Ryusuke Nakagawa

Pivotal role of cerebral interleukin-17|[ndash]|producing |[gamma]||[delta]|T cells in the delayed phase of ischemic brain injury

Lymphocyte recruitment and activation have been implicated in the progression of cerebral ischemia-reperfusion (I/R) injury, but the roles of specific lymphocyte subpopulations and cytokines during stroke remain to be clarified. Here we demonstrate that the infiltration of T cells into the brain, as well as the cytokines interleukin-23 (IL-23) and IL-17, have pivotal roles in the evolution of brain infarction and accompanying neurological deficits. Blockade of T cell infiltration into the brain by the immunosuppressant FTY720 reduced I/R-induced brain damage. The expression of IL-23, which was derived mostly from infiltrated macrophages, increased on day 1 after I/R, whereas IL-17 levels were elevated after day 3, and this induction of IL-17 was dependent on IL-23. These data, together with analysis of mice genetically disrupted for IL-17 and IL-23, suggest that IL-23 functions in the immediate stage of I/R brain injury, whereas IL-17 has an important role in the delayed phase of I/R injury during which apoptotic neuronal death occurs in the penumbra. Intracellular cytokine staining revealed that γδT lymphocytes, but not CD4+ helper T cells, were a major source of IL-17. Moreover, depletion of γδT lymphocytes ameliorated the I/R injury. We propose that T lymphocytes, including γδT lymphocytes, could be a therapeutic target for mitigating the inflammatory events that amplify the initial damage in cerebral ischemia.

Mechanisms of the Antimetastatic Effect in the Liver and of the Hepatocyte Injury Induced by α-Galactosylceramide in Mice

The role of mouse liver NK1.1 Ag+ T (NKT) cells in the antitumor effect of α-galactosylceramide (α-GalCer) has been unclear. We now show that, whereas α-GalCer increased the serum IFN-γ concentration and alanine aminotransferase activity in NK cell-depleted C57BL/6 (B6) mice and B6-beige/beige mice similarly to its effects in control B6 mice, its enhancement of the antitumor cytotoxicity of liver mononuclear cells (MNCs) was abrogated. Depletion of both NK and NKT cells in B6 mice reduced all these effects of α-GalCer. Injection of Abs to IFN-γ also inhibited the α-GalCer-induced increase in antitumor cytotoxicity of MNCs. α-GalCer induced the expression of Fas ligand on NKT cells in the liver of B6 mice. Whereas α-GalCer did not increase serum alanine aminotransferase activity in B6-lpr/lpr mice and B6-gld/gld mice, it increased the antitumor cytotoxicity of liver MNCs. The α-GalCer-induced increase in survival rate apparent in B6 mice injected intrasplenically with B16 tumor cells was abrogated in beige/beige mice, NK cell-depleted B6 mice, and B6 mice treated with Abs to IFN-γ. Depletion of CD8+ T cells did not affect the α-GalCer-induced antitumor cytotoxicity of liver MNCs but reduced the effect of α-GalCer on the survival of B6 mice. Thus, IFN-γ produced by α-GalCer-activated NKT cells increases both the innate antitumor cytotoxicity of NK cells and the adaptive antitumor response of CD8+ T cells, with consequent inhibition of tumor metastasis to the liver. Moreover, NKT cells mediate α-GalCer-induced hepatocyte injury through Fas-Fas ligand signaling.

Galectin-9 Induces Maturation of Human Monocyte-Derived Dendritic Cells

Maturation of dendritic cells (DCs) is critical for initiation of immune responses and is regulated by various stimulatory signals. We assessed the role of galectin (Gal)-9 in DC maturation. Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a dose-dependent manner, although Gal-9 had no or little effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-γ and IL-2) but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4+ T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were inhibited only slightly by lactose. We further found that a Gal-9 mutant that lacks β-galactoside binding activity reproduced the above activities and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the MAPK p38 and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or PI3K, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.

The mTOR pathway is highly activated in diabetic nephropathy and rapamycin has a strong therapeutic potential.

