Disruption of mitochondrial electron transport impairs urinary concentration via AMPK-dependent suppression of aquaporin 2
Carty et al. use a genetic mouse model to demonstrate that mitochondrial electron transport disruption impairs maximal urinary concentration via AMPK activation. The cover art features a merged immunofluorescence image, showing staining for total aquaporin 2 (green) and phosphorylated aquaporin 2 (red) in the kidney medulla of mice with collecting duct–specific inactivation of the ubiquinone (Q) binding protein QPC (UQCRQ).
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Physician-Scientist Development
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Abstract
The earliest MD-PhD programs were small and enrolled mostly men. Here, we show that since 2014 there has been a steady increase in the number of women in MD-PhD programs, the number of women reaching parity with men in 2023. This change was due to an increase in female applicants, a decrease in male applicants, and an increase in the acceptance rate for women, which had previously been lower than that for men. Data from the National MD-PhD Program Outcomes Study show that training duration has been similar for men and women, as have most choices of medical specialties and workplaces. However, women were less likely to have full-time faculty appointments, fewer had NIH grants, and those in the most recent graduation cohort at the time of the survey reported spending less time on research than men. Previously cited reasons for these differences include disproportionate childcare responsibilities, a paucity of role models, insufficient recognition, and gender bias. Institutions can and should address these obstacles, but training programs can help by preparing their graduates to succeed despite the systemic obstacles. The alternative is a persistent gender gap in the physician-scientist workforce, lost opportunities to benefit from diverse perspectives, and a diminished impact of valuable training resources.
Authors
Lawrence F. Brass, Myles H. Akabas
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Research Articles
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Abstract
Chronic activation of the adaptive immune system is a hallmark of atherosclerosis. As PI3Kδ is a key regulator of T and B cell differentiation and function, we hypothesized that alleviation of adaptive immunity by PI3Kδ inactivation may represent an attractive strategy counteracting atherogenesis. As expected, lack of hematopoietic PI3Kδ in atherosclerosis-prone Ldlr–/– mice resulted in lowered T and B cell numbers, CD4+ effector T cells, Th1 response, and immunoglobulin levels. However, despite markedly impaired peripheral pro-inflammatory Th1 cells and atheromatous CD4+ T cells, the unexpected net effect of hematopoietic PI3Kδ deficiency was aggravated vascular inflammation and atherosclerosis. Further analyses revealed that PI3Kδ deficiency impaired numbers, immunosuppressive functions, and stability of regulatory CD4+ T cells (Tregs), whereas macrophage biology remained largely unaffected. Adoptive transfer of wild-type Tregs fully restrained the atherosclerotic plaque burden in Ldlr–/– mice lacking hematopoietic PI3Kδ, whereas PI3Kδ-deficient Tregs failed to mitigate disease. Numbers of atheroprotective B-1 and pro-atherogenic B-2 cells as well as serum immunoglobulin levels remained unaffected by adoptively transferred wild-type Tregs. In conclusion, we demonstrate that hematopoietic PI3Kδ ablation promotes atherosclerosis. Mechanistically, we identified PI3Kδ signaling as a powerful driver of atheroprotective Treg responses, which outweigh PI3Kδ-driven pro-atherogenic effects of adaptive immune cells like Th1 cells.
Authors
Mario Zierden, Eva Maria Berghausen, Leoni Gnatzy-Feik, Christopher Millarg, Felix Simon Ruben Picard, Martha Kiljan, Simon Geißen, Apostolos Polykratis, Lea Zimmermann, Richard Julius Nies, Manolis Pasparakis, Stephan Baldus, Chanil Valasarajan, Soni Savai Pullamsetti, Holger Winkels, Marius Vantler, Stephan Rosenkranz
×Abstract
Cell cycle inhibitors have a long history as cancer treatment. Here, we report that these inhibitors combated cancer partially via the stimulator of IFN genes (STING) signaling pathway. We demonstrated that paclitaxel (microtubule stabilizer), palbociclib (cyclin-dependent kinase 4/6 inhibitor), and AZD1152 and GSK1070916 (aurora kinase B inhibitors) have anticancer functions beyond arresting the cell cycle. They consistently caused cytosolic DNA accumulation and DNA damage, which inadvertently triggered the cytosolic DNA sensor DEAD-box helicase 41 (DDX41) and activated STING to secrete pro-inflammatory senescence-associated secretory phenotype factors (SASPs). Interestingly, we found that DDX41 was a transcriptional target of HIF. Hypoxia induced expression of DDX41 through HIF-1, making hypoxic hepatocellular carcinoma (HCC) cells more sensitive to the antimitotic agents in STING activation and SASP production. The SASPs triggered immune cell infiltration in tumors for cancer clearance. The treatment with cell cycle inhibitors, especially paclitaxel, extended survival by perturbing mouse HCC growth when used in combination with anti–PD-1. We observed a trend that paclitaxel suppressed Sting wild-type HCC more effectively than Sting-KO HCC, suggesting that STING might contribute to the antitumor effects of paclitaxel. Our study revealed the immune-mediated tumor-suppressing properties of cell cycle inhibitors and suggested combined treatment with immunotherapy as a potential therapeutic approach.
