye. Instead, we found that IL-1β and high-mobility group box 1 strongly promoted IL-17 expression by γδT cells. Suppression of IL-1β and high-mobility group box 1, as well as depletion of γδT cells, reduced IL-17 levels and ameliorated experimental CNV. Our findings suggest the existence of a novel inflammatory cytokine network that promotes neovascularization in the eye.

Post-ischemic inflammation is an essential step in the progression of brain ischemia-reperfusion injury. However, the mechanism that activates infiltrating macrophages in the ischemic brain remains to be clarified. Here we demonstrate that peroxiredoxin (Prx) family proteins released extracellularly from necrotic brain cells induce expression of inflammatory cytokines including interleukin-23 in macrophages through activation of Toll-like receptor 2 (TLR2) and TLR4, thereby promoting neural cell death, even though intracellular Prxs have been shown to be neuroprotective. The extracellular release of Prxs in the ischemic core occurred 12 h after stroke onset, and neutralization of extracellular Prxs with antibodies suppressed inflammatory cytokine expression and infarct volume growth. In contrast, high mobility group box 1 (HMGB1), a well-known damage-associated molecular pattern molecule, was released before Prx and had a limited role in post-ischemic macrophage activation. We thus propose that extracellular Prxs are previously unknown danger signals in the ischemic brain and that its blocking agents are potent neuroprotective tools.

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.

There exists a hierarchy by which transcription factors can engage their target sites in chromatin, in that a subset of factors can bind transcriptionally silent, nucleosomal DNA, whereas most factors cannot, and this hierarchy is reflected, at least in part, in the developmental function of the factors. For example, transcription factors possessing the Forkhead box (Fox) DNAbinding domain contain an overall fold resembling that of linker histone and thus are structured to bind DNA, site specifically, in a nucleosomal context. Where tested, Fox factors bind early in the developmental or physiological activation of target genes, th ereby enabling the binding of other factors that cannot engage c hromatin on their own. To investigate the basis for early chromatin binding, we have used fluorescence recovery after photobleaching (FRAP) to analyze the mobility, in the live cell nucleus, of FoxA factors in comparison to linker histone and other transcription factors. We have further analyzed the factors for their ability to bind to chromatin in mitosis and thereby serve as epigenetic marks. The re sults indicate that the “pioneer” features of FoxA factors involve various chromatin-binding parameters seen in linker histones and that distinguish the factors with respect to their regulatory and mechanistic functions.

The CC chemokine eotaxin (CCL11) plays a major role in the recruitment and activation of eosinophils in allergic disorders. In the present study, we performed polymorphism screening of the coding and promoter regions of the eotaxin gene (SCYA11). A G to A single nucleotide substitution was detected at position 67, which resulted in a non-conservative amino acid change of Ala at position 23 to Thr (A23T) within the signal peptide. Two single nucleotide substitutions, ie, C to T at position −426 (−426C>T), and A to G at position −384 (−384A>G), were detected in the 5′-flanking regions. Significant linkage disequilibrium was observed between positions −426 and −384, and also between −384 and +67. No significant association was observed between these variations and susceptibility to asthma.

SummaryTransforming growth factor-β (TGF-β) has been shown to be required for Th17 cell differentiation via Smad-independent mechanisms. The molecular mechanism underlying this pathway remains to be clarified, however. We searched for genes regulated by TGF-β through the Smad-independent pathway by using Smad2 and Smad3 double-deficient T cells and identified the transcription factor Eomesodermin (Eomes), whose expression was suppressed by TGF-β via the c-Jun N-terminal kinase (JNK)-c-Jun signaling pathway. Inhibition of JNK strongly suppressed disease in an in vivo EAE model as well as in vitro Th17 cell induction. Overexpression of Eomes substantially suppressed Th17 cell differentiation, whereas ablation of Eomes expression could substitute for TGF-β in Th17 cell induction in primary T cells. Eomes suppressed Rorc and Il17a promoters by directly binding to the proximal region of these promoters. In conclusion, the suppression of Eomes by TGF-β via the JNK pathway is an important mechanism for Smad-independent Th17 cell differentiation.

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., The gut is populated by a myriad of microorganisms and how the immune system tolerates their presence is of great interest. Here, by studying colon morphology in multiple knockout mice, the authors demonstrate a potential role for prostaglandin E2 and SOCS1 in mediating immune tolerance.

