Publications

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) are known as an immune adjuvant. CpG DNAs coupled with a particular antigen enabling both CpG DNA and antigen delivery to the same antigen-presenting cell have been shown to be more effective. Based on our previous finding that β-(1→3)-d-glucan schizophyllan (SPG) can be used as a CpG DNA carrier, here we present the synthesis of an antigen-conjugated SPG and the characterization of the conjugate. Ovalbumin (OVA, 43 kDa) was used as a model antigen, and two OVA were conjugated to one SPG molecule (Mw = 150 000), denoted by OVA−SPG. Circular dichroism and gel electrophoresis showed that OVA−SPG could form a complex with a (dA)40-tailed CpG DNA at the 3' end (1668-(dA)40). When OVA−SPG was added to macrophages (J774.A1), the amount of the ingested OVA−SPG was increased compared with that of OVA itself, suggesting that Dectin-1 (proinflammatory nonopsonic receptor for β-glucans) is involved to ingest OVA−SPG. Furthermore, the complex of the conjugate and DNA was co-localized in the same vesicles, implying that OVA (antigen) and CpG DNA (adjuvant) were ingested into the cell at the same time. This paper shows that OVA−SPG can be used as a CpG DNA carrier to induce antigen-specific immune responses.

G-CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil-granulocyte lineage. We examined the role of stat3 in G-CSF signaling using the Cre-loxP recombination system. Although Stat3 was thought to be essential for the transduction of G-CSF-induced cell proliferation and differentiation signals, mice deficient for Stat3 in hematopoietic cells show neutrocytosis and infiltration of cells into the digestive tract. The number of progenitor cells in the neutrophil lineage is not changed, and G-CSF-induced proliferation of progenitor cells and prolonged neutrophil survival were observed in Stat3-deficient mice. In hematopoietic cells from Stat3-deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression was not induced by G-CSF stimulation. Stat3 null bone marrow cells displayed a significant activation of ERK1/2 under basal conditions, and the activation of ERK was enhanced and sustained by G-CSF stimulation. Furthermore, the augmented proliferation of Stat3-deficient bone marrow cells in response to G-CSF was dramatically decreased by addition of a MEK1 inhibitor. These results indicate that Stat3 functions as a negative regulator of G-CSF signaling by inducing SOCS3 expression, and ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G-CSF.

Dendritic cells (DC) are key players in the initiation and modulation of adaptive immune responses due to their ability to acquire and present antigen and stimulate T cells. For the induction of effector T cell functions, antigen must be presented by activated DC. In this study, we have compared uptake of antigen by mouse DC in the presence of different Toll-like receptor (TLR) agonists, which are potent inducers of DC activation. Here we show that the reduction in uptake of soluble antigen in the presence of the viral double-stranded RNA (dsRNA) analogues polyinosinic–polycytidylic acid and Ampligen is independent of TLR-mediated DC activation. A reduction in antigen uptake by bone marrow-derived and splenic DC was also observed in response to other RNA homopolymers such as polyinosinic and polyguanylic acids, which are known inhibitors of scavenger receptor-mediated endocytosis. Pinocytosis and mannose receptor-mediated uptake of soluble antigen were not affected by any of the tested nucleic acids. The reduction in antigen uptake by dsRNA did not negatively influence the T cell stimulating properties of the DC. In summary, we conclude that the decrease in antigen endocytosis observed in the presence of a variety of TLR agonists is independent of TLR signalling and is caused by competition for specific surface receptors that are involved in the uptake of these TLR agonists and the antigen.

No abstract is provided for this article.

Suppressor of cytokine signaling-1 (SOCS-1) is one of the negative-feedback regulators of Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling. We previously showed that SOCS-1 participates in LPS signaling, but it is not entirely clear yet how SOCS-1 suppresses LPS signaling. In this study, we demonstrate that SOCS-1 selectively inhibits LPS-induced IL-6 production through regulation of JAK–STAT but not production of TNF-α, granulocyte colony-stimulating factor, IFN-β, and other cytokines. We found that LPS directly activated Jak2 and Stat5, whereas SOCS-1 inhibited LPS-induced Jak2 and Stat5 activation. Furthermore, AG490, a Jak-specific inhibitor, and dominant negative Stat5 only reduced LPS-induced IL-6 production. Additionally, Stat5 interacted with p50, resulting in recruitment of Stat5 to the IL-6 promoter together with p50 in response to LPS stimulation. These findings suggest that the JAK–STAT pathway participates in LPS-induced IL-6 production and that SOCS-1 suppresses LPS signaling by regulating JAK–STAT.

<p>PDF file - 555K, Stainings of cytosolic DNA in Ara-C-treated BC2 cells and Yac-1 cells.</p>

No abstract is provided for this article.

