Dysfunctional Sars-CoV-2-M protein-specific cytotoxic T lymphocytes in patients recovering from severe COVID-19.

Although the importance of virus-specific cytotoxic T lymphocytes (CTL) in virus clearance is evident in COVID-19, the characteristics of virus-specific CTLs related to disease severity have not been fully explored. Here we show that the phenotype of virus-specific CTLs against immunoprevalent epitopes in COVID-19 convalescents might differ according to the course of the disease. We establish a cellular screening method that uses artificial antigen presenting cells, expressing HLA-A*24:02, the costimulatory molecule 4-1BBL, SARS-CoV-2 structural proteins S, M, and N and non-structural proteins ORF3a and nsp6/ORF1a. The screen implicates SARS-CoV-2 M protein as a frequent target of IFNγ secreting CD8+ T cells, and identifies M198-206 as an immunoprevalent epitope in our cohort of HLA-A*24:02 positive convalescent COVID-19 patients recovering from mild, moderate and severe disease. Further exploration of M198-206-specific CD8+ T cells with single cell RNA sequencing reveals public TCRs in virus-specific CD8+ T cells, and shows an exhausted phenotype with less differentiated status in cells from the severe group compared to cells from the moderate group. In summary, this study describes a method to identify T cell epitopes, indicate that dysfunction of virus-specific CTLs might be an important determinant of clinical outcomes.

Photoacoustic in vivo 3D imaging of tumor using a highly tumor-targeting probe under high-threshold conditions.

Three-dimensional (3D) representation of a tumor with respect to its size, shape, location, and boundaries is still a challenge in photoacoustic (PA) imaging using artificial contrast agents as probes. We carried out PA imaging of tumors in mice using 800RS-PMPC, which was obtained by coupling of 800RS, a near-infrared cyanine dye, with PMPC, a highly selective tumor-targeting methacrylate polymer having phosphorylcholine side chains, as a probe. The conjugate 800RS-PMPC forms compact nanoparticles (dDLS = 14.3 nm), retains the biocompatibility of the parent polymer (PMPC) and exhibits unprecedented PA performance. When applied to mice bearing a 6 × 3 × 3 mm3 tumor buried 6 mm beneath the skin, the probe 800RS-PMPC selectively accumulates in the tumor and emits PA signals that are strong enough to be unambiguously distinguished from noise signals of endogenous blood/hemoglobin. The PA image thus obtained under high-threshold conditions allows 3D characterization of the tumor in terms of its size, shape, location, and boundaries.

Biological reduction of nitroimidazole-functionalized gold nanorods for photoacoustic imaging of tumor hypoxia.

Tumor-selective accumulation of gold nanorods (GNR) has been demonstrated for visualization of tumor hypoxia by photoacoustic imaging. We prepared GNRs with hypoxia-targeting nitroimidazole units (G-NI) on their surface. Biological experiments revealed that G-NI produced a strong photoacoustic signal in hypoxic tumor cells and tissues.

Confinement of Singlet Oxygen Generated from Ruthenium Complex-Based Oxygen Sensor in the Pores of Mesoporous Silica Nanoparticles.

We synthesized mesoporous silica nanoparticles bearing ruthenium complexes in their pores (MSN-Ru) and characterized their photochemical properties. The ruthenium complexes that were immobilized in the pores showed oxygen-dependent phosphorescence, similar to the complexes that were not tethered to nanoparticles. Cellular imaging and in vivo experiments revealed that hypoxic cells and tissues could be visualized by monitoring the phosphorescence of MSN-Ru. Our most important finding was that the toxic effect of singlet oxygen (1O2), which was generated by excitation of the complexes, was effectively suppressed by the deactivation before leaking out from the pores. In addition, we observed a negligible toxic effect of the ruthenium complexes themselves due to the blockage of their direct interaction with intracellular biomolecules. Thus, MSN-Ru is a promising molecular probe of oxygen levels in living cells and tissues.

IgA regulates the composition and metabolic function of gut microbiota by promoting symbiosis between bacteria.