T(h) cells have long been divided into two subsets, T(h)1 and T(h)2; however, recently, T(h)17 and inducible regulatory T (iTreg) cells were identified as new T(h) cell subsets. Although T(h)1- and T(h)2-polarizing cytokines have been shown to suppress T(h)17 and iTreg development, transcriptional regulation of T(h)17 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 T(h)2 development, was repressed in T(h)17 and iTreg cells compared with T(h)1 and T(h)2 lineages. Gfi1 expression was enhanced by the IFN-gamma/STAT1 and IL-4/STAT6 pathways, whereas it was repressed by the transforming growth factor-beta1 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 gammat to the IL-17A promoter. In contrast, IL-17A expression was significantly enhanced in Gfi1-deficient T cells under T(h)17-promoting differentiation conditions as compared with wild-type T cells. In contrast, the impacts of Gfi1 in iTregs were not as strong as in T(h)17 cells. Taken together, these data strongly suggest that Gfi1 is a negative regulator of T(h)17 differentiation, which represents a novel mechanism for the regulation of T(h)17 development by cytokines.

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.

FoxA proteins are pioneer transcription factors, among the first to bind chromatin domains in development and enable gene activity. The Fox DNA-binding domain structurally resembles linker histone and binds nucleosomes stably. Using fluorescence recovery after photobleaching, we found that FoxA1 and FoxA2 move much more slowly in nuclei than other transcription factor types, including c-Myc, GATA-4, NF-1, and HMGB1. We find that slower nuclear mobility correlates with high nonspecific nucleosome binding, and point mutations that disrupt nonspecific binding markedly increase nuclear mobility. FoxA's distinct nuclear mobility is consistent with its pioneer activity in chromatin.

AIM: To investigate the clinical significance of BMP and activin membrane-bound inhibitor (BAMBI) which is a pseudoreceptor of transforming growth factor-beta (TGF-β) type I receptors and acts as a negative regulator of TGF-β signaling and expression aberrantly elevated in colorectal cancers (CRCs). We studied BAMBI expression in CRCs. METHODS: We studied BAMBI expression in 183 surgically resected CRCs by immunochemical and immunoblotting analyses using a generated monoclonal anti-BAMBI antibody. Commercially available anti-β-catenin and anti-p53 antibodies were also applied for immunochemical analyses as a comparison control. RESULTS: Immunohistochemical analysis revealed that BAMBI expression was observed in 148 (80.8%), and strong BAMBI expression was observed in 46% of the CRCs. Strong BAMBI expression was positively correlated with histological type, depth of invasion, lymph node metastases, and tumor node metastasis (TNM) stage (P < 0.05). Clear associations were found between BAMBI and β-catenin (P = 0.035) and p53 (P = 0.049) expression. In curatively resected CRC, 5-year recurrence-free survival was 51.9% (P = 0.037) for strong BAMBI expression compared to 79.8% for weak BAMBI expression. In the Cox’s multivariate analysis, lymph node metastases (RR 6.685; P < 0.001) and depth of invasion (RR 14.0; P = 0.013) were significant indicators for recurrence, and strong BAMBI expression (RR 2.26; P = 0.057) tended to be significant. CONCLUSION: BAMBI was linked to a potentially aggressive tumor phenotype and predicted tumor recurrence and cancer-related death in CRC. BAMBI expression might be applicable in the routine clinical setting of CRC.

Groucho-related (Gro/TLE/Grg) corepressors meditate embryonic segmentation, dorsal-ventral patterning, neurogenesis, and Notch and Wnt signaling. Although Gro/TLE/Grgs disrupt activator complexes and recruit histone deacetylases (HDAC), activator complexes can be disrupted in various ways, HDAC recruitment does not account for full corepressor activity, and a direct role for Gro/TLE/Grg binding and altering chromatin structure has not been explored. Using diverse chromatin substrates in vitro, we show that Grg3 creates higher-order, condensed complexes of polynucleosome arrays. Surprisingly, such complexes are in an open, exposed configuration. We find that chromatin binding enables Grg3 recruitment by a transcription factor and the creation of a closed, poorly accessible domain spanning three to four nucleosomes. Targeted recruitment of Grg3 blankets a similar-sized region in vivo, impairing activator recruitment and repressing transcription. These activities of a Groucho protein represent a newly discovered mechanism which differs from that of other classes of corepressors.

