Kouetsu Ogasawara

Distinct and Essential Roles of Transcription Factors IRF-3 and IRF-7 in Response to Viruses for IFN-α/β Gene Induction

Induction of the interferon (IFN)-alpha/beta gene transcription in virus-infected cells is an event central to innate immunity. Mice lacking the transcription factor IRF-3 are more vulnerable to virus infection. In embryonic fibroblasts, virus-induced IFN-alpha/beta gene expression levels are reduced and the spectrum of the IFN-alpha mRNA subspecies altered. Furthermore, cells additionally defective in IRF-7 expression totally fail to induce these genes in response to infections by any of the virus types tested. In these cells, a normal profile of IFN-alpha/beta mRNA induction can be achieved by coexpressing both IRF-3 and IRF-7. These results demonstrate the essential and distinct roles of thetwo factors, which together ensure the transcriptional efficiency and diversity of IFN-alpha/beta genes for the antiviral response.

Multistage Regulation of Th1-Type Immune Responses by the Transcription Factor IRF-1

Eradication of a given pathogen is dependent on the selective differentiation of T helper (Th) cells into Th1 or Th2 types. We show here that T cells from mice lacking the transcription factor IRF-1 fail to mount Th1 responses and instead exclusively undergo Th2 differentiation in vitro. Compromised Th1 differentiation is found to be associated with defects in multiple cell types, namely impaired production of interleukin-12 by macrophages, hyporesponsiveness of CD4+ T cells to interleukin-12, and defective development of natural killer cells. These results indicate the involvement of IRF-1 in multiple stages of the Th1 limb of the immune response.

Requirement for IRF-1 in the microenvironment supporting development of natural killer cells

Natural killer (NK) cells are critical for both innate and adaptive immunity. The development of NK cells requires interactions between their progenitors and the bone-marrow microenvironment; however, little is known about the molecular nature of such interactions. Mice that do not express the transcription factor interferon-regulatory factor-1 (IRF-1; such mice are IRF-1−/− mice) have been shown to exhibit a severe NK-cell deficiency,. Here we demonstrate that the lack of IRF-1 affects the radiation-resistant cells that constitute the microenvironment required for NK-cell development, but not the NK-cell progenitors themselves. We also show that IRF-1−/− bone-marrow cells can generate functional NK cells whencultured with the cytokine interleukin-15 (refs 9-12) and that the interleukin-15 gene is transcriptionally regulated by IRF-1. These results reveal, for the first time, a molecular mechanism by which the bone-marrow microenvironment supports NK-cell development.

Impairment of NK Cell Function by NKG2D Modulation in NOD Mice

Nonobese diabetic (NOD) mice, a model of insulin-dependent diabetes mellitus, have a defect in natural killer (NK) cell-mediated functions. Here we show impairment in an activating receptor, NKG2D, in NOD NK cells. While resting NK cells from C57BL/6 and NOD mice expressed equivalent levels of NKG2D, upon activation NOD NK cells but not C57BL/6 NK cells expressed NKG2D ligands, which resulted in downmodulation of the receptor. NKG2D-dependent cytotoxicity and cytokine production were decreased because of receptor modulation, accounting for the dysfunction. Modulation of NKG2D was mostly dependent on the YxxM motif of DAP10, the NKG2D-associated adaptor that activates phosphoinositide 3 kinase. These results suggest that NK cells may be desensitized by exposure to NKG2D ligands.

NKG2D-mediated Natural Killer Cell Protection Against Cytomegalovirus Is Impaired by Viral gp40 Modulation of Retinoic Acid Early Inducible 1 Gene Molecules

Natural killer (NK) cells play a critical role in the innate immune response against cytomegalovirus (CMV) infections. Although CMV encodes several gene products committed to evasion of adaptive immunity, viral modulation of NK cell activity is only beginning to be appreciated. A previous study demonstrated that the mouse CMV m152-encoded gp40 glycoprotein diminished expression of ligands for the activating NK cell receptor NKG2D on the surface of virus-infected cells. Here we have defined the precise ligands that are affected and have directly implicated NKG2D in immune responses to CMV infection in vitro and in vivo. Murine CMV (MCMV) infection potently induced transcription of all five known retinoic acid early inducible 1 (RAE-1) genes (RAE-1α, RAE-1β, RAE-1δ, RAE-1ɛ, and RAE-1γ), but not H-60. gp40 specifically down-regulated the cell surface expression of all RAE-1 proteins, but not H-60, and diminished NK cell interferon γ production against CMV-infected cells. Consistent with previous findings, a m152 deletion mutant virus (Δm152) was less virulent in vivo than the wild-type Smith strain of MCMV. Treatment of BALB/c mice with a neutralizing anti-NKG2D antibody before infection increased titers of Δm152 virus in the spleen and liver to levels seen with wild-type virus. These experiments demonstrate that gp40 impairs NK cell recognition of virus-infected cells through disrupting the RAE-1–NKG2D interaction.

