No description available.

Chronic kidney disease (CKD), a reduction in kidney function, has reached pandemic proportions and imposes a major healthcare burden worldwide. A hallmark of CKD is the accumulation of several chemical compounds, called uremic toxins, which inflict systemic and renal-specific damage. Of the known uremic toxins, kynurenine (Kyn) is known to be particularly vasculotoxic and is implicated in several complications of CKD. Indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes the first step in the metabolism of Tryptophan (Trp), regulates immune response to inflammatory cytokines in tissues. IDO plays a role in apoptosis and damage during acute kidney injury (AKI), a transient decrease in kidney function. During metabolism of Trp, IDO generates Kyn, a uremic solute, and therefore IDO may play a role in the brain and kidney damage due to accumulation of Kyn. The objective of the current study was to investigate the role and regulation of IDO in CKD pathology. Studies were performed to determine whether IDO is protective or pathologic and to find how IDO is regulated in the kidney during CKD. IDO in renopathology was examined using murine models of CKD. CKD was induced via a 0.2% adenine-supplemented diet (AD) model for 21 days. IDO regulation was examined using an Indoxyl Sulfate (IS)-specific solute model. Renal function in the IDO+/+ and IDO-/- AD mice was assessed through weekly measurement of blood urea nitrogen (BUN). H&E and Masson’s trichrome stains were used to assess percentages of glomerulosclerosis (GS) and immune infiltration (II), and combined interstitial fibrosis and tubular atrophy (IFTA) score in IDO+/+ and IDO-/- mice with and without CKD. IDO protein concentration in the kidneys of all mice with and without CKD and IDO+/+ IS mice was determined via immunoblotting. Patients with kidney disease suffer from neuropsychological disorders and neurocognitive decline. The effects of uremic solutes on the CNS was examined using immortalized human umbilical endothelial vein cells (HUVEC-TERT), in vitro. Cell proliferation and viability, in the presence of IS, were measured by BrdU and Alamar blue assays, respectively. In both IDO+/+ and IDO-/-, 21 days of AD results in significant deterioration of renal function. The average IFTA score and percentage of II in IDO-/- mice increased with AD compared to ND (p<0.05, p<0.001). IDO expression was seen sporadically in the glomeruli and walls of major vessels in the kidneys of 4d AD IDO+/+ mice, and in the tubules and vessel walls in the kidneys of 14d AD IDO+/+ mice. In IDO+/+ ND mice, endogenous IDO protein expression was undetectable at a signal intensity of 119.86 ± 268.01, whereas IDO+/+ AD mice showed a 370-fold higher level of IDO protein expression compared to IDO+/+ ND (p<0.001). IDO-/- AD IDO protein expression was 9.5-fold higher than in IDO-/- AD (p<0.05). IDO expression was found to be 58-fold higher in IDO+/+ mice with IS treatment (p<0.05). In the IS mice, non-significant trends toward decrease in cellular proliferation and viability with time were also observed (p=ns). IDO is upregulated at the protein level both in a CKD model and directly by the uremic solute, IS. IDO appears to be protective in the kidney during CKD, given the trend toward increased percentage of GS and II in IDO-/- compared to IDO+/+ mice with CKD, though there is little difference seen in total kidney IFTA. IDO upregulation is linked to increased apoptosis. Blocking uremic solute production would therefore prevent IDO protein upregulation and reduce apoptosis, alleviating renal damage during CKD.

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第17862号 / 薬博第793号 / 新制||薬||236(附属図書館) / 30682 / 京都大学大学院薬学研究科医療薬科学専攻 / (主査)教授 髙倉 喜信, 教授 橋田 充, 教授 佐治 英郎 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

No description available.

Yes / Alopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss. / Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945).

