Ayako Taguchi

The Absence of IDO Upregulates Type I IFN Production, Resulting in Suppression of Viral Replication in the Retrovirus-Infected Mouse

Indoleamine 2,3-dioxygenase, the l-tryptophan–degrading enzyme, plays a key role in the powerful immunomodulatory effects on several different types of cells. Because modulation of IDO activities after viral infection may have great impact on disease progression, we investigated the role of IDO following infection with LP-BM5 murine leukemia virus. We found suppressed BM5 provirus copies and increased type I IFNs in the spleen from IDO knockout (IDO−/−) and 1-methyl-d-l-tryptophan–treated mice compared with those from wild-type (WT) mice. Additionally, the number of plasmacytoid dendritic cells in IDO−/− mice was higher in the former than in the WT mice. In addition, neutralization of type I IFNs in IDO−/− mice resulted in an increase in LP-BM5 viral replication. Moreover, the survival rate of IDO−/− mice or 1-methyl-d-l-tryptophan–treated mice infected with LP-BM5 alone or with both Toxoplasma gondii and LP-BM5 was clearly greater than the survival rate of WT mice. To our knowledge, the present study is the first report to observe suppressed virus replication with upregulated type I IFN in IDO−/− mice, suggesting that modulation of the IDO pathway may be an effective strategy for treatment of virus infection.

Ability of IDO To Attenuate Liver Injury in α-Galactosylceramide–Induced Hepatitis Model

IDO converts tryptophan to l-kynurenine, and it is noted as a relevant molecule in promoting tolerance and suppressing adaptive immunity. In this study, we examined the effect of IDO in α-galactosylceramide (α-GalCer)–induced hepatitis. The increase in IDO expression in the liver of wild-type (WT) mice administered α-GalCer was confirmed by real-time PCR, Western blotting, and IDO immunohistochemical analysis. The serum alanine aminotransferase levels in IDO-knockout (KO) mice after α-GalCer injection significantly increased compared with those in WT mice. 1-Methyl-d-tryptophan also exacerbated liver injury in this murine hepatitis model. In α-GalCer–induced hepatitis models, TNF-α is critical in the development of liver injury. The mRNA expression and protein level of TNF-α in the liver from IDO-KO mice were more enhanced compared with those in WT mice. The phenotypes of intrahepatic lymphocytes from WT mice and IDO-KO mice treated with α-GalCer were analyzed by flow cytometry, and the numbers of CD49b+ and CD11b+ cells were found to have increased in IDO-KO mice. Moreover, as a result of the increase in the number of NK cells and macrophages in the liver of IDO-KO mice injected with α-GalCer, TNF-α secretion in these mice was greater than that in WT mice. Deficiency of IDO exacerbated liver injury in α-GalCer–induced hepatitis. IDO induced by proinflammatory cytokines may decrease the number of TNF-α–producing immune cells in the liver. Thus, IDO may suppress overactive immune response in the α-GalCer–induced hepatitis model.

Upregulation of indoleamine 2,3-dioxygenase in hepatocyte during acute hepatitis caused by hepatitis B virus-specific cytotoxic T lymphocytes in vivo.

Background/Aims: Indoleamine‐2,3‐dioxygenase (IDO) is a tryptophan‐catabolizing enzyme inducing suppression of T‐cell function and immune tolerance. In hepatitis B virus (HBV) transgenic (Tg) mice, the adoptive transfer of HBV‐specific cytotoxic T lymphocytes (CTL) causes a necroinflammatory liver disease that is histologically similar to acute viral hepatitis in man. The present study aimed to determine IDO expression in the liver and hepatocytes during an acute hepatitis model.

