Shigeru Kakuta

IL-17 Plays an Important Role in the Development of Experimental Autoimmune Encephalomyelitis

IL-17 is a proinflammatory cytokine that activates T cells and other immune cells to produce a variety of cytokines, chemokines, and cell adhesion molecules. This cytokine is augmented in the sera and/or tissues of patients with contact dermatitis, asthma, and rheumatoid arthritis. We previously demonstrated that IL-17 is involved in the development of autoimmune arthritis and contact, delayed, and airway hypersensitivity in mice. As the expression of IL-17 is also augmented in multiple sclerosis, we examined the involvement of this cytokine in these diseases using IL-17−/− murine disease models. We found that the development of experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis, was significantly suppressed in IL-17−/− mice; these animals exhibited delayed onset, reduced maximum severity scores, ameliorated histological changes, and early recovery. T cell sensitization against myelin oligodendrocyte glycoprotein was reduced in IL-17−/− mice upon sensitization. The major producer of IL-17 upon treatment with myelin digodendrocyte glycopritein was CD4+ T cells rather than CD8+ T cells, and adoptive transfer of IL-17−/− CD4+ T cells inefficiently induced EAE in recipient mice. Notably, IL-17-producing T cells were increased in IFN-γ−/− cells, while IFN-γ-producing cells were increased in IL-17−/− cells, suggesting that IL-17 and IFN-γ mutually regulate IFN-γ and IL-17 production. These observations indicate that IL-17 rather than IFN-γ plays a crucial role in the development of EAE.

Differential Roles of Interleukin-17A and -17F in Host Defense against Mucoepithelial Bacterial Infection and Allergic Responses

Interleukin-17A (IL-17A) is a cytokine produced by T helper 17 (Th17) cells and plays important roles in the development of inflammatory diseases. Although IL-17F is highly homologous to IL-17A and binds the same receptor, the functional roles of this molecule remain largely unknown. Here, we demonstrated with Il17a(-/-), Il17f(-/-), and Il17a(-/-)Il17f(-/-) mice that IL-17F played only marginal roles, if at all, in the development of delayed-type and contact hypersensitivities, autoimmune encephalomyelitis, collagen-induced arthritis, and arthritis in Il1rn(-/-) mice. In contrast, both IL-17F and IL-17A were involved in host defense against mucoepithelial infection by Staphylococcus aureus and Citrobacter rodentium. IL-17A was produced mainly in T cells, whereas IL-17F was produced in T cells, innate immune cells, and epithelial cells. Although only IL-17A efficiently induced cytokines in macrophages, both cytokines activated epithelial innate immune responses. These observations indicate that IL-17A and IL-17F have overlapping yet distinct roles in host immune and defense mechanisms.

Involvement of tumor necrosis factor-related apoptosis-inducing ligand in surveillance of tumor metastasis by liver natural killer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various tumor cells in vitro, but its physiological role in tumor surveillance remains unknown. Here, we report that TRAIL is constitutively expressed on murine natural killer (NK) cells in the liver and plays a substantial role in suppressing tumor metastasis. Freshly isolated NK cells, but not natural killer T cells or ordinary T cells, from the liver expressed cell surface TRAIL, which was responsible for spontaneous cytotoxicity against TRAIL-sensitive tumor cells in vitro along with perforin and Fas ligand (FasL). Administration of neutralizing monoclonal antibody against TRAIL significantly increased experimental liver metastases of several TRAIL-sensitive tumor cell lines. Such an anti-metastatic effect of TRAIL was not observed in NK cell–depleted mice or interferon-γ–deficient mice, the latter of which lacked TRAIL on liver NK cells. These findings provide the first evidence for the physiological function of TRAIL as a tumor suppressor.

