Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose importer, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and post-natal maintenance of the disc. Surprisingly, primary NP cells treated with two well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We show in vitro that NP cells mitigate GLUT1 loss by rewiring glucose import through GLUT3. Noteworthy, we demonstrate that substrates, such as glutamine and palmitate, do not compensate for glucose restriction resulting from dual inhibition of GLUT1/3 and inhibition compromises long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated two NP-specific knockout mice: Krt19CreERT; Glut1f/f and Foxa2Cre; Glut1f/f. Noteworthy, there were no apparent defects in post-natal disc health or development and maturation in mutant mice. Microarray analysis confirmed that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells.
Shira N. Johnston, Elizabeth S. Silagi, Vedavathi Madhu, Duc H. Nguyen, Irving M. Shapiro, Makarand V. Risbud
Cisplatin is a widely used chemotherapy drug but it induces both acute and chronic kidney diseases (CKD) in cancer patients. The pathogenesis of cisplatin-induced CKD is unclear and effective renoprotective approaches are not available. Here, we report that repeated low-dose cisplatin (RLDC) treatment of C57BL/6 mice induced chronic cellular senescence in kidney tubules, accompanied with tubular degeneration and pro-fibrotic phenotype transformation that culminated in maladaptive repair and renal fibrosis. Suppression of tubular senescence by senolytic drugs ABT-263 and Fisetin attenuated renal fibrosis and improved tubular repair as indicated by restoration of tubular regeneration and renal function. In vitro, RLDC also induced senescence in mouse proximal tubular BUMPT cells. ABT-263 eliminated senescent BUMPT cells following RLDC treatment, reversed the pro-fibrotic phenotype of the cells and increased their clonogenic activity. Moreover, ABT-263 alleviated the paracrine effect of RLDC-treated BUMPT cells on fibroblasts for fibrosis. Consistently, knockdown of p16 suppressed post-RLDC senescence and fibrotic changes in BUMPT cells, and alleviated their paracrine effects on renal fibroblast proliferation. These results indicate that persistent induction of tubular senescence plays an important role in promoting cisplatin-induced CKD. Targeting senescent tubular cells may be efficient to improve kidney repair for the prevention and treatment of cisplatin-induced CKD.
Siyao Li, Man J. Livingston, Zhengwei Ma, Xiaoru Hu, Lu Wen, Han-Fei Ding, Daohong Zhou, Zheng Dong
Mitochondrial dysfunction at birth predicts bronchopulmonary dysplasia (BPD) in extremely low birth weight (ELBW) infants. Recently, nebulized thyroid hormone (TH), given as triiodothyronine (T3) was noted to decrease pulmonary fibrosis in adult animals through improved mitochondrial function. We hypothesized that TH may have similar effects on hyperoxia-induced neonatal lung injury and mitochondrial dysfunction. To determine whether intranasal T3 decreases neonatal hyperoxic lung injury in newborn mice, T3 improves mitochondrial function in lung homogenates, neonatal murine lung fibroblasts (NMLF) and umbilical cord-derived mesenchymal stem cells (MSCs) obtained from ELBW infants, and whether neonatal hypothyroxinemia is associated with BPD in ELBW infants. Inhaled T3 (given intranasally) attenuated hyperoxia-induced lung injury and mitochondrial dysfunction in newborn mice. T3 also reduced bioenergetic deficits in UC-MSCs obtained both from infants with no/mild BPD and those with moderate/severe BPD. T3 also increased PGC1α content in lung homogenates of mice exposed to hyperoxia as well as mitochondrial potential in both NMLF and UC-MSCs. ELBW infants who died or developed moderate/severe BPD had lower TT4 compared to survivors with no/mild BPD. TH signaling and function may play a critical role in neonatal lung injury and inhaled T3 supplementation may be useful as a therapeutic strategy for BPD.
Bianca M. Vamesu, Teodora Nicola, Rui Li, Snehashis Hazra, Sadis Matalon, Naftali Kaminski, Namasivayam Ambalavanan, Jegen Kandasamy
Sepsis is a lethal syndrome characterized by systemic inflammation and abnormal coagulation. Despite therapeutic advances, sepsis mortality remains substantially high. Herein, we investigated the role of the plasminogen/plasmin (Plg/Pla) system during sepsis. Plasma levels of Plg were significantly lower in mice subjected to severe compared with non-severe sepsis, whereas systemic levels of IL-6, a marker of sepsis severity, were higher in severe sepsis. Plg levels correlated negatively with IL-6 in both septic mice and patients while the plasminogen activator inhibitor-1 (PAI-1) correlated positively with IL-6. Plg deficiency render mice susceptible to non-severe sepsis induced by cecal ligation and puncture (CLP), showing higher numbers of neutrophils and M1 macrophages, liver fibrin(ogen) deposition, lower efferocytosis and increased IL-6 and neutrophil extracellular traps (NETs) release associated with organ damage. Conversely, inflammatory features, fibrin(ogen) and organ damage were substantially reduced, and efferocytosis was increased by exogenous Pla given during CLP and LPS-induced endotoxemia. Plg or Pla protected mice from sepsis-induced lethality and enhanced the protective effect of antibiotics. Mechanistically, Plg/Pla afforded protection was associated with regulation of NET release, requiring Pla-protease activity and lysine binding sites. Altogether, Plg/Pla are important host protective players during sepsis, controlling local and systemic inflammation and collateral organ damage.