Immunoglobulin A (IgA) promotes health by regulating the composition and function of gut microbiota, but the molecular requirements for such homeostatic IgA function remain unknown. We found that a heavily glycosylated monoclonal IgA recognizing ovalbumin coats Bacteroides thetaiotaomicron (B. theta), a prominent gut symbiont of the phylum Bacteroidetes. In vivo, IgA alters the expression of polysaccharide utilization loci (PUL), including a functionally uncharacterized molecular family provisionally named Mucus-Associated Functional Factor (MAFF). In both mice and humans, MAFF is detected predominantly in mucus-resident bacteria, and its expression requires the presence of complex microbiota. Expression of the MAFF system facilitates symbiosis with other members of the phylum Firmicutes and promotes protection from a chemically induced model of colitis. Our data reveal a novel mechanism by which IgA promotes symbiosis and colonic homeostasis.

Tracking the Oxygen Status in the Cell Nucleus with a Hoechst-Tagged Phosphorescent Ruthenium Complex.

Molecular oxygen in living cells is distributed and consumed inhomogeneously, depending on the activity of each organelle. Therefore, tractable methods that can be used to monitor the oxygen status in each organelle are needed to understand cellular function. Here we report the design of a new oxygen-sensing probe for use in the cell nucleus. We prepared "Ru-Hoechsts", each consisting of a phosphorescent ruthenium complex linked to a Hoechst 33258 moiety, and characterized their properties as oxygen sensors. The Hoechst unit shows strong DNA-binding properties in the nucleus, and the ruthenium complex shows oxygen-dependent phosphorescence. Thus, Ru-Hoechsts accumulated in the cell nucleus and showed oxygen-dependent signals that could be monitored. Of the Ru-Hoechsts prepared in this study, Ru-Hoechst b, in which the ruthenium complex and the Hoechst unit were linked through a hexyl chain, showed the most suitable properties for monitoring the oxygen status. Ru-Hoechsts are probes with high potential for visualizing oxygen fluctuations in the nucleus.

Aggregate Formation of Oligonucleotides that Assist Molecular Imaging for Tracking of the Oxygen Status in Tumor Tissue.

The use of DNA aggregates could be a promising strategy for the molecular imaging of biological functions. Herein, phosphorescent oligodeoxynucleotides were designed with the aim of visualizing oxygen fluctuation in tumor cells. DNA-ruthenium conjugates (DRCs) that consisted of oligodeoxynucleotides, a phosphorescent ruthenium complex, a pyrene unit for high oxygen responsiveness, and a nitroimidazole unit as a tumor-targeting unit were prepared. In general, oligonucleotides have low cell permeability because of their own negative charges; however, the DRC formed aggregates in aqueous solution due to the hydrophobic pyrene and nitroimidazole groups, and smoothly penetrated the cellular membrane to accumulate in tumor cells in a hypoxia-selective manner. The oxygen-dependent phosphorescence of DRC in cells was also observed. In vivo experiments revealed that aggregates of DRC accumulated in hypoxic tumor tissue that was transplanted into the left leg of mice, and showed that oxygen fluctuations in tumor tissue could be monitored by tracking of the phosphorescence emission of DRC.

Confined Singlet Oxygen in Mesoporous Silica Nanoparticles: Selective Photochemical Oxidation of Small Molecules in Living Cells.

Chemical conversion of specific bioactive molecules by external stimuli in living cells is a powerful noninvasive tool for clarification of biomolecular interactions and to control cellular functions. However, in chaotic biological environments, it has been difficult to induce arbitrary photochemical reactions on specific molecules because of their poor molecular selectivity. Here we report a selective and nontoxic photochemical reaction system utilizing photoactivated mesoporous silica nanoparticles to control biological functions. Methylene blue modification within nanoparticle pores for photosensitization produced singlet oxygen confined to the pore that could mediate selective oxidation of small molecules without any damage to living cells. This intracellular photochemical system produced bioactive molecules in situ and remotely controlled the cell cycle phase. We also confirmed that this photoreaction could be applied to control cell cycle phase in tumor tissue transplanted in mice. The cell cycle phase in the cells in mice, to which our system was administered, was arrested at the G2/M phase upon photoirradiation. We demonstrate a simple and promising method for the exogenous conversion of an intracellular biomolecule to another functional compound.

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.

