Toshiaki Ohteki

Cloning of adiponectin receptors that mediate antidiabetic metabolic effects

Corrigendum (2004)10.1038/nature03091Adiponectin (also known as 30-kDa adipocyte complement-related protein; Acrp30) is a hormone secreted by adipocytes that acts as an antidiabetic and anti-atherogenic adipokine. Levels of adiponectin in the blood are decreased under conditions of obesity, insulin resistance and type 2 diabetes. Administration of adiponectin causes glucose-lowering effects and ameliorates insulin resistance in mice. Conversely, adiponectin-deficient mice exhibit insulin resistance and diabetes. This insulin-sensitizing effect of adiponectin seems to be mediated by an increase in fatty-acid oxidation through activation of AMP kinase and PPAR-α. Here we report the cloning of complementary DNAs encoding adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) by expression cloning. AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver. These two adiponectin receptors are predicted to contain seven transmembrane domains, but to be structurally and functionally distinct from G-protein-coupled receptors. Expression of AdipoR1/R2 or suppression of AdipoR1/R2 expression by small-interfering RNA supports our conclusion that they serve as receptors for globular and full-length adiponectin, and that they mediate increased AMP kinase and PPAR-α ligand activities, as well as fatty-acid oxidation and glucose uptake by adiponectin.

Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow.

Lymphoid tissue plasmacytoid and conventional dendritic cells (DCs) are continuously regenerated from hematopoietic stem cells. The cytokine dependence and biology of plasmacytoid and conventional DCs suggest that regeneration might proceed through common DC-restricted developmental intermediates. By selecting for cytokine receptor expression relevant to DC development, we identify here highly cycling Lin−c-KitintFlt3+M-CSFR+ cells with a distinct gene-expression profile in mouse bone marrow that, on a clonal level in vitro and as a population both in vitro and in vivo, efficiently generated plasmacytoid and conventional DCs but no other lineages, which increased in number after in vivo injection of the cytokine Flt3 ligand. These clonogenic common DC progenitors thus define a cytokine-regulated DC developmental pathway that ensures the supply of various DC populations.

T Cell-Specific Loss of Pten Leads to Defects in Central and Peripheral Tolerance

PTEN, a tumor suppressor gene, is essential for embryogenesis. We used the Cre-loxP system to generate a T cell-specific deletion of the Pten gene (Pten(flox/-) mice). All Pten(flox/-) mice develop CD4+ T cell lymphomas by 17 weeks. Pten(flox/-) mice show increased thymic cellularity due in part to a defect in thymic negative selection. Pten(flox/-) mice exhibit elevated levels of B cells and CD4+ T cells in the periphery, spontaneous activation of CD4+ T cells, autoantibody production, and hypergammaglobulinemia. Pten(flox/-) T cells hyperproliferate, are autoreactive, secrete increased levels of Th1/Th2 cytokines, resist apoptosis, and show increased phosphorylation of PKB/Akt and ERK. Peripheral tolerance to SEB is also impaired in Pten(flox/-) mice. PTEN is thus an important regulator of T cell homeostasis and self-tolerance.

Selective contribution of IFN-α/β signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection

A complex mechanism may be operational for dendritic cell (DC) maturation, wherein Toll-like receptor and other signaling pathways may be coordinated differently depending on the nature of the pathogens, in order for DC maturation to be most effective to a given threat. Here, we show that IFN-α/β signaling is selectively required for the maturation of DCs induced by double-stranded RNA or viral infection in vitro. Interestingly, the maturation is still observed in the absence of either of the two target genes of IFN-α/β, TLR3 and PKR (double-stranded-RNA-dependent protein kinase R), indicating the complexity of the IFN-α/β-induced transcriptional program in DCs. We also show that the DCs stimulated in vivo by these agents can migrate into the T cell zone of the spleen but fail to mature without the IFN signal. The immune system may have acquired the selective utilization of this cytokine system, which is essential for innate antiviral immunity, to effectively couple with the induction of adaptive immunity.