Diabetic nephropathy (DN) associated with type 2 diabetes is the most common cause of end-stage renal disease (ESRD) and a serious health issue in the world. Currently, molecular basis for DN has not been established and only limited clinical treatments are effective in abating the progression to ESRD associated with DN. Here we found that diabetic db/db mice which lack the leptin receptor signaling can be used as a model of ESRD associated with DN. We demonstrated that p70S6-kinase was highly activated in mesangial cells in diabetic obese db/db mice. Furthermore, systemic administration of rapamycin, a specific and potent inhibitor of mTOR, markedly ameliorated pathological changes and renal dysfunctions. Moreover, rapamycin treatment shows a significant reduction in fat deposits and attenuates hyperinsulinemia with few side effects. These results indicate that mTOR activation plays a pivotal role in the development of ESRD and that rapamycin could be an effective therapeutic agent for DN.

Antitumor activity of alpha-galactosylceramide, KRN7000, in mice with the melanoma B16 hepatic metastasis and immunohistological study of tumor infiltrating cells.

Liver metastasis of primary tumors is clinically a major problem. We examined the antitumor activity of KRN7000, an alpha-galactosylceramide, in mice with liver metastasis of the B16 melanoma. KRN7000 significantly inhibited tumor growth in the liver, and its potency was similar to that of interleukin-12. The KRN7000 administration resulted in a high percentage of cured mice, which acquired tumor-specific immunity. To study what kinds of antitumor effector cells participated in killing tumor cells, we then performed immunohistological analysis of tumor-infiltrating cells, and found that KRN7000 induced marked invasion of NK1.1+ cells, CD8+ cells, and F4/80+ cells (macrophages) into B16 tumor nodules. In addition, it appeared that KRN7000-treated, liver-associated macrophages possessed strong lytic activity against tumor cells. These results suggest that NK cells, NK1.1+ T (NKT) cells, cytotoxic T lymphocytes, and macrophages play an important role in killing tumor cells in the liver, and that KRN7000 may be useful for the treatment of cancer liver metastasis.

Age-Associated Augmentation of the Synthetic Ligand- Mediated Function of Mouse NK1.1 Ag+ T Cells: Their Cytokine Production and Hepatotoxicity In Vivo and In Vitro

We recently reported that the direct antitumor effectors in the liver induced by α-galactosylceramide (α-GalCer) are NK cells that are activated by the IFN-γ produced from NK1.1 Ag+ T cells (NKT cells) specifically stimulated with α-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the α-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-γ and IL-4 concentrations as well as alanine aminotransferase levels after the α-GalCer injection increased in an age-dependent manner. An α-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of α-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-γ and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1+ cells. Furthermore, liver mononuclear cells from old mice stimulated with α-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most α-GalCer-injected old mice, but no young mice, died, while anti-IFN-γ Ab pretreatment completely inhibited mouse mortality. However, α-GalCer-induced hepatic injury did not improve at all by anti-IFN-γ Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-γ is responsible for α-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-γ.

A Major Lipid Raft Protein Raftlin Modulates T Cell Receptor Signaling and Enhances Th17-Mediated Autoimmune Responses

The membrane microdomains known as lipid rafts have been shown to act as platforms for the initiation of various receptor signals. Through proteomic analysis, we have identified a novel protein termed Raftlin (raft-linking protein) as a major protein in lipid rafts. To determine the physiological and immunological functions of Raftlin in mammals, we generated Raftlin-deficient mice, as well as Raftlin-transgenic (Tg) mice. Although Raftlin was originally identified in B cells, we observe no severe abnormalities in the B cells of these mice, presumably due to a high expression of Raftlin-homologue (Raftlin-2). T cells, in contrast, expressed a substantial amount of Raftlin but no Raftlin-2. In Raftlin-deficient mice, T cell-dependent Ab production was reduced, and experimental autoimmune encephalomyelitis, a Th17-dependent autoimmune disease model, was ameliorated. In Raftlin-Tg mice, in contrast, Ab production was enhanced and experimental autoimmune encephalomyelitis was more severe. Cytokine production, especially that of IL-17, was reduced in Raftlin-deficient T cells, while it was enhanced in Raftlin-Tg T cells. We found that these changes were associated with the strength of the TCR-mediated signals. Importantly, localization of Lck protein in the lipid rafts was enhanced by Raftlin overexpression and reduced by Raftlin deficiency. These data indicate that Raftlin modulates TCR signals and is necessary for the fine-tuning of T cell-mediated immune responses.