Pharmacokinetics of Chiral Dendrimer-Triamine-Coordinated Gd-MRI Contrast Agents Evaluated by in Vivo MRI and Estimated by in Vitro QCM.

Recently, we developed novel chiral dendrimer-triamine-coordinated Gd-MRI contrast agents (Gd-MRI CAs), which showed longitudinal relaxivity (r1) values about four times higher than that of clinically used Gd-DTPA (Magnevist(®), Bayer). In our continuing study of pharmacokinetic differences derived from both the chirality and generation of Gd-MRI CAs, we found that the ability of chiral dendrimer Gd-MRI CAs to circulate within the body can be directly evaluated by in vitro MRI (7 T). In this study, the association constants (K(a)) of chiral dendrimer Gd-MRI CAs to bovine serum albumin (BSA), measured and calculated with a quartz crystal microbalance (QCM) in vitro, were found to be an extremely easy means for evaluating the body-circulation ability of chiral dendrimer Gd-MRI CAs. The K(a) values of S-isomeric dendrimer Gd-MRI CAs were generally greater than those of R-isomeric dendrimer Gd-MRI CAs, which is consistent with the results of our previous MRI study in vivo.

Water-soluble phosphorescent ruthenium complex with a fluorescent coumarin unit for ratiometric sensing of oxygen levels in living cells.

Dual emission was applied to a molecular probe for the ratiometric sensing of oxygen concentration in a living system. We prepared ruthenium complexes possessing a coumarin unit (Ru-Cou), in which the (3)MLCT phosphorescence of the ruthenium complex was efficiently quenched by molecular oxygen, whereas the coumarin unit emitted constant fluorescence independent of the oxygen concentration. The oxygen status could be determined precisely from the ratio of phosphorescence to fluorescence. We achieved the molecular imaging of cellular oxygen levels using Ru-Cou possessing an alkyl chain, which provided appropriate lipophilicity to increase cellular uptake.

Phosphorescent ruthenium complexes with a nitroimidazole unit that image oxygen fluctuation in tumor tissue.

Understanding oxygen fluctuation in a cancerous tumor is important for effective treatment, especially during radiotherapy. In this paper, ruthenium complexes bearing a nitroimidazole group are shown to report the oxygen status in tumor tissue directly. The nitroimidazole group was known to be accumulated in hypoxic tumor tissues. On the other hand, the ruthenium complex showed strong phosphorescence around 600 nm. The emission of ruthenium is quenched instantaneously by molecular oxygen due to energy transfer between triplet states of oxygen and ruthenium complex, but the emission is then recovered by the removal of oxygen. Thus, we could observe oxygen fluctuation in tumor tissue in a real-time manner by monitoring the phosphorescence of the ruthenium complex. The versatility of the probe is demonstrated by monitoring oxygen fluctuation in living cells and tumor tissue planted in mice. The ruthenium complex promptly penetrated plasma membrane and accumulated in cells to emit its oxygen-dependent phosphorescence. In vivo experiments revealed that the oxygen level in tumor tissue seems to fluctuate at the sub-minute timescale.

Ruthenium complexes with hydrophobic ligands that are key factors for the optical imaging of physiological hypoxia.

The phosphorescence emission of ruthenium complexes was applied to the optical imaging of physiological hypoxia. We prepared three complexes with hydrophobic substituents on the phenanthroline ligand and characterized their emission, which was quenched by molecular oxygen. Among the complexes synthesized in this study, a pyrene chromophore-linked ruthenium complex, Ru-Py, exhibited optimal properties for the imaging of hypoxia; the prolonged lifetime of the triplet excited state of the ruthenium chromophore, which was induced by efficient energy distribution and transfer from the pyrene unit, provided the highest sensitivity towards molecular oxygen. The introduction of hydrophobic pyrene increased the lipophilicity of the complex, leading to enhanced cellular uptake. Consequently, the bright phosphorescence of Ru-Py was seen in the cytoplasm of viable hypoxic cells, whereas the signal from aerobic cells was markedly weaker. Thus, we could clearly discriminate between hypoxic and aerobic cells by monitoring the phosphorescence emission. Furthermore, Ru-Py was applied to optical imaging in live mice. An intramuscular injection of Ru-Py successfully visualized ischemia-based hypoxia, which was constructed by leg banding.