Dectin-2 Recognition of α-Mannans and Induction of Th17 Cell Differentiation Is Essential for Host Defense against Candida albicans

Dectin-2 (gene symbol Clec4n) is a C-type lectin expressed by dendritic cells (DCs) and macrophages. However, its functional roles and signaling mechanisms remain to be elucidated. Here, we generated Clec4n(-/-) mice and showed that this molecule is important for host defense against Candida albicans (C. albicans). Clec4n(-/-) DCs had virtually no fungal alpha-mannan-induced cytokine production. Dectin-2 signaling induced cytokines through an FcRgamma chain and Syk-CARD9-NF-kappaB-dependent signaling pathway without involvement of MAP kinases. The yeast form of C. albicans induced interleukin-1beta (IL-1beta) and IL-23 secretion in a Dectin-2-dependent manner. In contrast, cytokine production induced by the hyphal form was only partially dependent on this lectin. Both yeast and hyphae induced Th17 cell differentiation, in which Dectin-2, but not Dectin-1, was mainly involved. Because IL-17A-deficient mice were highly susceptible to systemic candida infection, this study suggests that Dectin-2 is important in host defense against C. albicans by inducing Th17 cell differentiation.

Loss of DExD/H Box RNA Helicase LGP2 Manifests Disparate Antiviral Responses

The DExD/H box RNA helicase retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) are key intracellular receptors that recognize virus infection to produce type I IFN. A third helicase gene, Lgp2, is homologous to Rig-I and Mda5 but lacks a caspase activation and recruitment domain. We generated Lgp2-deficient mice and report that the loss of this gene greatly sensitizes cells to cytosolic polyinosinic/polycytidylic acid-mediated induction of type I IFN. However, negative feedback inhibition of IFN-β transcription was found to be normal in the absence of LGP2, indicating that LGP2 is not the primary negative regulator of type I IFN production. Our data further indicate that Lgp2−/− mice exhibited resistance to lethal vesicular stomatitis virus infection, a virus whose replicative RNA intermediates are recognized specifically by RIG-I rather than by MDA5 to trigger the production of type I IFN. However, mice lacking LGP2 were observed to exhibit a defect in type I IFN production in response to infection by the encephalomyocarditis virus, the replication of which activates MDA5-dependent innate immune responses. Collectively, our data indicate a disparate regulatory role for LGP2 in the triggering of innate immune signaling pathways following RNA virus infection.

The Muscle Protein Dok-7 Is Essential for Neuromuscular Synaptogenesis

The formation of the neuromuscular synapse requires muscle-specific receptor kinase (MuSK) to orchestrate postsynaptic differentiation, including the clustering of receptors for the neurotransmitter acetylcholine. Upon innervation, neural agrin activates MuSK to establish the postsynaptic apparatus, although agrin-independent formation of neuromuscular synapses can also occur experimentally in the absence of neurotransmission. Dok-7, a MuSK-interacting cytoplasmic protein, is essential for MuSK activation in cultured myotubes; in particular, the Dok-7 phosphotyrosine-binding domain and its target in MuSK are indispensable. Mice lacking Dok-7 formed neither acetylcholine receptor clusters nor neuromuscular synapses. Thus, Dok-7 is essential for neuromuscular synaptogenesis through its interaction with MuSK.

Requirement for natural killer T (NKT) cells in the induction of allograft tolerance

In this study, we investigated the role of Vα14 natural killer T (NKT) cells in transplant immunity. The ability to reject allografts was not significantly different between wild-type (WT) and Vα14 NKT cell-deficient mice. However, in models in which tolerance was induced against cardiac allografts by blockade of lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions, long-term acceptance of the grafts was observed only in WT but not Vα14 NKT cell-deficient mice. Adoptive transfer with Vα14 NKT cells restored long-term acceptance of allografts in Vα14 NKT cell-deficient mice. The critical role of Vα14 NKT cells to mediate immunosuppression was also observed in vitro in mixed lymphocyte cultures in which lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions were blocked. Experiments using IL-4- or IFN-γ-deficient mice suggested a critical contribution of IFN-γ to the Vα14 NKT cell-mediated allograft acceptance in vivo. These results indicate a critical contribution of Vα14 NKT cells to the induction of allograft tolerance and provide a useful model to investigate the regulatory role of Vα14 NKT cells in various immune responses.