Authors
Po Yee Wong, Cerise Yuen Ki Chan, Helen Do Gai Xue, Chi Ching Goh, Jacinth Wing Sum Cheu, Aki Pui Wah Tse, Misty Shuo Zhang, Yan Zhang, Carmen Chak Lui Wong
×Abstract
Sjögren’s disease is a chronic autoimmune disease characterized by symptoms of oral and ocular dryness and extraglandular manifestations. Mouth dryness is not only due to reduced saliva volume, but also to alterations in the quality of salivary mucins in patients with Sjögren’s disease. Mucins play a leading role in mucosa hydration and protection, where sulfated and sialylated oligosaccharides retain water molecules at the epithelial surface. The correct localization of glycosyltransferases and sulfotransferases within the Golgi apparatus determines adequate O-glycosylation and sulfation of mucins, which depends on specific golgins that tether enzyme-bearing vesicles. Here, we show that a golgin called Giantin was mislocalized in salivary glands from patients with Sjögren’s disease and formed protein complexes with Gal3-O-sulfotransferases (Gal3STs), which changed their localization in Giantin-knockout and -knockdown cells. Our results suggest that Giantin could tether Gal3ST-bearing vesicles and that its altered localization could affect Gal3ST activity, explaining the decreased sulfation of MUC5B observed in salivary glands from patients with Sjögren’s disease.
Authors
Matilde Nuñez, Patricia Carvajal, Sergio Aguilera, María-José Barrera, Soledad Matus, Alicia Couto, Malena Landoni, Gaelle Boncompain, Sergio González, Claudio Molina, Karina Pino, Sebastián Indo, Lourdes Figueroa, María-Julieta González, Isabel Castro
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Susceptibility to inflammatory bowel diseases (IBDs), Crohn’s disease (CD), and ulcerative colitis (UC) is linked with loss of intestinal epithelial barrier integrity and mitochondria dysfunction. Steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) domain-containing protein 7 (STARD7) is a phosphatidylcholine-specific (PC-specific) lipid transfer protein that transports PC from the ER to the mitochondria, facilitating mitochondria membrane stabilization and respiration function. The aim of this study was to define the contribution of STARD7 in the regulation of the intestinal epithelial mitochondrial function and susceptibility to colitis. In silico analyses identified significantly reduced expression of STARD7 in patients with UC, which was associated with downregulation of metabolic function and a more severe disease phenotype. STARD7 was expressed in intestinal epithelial cells, and STARD7 knockdown resulted in deformed mitochondria and diminished aerobic respiration. Loss of mitochondria function was associated with reduced expression of tight junction proteins and loss of intestinal epithelial barrier integrity that could be recovered by AMPK activation. Stard7+/– mice were more susceptible to the development of DSS-induced and Il10–/– spontaneous models of colitis. STARD7 is critical for intestinal epithelial mitochondrial function and barrier integrity, and loss of STARD7 function increases susceptibility to IBD.
Authors
Jazib Uddin, Ankit Sharma, David Wu, Sunil Tomar, Varsha Ganesan, Paula E. Reichel, Lakshmi Narasimha Rao Thota, Rodolfo I. Cabrera-Silva, Sahiti Marella, Gila Idelman, Hock L. Tay, Arturo Raya-Sandino, Mack B. Reynolds, Srikanth Elesela, Yael Haberman, Lee A. Denson, Charles A. Parkos, Mary X.D. O’Riordan, Nicholas W. Lukacs, David N. O’Dwyer, Senad Divanovic, Asma Nusrat, Timothy E. Weaver, Simon P. Hogan
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Human periosteal skeletal stem cells (P-SSCs) are critical for cortical bone maintenance and repair. However, their in vivo identity, molecular characteristics, and specific markers remain unknown. Here, single-cell sequencing revealed human periosteum contains SSC clusters expressing known SSC markers, podoplanin (PDPN) and PDGFRA. Notably, human P-SSCs, but not bone marrow SSCs, selectively expressed identified markers low density lipoprotein receptor-related protein 1 (LRP1) and CD13. These LRP1+CD13+ human P-SSCs were perivascular cells with high osteochondrogenic but minimal adipogenic potential. Upon transplantation into bone injuries in mice, they preserved self-renewal capability in vivo. Single-cell analysis of mouse periosteum further supported the preferential expression of LRP1 and CD13 in Prx1+ P-SSCs. When Lrp1 was conditionally deleted in Prx1 lineage cells, it led to severe bone deformity, short stature, and periosteal defects. By contrast, local treatment with an LRP1 agonist at the injury sites induced early P-SSC proliferation and bone healing. Thus, human and mouse periosteum contains unique osteochondrogenic stem cell subsets, and these P-SSCs express specific markers, LRP1 and CD13, with a regulatory mechanism through LRP1 that enhances P-SSC function and bone repair.