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.

Interleukin-13 damages intestinal mucosa via TWEAK and Fn14 in mice-a pathway associated with ulcerative colitis.

BACKGROUND & AIMS: TWEAK, a member of the tumor necrosis factor (TNF) superfamily, promotes intestinal epithelial cell injury and signals through the receptor Fn14 following irradiation-induced tissue damage and during development of colitis in mice. Interleukin (IL)-13, an effector of tissue damage in similar models, has been associated with the pathogenesis of ulcerative colitis (UC). We investigated interactions between TWEAK and IL-13 following mucosal damage in mice. METHODS: We compared patterns of gene expression in intestinal tissues from wild-type and TWEAK knockout mice following γ-irradiation. Intestinal explants from these mice were used to detect cell damage induced by IL-13 and TNF-α. Levels of messenger RNA for IL-13, TWEAK, and Fn14 were measured in mucosal samples from patients with UC. RESULTS: Based on gene expression analysis, TWEAK mediates γ-irradiation-induced epithelial cell cycle arrest and apoptosis. However, TWEAK alone did not induce damage or apoptosis of primary intestinal epithelial cells. On the other hand, exogenous IL-13 activated caspase-3 in naïve intestinal explants; this process required TWEAK, Fn14, and secretion of endogenous TNF-α which was mediated by ADAM17. Conversely, activation of caspase by exogenous TNF-α required IL-13, TWEAK, and Fn14. In mucosa from patients with UC, messenger RNA levels of IL-13, TWEAK, and Fn14 increased with level of disease severity. CONCLUSIONS: IL-13-induced damage of intestinal epithelial cells requires TWEAK, its receptor (Fn14), and TNF-α. IL-13, TNF-α, TWEAK, and Fn14 could perpetuate and aggravate intestinal inflammation in patients with UC.

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.

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.

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.

Thioredoxin binding protein 2 modulates natural killer T cell-dependent innate immunity in the liver: possible link to lipid metabolism.

Thioredoxin binding protein 2 (TBP2) plays a regulatory role in lipid metabolism and immune regulation. We previously reported the effect of TBP2 loss-of-function on lipid metabolism using TBP2 knockout (TBP2KO) mice. In this study, we employed TBP2 transgenic (TBP2TG) mice to analyze the in vivo effect of TBP2 gain-of-function. We revealed a decrease in the percentage of hepatic natural killer T (NKT) cells in TBP2KO mice and an increase in the percentage of hepatic NKT cells in TBP2TG mice. The TBP2KO mice were resistant to concanavalin A (ConA)-induced hepatitis, but they were highly susceptible to other types of hepatitis. TBP2 modulates lipid metabolism as well as NKT cell activity. Moreover, TBP2 expression was increased significantly in klotho-deficient mice, which exhibit a syndrome resembling aging human phenotypes. TBP2 may play multiple roles in lipid metabolism, innate immunity, and aging.

Direct association of thioredoxin-1 (TRX) with macrophage migration inhibitory factor (MIF): regulatory role of TRX on MIF internalization and signaling.

Thioredoxin-1 (TRX) is a small (14 kDa) multifunctional protein with the redox-active site Cys-Gly-Pro-Cys. Macrophage migration inhibitory factor (MIF) is a 12 kDa cytokine belonging to the TRX family. Historically, when we purified TRX from the supernatant of ATL-2 cells, a 12 kDa protein was identified along with TRX, which was later proved to be MIF. Here, we show that TRX and MIF form a complex in the cell and the culture supernatant of ATL-2 cells. Using a BIAcore assay, we confirmed that TRX has a specific affinity with MIF. We also found that extracellular MIF was more effectively internalized into the ATL-2 cells expressing TRX on the cell surface, than the Jurkat T cells which do not express surface TRX. Moreover, anti-TRX antibody blocked the MIF internalization, suggesting that the cell surface TRX is involved in MIF internalization into the cells. Furthermore, anti-TRX antibody inhibited MIF-mediated enhancement of TNF-alpha production from macrophage RAW264.7 cells. These results suggest that the cell surface TRX serves as one of the MIF binding molecules or MIF receptor component and inhibits MIF-mediated inflammatory signals.