Increased type I interferon (IFN-I) production and IFN-stimulated gene (ISG) expression are linked to the pathogenesis of systemic lupus erythematosus (SLE). Although the mechanisms responsible for dysregulated IFN-I production in SLE remain unclear, autoantibody-mediated uptake of endogenous nucleic acids is thought to play a role. 2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane) induces a lupus-like disease in mice characterized by immune complex nephritis with autoantibodies to DNA and ribonucleoproteins. We recently reported that TMPD also causes increased ISG expression and that the development of the lupus is completely dependent on IFN-I signaling (Nacionales, D.C., K.M. Kelly-Scumpia, P.Y. Lee, J.S. Weinstein, R. Lyons, E. Sobel, M. Satoh, and W.H. Reeves. 2007. Arthritis Rheum. 56:3770–3783). We show that TMPD elicits IFN-I production, monocyte recruitment, and autoantibody production exclusively through a Toll-like receptor (TLR) 7– and myeloid differentiation factor 88 (MyD88)–dependent pathway. In vitro studies revealed that TMPD augments the effect of TLR7 ligands but does not directly activate TLR7 itself. The effects of TMPD were amplified by the Y-linked autoimmune acceleration cluster, which carries a duplication of the TLR7 gene. In contrast, deficiency of Fcγ receptors (FcγRs) did not affect the production of IFN-I. Collectively, the data demonstrate that TMPD-stimulated IFN-I production requires TLR7/MyD88 signaling and is independent of autoantibody-mediated uptake of ribonucleoproteins by FcγRs.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by inflammation in multiple organs. A few treatments for SLE currently exist, including antimalarials, glucocorticoids, immunosuppressants, and two recently approved antibody agents; however, an unmet medical need remains for SLE. In addition, developing new drugs targeting SLE is a challenge since no specific biomarkers exist for the prediction of disease progression or drug response. A new drug candidate, E6742, is a specific antagonist of the toll-like receptors 7/8. To address the challenges for drug development in SLE, the process of developing E6742 utilizes a unique system of the Japan Agency for Medical Research and Development (AMED), the Cyclic Innovation for Clinical Empowerment (CiCLE) program. In the CiCLE program, a Phase 1 study in healthy adults was completed (NCT04683185) and a Phase 1/2 study in patients with SLE is on-going (NCT05278663). One of the potential benefits of this program is to conduct academia-led clinical research to identify specific biomarkers for E6742 in parallel with clinical studies (UMIN000042037). The aim of this review is to present current progress within the strategic collaboration of the AMED CiCLE program that optimize clinical development for patients with SLE.

The innate immune system evolved to recognize conserved microbial products, termed pathogen-associated molecular patterns (PAMPs), which are invariant among diverse groups of microorganisms. PAMPs are recognized by a set of germ-line encoded pattern recognition receptors (PRRs). Among the best characterized PAMPs are bacterial lipopolysaccharide (LPS), peptidoglycan (PGN), mannans, and other constituents of bacterial and fungal cell walls, as well as bacterial DNA. Recognition of bacterial DNA is the most enigmatic of these, as it depends on a particular sequence motif, called the CpG motif, in which an unmethylated CpG present in a particular sequence context accounts for a potent immunostimulatory activity of CpG DNA. Receptor(s) of the innate immune system that mediate recognition of CpG DNA are currently unknown. Here, we report that recognition of CpG DNA requires MyD88, an adaptor protein involved in signal transduction by the Toll-like receptors (TLRs), essential components of innate immune recognition in both Drosophila and mammals [1,2]. Signaling induced by CpG DNA was found to be unaffected in cells deficient in TLR2 or TLR4, suggesting that some other member of the Toll family mediates recognition of bacterial DNA.

Helminth parasites remain one of the most common causes of infections worldwide, yet little is still known about the immune signaling pathways that control their expulsion. C57BL/6 mice are chronically susceptible to infection with the gastrointestinal helminth parasite Heligmosomoides polygyrus. In this article, we report that C57BL/6 mice lacking the adapter protein MyD88, which mediates signaling by TLRs and IL-1 family members, showed enhanced immunity to H. polygyrus infection. Alongside increased parasite expulsion, MyD88-deficient mice showed heightened IL-4 and IL-17A production from mesenteric lymph node CD4(+) cells. In addition, MyD88(-/-) mice developed substantial numbers of intestinal granulomas around the site of infection, which were not seen in MyD88-sufficient C57BL/6 mice, nor when signaling through the adapter protein TRIF (TIR domain-containing adapter-inducing IFN-β adapter protein) was also ablated. Mice deficient solely in TLR2, TLR4, TLR5, or TLR9 did not show enhanced parasite expulsion, suggesting that these TLRs signal redundantly to maintain H. polygyrus susceptibility in wild-type mice. To further investigate signaling pathways that are MyD88 dependent, we infected IL-1R1(-/-) mice with H. polygyrus. This genotype displayed heightened granuloma numbers compared with wild-type mice, but without increased parasite expulsion. Thus, the IL-1R-MyD88 pathway is implicated in inhibiting granuloma formation; however, protective immunity in MyD88-deficient mice appears to be granuloma independent. Like IL-1R1(-/-) and MyD88(-/-) mice, animals lacking signaling through the type 1 IFN receptor (i.e., IFNAR1(-/-)) also developed intestinal granulomas. Hence, IL-1R1, MyD88, and type 1 IFN receptor signaling may provide pathways to impede granuloma formation in vivo, but additional MyD88-mediated signals are associated with inhibition of protective immunity in susceptible C57BL/6 mice.

Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes.