Aoi Son

Thioredoxin-1 Ameliorates Myosin-Induced Autoimmune Myocarditis by Suppressing Chemokine Expressions and Leukocyte Chemotaxis in Mice

Background—Cardiac myosin–induced myocarditis is an experimental autoimmune myocarditis (EAM) model used to investigate autoimmunological mechanisms in inflammatory heart diseases and resembles fulminant myocarditis in humans. We investigated the therapeutic role of thioredoxin-1 (TRX-1), a redox-regulatory protein with antioxidant and antiinflammatory effects, in murine EAM. Methods and Results—EAM was generated in 5-week-old male BALB/c mice by immunization with porcine cardiac myosin at days 0 and 7. Recombinant human TRX-1 (rhTRX-1), C32S/C35S mutant rhTRX-1, or saline was administered intraperitoneally every second day from day 0 to 20. In addition, rabbit anti-mouse TRX-1 serum or normal rabbit serum was administered intraperitoneally on days −1, 2, and 6. Animals were euthanized on day 21. Histological analysis of the heart showed that TRX-1 significantly reduced the severity of EAM, whereas mutant TRX-1 failed to have such an effect, and anti–TRX-1 antibody enhanced the disease markedly. Immunohistochemical analysis showed that TRX-1 significantly suppressed cardiac macrophage inflammatory protein (MIP)-1&agr;, MIP-2, and 8-hydroxydeoxyguanosine expression and macrophage infiltration into the heart in EAM. Although serum levels of MIP-1&agr; were not suppressed by TRX-1 until day 21, both an in vitro chemotaxis chamber assay and an in vivo air pouch model showed that TRX-1 significantly suppressed MIP-1&agr;– or MIP-2–induced leukocyte chemotaxis. However, real-time reverse transcription–polymerase chain reaction showed that TRX-1 failed to decrease chemokine receptor expression increased in the bone marrow cells of EAM mice. Conclusions—TRX-1 attenuates EAM by suppressing chemokine expressions and leukocyte chemotaxis in mice.

Physical and functional interaction of transmembrane thioredoxin-related protein with major histocompatibility complex class I heavy chain: redox-based protein quality control and its potential relevance to immune responses.

In the endoplasmic reticulum (ER), a variety of oxidoreductases classified in the thioredoxin superfamily have been found to catalyze the formation and rearrangement of disulfide bonds. However, the precise function and specificity of the individual thioredoxin family proteins remain to be elucidated. Here, we characterize a transmembrane thioredoxin-related protein (TMX), a membrane-bound oxidoreductase in the ER. TMX exists in a predominantly reduced form and associates with the molecular chaperon calnexin, which can mediate substrate binding. To determine the target molecules for TMX, we apply a substrate-trapping approach based on the reaction mechanism of thiol-disulfide exchange, identifying major histocompatibility complex (MHC) class I heavy chain (HC) as a candidate substrate. Unlike the classical ER oxidoreductases such as protein disulfide isomerase and ERp57, TMX seems not to be essential for normal assembly of MHC class I molecules. However, we show that TMX-class I HC interaction is enhanced during tunicamycin-induced ER stress, and TMX prevents the ER-to-cytosol retrotranslocation of misfolded class I HC targeted for proteasomal degradation. These results suggest a specific role for TMX and its mechanism of action in redox-based ER quality control.

Redox regulation of mast cell histamine release in thioredoxin-1 （TRX） transgenic mice

Thioredoxin-1 (TRX) is a stress-inducible redox-regulatory protein with antioxidative and anti-inflammatory effects. Here we show that the release of histamine from mast cells elicited by cross-linking of high-affinity receptor for IgE (FcεRI) was significantly suppressed in TRX transgenic (TRX-tg) mice compared to wild type (WT) mice. Intracellular reactive oxygen species (ROS) of mast cells stimulated by IgE and antigen was also reduced in TRX-tg mice compared to WT mice. Whereas there was no difference in the production of cytokines (IL-6 and TNF-α) from mast cells in response to 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) stimulation in TRX-tg and WT mice. Immunological status of TRX-tg mice inclined to T helper (Th) 2 dominant in primary immune response, although there was no difference in the population of dendritic cells (DCs) and regulatory T cells. We conclude that the histamine release from mast cells in TRX-tg mice is suppressed by inhibition of ROS generation. As ROS are involved in mast cell activation and facilitate mediator release, TRX may be a key signaling molecule regulating the early events in the IgE signaling in mast cells and the allergic inflammation.

Dendritic cells derived from TBP-2-deficient mice are defective in inducing T cell responses

Thioredoxin‐binding protein‐2 (TBP‐2), also known as vitamin D3‐up‐regulated protein 1 (VDUP1), was identified as an endogenous molecule interacting with thioredoxin (TRX). Here, we show that dendritic cells (DC) derived from TBP‐2‐deficient mice are defective in the function of T cell activation. To compare TBP‐2–/– DC function with wild‐type (WT) DC, we stimulated DC with lipopolysaccharide (LPS). Although TBP‐2–/– DC and WT DC expressed comparable levels of MHC class II and costimulatory molecules such as CD40, CD80 and CD86, the IL‐12p40, IL‐12p70 and IL‐6 productions of TBP‐2–/– DC were attenuated. In a mixed leukocyte reaction (MLR), the concentrations of IL‐2, IFN‐γ, IL‐4 and IL‐10 in the culture supernatant of MLR with TBP‐2–/– DC were significantly lower than those in the cultures with WT DC. In MLR also, as with LPS stimulation, IL‐12p40 and IL‐12p70 production from TBP‐2–/– DC was less than that from WT DC. Proliferation of T cells cultured with TBP‐2–/– DC was poorer than that with WT DC. In vivo delayed‐type hypersensitivity responses in TBP‐2–/– mice immunized with ovalbumin were significantly reduced compared to WT mice. These results indicate that TBP‐2 plays a crucial role in DC to induce T cell responses.

Thioredoxin and thioredoxin binding protein 2 in the liver

Thioredoxin (TRX) is a 12‐kDa protein with redox‐active dithiol in the active site ‐Cys‐Gly‐Pro‐Cys‐ and constitutes a major thiol reducing system. TRX protects cells from stress‐induced damage through antioxidative, antiapoptotic, and anti‐inflammatory effect. In animal models, thioacetamide (TAA)‐induced acute hepatitis and TAA‐induced liver fibrosis was attenuated in TRX transgenic (TRXTG) mice. Plasma level of TRX is a good marker for hepatitis and nonalcoholic steatohepatitis (NASH) in human patients. Recently, we identified TRX binding protein 2 (TBP2) in a yeast two‐hybrid screening. TBP2 regulates both the expression and reducing activity of TRX as well as cell growth. TBP2 knockout (TBP2KO) mice showed disorder in lipid metabolism. TBP2 plays a multiple role on cell growth and lipid and glucose metabolism. Thus, TRX and TBP2 play important roles in the pathophysiology of liver diseases, including NASH, indicating that ratio of TRX and TBP2 expression could be a novel marker of liver diseases like NASH.

Differential roles of Annexin A1 (ANXA1/lipocortin-1/lipomodulin) and thioredoxin binding protein-2 (TBP-2/VDUP1/TXNIP) in glucocorticoid signaling of HTLV-I-transformed T cells.

Glucocorticoid (GC) is widely used for therapeutic purposes in immunological and hematological disorders. Annexin A1 (ANXA1/lipocortin-1/lipomodulin), a GC-inducible molecule, was regarded as a vital anti-inflammatory mediator of GC. Thioredoxin binding protein-2 (TBP-2/VDUP1/TXNIP), a regulator of redox reactions, cell growth and lipid metabolism, was also reportedly induced by GC. HTLV-I infected T cells undergo the transition from the IL-2 dependent to IL-2 independent growth during the long-term culture in vitro. We found that these T cells responded to GC with growth arrest and apoptosis in the IL-2 dependent growth stage, whereas they failed to respond to GC after their growth had shifted into the IL-2 independent stage. Here we employed these T cell lines and studied the roles of ANXA1 and TBP-2 in mediating GC-induced apoptosis. In GC-sensitive T cells, ANXA1 expression was negligible and unaffected by GC treatment, whereas TBP-2 was expressed and induced by GC treatment. In GC-resistant T cells, however, ANXA1 was highly expressed regardless of GC treatment and promoted cellular proliferation. In contrast, TBP-2 expression was lost and could not mediate the GC-induced apoptosis. In conclusion, these results suggest that TBP-2, but not ANXA1, is directly involved in the switching of GC sensitivity and GC resistance in HTLV-I infected T cell lines, whereas ANXA1 may be a biomarker indicative of the advanced stage of the transformation.