Critical contribution of IFN‐γ and NK cells, but not perforin‐mediated cytotoxicity, to anti‐metastatic effect of α‐galactosylceramide

The glycolipid α ‐galactosylceramide (α ‐GalCer), which is presented by CD1d and specifically activates Vα 14 NKT cells, exerts a potent anti‐metastatic effect when administered in vivo. In this study, we demonstrated that α ‐GalCer administration led to rapid elimination of NKT cells by apoptosis in the liver and spleen, after they produced IFN‐γ  and IL‐4. In contrast, a more prolonged secretion of IFN‐γ  was observed by liver and splenic NK cells after α ‐GalCer administration. Cytotoxic activity of liver mononuclear cells was not augmented 3h after α ‐GalCer administration, but was increased at 24 h when NKT cells were mostly depleted. The α ‐GalCer‐induced cytotoxic activity was abolished in IFN‐γ ‐deficient and NK cell‐depleted mice as well as CD1‐deficient mice, suggesting that the α ‐Galcer‐induced cytotoxicity was mainly mediated by IFN‐γ ‐activated NK cells. While the α ‐GalCer‐induced cytotoxicity in vitro was mostly perforin dependent, anti‐metastatic effect of α ‐GalCer was impaired in NK cell‐depleted or IFN‐γ ‐deficient mice but not in perforin‐deficient mice. Collectively, these results indicated that the anti‐metastatic effect of α ‐GalCer is mainly mediated by NK cells, which are activated secondarily by IFN‐γ  produced by α ‐GalCer‐activated NKT cells, in aperforin‐independent manner.

Dynamic Functional Relay between Insulin Receptor Substrate 1 and 2 in Hepatic Insulin Signaling during Fasting and Feeding

Insulin receptor substrate (Irs) mediates metabolic actions of insulin. Here, we show that hepatic Irs1 and Irs2 function in a distinct manner in the regulation of glucose homeostasis. The PI3K activity associated with Irs2 began to increase during fasting, reached its peak immediately after refeeding, and decreased rapidly thereafter. By contrast, the PI3K activity associated with Irs1 began to increase a few hours after refeeding and reached its peak thereafter. The data indicate that Irs2 mainly functions during fasting and immediately after refeeding, and Irs1 functions primarily after refeeding. In fact, liver-specific Irs1-knockout mice failed to exhibit insulin resistance during fasting, but showed insulin resistance after refeeding; conversely, liver-specific Irs2-knockout mice displayed insulin resistance during fasting but not after refeeding. We propose the concept of the existence of a dynamic relay between Irs1 and Irs2 in hepatic insulin signaling during fasting and feeding.

A Tecpr1-Dependent Selective Autophagy Pathway Targets Bacterial Pathogens

Selective autophagy of bacterial pathogens represents a host innate immune mechanism. Selective autophagy has been characterized on the basis of distinct cargo receptors but the mechanisms by which different cargo receptors are targeted for autophagic degradation remain unclear. In this study we identified a highly conserved Tectonin domain-containing protein, Tecpr1, as an Atg5 binding partner that colocalized with Atg5 at Shigella-containing phagophores. Tecpr1 activity is necessary for efficient autophagic targeting of bacteria, but has no effect on rapamycin- or starvation-induced canonical autophagy. Tecpr1 interacts with WIPI-2, a yeast Atg18 homolog and PI(3)P-interacting protein required for phagophore formation, and they colocalize to phagophores. Although Tecpr1-deficient mice appear normal, Tecpr1-deficient MEFs were defective for selective autophagy and supported increased intracellular multiplication of Shigella. Further, depolarized mitochondria and misfolded protein aggregates accumulated in the Tecpr1-knockout MEFs. Thus, we identify a Tecpr1-dependent pathway as important in targeting bacterial pathogens for selective autophagy.