Gfi1 negatively regulates Th17 differentiation by inhibiting RORγt activity

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.

Essential Role of Bystander Cytotoxic CD122+CD8+ T Cells for the Antitumor Immunity Induced in the Liver of Mice by α-Galactosylceramide

We recently reported that NK cells and CD8+ T cells contribute to the antimetastatic effect in the liver induced by α-galactosylceramide (α-GalCer). In the present study, we further investigated how CD8+ T cells contribute to the antimetastatic effect induced by α-GalCer. The injection of anti-CD8 Ab into mice 3 days before α-GalCer injection (2 days before intrasplenic injection of B16 tumors) did not inhibit IFN-γ production nor did it reduce the NK activity of liver mononuclear cells after α-GalCer stimulation. However, it did cause a reduction in the proliferation of liver mononuclear cells and mouse survival time. Furthermore, although the depletion of NK and NKT cells (by anti-NK1.1 Ab) 2 days after α-GalCer injection no longer decreased the survival rate of B16 tumor-injected mice, the depletion of CD8+ T cells did. CD122+CD8+ T cells in the liver increased after α-GalCer injection, and antitumor cytotoxicity of CD8+ T cells in the liver gradually increased until day 6. These CD8+ T cells exhibited an antitumor cytotoxicity toward not only B16 cells, but also EL-4 cells, and their cytotoxicity significantly decreased by the depletion of CD122+CD8+ T cells. The critical, but bystander role of CD122+CD8+ T cells was further confirmed by adoptive transfer experiments into CD8+ T cell-depleted mice. Furthermore, it took 14 days after the first intrasplenic B16/α-GalCer injection for the mice to generate CD8+ T cells that can reject s.c. rechallenged B16 cells. These findings suggest that α-GalCer activates bystander antitumor CD122+CD8+ T cells following NK cells and further induces an adaptive antitumor immunity due to tumor-specific memory CD8+ CTLs.

SOCS3 in T and NKT Cells Negatively Regulates Cytokine Production and Ameliorates ConA-Induced Hepatitis

Suppressor of cytokine signaling 3 (SOCS3), a negative-feedback molecule for cytokine signaling, has been implicated in protection against liver injury. Previous studies have shown that overexpression of SOCS3 in the liver by adenovirus or membrane permeable recombinant protein protected the liver from various injuries. However it remained uncertain in which type of cells SOCS3 suppresses liver injury. In this study, we demonstrated that forced expression of SOCS3 in T and NKT cells suppressed ConA-induced hepatitis using T and NKT cell-specific SOCS3 transgenic (Lck-SOCS3 Tg) mice. IFN-γ and IL-4 production was reduced in Lck-SOCS3 Tg mice as well as splenocytes treated with ConA. IFN-γ and IL-4 levels were also reduced in Lck-SOCS3 Tg mice administrated with α-galactosylceramide, suggesting that SOCS3 in NKT cells has suppressive function. Sustained expression of SOCS3 in an NKT cell line also resulted in reduced expression of various cytokines and transcription factors. In contrast, T and NKT cell-specific SOCS3 conditional knockout (Lck-SOCS3 cKO) mice were hypersensitive to ConA-mediated hepatitis. Isolated SOCS3-deficient NKT cells produced higher levels of IFN-γ and IL-4. These data indicate that SOCS3 plays a negative regulatory role in NKT cell activation and that forced expression of SOCS3 in NKT cells is effective in preventing hepatitis.