Thioredoxin binding protein-2 mediates metabolic adaptation in response to lipopolysaccharide in vivo

Objectives:Endotoxin triggers a reorganization of the energy metabolic pathway, including the promotion of fatty acid utilization to adapt to a high energy demand during endotoxemia. However, the factors responsible for the metabolic adaptation and characteristic pathologies resulting from defective utilization fatty acids during endotoxin response have not been fully clarified. The thioredoxin binding protein-2 (TBP-2) knockout (TBP-2−/−) mouse is an animal model of fatty acid oxidation disorder. The aim of this study was to determine whether and how TBP-2 is involved in metabolic regulation in a lipopolysaccharide (LPS)-induced endotoxemia model in mice. Design:Prospective animal trial. Setting:Research laboratory. Subjects:TBP-2−/− and wild control mice. Intervention:TBP-2−/− and wild control mice were intraperitoneally injected with LPS. Mortality, serum levels of markers of hepatorenal injuries, cytokines, insulin, glucose and lipid derivatives, and the hepatic signaling pathway regulating gluconeogenesis were investigated. Measurements and Main Results:Following the administration of LPS, TBP-2−/− mice showed a predisposition for death without any significant elevation of inflammatory cytokines, compared to the wild mice. LPS-challenged TBP-2−/− mice showed fat deposition in the liver and kidney, organ injuries, glycogen depletion, and elevation of serum lipid derivatives such as free fatty acids, triglyceride and cholesterol. Hyperinsulinemia and hypoglycemia were observed in TBP-2−/− mice after LPS injection. Death due to the LPS administration was prevented by supplementation of glucose. Phosphorylation of Akt and FoxO1, an inhibitory pathway of gluconeogenesis in the liver of LPS-challenged TBP-2−/− mice was demonstrated, suggesting the enhancement of insulin signaling. Conclusions:TBP-2 is involved in metabolic control during LPS-induced endotoxemia. After the LPS challenge, TBP-2−/− mice showed several characteristic aspects, such as hepatorenal injuries, and dysregulation of the lipid and glucose metabolisms. Furthermore, hypoglycemia promoted by hyperinsulinemia may be a critical risk factor for mortality in circumstances in which fatty acid utilization is impaired during endotoxemia.

The Protective Role of the Transmembrane Thioredoxin-Related Protein TMX in Inflammatory Liver Injury

AIMS Accumulating evidence indicates that oxidative stress is associated with inflammation, and the cellular redox status can determine the sensitivity and the final outcome in response to inflammatory stimuli. To control the redox balance, mammalian cells contain a variety of oxidoreductases belonging to the thioredoxin superfamily. The large number of these enzymes suggests a complex mechanism of redox regulation in mammals, but the precise function of each family member awaits further investigations. RESULTS We generated mice deficient in transmembrane thioredoxin-related protein (TMX), a transmembrane oxidoreductase in the endoplasmic reticulum (ER). When exposed to lipopolysaccharide (LPS) and d-(+)-galactosamine (GalN) to induce inflammatory liver injury, mutant mice were highly susceptible to the toxicants and developed severe liver damage. LPS-induced production of inflammatory mediators was equivalent in both wild-type and TMX(-/-) mice, whereas neutralization of the proinflammatory cytokine tumor necrosis factor-α suppressed the toxic effects of LPS/GalN in the mutant mice. Liver transcriptional profiles revealed enhanced activation of the p53-signaling pathway in the TMX(-/-) mice after LPS/GalN treatment. Furthermore, TMX deficiency also caused increased sensitivity to thioacetamide, which exerts its hepatotoxicity through the generation of reactive oxygen species. INNOVATION The present study is the first to address the role of the oxidoreductase TMX in inflammatory liver injury. The phenotype of mice deficient in TMX suggests a functional link between redox regulation in the ER and susceptibility to oxidative tissue damage. CONCLUSION We conclude that TMX plays a major role in host defense under the type of inflammatory conditions associated with oxidative stress.

Human thioredoxin-1 attenuates the rate of lipopolysaccharide-induced preterm delivery in mice in association with its anti-inflammatory effect

Background:Maternal intrauterine infection/inflammation represents the major etiology of preterm delivery and the leading cause of neonatal mortality and morbidity. The aim of this study was to investigate the anti-inflammatory properties of thioredoxin-1 in vivo and its potential ability to attenuate the rate of inflammation-induced preterm delivery.Methods:Two intraperitoneal injections of lipopolysaccharide from Escherichia coli were administered in pregnant mice on gestational day 15, with a 3-h interval between the injections. From either 1 h before or 1 h after the first lipopolysaccharide injection, mice received three intravenous injections of either recombinant human thioredoxin-1, ovalbumin, or vehicle, with a 3-h interval between injections.Results:Intraperitoneal injection of lipopolysaccharide induced a rise of tumor necrosis factor-α, interferon-γ, monocyte chemotactic protein 1, and interleukin-6 in maternal serum levels and provoked preterm delivery. Recombinant human thoredoxin-1 prevented the rise in these proinflammatory cytokine levels. After the inflammatory challenge, placentas exhibited severe maternal vascular dilatation and congestion and a marked decidual neutrophil activation. These placental pathological findings were ameliorated by recombinant human thioredoxin-1, and the rate of inflammation-induced preterm delivery was attenuated.Conclusion:Thioredoxin-1 may thus represent a novel effective treatment to delay inflammation-induced preterm delivery.