Microparticles have been manipulated in a microfluidic channel by means of negative dielectrophoresis (n-DEP), and the approach applied to a heterogeneous immunoassay system. A microfluidic device, with three-dimensional (3-D) microelectrodes fabricated on two substrates, was used to manipulate particle flow in the channel and to capture the particles in the caged area that was enclosed by the collector electrodes. Polystyrene microparticles (6 μm diameters) modified with anti-mouse immunoglobulin G (IgG) were manipulated and captured in the caged area when surrounded by intense n-DEP electric fields. Specifically, particles were trapped when AC voltages with amplitudes of 6–15 V peak and frequencies over 500 kHz were applied to the two facing microelectrodes. A heterogeneous sandwich immunoassay was achieved by successively injecting a sample solution containing mouse antigen (IgG), and a solution containing a secondary antibody with a signal source (FITC-labeled anti-mouse IgG antibody), into the channel. The fluorescence intensity from captured particles in the caged area increased with increasing concentrations (10 ng/ml to 10 μg/ml) of mouse IgG. The described system enables mouse IgG to be assayed in 40 min. Thus, the automatic separation of free fractions from desired analytes and labeled antibodies can be achieved using a microfluidic device based on n-DEP.

We recently demonstrated that glycyrrhizin (GL) and its derivatives down-regulate TNFalpha- and IL-4-induced eotaxin 1 production by the human fetal lung fibroblast line HFL-1 at protein or mRNA levels. In particular, the GL derivative hetero-30-OH-GL (3beta-[(2-O-beta-D-glucopyranuronosyl-beta-D-glucopyranuronosyl)oxy]-olean-11,13(18)-dien-30-ol) showed marked inhibition of eotaxin 1 production with less cytotoxicity than 18beta-GL. To identify the molecular mechanism of this effect, we focused on the inhibition of the transcriptional factors NF-kappaB and signal transducer and activator of transcription 6 (STAT6), which regulate eotaxin 1 gene activation. STAT6 phosphorylation and translocation of phospho-STAT6 from cytosol to nuclei were slightly inhibited by 18beta-GL and significantly inhibited by hetero-30-OH-GL. While IkappaBalpha degradation and translocation of NF-kappaB p65 to nuclei were not significantly affected by either compound, the stability of eotaxin-1 mRNA was decreased with hetero-30-OH-GL. In addition, eotaxin 1 promoter activity was markedly inhibited by hetero-30-OH-GL. Electrophoretic mobility shift assay (EMSA) confirmed these results. Thus, hetero-30-OH-GL significantly inhibited eotaxin 1 expression by the selective inhibition of IL-4 signal transduction as well as by enhanced mRNA degradation.

Background: Thymus and activation-regulated chemokine (TARC) plays an important role in the pathogenesis of atopic dermatitis (AD). We recently detected the single nucleotide polymorphism (SNP) (−431C>T) in the 5′-flanking region of TARC gene. Objectives: To examine whether the −431C>T SNP of the TARC gene is associated with susceptibility to AD and whether it affects the promoter activity of the TARC gene. Methods: We investigated the genotype and allele frequencies of the SNP in 193 AD patients and 158 healthy controls by polymerase chain reaction-restriction fragment length polymorphism method. We compared the promoter activities between TARC promoter carrying 431C and that carrying −431T by transient-transfection assay in DJM-1 cell line. Results: There were no significant differences in genotype or allele frequencies between AD patients and controls (genotype: P = 0.38, allele: P = 0.22). Luciferase activity was higher in −431T constructs than in −431C constructs (2.3-fold, P = 9.5 × 10−6). Conclusion: These results suggest that the −431C>T SNP of the TARC gene enhances the promoter activity of TARC gene but is not associated with susceptibility to AD in Japanese population.

The Wnt signaling pathway is activated in most human colorectal tumors. Mutational inactivation in the tumor suppressor adenomatous polyposis coli (APC), as well as activation of beta-catenin, causes the accumulation of beta-catenin, which in turn associates with the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors and activates transcription of their target genes. Here we show that beta-catenin activates transcription of the BMP and activin membrane-bound inhibitor (BAMBI)/NMA gene. The expression level of BAMBI was found to be aberrantly elevated in most colorectal and hepatocellular carcinomas relative to the corresponding non-cancerous tissues. Expression of BAMBI in colorectal tumor cell lines was repressed by a dominant-negative mutant of TCF-4 or by an inhibitor of beta-catenin-TCF interaction, suggesting that beta-catenin is responsible for the aberrant expression of BAMBI in colorectal tumor cells. Furthermore, overexpression of BAMBI inhibited the response of tumor cells to transforming growth factor-beta signaling. These results suggest that beta-catenin interferes with transforming growth factor-beta-mediated growth arrest by inducing the expression of BAMBI, and this may contribute to colorectal and hepatocellular tumorigenesis.