Marked increases in hippocampal neuron indoleamine 2, 3-dioxygenase via IFN-γ-independent pathway following transient global ischemia in mouse

Indoleamine 2, 3-dioxygenase (IDO), which catabolizes L-tryptophan (L-TRP) to L-kynurenine (L-KYN), is an immunoregulatory factor that is up-regulated via an interferon-gamma (IFN-gamma)-dependent and/or -independent mechanism. In this study, we investigated the localization of IDO and whether induction of IDO expression is an IFN-gamma-dependent and/or -independent mechanism in the CNS after cerebral ischemia. The expressions of IDO protein and mRNA were investigated at different time points following cerebral ischemia using immunohistochemistry, immunofluorescence and RT-PCR. Hippocampal neuron IDO mRNA and immunohistochemical staining were significantly up-regulated 72h after transient global ischemia. Although IFN-gamma is a dominant inducer of IDO, hippocampal neuron IDO was clearly up-regulated in IFN-gamma KO mice. In summary, this is the first finding that up-regulation of IDO in hippocampal neurons after transient global ischemia occurs via INF-gamma-independent mechanisms.

Folate antagonist, methotrexate induces neuronal differentiation of human embryonic stem cells transplanted into nude mouse retina

Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuroretinal cells. However, the transplanted ES cells also have a tumorigenic activity as they have the ability for multipotent differentiation to various types of tissues. In the present study, human ES (hES) cells were transplanted into adult nude mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-D-aspartate (NMDA) administration. After the transplantation of hES cells, the folate antagonist, methotrexate (MTX) was administrated in order to control the differentiation of the transplanted hES cells. Neuronal differentiation and teratogenic potential of hES cells were examined immunohistochemically 5 weeks after transplantation. The proliferative activity of transplanted cells was determined by both the mitotic index and the Ki-67 proliferative index. Disappearance of Oct-4-positive hES cells showing undifferentiated morphology was observed after intraperitoneal MTX treatment daily, for 15 days. Decreased mitotic and Ki-67 proliferative indices, and increased neuronal differentiation were detected in the surviving hES cells after the MTX treatment. These results suggest two important effects of intraperitoneal MTX treatment for hES cells transplanted into nude mouse retina: (1) MTX treatment following transplantation induces neuronal differentiation, and (2) MTX decreases proliferative activity and tumorigenic potential.

Localization and spatiotemporal expression of IDO following transient forebrain ischemia in gerbils.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine pathway that converts L-tryptophan to L-kynurenine. Transient forebrain ischemia initiates a series of cellular events that lead to the delayed neuronal degeneration of several brain regions. The goal of this study was to determine the localization of IDO in gerbil brain, and analyze the spatiotemporal expression of IDO in a transient forebrain ischemic model. Expression of IDO in the normal gerbil brain was observed by using immunohistochemistry. Time-course of the expression of IDO following transient forebrain ischemic gerbils was examined by immunohistochemistry, combined with hematoxylin and eosin staining for morphological analysis, and in situ terminal dUTP-biotin nick end labeling of DNA fragments (TUNEL) method. In normal gerbils, IDO immunostaining was observed in thalamus, hypothalamus and amygdaloid nucleus. IDO expression was negative in the cingulate cortex, hippocampal CA1 region and parietal cortex. Following transient ischemia, we observed a time-dependent increase of IDO expression in CA1, cingulate cortex and hypothalamus. The peak of IDO expression in CA1 and cingulate cortex occurred at 48 h after ischemic insult and diminished by 2 weeks. TUNEL staining was observed only in the CA1 region at 72 and 96 h after transient ischemia. Thus, IDO protein is present in specific regions in gerbil brain, and dynamic changes of IDO expression was observed in some neurons following transient ischemia.