Juliana P. Vago, Isabella Zaidan, Luiza O. Perucci, Larissa F. Brito, Lívia C.R. Teixeira, Camila M.S. Silva, Thaís C. Miranda, Eliza M. Melo, Alexandre S. Bruno, Celso M. Queiroz-Junior, Michelle A. Sugimoto, Luciana P. Tavares, Laís C. Grossi, Isabela N. Borges, Nagyung Baik, André Talvani, Raphael G. Ferreira, José C. Alves-Filho, Vandack Nobre, Mauro M. Teixeira, Robert J. Parmer, Lindsey A. Miles, Lirlândia P. Sousa
Metabolic crosstalk from skeletal muscle to multiple organs is important for maintaining homeostasis, and its dysregulation can lead to various diseases. Chronic glucocorticoid administration often induces muscle atrophy and metabolic disorders such as diabetes and central obesity; however, the detailed underlying mechanism remains unclear. We previously reported that the deletion of glucocorticoid receptor (GR) in skeletal muscle increases muscle mass and reduces fat mass through muscle–liver–fat communication under physiological conditions. In this study, we show that muscle GR signaling plays a crucial role in accelerating obesity through the induction of hyperinsulinemia. Fat accumulation in liver and adipose tissue, muscle atrophy, hyperglycemia, and hyperinsulinemia induced by chronic corticosterone (CORT) treatment improved in muscle-specific GR knockout (GRmKO) mice. Such CORT-induced fat accumulation was alleviated by suppressing insulin production (streptozotocin injection), indicating that hyperinsulinemia enhanced by muscle GR signaling promotes obesity. Strikingly, glucose intolerance and obesity in ob/ob mice without CORT treatment were also improved in GRmKO mice, indicating that muscle GR signaling contributes to obesity-related metabolic changes, regardless of systemic glucocorticoid levels. Thus, this study provides new insight for the treatment of obesity and diabetes by targeting muscle GR signaling.
Hiroki Yamazaki, Masaaki Uehara, Noritada Yoshikawa, Akiko Kuribara-Souta, Motohisa Yamamoto, Yasuko Hirakawa, Yasuaki Kabe, Makoto Suematsu, Hirotoshi Tanaka
Primary Sjogren’s syndrome (pSS) is a systemic autoimmune inflammatory disease mainly defined by T cell–dominated destruction of exocrine glands. Currently, CD8+T cells were closely related to the pathogenesis of pSS. However, the single-cell immune profiling of pSS and molecular signatures of pathogenic CD8+T cells have not been well elucidated. Our multiomics investigation identified that both T cell and B cell, especially CD8+T cells, were undergoing significant clonal expansion in pSS patients. TCR clonality analysis revealed that peripheral granzyme (GZM) K+CXCR6+CD8+T cells had higher proportion of shared clones with CD69+CD103-CD8+ tissue resident memory T (TRM) cells in labial glands in pSS. CD69+CD103-CD8+TRM cells featured by high expression of GZMK were more active and cytotoxic in pSS compared with their CD103+ counterparts. Peripheral GZMK+CXCR6+CD8+T cells with higher CD122 expression were increased and harbored a gene signature similar to TRM cells in pSS. Consistently, IL-15 was significantly elevated in pSS plasma and showed the capacity to promote differentiation of CD8+T cells into GZMK+CXCR6+CD8+T cells in a STAT5 dependent manner. Taken together, we depicted the immune landscape of pSS and further conducted comprehensive bioinformatics analysis and in vitro experimental investigation to characterize the pathogenic role and differentiation trajectory of CD8+TRM cells in pSS.
Ting Xu, Hao-Xian Zhu, Xing You, Jin-Fen Ma, Xin Li, Pan-Yue Luo, Yang Li, Zhe-Xiong Lian, Cai-Yue Gao
This study aimed to enhance anti-tumor immune responses to pancreatic cancer via antibody-based blockade of IL-6 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Mice bearing subcutaneous or orthotopic pancreatic tumors were treated with blocking antibodies to IL 6 and/or CTLA-4. In both tumor models, dual IL-6 and CTLA-4 blockade significantly inhibited tumor growth. Additional investigations revealed that dual therapy induced an overwhelming infiltration of T cells into the tumor as well as changes in CD4+ T cell subsets. Dual blockade therapy elicited CD4+ T cells to secrete increased IFN-γ in vitro. Likewise, in vitro stimulation of pancreatic tumor cells with IFN-γ profoundly increased tumor cell production of CXCR3 specific chemokines, even in the presence of IL-6. In vivo blockade of CXCR3 prevented orthotopic tumor regression in the presence of the combination treatment, demonstrating a dependence on the CXCR3 axis for anti-tumor efficacy. Both CD4+ and CD8+ T cells were required for the anti-tumor activity of this combination therapy, as their in vivo depletion via antibodies impaired outcomes. These data represent the first report of IL-6 and CTLA 4 blockade as a means to regress pancreatic tumors with defined operative mechanisms of efficacy. Given these results, this therapeutic combination has potential for immediate clinical translation.