A Novel Motor, KIF13A, Transports Mannose-6-Phosphate Receptor to Plasma Membrane through Direct Interaction with AP-1 Complex

Intracellular transport mediated by kinesin superfamily proteins (KIFs) is a highly regulated process. The molecular mechanism of KIFs binding to their respective cargoes remains unclear. We report that KIF13A is a novel plus end-directed microtubule-dependent motor protein and associates with beta 1-adaptin, a subunit of the AP-1 adaptor complex. The cargo vesicles of KIF13A contained AP-1 and mannnose-6-phosphate receptor (M6PR). Overexpression of KIF13A resulted in mislocalization of the AP-1 and the M6PR. Functional blockade of KIF13A reduced cell surface expression of the M6PR. Thus, KIF13A transports M6PR-containing vesicles and targets the M6PR from TGN to the plasma membrane via direct interaction with the AP-1 adaptor complex.

Antiviral response by natural killer cells through TRAIL gene induction by IFN–α/β

Natural killer (NK) cells play an important role in early defense against viral infection. The cytotoxic activity of NK cells is increased by interferon‐α/β (IFN‐α/β), produceden masse in virally infected cells. However, the mechanism(s) by which IFN‐α/β contribute to the NK‐cell‐mediated antiviral response is not well understood. Here we provide evidence that the cytotoxicity of NK cells is enhanced by IFN‐α/β through induction of TNF‐related apoptosis‐inducing ligand (TRAIL). Isolation and analysis of the murine TRAIL promoter revealed the presence of an IFN‐stimulated response element (ISRE), which binds to the transcription factor ISGF3 (interferon stimulated gene factor‐3). This promoter is indeed activated by IFN‐β in ISGF3‐dependent manner. We also show that virally infected cells, but not uninfected cells, are susceptible to TRAIL‐mediated cytotoxicity in vitro, and that the TRAIL expressed in NK cells is indeed crucial in limiting virus replication in vivo. Thus, our study reveals a new molecular link between IFN‐α/β signaling and activation of NK cells in antiviral response of the host.

CD8+ T Cell–Mediated Skin Disease in Mice Lacking IRF-2, the Transcriptional Attenuator of Interferon-α/β Signaling

Abstract The balanced action of cytokines is known to be critical for the maintenance of homeostatic immune responses. Here, we report the development of an inflammatory skin disease involving CD8 + T cells, in mice lacking the transcription factor, interferon regulatory factor-2 (IRF-2). CD8 + T cells exhibit in vitro hyper-responsiveness to antigen stimulation, accompanied with a notable upregulation of the expression of genes induced by interferon-α/β (IFN-α/β). Furthermore, both disease development and CD8 + T cell abnormality are suppressed by the introduction of nullizygosity to the genes that positively regulate the IFN-α/β signaling pathway. IRF-2 may represent a unique negative regulator, attenuating IFN-α/β-induced gene transcription, which is necessary for balancing the beneficial and harmful effects of IFN-α/β signaling in the immune system.

Cutting Edge: Toll-Like Receptor Signaling in Macrophages Induces Ligands for the NKG2D Receptor

Macrophages recognize the presence of infection by using the Toll-like receptor (TLR) family of proteins that detect ligands on bacterial, viral, and fungal pathogens. We show that murine macrophages stimulated with pathogen products known to signal through TLRs express ligands for the NKG2D receptor, found on NK cells, activated CD8+ T cells and activated macrophages. TLR signaling, through the MyD88 adaptor, up-regulates transcription of the retinoic acid early inducible-1 (RAE-1) family of NKG2D ligands, but not H-60 or murine UL16-binding protein-like transcript-1. RAE-1 proteins are found on the surface of activated, but not resting, macrophages and can be detected by NKG2D on NK cells resulting in down-regulation of this receptor both in vitro and in vivo. RAE-1-NKG2D interactions provide a mechanism by which NK cells and infected macrophages communicate directly during an innate immune response to infection.

Function of NKG2D in natural killer cell-mediated rejection of mouse bone marrow grafts.

Irradiation-resistant natural killer (NK) cells in an F1 recipient can reject parental bone marrow, and host NK cells can also prevent engraftment of allogeneic bone marrow. We show here that repopulating bone marrow cells in certain mouse strains expressed retinoic acid early inducible 1 proteins, which are ligands for the activating NKG2D NK cell receptor. Treatment with a neutralizing antibody to NKG2D prevented rejection of parental BALB/c bone marrow in (C57BL/6 × BALB/c) F1 recipients and allowed engraftment of allogeneic BALB.B bone marrow in C57BL/6 recipients. Additionally, bone marrow from C57BL/6 mice transgenic for retinoic acid early inducible 1ε was rejected by syngeneic mice but was accepted after treatment with antibody to NKG2D. If other stem cells or tissues upregulate expression of NKG2D ligands after transplantation, NKG2D may contribute to graft rejection in immunocompetent hosts.