Saho Maruyama

Identification of IFN Regulatory Factor-1 Binding Site in IL-12 p40 Gene Promoter

IL-12 is a heterodimer composed of p40 and p35 and is a key cytokine that functions to protect the host from viral and microbial infections. IL-12 links the innate immune system with the acquired immune system during infection, and induces differentiation of type 1 T cells that play an important role in the eradication of microbes. The induction of the IL-12 p40 gene is regulated by NF-κB in the presence of IFN-γ. IFN-γ induces IFN regulatory factor-1 (IRF-1), which in turn induces the transcription of the IL-12 p40 gene. However, the IRF-1 binding site in the promoter region of the IL-12 p40 gene has not yet been formally determined. In the present study, we demonstrated that IRF-1 directly binds to the IL-12 p40 gene promoter and identified its binding site. The IRF-1 binding site in the promoter region of the IL-12 p40 gene is shown to be in the −72 to −58 area of the 5′-upstream region. The −63 to −61 position is the critical site within this region for the binding of IRF-1 to the IL-12 p40 gene promoter. While IFN-γ must be present for IL-12 p40 gene induction, the p35 gene is strongly induced by LPS, even in the absence of IFN-γ, and therefore the induction of the p35 gene is IRF-1 independent.

The Menin–Bach2 axis is critical for regulating CD4 T-cell senescence and cytokine homeostasis

Although CD4 T-cell senescence plays an important role in immunosenescence, the mechanism behind this process remains unclear. Here we show that T cell-specific Menin deficiency results in the premature senescence of CD4 T cells, which is accompanied by the senescence-associated secretory phenotype after antigenic stimulation and dysregulated cytokine production. Menin is required for the expansion and survival of antigen-stimulated CD4 T cells in vivo and acts by targeting Bach2, which is known to regulate immune homeostasis and cytokine production. Menin binds to the Bach2 locus and controls its expression through maintenance of histone acetylation. Menin binding at the Bach2 locus and the Bach2 expression are decreased in the senescent CD4 T cells. These findings reveal a critical role of the Menin-Bach2 pathway in regulating CD4 T-cell senescence and cytokine homeostasis, thus indicating the involvement of this pathway in the inhibition of immunosenescence.

Down-Regulation of IL-12 p40 Gene in Plasmodium berghei-Infected Mice

We analyzed the mechanism that causes suppression of IL-12 p40 gene induction during Plasmodium berghei infection. Although IL-12 together with IFN-γ plays an important role in protection against pathogenic infection, the IL-12 p70 protein production of infected macrophages is lower than that by the uninfected macrophages. We showed in the present study that the induction of IL-12 p40 gene but not IL-12 p35 gene in macrophages of P. berghei-infected mice was profoundly inhibited. The inhibition was induced by interaction with macrophages that had contacted with P. berghei-infected erythrocytes and was mediated by a soluble factor, IL-10. There was comparable activation of NF-κB in uninfected and infected cells. The induction of IFN-regulatory factor-1 gene was comparable in transcription level in uninfected and infected cells, while the unidentified complex formation of IFN-regulatory factor-1 was observed in infected cells. Therefore, the inhibition of the IL-12 p40 gene induction appeared to be regulated at transcriptional regulation level of the gene.

Modulation of the Immune System by Listeria monocytogenes-Mediated Gene Transfer into Mammalian Cells

In this study, we established a method for Listeria monocytogenes (Lm)‐mediated gene transfer into mammalian cells to manipulate the immune response of the host during infection by pathogens. We used the Lm‐mediated gene transfer method in an in vivo study to manipulate host immune responses against Leishmania major (L. major)‐infection. The injection of Lm modulated the susceptible host into a resistant state against L. major‐infection. A more efficient protective effect was obtained with the injection of IL‐12‐cDNA containing Lm, and the protective effect was stronger than that of the resistant strain. The protective mechanism of Lm‐injection against L. major‐infection observed here appeared to be a result of the activation of the local immune system by the Lm‐mediated gene transfer method. The present study is the first demonstration that a gene introduced into a host by Lm works to modulate the murine host immune response against infections in vivo. Since this system strongly induces Th1 responses and suppresses Th2 responses in infected hosts, the system can be used for controlling infectious diseases and for protection against allergic responses in the future.

