Hiroaki Hemmi

The Roles of Two IκB Kinase-related Kinases in Lipopolysaccharide and Double Stranded RNA Signaling and Viral Infection

Viral infection and stimulation with lipopolysaccharide (LPS) or double stranded RNA (dsRNA) induce phosphorylation of interferon (IFN) regulatory factor (IRF)-3 and its translocation to the nucleus, thereby leading to the IFN-β gene induction. Recently, two IκB kinase (IKK)–related kinases, inducible IκB kinase (IKK-i) and TANK-binding kinase 1 (TBK1), were suggested to act as IRF-3 kinases and be involved in IFN-β production in Toll-like receptor (TLR) signaling and viral infection. In this work, we investigated the physiological roles of these kinases by gene targeting. TBK1-deficient embryonic fibroblasts (EFs) showed dramatic decrease in induction of IFN-β and IFN-inducible genes in response to LPS or dsRNA as well as after viral infection. However, dsRNA-induced expression of these genes was residually detected in TBK1-deficient cells and intact in IKK-i–deficient cells, but completely abolished in IKK-i/TBK1 doubly deficient cells. IRF-3 activation, in response not only to dsRNA but also to viral infection, was impaired in TBK1-deficient cells. Together, these results demonstrate that TBK1 as well as, albeit to a lesser extent, IKK-i play a crucial role in the induction of IFN-β and IFN-inducible genes in both TLR-stimulated and virus-infected EFs.

Interferon-α and Interleukin-12 Are Induced Differentially by Toll-like Receptor 7 Ligands in Human Blood Dendritic Cell Subsets

Dendritic cells (DCs) play a crucial role in the immune responses against infections by sensing microbial invasion through toll-like receptors (TLRs). In humans, two distinct DC subsets, CD11c− plasmacytoid DCs (PDCs) and CD11c+ myeloid DCs (MDCs), have been identified and can respond to different TLR ligands, depending on the differential expression of cognate TLRs. In this study, we have examined the effect of TLR-7 ligands on human DC subsets. Both subsets expressed TLR-7 and could respond to TLR-7 ligands, which enhanced the survival of the subsets and upregulated the surface expression of costimulatory molecules such as CD40, CD80, and CD86. However, the cytokine induction pattern was distinct in that PDCs and MDCs produced interferon (IFN)-α and interleukin (IL)-12, respectively. In response to TLR-7 ligands, the Th1 cell supporting ability of both DC subsets was enhanced, depending on the cytokines the respective subsets produced. This study demonstrates that TLR-7 exerts its biological effect in a DC subset-specific manner.

IκB kinase-α is critical for interferon-α production induced by Toll-like receptors 7 and 9

The Toll-like receptor (TLR) family has important roles in microbial recognition and dendritic cell activation. TLRs 7 and 9 can recognize nucleic acids and trigger signalling cascades that activate plasmacytoid dendritic cells to produce interferon-α (IFN-α) (refs 7, 8). TLR7/9-mediated dendritic cell activation is critical for antiviral immunity but also contributes to the pathogenesis of systemic lupus erythematosus, a disease in which serum IFN-α levels are elevated owing to plasmacytoid dendritic cell activation. TLR7/9-induced IFN-α induction depends on a molecular complex that contains a TLR adaptor, MyD88, and IFN regulatory factor 7 (IRF-7) (refs 10–14), but the underlying molecular mechanisms are as yet unknown. Here we show that IκB kinase-α (IKK-α) is critically involved in TLR7/9-induced IFN-α production. TLR7/9-induced IFN-α production was severely impaired in IKK-α-deficient plasmacytoid dendritic cells, whereas inflammatory cytokine induction was decreased but still occurred. Kinase-deficient IKK-α inhibited the ability of MyD88 to activate the Ifna promoter in synergy with IRF-7. Furthermore, IKK-α associated with and phosphorylated IRF-7. Our results identify a role for IKK-α in TLR7/9 signalling, and highlight IKK-α as a potential target for manipulating TLR-induced IFN-α production.

Involvement of the Toll-Like Receptor 9 Signaling Pathway in the Induction of Innate Immunity by Baculovirus

ABSTRACT We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c+ dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-α) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-α by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.

Activation of TBK1 and IKKε Kinases by Vesicular Stomatitis Virus Infection and the Role of Viral Ribonucleoprotein in the Development of Interferon Antiviral Immunity

ABSTRACT Mounting an immune response to a viral pathogen involves the initial recognition of viral antigens through Toll-like receptor-dependent and -independent pathways and the subsequent triggering of signal transduction cascades. Among the many cellular kinases stimulated in response to virus infection, the noncanonical IKK-related kinases TBK1 and IKKε have been shown to phosphorylate and activate interferon regulatory factor 3 (IRF-3) and IRF-7, leading to the production of alpha/beta interferons and the development of a cellular antiviral state. In the present study, we examine the activation of TBK1 and IKKε kinases by vesicular stomatitis virus (VSV) infection in human lung epithelial A549 cells. We demonstrate that replication-competent VSV is required to induce activation of the IKK-related kinases and provide evidence that ribonucleoprotein (RNP) complex of VSV generated intracellularly during virus replication can activate TBK1 and IKKε activity. In TBK1-deficient cells, IRF-3 and IRF-7 activation is significantly reduced, although transcriptional upregulation of IKKε following treatment with VSV, double-stranded RNA, or RNP partially compensates for the loss of TBK1. Biochemical analyses with purified TBK1 and IKKε kinases in vitro demonstrate that the two kinases exhibit similar specificities with respect to IRF-3 and IRF-7 substrates and both kinases target serine residues that are important for full transcriptional activation of IRF-3 and IRF-7. These data suggest that intracellular RNP formation contributes to the early recognition of VSV infection, activates the catalytic activity of TBK1, and induces transcriptional upregulation of IKKε in epithelial cells. Induction of IKKε potentially functions as a component of the amplification mechanism involved in the establishment of the antiviral state.