Authors
Youngjae Jeong, Lorenzo Deveza, Laura Ortinau, Kevin Lei, John R. Dawson, Dongsu Park
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Daptomycin is a last-resort lipopeptide antibiotic that disrupts cell membrane (CM) and peptidoglycan homeostasis. Enterococcus faecalis has developed a sophisticated mechanism to avoid daptomycin killing by redistributing CM anionic phospholipids away from the septum. The CM changes are orchestrated by a 3-component regulatory system, designated LiaFSR, with a possible contribution of cardiolipin synthase (Cls). However, the mechanism by which LiaFSR controls the CM response and the role of Cls are unknown. Here, we show that cardiolipin synthase activity is essential for anionic phospholipid redistribution and daptomycin resistance since deletion of the 2 genes (cls1 and cls2) encoding Cls abolished CM remodeling. We identified LiaY, a transmembrane protein regulated by LiaFSR, and Cls1 as important mediators of CM remodeling required for redistribution of anionic phospholipid microdomains. Together, our insights provide a mechanistic framework on the enterococcal response to cell envelope antibiotics that could be exploited therapeutically.
Authors
April H. Nguyen, Truc T. Tran, Diana Panesso, Kara S. Hood, Vinathi Polamraju, Rutan Zhang, Ayesha Khan, William R. Miller, Eugenia Mileykovskaya, Yousif Shamoo, Libin Xu, Heidi Vitrac, Cesar A. Arias
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Disruption of the circadian clock in skeletal muscle worsens local and systemic health, leading to decreased muscle strength, metabolic dysfunction, and aging-like phenotypes. Whole-body knockout mice that lack Bmal1, a key component of the molecular clock, display premature aging. Here, by using adeno-associated viruses, we rescued Bmal1 expression specifically in the skeletal muscle fibers of Bmal1-KO mice and found that this engaged the circadian clock and clock output gene expression, contributing to extended lifespan. Time course phenotypic analyses found that muscle strength, mobility, and glucose tolerance were improved with no effects on muscle mass or fiber size or type. A multiomics approach at 2 ages further determined that restored muscle Bmal1 improved glucose handling pathways while concomitantly reducing lipid and protein metabolic pathways. The improved glucose tolerance and metabolic flexibility resulted in the systemic reduction of inflammatory signatures across peripheral tissues, including liver, lung, and white adipose fat. Together, these findings highlight the critical role of muscle Bmal1 and downstream target genes for skeletal muscle homeostasis with considerable implications for systemic health.
Authors
Miguel A. Gutierrez-Monreal, Christopher A. Wolff, Eduardo E. Rijos, Mark R. Viggars, Collin M. Douglas, Vishwajeeth Pagala, Junmin Peng, Liam C. Hunt, Haocheng Ding, Zhiguang Huo, Fabio Demontis, Karyn A. Esser
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Accumulation of extracellular matrix (ECM) proteins in trabecular meshwork (TM), which leads to increased outflow resistance of aqueous humor and consequently high intraocular pressure, is a major cause of primary open-angle glaucoma (POAG). According to our preliminary research, the RapGAP protein superfamily member, signal-induced proliferation-associated 1-like 1 protein (SIPA1L1), which is involved in tissue fibrosis, may have an impact on POAG by influencing ECM metabolism of TM. This study aims to confirm these findings and identify effects and cellular mechanisms of SIPA1L1 on ECM changes and phagocytosis in human TM (HTM) cells. Our results showed that the expression of SIPA1L1 in HTM cells was significantly increased by TGF-β2 treatment in label-free quantitative proteomics. The aqueous humor and TM cell concentration of SIPA1L1 in POAG patients was higher than that of control. In HTM cells, TGF-β2 increased expression of SIPA1L1 along with accumulation of ECM, RhoA, and p-cofilin 1. The effects of TGF-β2 were reduced by si-SIPA1L1. TGF-β2 decreased HTM cell phagocytosis by polymerizing cytoskeletal actin filaments, while si-SIPA1L1 increased phagocytosis by disassembling actin filaments. Simultaneously, overexpressing SIPA1L1 alone exhibited comparable effects to that of TGF-β2. Our studies demonstrate that SIPA1L1 not only promotes the production of ECM, but also inhibits its removal by reducing phagocytosis. Targeting SIPA1L1 degradation may become a significant therapy for POAG.
Authors
Chenyu Xu, Jiahong Wei, Dan Song, Siyu Zhao, Mingmin Hou, Yuchen Fan, Li Guo, Hao Sun, Tao Guo
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Immune evasion by tumors is promoted by low T cell infiltration, ineffective T cell activity directed against the tumor, and reduced tumor antigen presentation. The TET2 DNA dioxygenase gene is frequently mutated in hematopoietic malignancies and loss of TET enzymatic activity is found in a variety of solid tumors. We showed previously that vitamin C (VC), a cofactor of TET2, enhances tumor-associated T cell recruitment and checkpoint inhibitor therapy responses in a TET2-dependent manner. Using single-cell RNA sequencing (scRNA-seq) analysis performed on B16-OVA melanoma tumors, we have shown here that an additional function for TET2 in tumors is to promote expression of certain antigen presentation machinery genes, which is potently enhanced by VC. Consistently, VC promoted antigen presentation in cell-based and tumor assays in a TET2-dependent manner. Quantifying intercellular signaling from the scRNA-seq dataset showed that T cell–derived IFN-γ–induced signaling within the tumor and tumor microenvironment requires tumor-associated TET2 expression, which is enhanced by VC treatment. Analysis of patient tumor samples indicated that TET activity directly correlates with antigen presentation gene expression and with patient outcomes. Our results demonstrate the importance of tumor-associated TET2 activity as a critical mediator of tumor immunity, which is augmented by high-dose VC therapy.