Bach2–Batf interactions control Th2-type immune response by regulating the IL-4 amplification loop

Although Bach2 has an important role in regulating the Th2-type immune response, the underlying molecular mechanisms remain unclear. We herein demonstrate that Bach2 associates with Batf and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2–Batf complex antagonizes the recruitment of the Batf–Irf4 complex to AP-1 motifs and suppresses Th2 cytokine production. Furthermore, we find that Bach2 regulates the Batf and Batf3 expressions via two distinct pathways. First, Bach2 suppresses the maintenance of the Batf and Batf3 expression through the inhibition of IL-4 production. Second, the Bach2–Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf–Irf4 complex. These findings suggest that IL-4 and Batf form a positive feedback amplification loop to induce Th2 cell differentiation and the subsequent Th2-type immune response, and Bach2–Batf interactions are required to prevent an excessive Th2 response.

Attenuated Listeria infection activates natural killer cell cytotoxicity to regress melanoma growth in vivo

Listeria monocytogenes infection induces various types of immune responses. The Lm‐induced immunity not only protects the hosts against Lm infection but also has a therapeutic effect on other diseases such as tumors and infectious diseases. In the present study, we sought to identify the cells and molecules that are primarily responsible for the Lm‐induced antitumor immune response. We investigated the mechanism of the antitumor immune response induced by Lm infection using melanoma cells and various types of gene‐manipulated mice and B16F10 melanoma cells. Melanoma cells were implanted into mice intrasplenically or intraperitoneally. Lm infection of mice remarkably suppressed the growth of transplanted melanoma. The suppression of melanoma growth was due to the augmented NK cytotoxicity. The Lm‐induced NK activation against melanoma was type I interferon‐ and signal transducer and activator of transcription (STAT)1‐dependent but independent of IL‐12 and IFN‐γ. In contrast to avirulent Listeria innocua and hly− Lm failed to induce NK activation, a mutant Lm strain with minimal hemolytic activity and with normal accessibility to cytoplasm‐induced NK activation. We demonstrated that the attenuated Lm entrance into the cytoplasm induces the production of type I IFN followed by the activation of NK cells, which is essential for the Lm‐induced antitumor response.

Gfi1, a transcriptional repressor, inhibits the induction of the T helper type 1 programme in activated CD4 T cells

A transcriptional repressor Gfi1 promotes T helper type 2 (Th2) cell development and inhibits Th17 and inducible regulatory T‐cell differentiation. However, the role of Gfi1 in regulating Th1 cell differentiation and the Th1‐type immune response remains to be investigated. We herein demonstrate that Gfi1 inhibits the induction of the Th1 programme in activated CD4 T cells. The activated Gfi1‐deficient CD4 T cells spontaneously develop into Th1 cells in an interleukin‐12‐ and interferon‐γ‐independent manner. The increase of Th1‐type immune responses was confirmed in vivo in Gfi1‐deficient mice using a murine model of nickel allergy and delayed‐type hypersensitivity (DTH). The expression levels of Th1‐related transcription factors were found to increase in Gfi1‐deficient activated CD4 T cells. Tbx21, Eomes and Runx2 were identified as possible direct targets of Gfi1. Gfi1 binds to the Tbx21, Eomes and Runx2 gene loci and reduces the histone H3K4 methylation levels in part by modulating Lsd1 recruitment. Together, these findings demonstrate a novel regulatory role of Gfi1 in the regulation of the Th1‐type immune response.