Eimeria falciformis ist ein Apicomplexa-Parasit, welcher das Blinddarmepithel der Maus befällt. Aufgrund des monoxenen Lebenszyklus in einem exzellent-erforschten Wirt, bietet sich E. falciformis als Modellorganismus an, um Wirts-Parasit-Interaktionen zu untersuchen. Im Rahmen dieser Arbeit wurden mit Hilfe von Genexpressionsanalysen bei E. falciformis-infizierten Zellen und Mäusen Wirtsfaktoren identifiziert, welche für die in vitro bzw. in vivo Entwicklung des Parasiten vonnöten sind. Der Transkriptionsfaktor c-FOS (FBJ osteosarcoma oncogene) zeigte eine erhöhte Expression bei der Infektion einer Epithelzelllinie mit E. falciformis. C-FOS ist ein Bestandteil des AP-1 (activator protein 1) Komplexes, welcher die Transkription zahlreicher Gene unterschiedlichster Funktion steuert. Unsere Ergebnisse zeigen, dass die Entwicklung von E. falciformis in Zellen, welche den Transkriptionsfaktor nicht besitzen (c-FOS knockout Zellen) beeinträchtigt war. Diese Beobachtung betont eine mögliche Ausbeutung des Transkriptionsfaktors des Wirtes durch den Parasiten. In E. falciformis-infizierten Mäusen war die Expression des Enzyms Indoleamin 2,3-Dioxygenase (IDO1) bemerkenswert induziert. IDO1 katalysiert die erste und geschwindigkeits-bestimmende Reaktion des Tryptophan-Abbaus innerhalb des Kynurenin-Stoffwechselweges. Wir zeigen in dieser Studie, dass in den E. falciformis-infizierten Epithelzellen IDO1 IFN-gamma abhängig induziert wird. Das Wachstum des Parasiten war zudem beeinträchtigt in IDO1-/- Mäusen sowie in Mäusen, in welchen zwei weiterer Enzyme des Kynurenin-Stoffwechselweges pharmakologisch inhibiert wurden. Bemerkenswerterweise konnte das Parasitenwachstum in IDO1-/- Mäusen durch Gabe von Xanthurensäure, ein Nebenprodukt des Tryptophan-Abbaus, auf Wildtyp-Niveau angehoben werden. Diese Daten demonstrieren, dass sich der intrazelluläre Parasit E. falciformis die wirtseigenen Verteidigungsmechanismen (IFN-gamma, IDO1) für seine eigene Entwicklung zu Nutze macht. / Eimeria falciformis is a highly host- and tissue-specific parasite of murine caecum epithelium. Its monoxenous life cycle in a well-investigated host makes it an excellent model to examine parasite-host interactions. To identify the host determinants of the parasite infection, this work involved the comparative in vitro and in vivo analyses of mouse gene modulation by E. falciformis. The in vitro microarray analyses identified the transcription factor FBJ osteosarcoma oncogene (c-FOS) as highly induced during E. falciformis infection. C-FOS is part of the activator protein 1 (AP-1) complex, which controls the transcription of genes involved in various biological processes. We show that infection of c-FOS-deficient mouse cells results in an impaired development of E. falciformis, highlighting an exploitation of the host transcription factor by an apicomplexan parasite. Our ex vivo gene expression analyses using mouse caecum cells revealed a substantial modulation of the host transcriptome. The indoleamine 2,3-dioxygenase 1 (IDO1), the first and rate-limiting enzyme of tryptophan catabolism in the kynurenine pathway, was one of the most up-regulated epithelial transcripts. Induction of IDO1 supposedly depletes tryptophan in host cells, which is proposed to inhibit the in vitro growth of pathogens auxotrophic for this essential amino acid. We show that E. falciformis induces IDO1 in the epithelial cells in an IFN-gamma-dependent manner. The absence or inhibition of IDO1 and two downstream enzymes of the pathway in the mouse impairs parasite growth. Noticeably, the parasite development was entirely rescued by xanthurenic acid, a by-product of tryptophan catabolism. These data demonstrate contrasting roles of IFN-gamma signaling and a conceptual subversion of the host defense (IFN-gamma, IDO1) by an intracellular pathogen for progression of its natural life cycle.

Amino acid availability plays an important role in modulating the activity of T-cells. One of the pathways employed by T-cells to sense nutrient levels is the “mammalian target of rapamycin” (mTOR) pathway that is inhibited in response to nutrient depletion. Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme along the tryptophan catabolising kynurenine pathway. T-cells are very sensitive to lack of this essential amino acid in their microenvironment and this confers strong immunomodulatory properties to cells expressing active IDO. It therefore has a significant physiological role as a homeostatic mechanism used in mammalian organisms to dampen excessive activation of the immune system but is also used as an immune evasion mechanism by many cancers. In this study, we investigated the IDO inhibitory properties and mechanism of action of the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) that potentially forms a negative feedback loop in the kynurenine pathway. We studied the molecule in enzymatic assays, in live cells and discovered that it inhibits IDO in an indirect way via the formation of hydrogen peroxide. Secondly, we looked at the effects of tryptophan and its metabolites on T-cell proliferation and mTOR activity, and discovered a metabolite that inhibits T-cell proliferation. Lastly we examined mechanisms of T-cell suppression employed by myeloid derived suppressor cells (MDSCs), focusing on their ability to deplete amino acids from their microenvironment. We were able to exclude tryptophan consumption as a suppressive mechanism and established that by manipulating extracellular concentrations of several amino acids other than arginine and cysteine – that are known to be utilised by MDSCs - we were able to reduce their inhibitory properties. In summary, we have described in detail how 3-HAA inhibits IDO in in vitro assays, outlined how some tryptophan metabolites can inhibit T-cell proliferation, and clarified aspects of suppressive mechanism employed by MDSCs.