TLR signalling and the function of dendritic cells.

The recognition of microbes by innate immune cells initiates activation of the whole immune system. Toll-like receptors (TLRs) are known to recognize various components of invading pathogens. At present, the natural ligands for almost all TLR members have been identified. TLRs are expressed on many types of cells including macrophages and dendritic cells (DCs). The recognition of invading microbes by TLRs on DCs induces proinflammatory cytokine production and enhanced antigen presentation to naive T cells, and finally activates antigen-specific adaptive immune responses. The sequential activation of innate and subsequent adaptive immunity are crucial steps to eradicate invading pathogens. Recently, the TLR signalling pathway has been intensively investigated. Accumulating evidence indicates that, at least, four adaptor molecules are involved in TLR signalling and provide their signalling specificities. Distinct TLR ligands provide distinct activation status and cytokine production patterns for antigen presenting cells, resulting in the induction of differential immune responses. Thus, TLRs are critical molecules to induce not only inflammatory responses but also fine-tuned adaptive immune responses depending on invading pathogens.

Innate Vα14+ natural killer T cells mature dendritic cells, leading to strong adaptive immunity

Summary: The observation that the glycolipid α‐galactosylceramide (α‐GalCer) is a potent stimulator of natural killer T (NKT) cells has provided an important means for investigating NKT cell biology. α‐GalCer is presented on CD1d to the invariant NKT receptor, leading to interleukin‐12 (IL‐12) production by dendritic cells (DCs) and to NK cell activation. We review our research on the tumor‐protective properties of α‐GalCer, particularly the major role played by DCs. We compared administration of α‐GalCer on mature DCs with soluble glycolipid and found that DCs induced more prolonged interferon‐γ (IFN‐γ) production by NKT cells and better protection against B16 melanoma. Human α‐GalCer‐loaded DCs also expanded NKT cell numbers in cancer patients. α‐GalCer‐activated NKT cells were then found to induce DC maturation in vivo. The maturing DCs produced IL‐12, upregulated co‐stimulatory molecules, and induced adaptive immunity to captured cellular antigens, including prolonged, combined CD4+/CD8+ T‐cell immunity to dying tumor cells. Surprisingly, co‐stimulator‐poor tumor cells, if directly loaded with α‐GalCer (‘tumor/Gal’) and injected intravenously, also induced strong NKT‐ and NK‐cell responses. The latter killed the tumor/Gal, which were subsequently cross presented by CD1d on DCs to elicit DC maturation and prolonged adaptive T‐cell immunity, which lasted 6–12 months. These findings help explain tumor protection via α‐GalCer and urge development of the DC‐NKT axis to provide innate and adaptive immunity to human cancers.

Osteoclast-specific Dicer gene deficiency suppresses osteoclastic bone resorption.

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010.

Osteoblastic bone formation is induced by using nanogel-crosslinking hydrogel as novel scaffold for bone growth factor

Bone regeneration for the defects in revision surgery of joint replacement is an increasingly important issue. To repair bone defects, bone cell activation by growth factors using synthetic resorbable scaffold is a useful and safe option. We examine the efficiency of nanogel‐crosslinking hydrogel as a novel synthetic scaffold for BMP to stimulate osteoblasts and to induce bone formation. Cholesterol‐bearing pullulan nanogel‐crosslinking hydrogel (CHPA/Hydrogel) was used to deliver BMP. The CHPA hydrogel pellets were implanted in vivo. Single implantation of CHPA/hydrogel containing low amounts of BMP induced osteoblastic activation and new bone formation in vivo. Furthermore, nanogel in a disc shape established recruitment of osteoblastic cells that vigorously formed bone to heal the calvarial defects, which did not heal spontaneously without it. In conclusion, CHPA/hydrogel serves as an efficient and versatile scaffold for the stimulation of osteoblasts to form bone and to repair defects via delivery of BMP. J. Cell. Physiol. 220: 1–7, 2009.

Sympathetic control of bone mass regulated by osteopontin

The sympathetic nervous system suppresses bone mass by mechanisms that remain incompletely elucidated. Using cell-based and murine genetics approaches, we show that this activity of the sympathetic nervous system requires osteopontin (OPN), a cytokine and one of the major members of the noncollagenous extracellular matrix proteins of bone. In this work, we found that the stimulation of the sympathetic tone by isoproterenol increased the level of OPN expression in the plasma and bone and that mice lacking OPN (OPN-KO) suppressed the isoproterenol-induced bone loss by preventing reduced osteoblastic and enhanced osteoclastic activities. In addition, we found that OPN is necessary for changes in the expression of genes related to bone resorption and bone formation that are induced by activation of the sympathetic tone. At the cellular level, we showed that intracellular OPN modulated the capacity of the β2-adrenergic receptor to generate cAMP with a corresponding modulation of cAMP-response element binding (CREB) phosphorylation and associated transcriptional events inside the cell. Our results indicate that OPN plays a critical role in sympathetic tone regulation of bone mass and that this OPN regulation is taking place through modulation of the β2-adrenergic receptor/cAMP signaling system.