Authors
Meng Cheng, Angel Ka Yan Chu, Zhijun Li, Shiyue Yang, Matthew D. Smith, Qi Zhang, Nicholas G. Brown, William F. Marzluff, Nabeel Bardeesy, J. Justin Milner, Joshua D. Welch, Yue Xiong, Albert S. Baldwin
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Mutations in the CLCNKB gene (1p36), encoding the basolateral chloride channel ClC-Kb, cause type 3 Bartter syndrome. We identified a family with a mixed Bartter/Gitelman phenotype and early-onset kidney failure and by employing a candidate gene approach, identified what we believe is a novel homozygous mutation (CLCNKB c.499G>T [p.Gly167Cys]) in exon 6 of CLCNKB in the index patient. We then validated these results with Sanger and whole-exome sequencing. Compared with wild-type ClC-Kb, the Gly167Cys mutant conducted less current and exhibited impaired complex N-linked glycosylation in vitro. We demonstrated that loss of Gly-167, rather than gain of a mutant Cys, impairs complex glycosylation, but that surface expression remains intact. Moreover, Asn-364 was necessary for channel function and complex glycosylation. Morphologic evaluation of human kidney biopsies revealed typical basolateral localization of mutant Gly167Cys ClC-Kb in cortical distal tubular epithelia. However, we detected attenuated expression of distal sodium transport proteins, changes in abundance of distal tubule segments, and hypokalemia-associated intracellular condensates from the index patient compared with control nephrectomy specimens. The present data establish what we believe are novel regulatory mechanisms of ClC-Kb activity and demonstrate nephron remodeling in humans, caused by mutant ClC-Kb, with implications for renal electrolyte handling, blood pressure control, and kidney disease.
Authors
Yogita Sharma, Robin Lo, Viktor N. Tomilin, Kotdaji Ha, Holly Deremo, Aishwarya V. Pareek, Wuxing Dong, Xiaohui Liao, Svetlana Lebedeva, Vivek Charu, Neeraja Kambham, Kerim Mutig, Oleh Pochynyuk, Vivek Bhalla
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Hermansky-Pudlak syndrome (HPS), particularly types 1 and 4, is characterized by progressive pulmonary fibrosis, a major cause of morbidity and mortality. However, the precise mechanisms driving pulmonary fibrosis in HPS are not fully elucidated. Our previous studies suggested that CHI3L1-driven fibroproliferation may be a notable factor in HPS-associated fibrosis. This study aimed to explore the role of CHI3L1-CRTH2 interaction on type 2 innate lymphoid cells (ILC2s) and explored the potential contribution of ILC2-fibroblast crosstalk in the development of pulmonary fibrosis in HPS. We identified ILC2s in lung tissues from patients with idiopathic pulmonary fibrosis and HPS. Using bleomycin-challenged WT and Hps1–/– mice, we observed that ILC2s were recruited and appeared to contribute to fibrosis development in the Hps1–/– mice, with CRTH2 playing a notable role in ILC2 accumulation. We sorted ILC2s, profiled fibrosis-related genes and mediators, and conducted coculture experiments with primary lung ILC2s and fibroblasts. Our findings suggest that ILC2s may directly stimulate the proliferation and differentiation of primary lung fibroblasts partially through amphiregulin-EGFR–dependent mechanisms. Additionally, specific overexpression of CHI3L1 in the ILC2 population using the IL-7Rcre driver, which was associated with increased fibroproliferation, indicates that ILC2-mediated, CRTH2-dependent mechanisms might contribute to optimal CHI3L1-induced fibroproliferative repair in HPS-associated pulmonary fibrosis.
Authors
Parand Sorkhdini, Kiran Klubock-Shukla, Selena Sheth, Dongqin Yang, Alina Xiaoyu Yang, Carmelissa Norbrun, Wendy J. Introne, Bernadette R. Gochuico, Yang Zhou
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Osteoclasts are the sole bone-resorbing cells and are formed by the fusion of osteoclast precursor cells (OCPs) derived from myeloid lineage cells. Animal studies reveal that circulating OCPs (cOCPs) in blood travel to bone and fuse with bone-resident osteoclasts. However, the characteristics of human cOCPs and their association with bone diseases remain elusive. We have identified and characterized human cOCPs and found a positive association between cOCPs and osteoclast activity. Sorted cOCPs have a higher osteoclastogenic potential than other myeloid cells and effectively differentiate into osteoclasts. cOCPs exhibit distinct morphology and transcriptomic signatures. The frequency of cOCPs in the blood varies among treatment-naive postmenopausal women and has an inverse correlation with lumbar spine bone density and a positive correlation with serum CTX, a bone resorption marker. The increased cOCPs in treatment-naive patients with osteoporosis were significantly diminished by denosumab, a widely used antiresorptive therapy. Our study reveals the distinctive identity of human cOCPs and the potential link between the dynamic regulation of cOCPs and osteoporosis and its treatment. Taken together, our study enhances our understanding of human cOCPs and highlights a potential opportunity to measure cOCPs through a simple blood test, which could potentially identify high-risk individuals.