O câncer de mama é o tipo de câncer mais comum entre as mulheres e a sobrevivência de pacientes por ele acometidos está aumentando graças a diversas novas abordagens relacionadas ao seu diagnóstico precoce e a tratamentos mais eficientes. A enzima indoleamina- 2,3 dioxigenase (IDO) é expressa por diversas células e também por células tumorais. Esta enzima atua inibindo a proliferação de linfócitos T, comprometendo a atividade citotóxica do mesmo. O 1-metil-DL-triptofano (1MT) é um inibidor competitivo da IDO, que bloqueia seu efeito imunossupressor e poderia cooperar com a quimioterapia na regressão de tumores. Por este motivo, além de evidenciar a expressão da IDO em células tumorais mamárias em tecido e em cultivo, este trabalho também teve como objetivo verificar o efeito &ldquo;in vitro&rdquo; da associação do 1-metil-DL-triptofano (1MT) ao quimioterápico paclitaxel (taxol&reg;), como método de atenuação no crescimento tumoral. Acredita-se que isto possibilitaria a restauração da capacidade de proliferação dos linfócitos T e de sua capacidade de resposta citotóxica. Os resultados demonstraram que a IDO é expressa no tecido mamário, com alta concentração no estroma tumoral. Os cultivos suplementados mostraram que as diferenças mais significativas na expressão da IDO foram observadas no grupo tratado com paclitaxel associado ao 1-metil-DL-triptofano após suplementação contínua com os fatores mencionados, onde houve redução de 12,06% para 3,56% na expressão da enzima. Dessa forma pode-se sugerir que esta associação foi mais eficaz na contenção dessa expressão o que poderia levar à uma restauração da capacidade de resposta celular dos linfócitos T contra o tumor mamário. Tal resultado poderia colaborar no desenvolvimento de uma nova estratégia terapêutica no tratamento de pessoas afetadas pelo câncer de mama / Breast cancer is the most common type of cancer among women and the survival of patients affected by it is increasing, mainly dueto several new approaches in early diagnosis and more effective treatments. The enzyme indoleamine- 2,3 - dioxygenase (IDO) is expressed on many cells and also on tumor cells. This enzyme acts by inhibiting the proliferation of T lymphocytes, thus compromising their cytotoxic activity. The 1-methyl-DL-tryptophan (1MT) is a competitive inhibitor of IDO, which blocks its immunosuppressive effect and could collaborate with chemotherapy in tumor regression. Thus, besides highlighting the expression of IDO in tumor cells in mammary tissue and culture, this study also aimed to determine the &quot;in vitro&quot; effect of the association of 1-methyl-DL-tryptophan (1MT) and paclitaxel (Taxol&reg;) chemotherapy, as an attenuation approach to tumor growth. It is believed that it would allow the restoration of T-lymphocytes proliferation capability and their cytotoxic response. Results showed that IDO is expressed in breast tissue with a high concentration in the tumor stroma. The supplemented cultures showed that the most significant differences in the expression of IDO were observed in the group treated with paclitaxel associated with 1-methyl-DL-tryptophan continuous supplementation, reducing the enzyme expression from 12.06% to 3.56%. Therefore, it may be suggested that this association was more effective in reducing IDO expression and such outcome could collaborate in developing a new therapeutic strategy for breast cancer treatment

