Shigeaki Hida

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.

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.

IFN regulatory factor-2 deficiency revealed a novel checkpoint critical for the generation of peripheral NK cells.

NK cell development is far less understood compared with that of T and B cells despite the critical importance of NK cells in innate immunity. Mice lacking the transcription factor IFN regulatory factor-2 (IRF-2) are known to exhibit NK cell deficiency. However, the role of IRF-2 in NK cell development has remained unclear. In this study we found that NK cell deficiency in the periphery in IRF-2-deficient mice was due to selective loss of mature NK cells, but not to maturation arrest, and NK cells in these mice exhibited very immature surface phenotypes (CD11blowDx5low) with highly compromised NK receptor expression. In contrast, IRF-2-deficient NK cells in bone marrow (BM) showed relatively mature phenotypes (CD11blowDx5high) with less compromised NK receptor repertoire. Furthermore, BM NK cells in IRF-2-deficient mice were found to proliferate almost normally, but underwent accelerated apoptosis. These observations indicated that NK cell maturation could advance up to a late, but not the final, stage in the BM, whereas these cells were incapable of contributing to the peripheral NK cell pool due to premature death in the absence of IRF-2. In contrast, NK cell numbers and Ly49 expression were much more severely reduced in BM in IL-15-deficient mice than in IRF-2−/− mice. The differential peripheral and central NK cell deficiencies in IRF-2−/− mice thus revealed a novel late checkpoint for NK cell maturation, distinct from the early IL-15-dependent expansion stage.

Critical role of caspase-1 in vascular inflammation and development of atherosclerosis in Western diet-fed apolipoprotein E-deficient mice

OBJECTIVE Recent investigations have suggested that the inflammasome plays a role in the development of vascular inflammation and atherosclerosis; however, its precise role remains controversial. We produced double-deficient mice for apolipoprotien E (Apoe) and caspase-1 (Casp1), a key component molecule of the inflammasome, and investigated the effect of caspase-1 deficiency on vascular inflammation and atherosclerosis. METHODS AND RESULTS Atherosclerotic plaque areas in whole aortas and aortic root of Western diet (WD)-fed Apoe(-/-)Casp1(-/-) mice were significantly reduced compared to those in Apoe(-/-) mice. The amount of macrophages and vascular smooth muscle cells in the plaques was also reduced in Apoe(-/-)Casp1(-/-) mice. No significant differences in plasma lipid profiles and body weight change were observed between these mice. Expression of interleukin (IL)-1β in the plaques as well as plasma levels of IL-1β, IL-1α, IL-6, CCL2, and TNF-α, in Apoe(-/-)Casp1(-/-) mice were lower than those in Apoe(-/-) mice. In vitro experiments showed that calcium phosphate crystals induced caspase-1 activation and secretion of IL-1β and IL-1α in macrophages. CONCLUSION Our findings suggest that caspase-1 plays a critical role in vascular inflammation and atherosclerosis, and that modulation of caspase-1 could be a potential target for prevention and treatment of atherosclerosis.

Requirement of the IFN-alpha/beta-induced CXCR3 chemokine signalling for CD8+ T cell activation.

Background: Activation of both CD4+ T and CD8+ T cells is triggered by the engagement of the T cell antigen receptor (TCR) with MHC/peptide complexes on antigen‐presenting cells. This process also requires other molecular interactions, which transmit co‐stimulatory signals to these T cells. To ensure an effective immune response, distinct T cell subsets may additionally employ unique mechanism(s) for efficient activation.

Differences in interleukin 1 (IL-1), IL-6, tumor necrosis factor and IL-1 receptor antagonist production by human monocytes stimulated with muramyl dipeptide (MDP) and its stearoyl derivative, romurtide