Eotaxin is believed to play an important role in atopic dermatitis (AD) as a potent chemoattractant and activator of eosinophils and Th2 lymphocytes. The eotaxin gene is located at chromosome 17q21.1–q21.2, and linkage findings of AD on chromosome 17 were reported. Recently we have identified single nucleotide polymorphisms (SNPs) of eotaxin gene (−426C>T, −384A>G, 67G>A). To learn whether eotaxin gene SNPs are associated with susceptibility to AD or phenotypes of AD, we investigated the genotype frequencies at each SNP of the gene in AD patients and in controls. We examined 140 Japanese AD patients and 140 healthy Japanese individuals. Genotyping was performed using the polymerase chain reaction–restriction fragment length polymorphism method. No significant difference was observed in allele or genotype frequencies of any SNP between AD patients and controls. Serum immunoglobulin E (IgE) levels were significantly lower in CT and TT genotype than in CC (P=0.038) in −426C>T SNP, and lower in GG than in AA and AG with borderline significance (P=0.053) in −384A>G SNP in AD patients. Eotaxin gene SNPs in the promoter and exon regions are not associated with susceptibility to AD, but two of them in the promoter region are associated with phenotype of AD.

Th2-specific chemokine thymus and activation-regulated chemokine (TARC)/CC chemokine ligand (CCL)17 is highly implicated in the pathogenesis of Th-2-dominated allergic diseases such as bronchial asthma (BA) and atopic dermatitis (AD). We performed polymorphism screening of the coding and promoter regions of the TARC gene. We found two rare variations in the coding region of exon 3 (2134C>T and 2037G>A) and a single nucleotide polymorphism (SNP) in the 5'-flanking region (-431C>T). Individuals carrying the 431T allele showed significantly increased serum levels of TARC compared with those not carrying the 431T allele, suggesting that this SNP has functional significance. However, when the genotypes at the SNP site were determined for 158 healthy individuals, 105 patients with BA and 148 patients with AD, we observed no significant association of the SNP with susceptibility to BA or AD.

CCR4 is now known to be selectively expressed in Th2 cells. Since the bronchial epithelium is recognized as an important source of mediators fundamental to the manifestation of respiratory allergic inflammation, we studied the expression of two functional ligands for CCR4, i.e., macrophage-derived chemokine (MDC) and thymus- and activation-regulated chemokine (TARC), in bronchial epithelial cells. The bronchial epithelium of asthmatics and normal subjects expressed TARC protein, and the asthmatics showed more intense expression than the normal subjects. On the other hand, MDC expression was only weakly detected in the asthmatics, but the intensity was not significantly different from that of normal subjects. Combination of TNF-α and IL-4 induced expression of TARC protein and mRNA in bronchial epithelial A549 cells, which was slightly up-regulated by IFN-γ. The enhancement by IFN-γ was more pronounced in bronchial epithelial BEAS-2B cells, and a maximum production occurred with combination of TNF-α, IL-4, and IFN-γ. On the other hand, MDC was essentially not expressed in any of the cultures. Furthermore, expressions of TARC protein and mRNA were almost completely inhibited by glucocorticoids. These results indicate that the airway epithelium represents an important source of TARC, which potentially plays a role via a paracrine mechanism in the development of allergic respiratory diseases. Furthermore, the beneficial effect of inhaled glucocorticoids on asthma may be at least in part due to their direct inhibitory effect on TARC generation by the bronchial epithelium.