Menin Plays a Critical Role in the Regulation of the Antigen-Specific CD8+ T Cell Response upon Listeria Infection

Menin, a tumor suppressor protein, is encoded by the MEN1 gene in humans. Certain germinal mutations of MEN1 induce an autosomal-dominant syndrome that is characterized by concurrent parathyroid adenomas and several other tumor types. Although menin is also expressed in hematopoietic lineages, its role in CD8+ T cells remains unclear. We generated Meninflox/flox CD4-Cre (Menin-KO) mice by crossing Meninflox/flox mice with CD4-Cre transgenic (Tg) mice to determine the role of menin in CD8+ T cells. Wild-type (WT) and Menin-KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8+ T cells. Menin deficiency resulted in an impaired primary immune response by CD8+ T cells. On day 7, there were fewer Menin-KO OVA-specific CD8+ T cells compared with WT cells. Next, we adoptively transferred WT and Menin-KO OT-1 Tg CD8+ T cells into congenic recipient mice and infected them with L. monocytogenes expressing OVA to determine the CD8+ T cell–intrinsic effect. Menin-KO OT-1 Tg CD8+ T cells were outcompeted by the WT cells upon infection. Increased expression of Blimp-1 and T-bet, cell cycle inhibitors, and proapoptotic genes was observed in the Menin-KO OT-1 Tg CD8+ T cells upon infection. These data suggest that menin inhibits differentiation into terminal effectors and positively controls proliferation and survival of Ag-specific CD8+ T cells that are activated upon infection. Collectively, our study uncovered an important role for menin in the immune response of CD8+ T cells to infection.

Differential Effect of Listeria monocytogenes Infection on Cytokine Production and Cytotoxicity of CD8 T Cells

Bacterial infection induces a shift to type 1 CD4 T cell subset in an infected host and this shift is important for protection of the host from disease development. Many researchers think that the shift is antigen‐dependent, but we previously demonstrated an initial induction step for CD4 T cell subsets during Listeria monocytogenes (Lm) infection is antigen‐independent. Although Listeria is a TLR2 ligand, the immune system of the Lm‐infected host responded to the pathogen to induce expression of CD69 but not CD25 on CD4 T cells, CD8 T cells and B cells even in the absence of TLR2 or MyD88. The antigen‐independent activation of type 1 CD4 T cells accelerate the clearance of pathogens by activating innate immune cells with type 1 cytokines. Type 1 CD4 T cells and CD8 T cells also collaborate to protect the host from intracellular Lm infection. Since CD8 T cells function mainly as cytotoxic T cells and CD69‐positive CD8 T cells increase during Lm‐infection, cytotoxic activity of CD8 T cells was evaluated during Lm‐infection. Although CD8 T cells were activated to produce IFN‐γ, the cytotoxic function of CD8 T cells in Lymphocytic choriomeningitis virus (LCMV) p14 TCR‐transgenic mouse was not augmented by Lm‐infection. Therefore, Lm‐infection differentially influences on cytokine production and cytotoxicity of CD8 T cells.

The Transcriptional Repressor Gfi1 Plays a Critical Role in the Development of NKT1- and NKT2-Type iNKT Cells.

Gfi1 plays an important role in the development and maintenance of many hematopoietic linage cells. However, the impact of Gfi1-deficiency on the iNKT cell differentiation remains unclear. We herein demonstrate a critical role of Gfi1 in regulating the development of iNKT cell subsets. In the thymus of T cell-specific Gfi1-deficient mice, iNKT cells normally developed up to stage 2, while the number of stage 3 NK1.1pos iNKT cells was significantly reduced. Furthermore, CD4pos iNKT cells were selectively reduced in the peripheral organs of T cell-specific Gfi1-deficient mice. The α-GalCer-dependent production of IFN-γand Th2 cytokines, but not IL-17A, was severely reduced in T cell-specific Gfi1-deficient mice. In addition, a reduction of the α-GalCer-induced anti-tumor activity was observed in Gfi1-deficient mice. These findings demonstrate the important role of Gfi1 in regulating the development and function of NKT1- and NKT2-type iNKT cell subsets.