The Transcription Factor NF-ATc1 Regulates Lymphocyte Proliferation and Th2 Cytokine Production

NF-ATc1 is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In activated T cells, nuclear NF-AT binds to the promoter regions of multiple cytokine genes and induces their transcription. The role of NF-ATc1 was investigated in recombination activating gene-1 (RAG-1)-deficient blastocyst complementation assays using homozygous NF-ATc1-/- mutant ES cell lines. NF-ATc1-/-/RAG-1-/- chimeric mice showed reduced numbers of thymocytes and impaired proliferation of peripheral lymphocytes, but normal production of IL-2. Induction in vitro of Th2 responses, as demonstrated by a decrease in IL-4 and IL-6 production, was impaired in mutant T cells. These data indicate that NF-ATc1 plays roles in the development of T lymphocytes and in the differentiation of the Th2 response.

Regulation of IgA production by naturally occurring TNF/iNOS-producing dendritic cells

Immunoglobulin-A has an irreplaceable role in the mucosal defence against infectious microbes. In human and mouse, IgA-producing plasma cells comprise ∼20% of total plasma cells of peripheral lymphoid tissues, whereas more than 80% of plasma cells produce IgA in mucosa-associated lymphoid tissues (MALT). One of the most biologically important and long-standing questions in immunology is why this ‘biased’ IgA synthesis takes place in the MALT but not other lymphoid organs. Here we show that IgA class-switch recombination (CSR) is impaired in inducible-nitric-oxide-synthase-deficient (iNOS-/-; gene also called Nos2) mice. iNOS regulates the T-cell-dependent IgA CSR through expression of transforming growth factor-β receptor, and the T-cell-independent IgA CSR through production of a proliferation-inducing ligand (APRIL, also called Tnfsf13) and a B-cell-activating factor of the tumour necrosis factor (TNF) family (BAFF, also called Tnfsf13b). Notably, iNOS is preferentially expressed in MALT dendritic cells in response to the recognition of commensal bacteria by toll-like receptor. Furthermore, adoptive transfer of iNOS+ dendritic cells rescues IgA production in iNOS-/- mice. Further analysis revealed that the MALT dendritic cells are a TNF-α/iNOS-producing dendritic-cell subset, originally identified in mice infected with Listeria monocytogenes. The presence of a naturally occurring TNF-α/iNOS-producing dendritic-cell subset may explain the predominance of IgA production in the MALT, critical for gut homeostasis.

Synergistic Effect of Nod1 and Nod2 Agonists with Toll-Like Receptor Agonists on Human Dendritic Cells To Generate Interleukin-12 and T Helper Type 1 Cells

ABSTRACT A synthetic Nod2 agonist, muramyldipeptide (MDP), and two Nod1 agonists, FK565 and FK156, mimic the bacterial peptidoglycan moiety and are powerful adjuvants that induce cell-mediated immunity, especially delayed-type hypersensitivity. In this study, we used human dendritic cell (DC) cultures to examine possible T helper type 1 (Th1) responses induced by MDP and FK565/156 in combination with various synthetic Toll-like receptor (TLR) agonists, including synthetic lipid A (TLR4 agonist), the synthetic triacyl lipopeptide Pam3CSSNA (TLR2 agonist), poly(I:C) (TLR3 agonist), and CpG DNA (TLR9 agonist). Immature DCs derived from human monocytes expressed mRNAs for Nod1, Nod2, TLR2, TLR3, TLR4, and TLR9. The stimulation of DCs with MDP and FK565 in combination with lipid A, poly(I:C), and CpG DNA, but not with Pam3CSSNA, synergistically induced interleukin-12 (IL-12) p70 and gamma interferon (IFN-γ), but not IL-18, in culture supernatants and induced IL-15 on the cell surface. In correlation with the cytokine induction, an upregulation of the mRNA expression of these cytokine genes was observed. Notably, IL-12 p35 mRNA expression increased >1,000-fold upon stimulation with lipid A plus either MDP or FK565 compared with stimulation with each stimulant alone. In contrast, for the expression of CD83 and costimulatory molecules such as CD40, CD80, and CD86, no synergistic effects were observed upon stimulation with Nod plus TLR agonists. The culture supernatants of DCs stimulated with lipid A plus either MDP or FK565 activated human T cells to produce high levels of IFN-γ, and the activity was attributable to DC-derived IL-12. These findings suggest that Nod1 and Nod2 agonists in combination with TLR3, TLR4, and TLR9 agonists synergistically induce IL-12 and IFN-γ production in DCs to induce Th1-lineage immune responses.

Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion.

Type I interferons (IFNs), a family of cytokines, orchestrate numerous biological and cellular processes. Although it is well known that type I IFNs are essential for establishing the host antiviral state, their role in hematopoietic homeostasis has not been studied. Here we show that type I IFNs induce proliferation and exhaustion in hematopoietic stem cells (HSCs) and that interferon regulatory factor-2 (IRF2), a transcriptional suppressor of type I IFN signaling, preserves the self-renewal and multilineage differentiation capacity of HSCs. HSCs were substantially less abundant in the bone marrow of Irf2−/− as compared to Irf2+/− mice. Irf2−/− HSCs showed enhanced cell cycling status and failed to produce hematopoietic cells in competitive repopulation assays, and the reconstituting capacity of Irf2−/− HSCs was restored by disabling type I IFN signaling in these cells. In wild-type mice, injection of poly(I:C), an inducer of type I IFN signaling, or IFN-α induced HSC proliferation, and chronic type I IFN signaling further reduced the number of quiescent HSCs. Notably, combined poly(I:C) and 5-fluorouracil (5-FU) treatment allowed exogenous HSC engraftment and hematopoietic reconstitution in WT mice. Our findings provide insight into the molecular basis for the maintenance of HSC quiescence and may lead to improvements in bone marrow transplantation and type I IFN–based therapies for viral infection and cancer.

Critical role of IL-15–IL-15R for antigen-presenting cell functions in the innate immune response

Activation of dendritic cells (DCs) and macrophages by infectious agents leads to secretion of interleukin 12 (IL-12), which subsequently induces interferon-γ (IFN-γ) production by multiple cell types that include DCs and macrophages. In turn, IFN-γ acts on macrophages to augment IL-12 secretion and to produce nitric oxide (NO), which eradicates infected microbes. We show here that in cytokine common γ subunit–deficient and/or IL-2 receptor β–deficient mice, production of IL-12, IFN-γ and NO by DCs and macrophages was severely impaired, as was up-regulation of major histocompatibility complex class II and CD40. Similar phenotypes were observed in DCs and macrophages from IL-15–deficient mice but not in those from IL-2–deficient mice. This shows that the IL-15–IL-15R interaction is critical in early activation of antigen-presenting cells and plays an important role in the innate immune system.

Interleukin 15–dependent crosstalk between conventional and plasmacytoid dendritic cells is essential for CpG-induced immune activation

The function of interleukin 15 (IL-15) in unmethylated CpG oligodeoxynucleotide (CpG)–induced immune responses remains unknown. Here, in response to CpG, both wild-type and natural killer cell–depleted mice produced IL-12 and became resistant to a lethal dose of Listeria monocytogenes. In contrast, CpG-treated IL-15-deficient mice produced little IL-12 and succumbed to L. monocytogenes. CpG-stimulated conventional dendritic cells (cDCs) were the main producers of both IL-15 and IL-12, but cDCs did not produce IL-12 in the absence of plasmacytoid DCs (pDCs). The cDC-derived IL-15 induced CD40 expression by cDCs. Interaction between CD40 on cDCs and CD40 ligand on pDCs led to IL-12 production by cDCs. Thus, IL-15-dependent crosstalk between cDCs and pDCs is essential for CpG-induced immune activation.