Spatiotemporal expression of Hsp20 and its phosphorylation in hippocampal CA1 pyramidal neurons after transient forebrain ischemia

Abstract The goal of this study was to analyze the spatiotemporal expression of heat shock protein 20 (Hsp20) and its phosphorylation in gerbil brain after transient forebrain ischemia. Brain sections from Mongolian gerbil killed 24, 48, 72 and 96 hours and 2 weeks after ischemia (n=5 in each experimental group) were evaluated with immunohistochemical and in situ DNA end-labeling [terminal 2′-deoxyuridine 5′-triphosphate nick end-labeling (TUNEL)] techniques. Ischemia-associated Hsp20 expression was observed 24 and 48 hours later in the area of the stratum radiatum and then disappeared by 72 hours. This staining appeared along the lines of apical dendrites. Hsp20 staining in the stratum pyramidale was observed again 2 weeks after ischemia. Strong immunoreactivity for phosphorylation markers was observed in the stratum pyramidale 2 weeks after ischemia, whereas no staining was seen at either 24 or 48 hours after ischemia. Fragmented DNA was observed in nuclei and apical dendrites of CA1 pyramidal neurons by TUNEL method between 72 and 96 hours after reperfusion. The emerging expression of the Hsp20 protein within the restricted location of CA1 before the fragmented DNA transport suggests the strong relationship between Hsp20 and CA1 neuronal cell apoptosis. These findings imply a two-phase role of Hsp20 in brain ischemia: an acute phase before DNA fragmentation and a subacute phase 2 weeks after ischemia. The former may be associated with apoptosis with fragmented nuclear DNA transport into neuronal fibers and the latter associated with glial response to ischemic insult. Phosphorylation of Hsp20 might contribute to the subacute phase but not to an acute phase.

Localization of septin 8 in murine retina, and spatiotemporal expression of septin 8 in a murine model of photoreceptor cell degeneration

The septins, which form a conserved family of cytoskeletal GTP-binding proteins in mammals, comprise stable heteromeric complexes and have diverse roles in protein scaffolding, cytokinesis, vesicle trafficking and plasma membrane integrity following cell division. The goal of this study was to determine the localization of septin 8 in murine adult retina, and analyze the spatiotemporal expression of septin 8 in a murine model of photoreceptor cell degeneration. Expression of septin 8 in the normal retina of mouse and rat was observed by using immunohistochemistry and Western blotting. Furthermore, time course of the expression of septin 8 in mouse photoreceptor cell degeneration were examined by immunohistochemistry combined with hematoxylin and eosin staining, and in situ DNA fragment labeling method. In normal mouse and rat retina, localization of septin 8 is restricted in nuclei of photoreceptor cells. 96 h after intravitreal injection of cobalt chloride most photoreceptor cells lost septin 8 immunostaining at the same time as nuclear DNA fragmentation. The results of this study show that septin 8 protein is present in the specific location within the retina. Furthermore, the disappearance of septin 8 in the nuclei of photoreceptor cells is concomitant with nuclear DNA fragmentation. This suggests that loss of septin 8 could be a useful prognostic indicator for photoreceptor cell degeneration.

Indoleamine 2,3-dioxygenase 1 is upregulated in activated microglia in mice cerebellum during acute viral encephalitis.

Indoleamine 2,3-dioxygenase1 (IDO1) is the rate-limiting enzyme in the kynurenine pathway that converts l-tryptophan to l-kynurenine. Encephalomyocarditis virus (EMCV) can cause acute myocarditis in various animals including mice. Previously, IDO1 has been reported to have an important immunomodulatory function in immune-related diseases. However, the pathophysiological roles of IDO1 following acute viral infection of central nervous system are not fully understood. We observed that acute EMCV infection leads to a highly reproducible neuronal degeneration in mouse cerebellum. The goal of this study is to determine tissue/cell-specific and time-dependent expressions of IDO1 during acute EMCV infection in mouse cerebellum. IDO1 was up-regulated in microglia, which was recognized to be activated morphologically and positive for ionized calcium-binding adapter molecule 1 (Iba-1), a protein expressed in microglia, within EMCV-induced cerebellar lesions showing neuronal degeneration although the very weak expression of IDO1 is detected only in cytoplasm of Purkinje cells. No GFAP immunostaining was observed in EMCV-induced cerebellar lesions although many reactive astrocytes surrounding the lesions showed strongly positive immunostaining for GFAP 10 days after the viral inoculation. Thus, IDO1 expression may affect EMCV-induced neuronal degeneration in cerebellum.