Authors
Kaichi Kaneko, Jefferson Tsai, Deniece Meñez, Brian Oh, Andrew Junwoo Suh, Seyeon Bae, Masataka Mizuno, Akio Umemoto, Eugenia Giannopoulou, Takayuki Fujii, Yaxia Zhang, Emily M. Stein, Richard S. Bockman, Kyung-Hyun Park-Min
×Abstract
Brain arteriovenous malformations (bAVMs) are anomalies forming vascular tangles connecting the arteries and veins, which cause hemorrhagic stroke in young adults. Current surgical approaches are highly invasive, and alternative therapeutic methods are warranted. Recent genetic studies identified KRAS mutations in endothelial cells of bAVMs; however, the underlying process leading to malformation in the postnatal stage remains unknown. Here we established a mouse model of bAVM developing during the early postnatal stage. Among 4 methods tested, mutant KRAS specifically introduced in brain endothelial cells by brain endothelial cell–directed adeno-associated virus (AAV) and endothelial cell–specific Cdh5-CreERT2 mice successfully induced bAVMs in the postnatal period. Mutant KRAS led to the development of multiple vascular tangles and hemorrhage in the brain with increased MAPK/ERK signaling and growth in endothelial cells. Three-dimensional analyses in cleared tissue revealed dilated vascular networks connecting arteries and veins, similar to human bAVMs. Single-cell RNA-Seq revealed dysregulated gene expressions in endothelial cells and multiple cell types involved in the pathological process. Finally, we employed CRISPR/CasRx to knock down mutant KRAS expression, which efficiently suppressed bAVM development. The present model reveals pathological processes that lead to postnatal bAVMs and demonstrates the efficacy of therapeutic strategies with CRISPR/CasRx.
Authors
Shoji Saito, Yuka Nakamura, Satoshi Miyashita, Tokiharu Sato, Kana Hoshina, Masayasu Okada, Hitoshi Hasegawa, Makoto Oishi, Yukihiko Fujii, Jakob Körbelin, Yoshiaki Kubota, Kazuki Tainaka, Manabu Natsumeda, Masaki Ueno
×Abstract
BACKGROUND Left ventricular hypertrophy (LVH) and dyslipidemia are strong, independent predictors for cardiovascular disease, but their relationship is less well studied. A longitudinal lipidomic profiling of left ventricular mass (LVM) and LVH is still lacking.METHODS Using liquid chromatography–mass spectrometry (LC-MS), we repeatedly measured 1,542 lipids from 1,755 unique American Indians attending 2 exams (mean, 5 years apart). Cross-sectional associations of individual lipid species with LVM index (LVMI) were examined by generalized estimating equation (GEE), followed by replication in an independent biracial cohort (65% White, 35% Black). Baseline plasma lipids associated with LVH risk beyond traditional risk factors were identified by logistic GEE model in American Indians. Longitudinal associations between changes in lipids and changes in LVMI were examined by GEE, adjusting for baseline lipids, baseline LVMI, and covariates.RESULTS Multiple lipid species were significantly associated with LVMI or the risk of LVH in American Indians. Some lipids were confirmed in Black and White individuals. Moreover, some LVH-related lipids were inversely associated with risk of coronary heart disease (CHD). Longitudinal changes in several lipid species were significantly associated with changes in LVMI.CONCLUSION Altered fasting plasma lipidome and its longitudinal change over time were significantly associated with LVMI and risk for LVH in American Indians. Our results offer insight into the role of individual lipid species in LV remodeling and risk of LVH, independent of known risk factors.FUNDING This study was supported by the NIH grant (R01DK107532). The Strong Heart Study has been funded in whole or in part with federal funds from the National Heart, Lung, and Blood Institute, NIH, Department of Health and Human Services, under contract nos. 75N92019D00027, 75N92019D00028, 75N92019D00029, and 75N92019D00030.
Authors
Mingjing Chen, Zhijie Huang, Guanhong Miao, Jin Ren, Jinling Liu, Mary J. Roman, Richard B. Devereux, Richard R. Fabsitz, Ying Zhang, Jason G. Umans, Shelley A. Cole, Tanika N. Kelly, Oliver Fiehn, Jinying Zhao
×Abstract
Airway remodeling is a critical factor determining the pathogenesis and treatment sensitivity of severe asthma (SA) or uncontrolled asthma (UA). The activation of epithelial-mesenchymal trophic units (EMTUs) regulated by airway epithelial cells (AECs) has been proven to induce airway remodeling directly. However, the triggers for EMTU activation and the underlying mechanism of airway remodeling are not fully elucidated. Here, we screened the differentially expressed gene cathepsin C (CTSC; also known as dipeptidyl peptidase 1 [DPP-1]) in epithelia of patients with SA and UA using RNA-sequencing data and further verified the increased expression of CTSC in induced sputum of patients with asthma, which was positively correlated with severity and airway remodeling. Moreover, direct instillation of exogenous CTSC induced airway remodeling. Genetic inhibition of CTSC suppressed EMTU activation and airway remodeling in two asthma models with airway remodeling. Mechanistically, increased secretion of CTSC from AECs induced EMTU activation through the p38-mediated pathway, further inducing airway remodeling. Meanwhile, inhibition of CTSC also reduced the infiltration of inflammatory cells and the production of inflammatory factors in the lungs of asthmatic mice. Consequently, targeting CTSC with compound AZD7986 protected against airway inflammation, EMTU activation, and remodeling in the asthma model. Based on the dual effects of CTSC on airway inflammation and remodeling, CTSC is a potential biomarker and therapeutic target for SA or UA.