The rate of graft survival has dramatically increased using calcineurin inhibitors, however chronic graft rejection and risk of infection are difficult to manage. Induction of allograft-specific regulatory T-cells (Tregs) is considered an ideal way to achieve long-term tolerance for allografts. However, efficient in vitro methods for developing allograft-specific Tregs which is applicable to MHC full-mismatched cardiac transplant models have not been established. We compared antigen-nonspecific polyclonal-induced Tregs (iTregs) as well as antigen-specific iTregs and thymus-derived Tregs (nTregs) that were expanded via direct and indirect pathways. We found that iTregs induced via the indirect pathway had the greatest ability to prolong graft survival and suppress angiitis. Antigen-specific iTregs generated ex vivo via both direct and indirect pathways using dendritic cells from F1 mice also induced long-term engraftment without using MHC peptides. In antigen-specific Treg transferred models, activation of dendritic cells and allograft-specific CTL generation were suppressed. The present study demonstrated the potential of ex vivo antigen-specific Treg expansion for clinical cell-based therapeutic approaches to induce lifelong immunological tolerance for allogeneic cardiac transplants.

8 B~A ~ Increased Levels of a TH2-Type CC Chemokine Thymus and ~p-lr Activation-Regulated Chemokine (TARC) in Serum and Induced Sputum of Asthmatics Takashi Sekiya*, Hirokazu Yamada*, Masao Yamaguchi*, Akira lshii*, Kazuhiko Yamamoto*, Atsushi Morita§, Yasuyuki Sano¥ Osamu Yoshie~, Kouji Matsushima*, Koichi Hirai* *University of Tokyo, Tokyo, Japan §Shionogi Institute for Medical Science, Osaka, Japan ~q)oai Memorial Hospital, Tokyo, Japan ~Kinki University, Osaka, Japan BACKGROUND: Cytokines liberated by Th2 cells play a crucial role in the pathogenesis of bronchial asthma. Recent studies have demonstrated that CC chemokine receptor (CCR)4 is preferentially expressed in Th2 cells. These facts suggest the possible involvement of both of CCR4-specific ligands, i.e., thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), in pathogenesis of bronchial asthma via recruitment of Th2 cells at inflammatory sites. We explored the levels of TARC and MDC in serum and induced sputum of asthmatics. METHODS: Levels of TARC in serum (46 asthmatics and 26 normal individuals) and induced sputa (30 asthmatics and 6 normal individuals) were measured by a high-sensitive ELISA system. Levels of eotaxin and MDC were also measured by ELISA. RESULTS: TARC but not MDC was significantly increased in asthmatic sera (p

Freeze-fracture electron microscopy of the plasma membranes of Candida albicans yeast cells and red blood cells treated with amphotericin methyl ester and amphotericin B showed that amphotericin B (50 micrograms ml-1) caused extreme aggregation of intramembranous particles on the protoplasmic fracture face of the C. albicans membrane, and a marked reduction of the density of intramembranous particles. On the other hand, the rearrangement of intramembranous particles induced by amphotericin methyl ester (50 micrograms ml-1) produced elevations of the particle-free membrane domains toward the outside of the cells, so that the particles were aggregated in linear furrows surrounding these elevations on the protoplasmic fracture face, and the corresponding ridges on the exoplasmic fracture face. The density of intramembranous particles was greatly reduced on the protoplasmic fracture face. Both polyenes produced only small changes in the erythrocyte membranes at the same concentration. These results suggest that amphotericin methyl ester affects the ergosterol-containing membranes more than amphotericin B, and that ergosterol has a higher sensitivity for these two polyene antibiotics than cholesterol.

Freeze-fractures of the conidial wall surface of the deuteromycetous fungi Penicillium sp., Cladobotryum varium, Cladosporium macrocarpum, and Aspergillus niger and of the sporangiospore surface of the zygomycetous fungus Mycotypha microspora reveal rodlet fascicles. Shadow replicas and deep-etching of these cells demonstrate an outermost amorphous layer adjacent to the rodlet fascicles. In each case, individual rodlets are apparently composed of chains of subunits (approx 50 A diam) while interdigitated fascicles consist of clusters of rodlets arranged in parallel. The conidial wall of A. niger was separated into an outer and inner layer by passing the cells through a cell fractionator. Rodlets were present on the extracellular surface of the isolated and purified outer layer, but absent from the inner layer. The hyphal wall of A. niger was also isolated and purified. A comparison of the chemical composition of the three wall layers demonstrated distinct quantitative differences in total carbohydrate, monosaccharide, total hexosamine, peptide, and lipid content. On the basis of the gross chemical composition of the outer conidial wall layer, as well as data obtained from enzymatic digestion and various chemical treatments of this layer followed by ultrastructural examinations, it is suggested that rodlets of A. niger are mainly proteinaceous.