Michael Brandon Ware, Maggie Phillips, Christopher McQuinn, Mohammad Y. Zaidi, Hannah M. Knochelmann, Emily Greene, Brian S. Robinson, Cameron J. Herting, Thomas A. Mace, Zhengjia Chen, Chao Zhang, Matthew R. Farren, Amanda N. Ruggieri, Jacob S. Bowers, Reena Shakya, Alton Brad Farris, Gregory Young, William E. Carson III, Bassel El-Rayes, Chrystal M. Paulos, Gregory B. Lesinski
Multiple randomized, controlled clinical trials have yielded discordant results regarding the efficacy of convalescent plasma in outpatients, with some showing an approximate two-fold reduction in risk and others showing no effect. We quantified binding and neutralizing antibody levels in 492 of the 511 participants from the C3PO trial of a single unit of COVID-19 convalescent plasma (CCP) vs. saline infusion. In a subset of 70 participants, peripheral blood mononuclear cells were obtained to define the evolution of B and T cell responses through day 30. Binding and neutralizing antibody responses were measurably higher one hour post-infusion in recipients of CCP compared to saline plus multivitamin, but levels achieved by the native immune system by day 15 were much higher than seen immediately after CCP administration. Infusion of CCP did not block generation of the host antibody response or skew B or T cell phenotype or maturation. Activated CD4+ and CD8+ T cells were associated with more severe disease outcome. These data show that CCP leads to a measurable boost in anti-SARS-CoV-2 antibodies, but that the boost is modest and may not be sufficient to alter disease course.
John F. McDyer, Mahzad Azimpouran, Valerie L. Durkalski-Mauldin, Robert G. Clevenger, Sharon D. Yeatts, Xutao Deng, William Barsan, Robert Silbergleit, Nahed El Kassar, Iulia Popescu, Dimiter Dimitrov, Wei Li, Emily J. Lyons, Sophia C. Lieber, Mars Stone, Frederick K. Korley, Clifton W. Callaway, Larry J. Dumont, Philip J. Norris
Hypothalamic neurons regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). However, the molecular mechanisms that enable hypothalamic neurons to detect primary nutrients remain elusive. Here, we identified L-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor (LepR)-expressing neurons as being important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1–mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.
Gyujin Park, Kazuya Fukasawa, Tetsuhiro Horie, Yusuke Masuo, Yuka Inaba, Takanori Tatsuno, Takanori Yamada, Kazuya Tokumura, Sayuki Iwahashi, Takashi Iezaki, Katsuyuki Kaneda, Yukio Kato, Yasuhito Ishigaki, Michihiro Mieda, Tomohiro Tanaka, Kazuma Ogawa, Hiroki Ochi, Shingo Sato, Yun-Bo Shi, Hiroshi Inoue, Hojoon Lee, Eiichi Hinoi
Low capacity to produce reactive oxygen species (ROS) due to mutations in neutrophil cytosolic factor 1 (NCF1/p47phox), a component of NADPH oxidase 2 (NOX2) complex, is strongly associated with systemic lupus erythematosus in both humans and mouse models. Here, we aim to identify the key immune cell type(s) and cellular mechanisms driving lupus pathogenesis under the condition of NCF1-dependent ROS deficiency. Using a set of cell-specific Cre-deleter, the human NCF1-339 variant knock-in, and transgenic mouse strains, we show that low ROS production in plasmacytoid dendritic cells (pDCs) exacerbates both pristane-induced lupus and a newly established Yaa-related spontaneous model by promoting pDC accumulation in multiple organs during lupus development, accompanied by elevated IFNα levels and expression of IFN-stimulated genes. Mechanistic studies reveal that ROS deficiency enhances pDC generation through the AKT/mTOR pathway and CCR2-mediated migration to tissues, which together with hyperactivation of the redox-sensitive STING/IFNα/JAK1/STAT1 cascade further augments type I IFN responses. More importantly, by suppressing these pathways, restoration of NOX2-derived ROS specifically in pDCs protects against lupus. These discoveries explain the causative effect of dysfunctional NCF1 in lupus and demonstrate the protective role of pDC-derived ROS in disease development driven by NCF1-dependent ROS deficiency.
Huqiao Luo, Vilma Urbonaviciute, Amir Ata Saei, Hezheng Lyu, Massimiliano Gaetani, Ákos Végvári, Yanpeng Li, Roman A. Zubarev, Rikard Holmdahl
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