Authors
Lin Yuan, Qingwu Qin, Ye Yao, Long Chen, Huijun Liu, Xizi Du, Ming Ji, Xinyu Wu, Weijie Wang, Qiuyan Qin, Yang Xiang, Bei Qing, Xiangping Qu, Ming Yang, Xiaoqun Qin, Zhenkun Xia, Chi Liu
×Abstract
Graves’ disease (GD) is an autoimmune condition that can progress to Graves’ ophthalmopathy (GO), leading to irreversible damage to orbital tissues and potential blindness. The pathogenic mechanism is not fully understood. In this study, we conducted single-cell multi-omics analyses on healthy individuals, patients with GD without GO, newly diagnosed patients with GO, and treated patients with GO. Our findings revealed gradual systemic inflammation during GO progression, marked by overactivation of cytotoxic effector T cell subsets, and expansion of specific T cell receptor clones. Importantly, we observed a decline in the immunosuppressive function of activated Treg (aTreg) accompanied by a cytotoxic phenotypic transition. In vitro experiments revealed that dysfunction and transition of GO-autoreactive Treg were regulated by the yin yang 1 (YY1) upon secondary stimulation of thyroid stimulating hormone receptor (TSHR) under inflammatory conditions. Furthermore, adoptive transfer experiments of the GO mouse model confirmed infiltration of these cytotoxic Treg into the orbital lesion tissues. Notably, these cells were found to upregulate inflammation and promote pathogenic fibrosis of orbital fibroblasts (OFs). Our results reveal the dynamic changes in immune landscape during GO progression and provide direct insights into the instability and phenotypic transition of Treg, offering potential targets for therapeutic intervention and prevention of autoimmune diseases.
Authors
Zhong Liu, Shu-Rui Ke, Zhuo-Xing Shi, Ming Zhou, Li Sun, Qi-Hang Sun, Bing Xiao, Dong-Liang Wang, Yan-Jin Huang, Jin-Shan Lin, Hui-Shi Wang, Qi-Kai Zhang, Cai-Neng Pan, Xuan-Wei Liang, Rong-Xin Chen, Zhen Mao, Xian-Chai Lin
×Abstract
Sepsis-induced acute lung injury (ALI) is prevalent in patients with sepsis and has a high mortality rate. Peptidyl arginine deiminase 2 (PADI2) and PADI4 play crucial roles in mediating the host’s immune response in sepsis, but their specific functions remain unclear. Our study shows that Padi2–/– Padi4–/– double KO (DKO) improved survival, reduced lung injury, and decreased bacterial load in Pseudomonas aeruginosa (PA) pneumonia–induced sepsis mice. Using single-cell RNA-Seq (scRNA-Seq), we found that the deletion of Padi2 and Padi4 reduced the Nlrp3+ proinflammatory macrophages and fostered Chil3+ myeloid cell differentiation into antiinflammatory macrophages. Additionally, we observed the regulatory role of the NLRP3/Ym1 axis upon DKO, confirmed by Chil3 knockdown and Nlrp3-KO experiments. Thus, eliminating Padi2 and Padi4 enhanced the polarization of Ym1+ M2 macrophages by suppressing NLRP3, aiding in inflammation resolution and lung tissue repair. This study unveils the PADIs/NLRP3/Ym1 pathway as a potential target in treatment of sepsis-induced ALI.
Authors
Xin Yu, Yujing Song, Tao Dong, Wenlu Ouyang, Liujiazi Shao, Chao Quan, Kyung Eun Lee, Tao Tan, Allan Tsung, Katsuo Kurabayashi, Hasan B. Alam, Mao Zhang, Jianjie Ma, Yongqing Li
×Abstract
Urinary concentration is an energy-dependent process that minimizes body water loss by increasing aquaporin 2 (AQP2) expression in collecting duct (CD) principal cells. To investigate the role of mitochondrial (mt) ATP production in renal water clearance, we disrupted mt electron transport in CD cells by targeting ubiquinone (Q) binding protein QPC (UQCRQ), a subunit of mt complex III essential for oxidative phosphorylation. QPC-deficient mice produced less concentrated urine than controls, both at baseline and after type 2 vasopressin receptor stimulation with desmopressin. Impaired urinary concentration in QPC-deficient mice was associated with reduced total AQP2 protein levels in CD tubules, while AQP2 phosphorylation and membrane trafficking remained unaffected. In cultured inner medullary CD cells treated with mt complex III inhibitor antimycin A, the reduction in AQP2 abundance was associated with activation of 5′ adenosine monophosphate–activated protein kinase (AMPK) and was reversed by treatment with AMPK inhibitor SBI-0206965. In summary, our studies demonstrated that the physiological regulation of AQP2 abundance in principal CD cells was dependent on mt electron transport. Furthermore, our data suggested that oxidative phosphorylation in CD cells was dispensable for maintaining water homeostasis under baseline conditions, but necessary for maximal stimulation of AQP2 expression and urinary concentration.
