Keigyou Yoh

T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow

Mutually exclusive expression of T-bet and Eomes drives the development of distinct NK cell lineages with complementary functions.

Hyperglycemia induces oxidative and nitrosative stress and increases renal functional impairment in Nrf2-deficient mice

The transcription factor Nrf2 regulates the expression of antioxidant genes. Hyperglycemia‐induced oxidative stress is involved in the pathogenesis of diabetes and its complications. However, little is known about the protective role of Nrf2 in diabetes. To gain insight into the protective role of Nrf2 in diabetes we treated Nrf2 knockout (Nrf2 KO) mice with streptozotocin (STZ). The STZ Nrf2 KO mice did not develop renal hyperfiltration, which was observed in the STZ‐treated wild‐type (STZ WT) mice, but renal function gradually deteriorated over the 10‐week observation period. Urinary excretion of nitric oxide metabolites and the occurrence of 8‐nitroguanosine, which was detected in glomerular lesions, were increased in STZ Nrf2 KO mice during the early stages after treatment. In vivo electron paramagnetic resonance analysis revealed an accelerated rate of decay of the 3‐carbamoyl‐2,2,5,5‐tetramethylpyrrolidine‐1‐oxyl spin probe signal in STZ Nrf2 KO mice. The addition of superoxide dismutase prolonged the half‐life of the signal, which suggested that increased oxygen radical formation occurred in the STZ Nrf2 KO mice. These results suggested that hyperglycemia increased oxidative and nitrosative stress and accelerated renal injury in the Nrf2 KO mice and that Nrf2 serves as a defense factor against some diabetic complications.

MafB Is Essential for Renal Development and F4/80 Expression in Macrophages

ABSTRACT MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages, characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages, we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. mafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly, these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells, GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly, F4/80 expression in macrophages was suppressed in the homozygous mutant, although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells, MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages, whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation, renal tubule survival, and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages.

Nrf2 regulates the sensitivity of death receptor signals by affecting intracellular glutathione levels

Nrf2 is a basic leucine zipper transcriptional activator that is essential for the coordinate transcriptional induction of various antioxidant drug-metabolizing enzymes. Numerous studies have firmly established Nrf2s importance in protection from oxidative stress and certain chemical insults. Given the protective function of Nrf2, surprisingly few studies have focused on the relationship between Nrf2 and apoptosis. Therefore, we analysed how Nrf2 influences Fas signaling using Nrf2-deficient T cells. At a concentration of 1 μg/ml, the anti-Fas antibody induced 60% of cell death in Nrf2-deficient cultured thymocytes while, using the same treatment, only 40% of Nrf2 wild-type thymocytes died (P<0.05). Nrf2 deficiency enhances the sensitivity of Fas-mediated apoptosis in T cells. Next we examined the effect of Nrf2 deficiency during hepatocellular apoptosis in vivo. In comparison to wild-type mice, Nrf2-deficient mice displayed more severe hepatitis after induction with the anti-Fas antibody or tumor necrosis factor (TNF)-α. The enhanced sensitivity to anti-Fas or TNF-α stimulation was restored by preadministration of glutathione ethyl monoester, a compound capable of passing the cell membrane and upregulating the intracellular levels of glutathione. The results indicated that Nrf2 activity regulates the sensitivity of death signals by means of intracellular glutathione levels.

Overexpression of c-Maf Contributes to T-Cell Lymphoma in Both Mice and Human

c-Maf translocation or overexpression has been observed in human multiple myeloma. Although c-maf might function as an oncogene in multiple myeloma, a role for this gene in other cancers has not been shown. In this study, we have found that mice transgenic for c-Maf whose expression was direct to the T-cell compartment developed T-cell lymphoma. Moreover, we showed that cyclin D2, integrin beta(7), and ARK5 were up-regulated in c-Maf transgenic lymphoma cells. Furthermore, 60% of human T-cell lymphomas (11 of 18 cases), classified as angioimmunoblastic T-cell lymphoma, were found to express c-Maf. These results suggest that c-Maf might cause a type of T-cell lymphoma in both mice and humans and that ARK5, in addition to cyclin D2 and integrin beta(7), might be downstream target genes of c-Maf leading to malignant transformation.

