Makoto Kanoh

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.

Evaluation of mitogen-induced responses in marine mammal and human lymphocytes by in-vitro exposure of butyltins and non-ortho coplanar PCBs

The effects of exposure to butyltin compounds (BTs: tributyltin; TBT, dibutyltin; DBT and monobutyltin; MBT) and non-ortho coplanar PCBs (IUPAC 77, 126 and 169) on marine mammals and human lymphocyte were evaluated. Peripheral blood mononuclear cells (PBMCs) isolated from Dalls porpoises (Phocoenoides dalli), bottlenose dolphins (Tursiops truncatus), a California sealion (Zalophus californianus), a larga seal (Phocoa largha) and humans (Homo sapiens) were exposed at varying concentrations of BTs and coplanar PCBs. Concanavalin A (Con A)-stimulated mitogenesis found significantly suppressed (P<0.01) when the cells were exposed at 300 nM (89 ng/ml) of TBT and 330 nM of DBT (77 ng/ml), while MBT showed little cytotoxicity at treatment levels of up to 3,600 nM (620 ng/ml). BTs concentrations in the liver of Dalls porpoises from Japanese coastal waters ranged between 81-450 ng/g for TBT and 200-1,100 ng/g (wet wt.) for DBTs, which is greater than the cytotoxic levels registered in this study. In contrast, non-ortho coplanar PCBs did not suppress cell proliferation at concentrations of up to 30 nM (10 ng/ml). The residue levels of coplanar PCBs in the blubber of Dalls porpoises were 0.12-1.3 ng/g, which were one order of lower than those levels that do cell proliferation. When cells were exposed to a mixture of TBT/DBTand coplanar PCBs, the proliferation was significantly reduced to 33 nM DBT plus 34 nM CB-77 and 33 nM DBT plus 28 nM CB-169 mixtures, respectively. The investigations relating the contaminant-induced immunosuppression in marine mammals have been focused on persistent organochlorines such as PCBs. pesticides and dioxin compounds. However, this study suggested the possibility of BTs could also pose a serious threat to the immune functions in free-ranging marine mammals and humans.

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.

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.

Mechanisms of Neutralization of Endotoxin by Monoclonal Antibodies to O and R Determinants of Lipopolysaccharide

The protective potentials of antibodies to O and R core regions of lipopolysaccharide (LPS) have been amply substantiated (2, 7). However, the efficacy of antibodies to the toxic lipid A moiety itself is still ambiguous, and how antibodies to regions distal from lipid A can neutralize the toxicity remains to be clarified. We have compared the effects of mouse monoclonal antibodies (mAbs) of IgG class to these three regions of LPS on the biological activities as well as micellic structure of LPS, in order to shed light on the mechanism of neutralization of endotoxin by these antibodies.

Polymorphonuclear Leukocyte‐Inhibitory Factor of Bordetella pertussis

In vivo biologic effects of the polymorphonuclear leukocyte‐inhibitory factor (PIF) of Bordetella pertussis were tested by using two experimentally induced inflammatory processes in mice. The intravenous injection of a partially purified extract from phase I bacteria strongly inhibited the glycogen‐induced peritoneal infiltration of polymorphonuclear leukocytes (PMN) and the Arthus reactions, whereas little inhibitory activity was found in the extract from phase III bacteria. The activity was localized in the outer membrane of phase I bacteria, as was the in vitro PIF activity, and the two activities gave the same behavior in DEAE‐cellulose chromatography. Therefore the observed suppression of inflammatory processes in mice is probably due to the inhibitory action of PIF on the function of PMN in vivo.

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.