Indolamina-2,3-dioxigenase (IDO) e o catabolismo do triptofano estão envolvidos no controle da imunidade inata e adaptativa contra patógenos. Investigamos o papel da IDO na paracoccidiodomicose pulmonar (PCM) de animais suscetíveis (B10.A) e resistentes (A/J) ao fungo. Observou-se uma ação marcante da IDO ao início da doença de camundongos B10.A onde a enzima controla a carga fúngica mas, também, induz anergia de células TCD4+ e TCD8+, creditada em parte à expansão de células Treg e aumento de linfócitos em apoptose. Em camundongos A/J, a IDO controla a carga fúngica inicial, porém, o seu efeito supressor sobre linfócitos TCD4+ é somente observado na 8ª semana. Assim como em camundongos B10.A, a IDO mostrou-se indutora de células Treg e linfócitos em apoptose durante a imunidade desenvolvida por camundongos A/J. Em conclusão, foi demonstrado pela primeira vez que a IDO exerce um importante mecanismo microbicida e imunorregulador na PCM de hospedeiros resistentes e suscetíveis ao P. brasiliensis. / Indoleamine-2, 3-dioxygenase (IDO) and tryptophan catabolism are involved in the control of innate and adaptive immunity against pathogens. We investigated the role of IDO in the pulmonary paracoccidiodomycosis developed by susceptible (B10.A) and resistant (A/J) mice to the fungus. We verified that IDO plays a different effect in innate the immunity of B10.A and A/J mice. Early in the infection, IDO controlled the fungal loads but also induced anergy of CD4+ and CD8+ T cells of B10.A mice. T cell anergy was partially due to the expansion of Treg cells and increased apoptosis of lymphocytes. In resistant mice, IDO controlled the initial fungal loads, but exerted a suppressive effect on T lymphocytes only at week 8. As in B10.A mice IDO was shown to induce Treg cells and apoptosis of lymphocytes in the course of immune response developed by resistant mice. In conclusion our work showed for the first time that IDO play an important role in the fungicidal and immunoregulatory mechanisms developed by susceptible and resistant mice to P. brasiliensis infection.

O câncer de mama é o tipo de câncer mais comum entre as mulheres e a sobrevivência de pacientes por ele acometidos está aumentando graças a diversas novas abordagens relacionadas ao seu diagnóstico precoce e a tratamentos mais eficientes. A enzima indoleamina- 2,3 dioxigenase (IDO) é expressa por diversas células e também por células tumorais. Esta enzima atua inibindo a proliferação de linfócitos T, comprometendo a atividade citotóxica do mesmo. O 1-metil-DL-triptofano (1MT) é um inibidor competitivo da IDO, que bloqueia seu efeito imunossupressor e poderia cooperar com a quimioterapia na regressão de tumores. Por este motivo, além de evidenciar a expressão da IDO em células tumorais mamárias em tecido e em cultivo, este trabalho também teve como objetivo verificar o efeito &ldquo;in vitro&rdquo; da associação do 1-metil-DL-triptofano (1MT) ao quimioterápico paclitaxel (taxol&reg;), como método de atenuação no crescimento tumoral. Acredita-se que isto possibilitaria a restauração da capacidade de proliferação dos linfócitos T e de sua capacidade de resposta citotóxica. Os resultados demonstraram que a IDO é expressa no tecido mamário, com alta concentração no estroma tumoral. Os cultivos suplementados mostraram que as diferenças mais significativas na expressão da IDO foram observadas no grupo tratado com paclitaxel associado ao 1-metil-DL-triptofano após suplementação contínua com os fatores mencionados, onde houve redução de 12,06% para 3,56% na expressão da enzima. Dessa forma pode-se sugerir que esta associação foi mais eficaz na contenção dessa expressão o que poderia levar à uma restauração da capacidade de resposta celular dos linfócitos T contra o tumor mamário. Tal resultado poderia colaborar no desenvolvimento de uma nova estratégia terapêutica no tratamento de pessoas afetadas pelo câncer de mama / Breast cancer is the most common type of cancer among women and the survival of patients affected by it is increasing, mainly dueto several new approaches in early diagnosis and more effective treatments. The enzyme indoleamine- 2,3 - dioxygenase (IDO) is expressed on many cells and also on tumor cells. This enzyme acts by inhibiting the proliferation of T lymphocytes, thus compromising their cytotoxic activity. The 1-methyl-DL-tryptophan (1MT) is a competitive inhibitor of IDO, which blocks its immunosuppressive effect and could collaborate with chemotherapy in tumor regression. Thus, besides highlighting the expression of IDO in tumor cells in mammary tissue and culture, this study also aimed to determine the &quot;in vitro&quot; effect of the association of 1-methyl-DL-tryptophan (1MT) and paclitaxel (Taxol&reg;) chemotherapy, as an attenuation approach to tumor growth. It is believed that it would allow the restoration of T-lymphocytes proliferation capability and their cytotoxic response. Results showed that IDO is expressed in breast tissue with a high concentration in the tumor stroma. The supplemented cultures showed that the most significant differences in the expression of IDO were observed in the group treated with paclitaxel associated with 1-methyl-DL-tryptophan continuous supplementation, reducing the enzyme expression from 12.06% to 3.56%. Therefore, it may be suggested that this association was more effective in reducing IDO expression and such outcome could collaborate in developing a new therapeutic strategy for breast cancer treatment