EPR IMAGING OF REDUCING ACTIVITY IN Nrf2 TRANSCRIPTIONAL FACTOR-DEFICIENT MICE

Mice that lack the Nrf2 (NF-E2-related factor 2) transcription factor develop a lupus-like autoimmune nephritis. The tissue-reducing activity of Nrf2-deficient mice was evaluated using a combination of real-time EPR imaging and spin probe kinetic analysis. Substantial delay in the spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl-PROXYL) disappearance in the liver and kidneys of Nrf2-deficient mice was observed by EPR imaging. The half-life of the spin probe in the upper abdominal area was prolonged in both the Nrf2-deficient mice and in aged mice. The combination of Nrf2 deficiency and aging in female mice resulted in the most prolonged half-life of disappearance, which was four times longer than that of juvenile female mice with a wild-type genotype. These results indicate that the low reducing activity in these organs is brought about by both Nrf2 deficiency and the aging process, and it may play a key role in the onset of autoimmune nephritis. This combination of the EPR imaging and half-life analysis appears to be a very powerful tool in the real-time analysis of reducing activity.

Transcription Factors GATA-3 and RORγt Are Important for Determining the Phenotype of Allergic Airway Inflammation in a Murine Model of Asthma

In refractory asthma, neutrophils, rather than eosinophils, often predominate in the airways. Neutrophilic airway inflammation appears to be resistant to steroids and may be related to the Th17, rather than the Th2, cytokine milieu. However, the role of GATA-3 and RORγt, transcription factors for Th2 and Th17 cell differentiation, respectively, in the pathogenesis of steroid-insensitive asthma remains unclear. To examine the effect of GATA-3– and RORγt-overexpression backgrounds on airway inflammation and steroid sensitivity, we generated two strains of transgenic mice overexpressing GATA-3 or RORγt. Mice were sensitized and challenged with OVA. Some OVA-sensitized/challenged mice were treated with dexamethasone, anti–IL-17 Ab, CXCR2 antagonist, or anti–IL-6R Ab to demonstrate their therapeutic effects on airway inflammation. Although Ag-specific airway inflammation and hyperresponsiveness were induced in each mouse, the phenotype of inflammation showed a distinct difference that was dependent upon the genotype. GATA-3–overexpressing mice exhibited steroid-sensitive eosinophilic inflammation with goblet cell hyperplasia and mucus hyperproduction under Th2-biased conditions, and RORγt-overexpressing mice developed steroid-insensitive neutrophilic inflammation under Th17-biased conditions. The levels of keratinocyte-derived chemokine, MIP-2, IL-6, and other neutrophil chemotaxis-related mediators were significantly elevated in OVA-exposed RORγt-overexpressing mice compared with wild-type mice. Interestingly, airway hyperresponsiveness accompanied by neutrophilic airway inflammation in RORγt-overexpressing mice was effectively suppressed by anti–IL-17 Ab, CXCR2 antagonist, or anti–IL-6R Ab administration. In conclusion, our results suggest that the expression levels of GATA-3 and RORγt may be important for determining the phenotype of asthmatic airway inflammation. Furthermore, blockade of the Th17-signaling pathway may be a treatment option for steroid-insensitive asthma.

c-Maf expression in angioimmunoblastic T-cell lymphoma.