The immunostimulatory reagents muramyl dipeptide (MDP) and its stearoyl derivative romurtide [MDP-Lys(L18)] were assessed for cytokine inducing activity in human monocytes. Both MDP and romurtide stimulated the production of interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF) and IL-1 receptor antagonist (IL-1Ra). Kinetics study indicated that IL-1, TNF and IL-1Ra were induced after 4 h stimulation but IL-6 was produced at a later phase. Romurtide induced these cytokines for longer period that MDP. Dose-response study indicated that romurtide was far more potent than MDP in induction of IL-1, IL-6 and TNF. Although the magnitude of the IL-1 and IL-6 induction was almost the same, that of TNF induction was greater in romurtide-stimulated monocytes than in MDP-stimulated cells. Among IL-1, IL-1 beta appeared to be a major product. In contrast to other cytokines, IL-1Ra was induced by MDP and romurtide in a similar dose and time dependent manner with similar magnitude of response. These studies indicate that MDP and romurtide, especially romurtide, are very potent inducers of both immunostimulatory and immunosuppressive cytokines by human monocytes but with different efficacy and kinetics.

Critical role of Th17 cells in inflammation and neovascularization after ischaemia

AIMS Increasing evidence suggests that CD4(+) T cells contribute to neovascularization in ischaemic tissue. However, the T cell subset responsible for neovascularization after ischaemia remains to be determined. Here, we investigated the role of Th17 cells secreting interleukin (IL)-17, a newly identified subset of CD4(+) T cells, in the neovascularization after murine hindlimb ischaemia. METHODS AND RESULTS Unilateral hindlimb ischaemia was produced in wild-type (WT) C57BL/6 mice. Depletion of CD4(+) T cells resulted in significantly reduced blood flow perfusion in the ischaemic limbs. The expression of IL-17 and retinoic acid receptor-related orphan receptor γt (RORγt) was up-regulated in the ischaemic limbs. IL-17-deficient mice showed a significant reduction in blood flow perfusion, inflammatory cell infiltration, and production of angiogenic cytokines in the ischaemic limbs compared with WT mice. In bone marrow transplantation experiments, the absence of IL-17 specifically in bone marrow cells diminished the neovascularization after ischaemia. Furthermore, IL-17-deficient CD4(+) T cells transferred into the ischaemic limbs of T cell-deficient athymic nude mice evoked a significantly limited neovascularization compared with WT CD4(+) T cells. CONCLUSION These findings identify Th17 cells as a new angiogenic T cell subset and provide new insight into the mechanism by which T cells promote neovascularization after ischaemia.

Genetic Control Directed toward Spontaneous IFN-α/IFN-β Responses and Downstream IFN-γ Expression Influences the Pathogenesis of a Murine Psoriasis-Like Skin Disease

Psoriasis is an inflammatory skin disease, onset and severity of which are controlled by multiple genetic factors; aberrant expression of and responses to several cytokines including IFN-α/IFN-β and IFN-γ are associated with this “type 1” disease. However, it remains unclear whether genetic regulation influences these cytokine-related abnormalities. Mice deficient for IFN regulatory factor-2 (IRF-2) on the C57BL/6 background (IRF-2−/−BN mice) exhibited accelerated IFN-α/IFN-β responses leading to a psoriasis-like skin inflammation. In this study, we found that this skin phenotype disappeared in IRF-2−/− mice with the BALB/c or BALB/c × C57BL/6 F1 backgrounds. Genome-wide scan revealed two major quantitative trait loci controlled the skin disease severity. Interestingly, these loci were different from that for the defect in CD4+ dendritic cells, another IFN-α/IFN-β-dependent phenotype of the mice. Notably, IFN-γ expression as well as spontaneous IFN-α/IFN-β responses were up-regulated several fold spontaneously in the skin in IRF-2−/−BN mice but not in IRF-2−/− mice with “resistant” backgrounds. The absence of such IFN-γ up-regulation in IRF-2−/−BN mice lacking the IFN-α/IFN-β receptor or β2-microglobulin indicated that accelerated IFN-α/IFN-β signals augmented IFN-γ expression by CD8+ T cells in the skin. IFN-γ indeed played pathogenic roles as skin inflammation was delayed and was much more infrequent when IRF-2−/−BN mice lacked the IFN-γ receptor. Our current study thus revealed a novel genetic mechanism that kept the skin immune system under control and prevented skin inflammation through regulating the magnitude of IFN-α/IFN-β responses and downstream IFN-γ production, independently of CD4+ dendritic cells.