Authors
Joshua S. Carty, Ryoichi Bessho, Yvonne Zuchowski, Jonathan B. Trapani, Olena Davidoff, Hanako Kobayashi, Joseph T. Roland, Jason A. Watts, Andrew S. Terker, Fabian Bock, Juan Pablo Arroyo, Volker H. Haase
×Abstract
Leucine-zipper–like posttranslational regulator 1 (LZTR1) is a member of the BTB-Kelch superfamily, which regulates the RAS proteostasis. Autosomal dominant (AD) mutations in LZTR1 have been identified in patients with Noonan syndrome (NS), a congenital anomaly syndrome. However, it remains unclear whether LZTR1 AD mutations regulate the proteostasis of the RAS subfamily molecules or cause NS-like phenotypes in vivo. To elucidate the pathogenesis of LZTR1 mutations, we generated 2 LZTR1 mutation knock-in mice (Lztr1G245R/+ and Lztr1R409C/+), which correspond to the human p.G248R and p.R412C mutations, respectively. LZTR1-mutant male mice exhibit low birth weight, distinctive facial features, and cardiac hypertrophy. Cardiomyocyte size and the expression of RAS subfamily members, including MRAS and RIT1, were significantly increased in the left ventricles (LVs) of mutant male mice. LZTR1 AD mutants did not interact with RIT1 and functioned as dominant-negative forms of WT LZTR1. Multi-omics analysis revealed that the mitogen-activated protein kinase (MAPK) signaling pathway was activated in the LVs of mutant mice. Treatment with the MEK inhibitor trametinib ameliorated cardiac hypertrophy in mutant male mice. These results suggest that the MEK/ERK pathway is a therapeutic target for the NS-like phenotype resulting from dysfunction of RAS proteostasis by LZTR1 AD mutations.
Authors
Taiki Abe, Kaho Morisaki, Tetsuya Niihori, Miho Terao, Shuji Takada, Yoko Aoki
×Abstract
Circadian time of intake gates the cardioprotective effects of glucocorticoid administration in both healthy and infarcted hearts. The cardiomyocyte-specific glucocorticoid receptor (GR) and its cofactor, Krüppel-like factor 15 (KLF15), play critical roles in maintaining normal heart function in the long term and serve as pleiotropic regulators of cardiac metabolism. Despite this understanding, the cardiomyocyte-autonomous metabolic targets influenced by the concerted epigenetic action of the GR/KLF15 axis remain undefined. Here, we demonstrated the critical roles of the cardiomyocyte-specific GR and KLF15 in orchestrating a circadian-dependent glucose oxidation program within the heart. Combining integrated transcriptomics and epigenomics with cardiomyocyte-specific inducible ablation of GR or KLF15, we identified their synergistic role in the activation of adiponectin receptor expression (Adipor1) and the mitochondrial pyruvate complex (Mpc1/2), thereby enhancing insulin-stimulated glucose uptake and pyruvate oxidation. Furthermore, in obese diabetic (db/db) mice exhibiting insulin resistance and impaired glucose oxidation, light-phase prednisone administration, as opposed to dark-phase prednisone dosing, restored cardiomyocyte glucose oxidation and improved diastolic function. These effects were blocked by combined in vivo knockdown of GR and KLF15 levels in db/db hearts. In summary, this study leveraged the circadian-dependent cardioprotective effects of glucocorticoids to identify cardiomyocyte-autonomous targets for the GR/KLF15 axis in glucose metabolism.
Authors
Hima Bindu Durumutla, Ashok Daniel Prabakaran, Fadoua El Abdellaoui Soussi, Olukunle Akinborewa, Hannah Latimer, Kevin McFarland, Kevin Piczer, Cole Werbrich, Mukesh K. Jain, Saptarsi M. Haldar, Mattia Quattrocelli
×Abstract
Human studies linking metabolism with organism-wide physiologic function have been challenged by confounding, adherence, and precision. Here, we united physiologic and molecular phenotypes of metabolism during controlled dietary intervention to understand integrated metabolic-physiologic responses to nutrition. In an inpatient study of individuals who underwent serial 24-hour metabolic chamber experiments (indirect calorimetry) and metabolite profiling, we mapped a human metabolome onto substrate oxidation rates and energy expenditure across up to 7 dietary conditions (energy balance, fasting, multiple 200% caloric excess overfeeding of varying fat, protein, and carbohydrate composition). Diets exhibiting greater fat oxidation (e.g., fasting, high-fat) were associated with changes in metabolites within pathways of mitochondrial β-oxidation, ketogenesis, adipose tissue fatty acid liberation, and/or multiple anapleurotic substrates for tricarboxylic acid cycle flux, with inverse associations for diets with greater carbohydrate availability. Changes in each of these metabolite classes were strongly related to 24-hour respiratory quotient (RQ) and substrate oxidation rates (e.g., acylcarnitines related to lower 24-hour RQ and higher 24-hour lipid oxidation), underscoring links between substrate availability, physiology, and metabolism in humans. Physiologic responses to diet determined by gold-standard human metabolic chambers are strongly coordinated with biologically consistent, interconnected metabolic pathways encoded in the metabolome.