The oncogene c-Maf was recently found to be overexpressed in approximately 50% of multiple myeloma cases, and a role for c-Maf in promoting cyclin D2 expression has been postulated. We previously examined c-Maf expression in various T-cell lymphomas by reverse-transcription polymerase chain reaction and found extremely elevated c-Maf levels in angioimmunoblastic T-cell lymphoma (AILT). In this study, we examined T-cell lymphomas for c-Maf and cyclin expression immunohistochemically. Of 93 cases of T-cell lymphomas we investigated in the current study, c-Maf expression was seen in 23 out of 31 cases of AILT, 3 out of 11 of adult T-cell leukemia/lymphoma, 4 out of 19 of peripheral T-cell lymphoma, unspecified [PTCL(U)], and 0 out of 11 cases of mycosis fungoides, 0 out of 11 of anaplastic large cell lymphoma, and 1 out of 10 of extranodal NK/T-cell lymphoma, nasal type. Double immunostaining in AILT revealed that the majority of c-Maf–positive cells were also positive for CD43 (MT1), CD45RO (UCHL-1), and CD4 but were negative for CD20 (L26). Additionally, cyclins D1 and D2, which stimulate cell cycle progression, were overexpressed in a large number of the c-Maf–positive AILT samples. Quantitative reverse-transcription polymerase chain reaction analysis also showed that c-Maf was overexpressed in 8/31 cases of AILT, 0/19 cases of PTCL(U), 0/11 cases of anaplastic large cell lymphoma, 0/10 cases of extranodal NK/T-cell lymphoma, nasal type, and 2/8 cases of multiple myeloma, presenting significant difference between AILT and PTCL(U) (P=0.016, χ2 test).These findings strongly suggest that CD4-positive neoplastic T cells in AILT show c-Maf expression and provide new insight into the pathogenesis of AILT suggesting c-Maf to be a useful diagnostic marker for AILT.

Th1 and Type 1 Cytotoxic T Cells Dominate Responses in T-bet Overexpression Transgenic Mice That Develop Contact Dermatitis

Contact dermatitis in humans and contact hypersensitivity (CHS) in animal models are delayed-type hypersensitivity reactions mediated by hapten-specific T cells. Recently, it has become clear that both CD4+ Th1 and CD8+ type 1 cytotoxic T (Tc1) cells can act as effectors in CHS reactions. T-bet has been demonstrated to play an important role in Th1 and Tc1 cell differentiation, but little is known about its contribution to CHS. In the present study, we used C57BL/6 mice transgenic (Tg) for T-bet to address this issue. These Tg mice, which overexpressed T-bet in their T lymphocytes, developed dermatitis characterized by swollen, flaky, and scaly skin in regions without body hair. Skin histology showed epidermal hyperkeratosis, neutrophil, and lymphocyte infiltration similar to that seen in contact dermatitis. T-bet overexpression in Tg mice led to elevated Th1 Ig (IgG2a) and decreased Th2 Ig (IgG1) production. Intracellular cytokine analyses demonstrated that IFN-γ was increased in both Th1 and Tc1 cells. Furthermore, Tg mice had hypersensitive responses to 2,4-dinitrofluorobenzene, which is used for CHS induction. These results suggest that the level of expression of T-bet might play an important role in the development of contact dermatitis and that these Tg mice should be a useful model for contact dermatitis.

Transcription factor GATA-3 is essential for lens development

During vertebrate lens development, the anterior, ectoderm‐derived lens vesicle cells differentiate into a monolayer of epithelial cells that retain proliferative potential. Subsequently, they exit the cell cycle and give rise to posterior lens fiber cells that form the lens body. In the present study, we demonstrate that the transcription factor GATA‐3 is expressed in the posterior lens fiber cells during embryogenesis, and that GATA‐3 deficiency impairs lens development. Interestingly, expression of E‐cadherin, a premature lens vesicle marker, is abnormally prolonged in the posterior region of Gata3 homozygous mutant lenses. Furthermore, expression of γ‐crystallin, a differentiation marker for fiber cells, is reduced. This suppressed differentiation is accompanied by an abnormal cellular proliferation, as well as with diminished levels of the cell‐cycle inhibitors Cdkn1b/p27 and Cdkn1c/p57 and increased Ccnd2/cyclin D2 abundance. Thus, these observations suggest that GATA‐3 is essential for lens cells differentiation and proper cell cycle control. Developmental Dynamics 238:2280–2291, 2009.