Authors
Andrew S. Perry, Paolo Piaggi, Shi Huang, Matthew Nayor, Jane Freedman, Kari E. North, Jennifer E. Below, Clary B. Clish, Venkatesh L. Murthy, Jonathan Krakoff, Ravi V. Shah
×Abstract
Chronic rhinosinusitis with nasal polyps (CRSwNP) is an inflammatory upper airway disease, divided into eosinophilic CRSwNP (eCRSwNP) and noneosinophilic CRSwNP (neCRSwNP) according to eosinophilic levels. Neutrophils are major effector cells in CRSwNP, but their roles in different inflammatory environments remain largely unclear. We performed an integrated transcriptome analysis of polyp-infiltrating neutrophils from patients with CRSwNP, using healthy donor blood as a control. Additional experiments, including flow cytometry and in vitro epithelial cell and fibroblast culture, were performed to evaluate the phenotypic feature and functional role of neutrophils in CRSwNP. Single-cell RNA-sequencing analysis demonstrated that neutrophils could be classified into 5 functional subsets, with GBP5+ neutrophils occurring mainly in neCRSwNP and a high proportion of CXCL8+ neutrophils in both subendotypes. GBP5+ neutrophils exhibited significant IFN-I pathway activity in neCRSwNP. CXCL8+ neutrophils displayed increased neutrophil activation scores and mainly secreted oncostatin M (OSM), which facilitates communication with other cells. In vitro experiments showed that OSM enhanced IL-13– or IL-17–mediated immune responses in nasal epithelial cells and fibroblasts. Our findings indicate that neutrophils display transcriptional plasticity and activation when exposed to polyp tissue, contributing to CRSwNP pathogenesis by releasing OSM, which interacts with epithelial cells and fibroblasts depending on the inflammatory environment.
Authors
Chen Zhang, Qianqian Zhang, Jiani Chen, Han Li, Fuying Cheng, Yizhang Wang, Yingqi Gao, Yumin Zhou, Le Shi, Yufei Yang, Juan Liu, Kai Xue, Yaguang Zhang, Hongmeng Yu, Dehui Wang, Li Hu, Huan Wang, Xicai Sun
×Abstract
Opioid use may affect the HIV-1 reservoir and its reversal from latency. We studied 47 virally suppressed people with HIV (PWH) and observed that lower concentration of HIV-1 latency reversal agents (LRAs), used with small molecules that did not reverse latency, synergistically increased the magnitude of HIV-1 reactivation ex vivo, regardless of opioid use. This LRA boosting, which combined a second mitochondria-derived activator of caspases mimetic or low-dose PKC agonist with histone deacetylase inhibitors, generated more unspliced HIV-1 transcription than PMA with ionomycin (PMAi), the maximal known HIV-1 reactivator. LRA boosting associated with greater histone acetylation, modulated surface activation-induced markers, and altered T cell production of TNF-α, IL-2, and IFN-γ. HIV-1 reservoirs in PWH contained unspliced and polyadenylated virus mRNA, the ratios of which were greater in resting than total CD4+ T cells and corrected to 1:1 with PMAi exposure. We characterized treated suppressed HIV-1 infection as a period of inefficient, not absent, virus transcription. Multiply spliced HIV-1 transcripts and virion production did not consistently increase with LRA boosting, suggesting the presence of a persistent posttranscriptional block. LRA boosting can be leveraged to probe mechanisms of an effective cellular HIV-1 latency reversal program.
Authors
Tyler Lilie, Jennifer Bouzy, Archana Asundi, Jessica Taylor, Samantha Roche, Alex Olson, Kendyll Coxen, Heather Corry, Hannah Jordan, Kiera Clayton, Nina Lin, Athe Tsibris
×Abstract
The application of chimeric antigen receptor (CAR) T cell therapy in solid tumors is hindered by life-threatening toxicities resulting from on-target, off-tumor killing of nonmalignant cells that express low levels of the target antigen. Mesothelin (MSLN) has been identified as a target antigen for CAR T cell treatment of mesothelioma, lung, ovarian, and other cancers because of its high expression on tumor cells and limited expression on mesothelial cells. However, fatal off-tumor toxicity of high-affinity MSLN-targeting CAR T cells has been reported in multiple clinical trials. In this study, we constructed CARs using mutant variants of a single-domain nanobody that bind both human and mouse MSLN with a wide range of affinities and examined tumor responses and their toxicities from on-target, off-tumor interactions in mouse models. CAR T cells with low nanomolar affinity (equilibrium dissociation constant, KD) exhibited profound systemic expansion with no apparent infiltration into the tumor. With a gradual reduction of CAR affinity toward the micromolar KD, the expansion of CAR T cells became more restricted to tumors. Our preclinical studies demonstrated that high-affinity MSLN CARs were associated with fatal on-target, off-tumor toxicity and that affinity-tuned CARs rendered T cells more selective for MSLN-high tumors.
Authors
Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Sydney J. Trumper, Diella S. Babu, Irene M. Min, Jacqueline M. Tremblay, Charles B. Shoemaker, Moonsoo M. Jin
