Research Articles
Abstract
Excessive erythrocytosis (EE) is a major hallmark of patients suffering from chronic mountain sickness (CMS, also known as Monge’s disease) and is responsible for major morbidity and even mortality in early adulthood. We took advantage of unique populations, one living at high altitude (Peru) showing EE, with another population, at the same altitude and region, showing no evidence of EE (non-CMS). Through RNA-Seq, we identified and validated the function of a group of long noncoding RNAs (lncRNAs) that regulate erythropoiesis in Monge’s disease, but not in the non-CMS population. Among these lncRNAs is hypoxia induced kinase-mediated erythropoietic regulator (HIKER)/LINC02228, which we showed plays a critical role in erythropoiesis in CMS cells. Under hypoxia, HIKER modulated CSNK2B (the regulatory subunit of casein kinase 2). A downregulation of HIKER downregulated CSNK2B, remarkably reducing erythropoiesis; furthermore, an upregulation of CSNK2B on the background of HIKER downregulation rescued erythropoiesis defects. Pharmacologic inhibition of CSNK2B drastically reduced erythroid colonies, and knockdown of CSNK2B in zebrafish led to a defect in hemoglobinization. We conclude that HIKER regulates erythropoiesis in Monge’s disease and acts through at least one specific target, CSNK2B, a casein kinase.
Authors
Priti Azad, Dan Zhou, Hung-Chi Tu, Francisco C. Villafuerte, David Traver, Tariq M. Rana, Gabriel G. Haddad
Abstract
Herpes simplex virus type 2 (HSV-2) coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well defined. HSV-2 recurrences result in an influx of activated CD4+ T cells to sites of viral replication and an increase in activated CD4+ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4+ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2–infected and bystander 2D10 cells. Bulk and single-cell RNA-Seq studies of activated primary human CD4+ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2–infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms, including upregulation of MALAT1 to release epigenetic silencing.
Authors
Carl A. Pierce, Lip Nam Loh, Holly R. Steach, Natalia Cheshenko, Paula Preston-Hurlburt, Fengrui Zhang, Stephanie Stransky, Leah Kravets, Simone Sidoli, William Philbrick, Michel Nassar, Smita Krishnaswamy, Kevan C. Herold, Betsy C. Herold
Abstract
Characterized by the accumulation of somatic mutations in blood cell lineages, clonal hematopoiesis of indeterminate potential (CHIP) is frequent in aging and involves the expansion of mutated hematopoietic stem and progenitor cells (HSC/Ps) that leads to an increased risk of hematologic malignancy. However, the risk factors that contribute to CHIP-associated clonal hematopoiesis (CH) are poorly understood. Obesity induces a proinflammatory state and fatty bone marrow (FBM), which may influence CHIP-associated pathologies. We analyzed exome sequencing and clinical data for 47,466 individuals with validated CHIP in the UK Biobank. CHIP was present in 5.8% of the study population and was associated with a significant increase in the waist-to-hip ratio (WHR). Mouse models of obesity and CHIP driven by heterozygosity of Tet2, Dnmt3a, Asxl1, and Jak2 resulted in exacerbated expansion of mutant HSC/Ps due in part to excessive inflammation. Our results show that obesity is highly associated with CHIP and that a proinflammatory state could potentiate the progression of CHIP to more significant hematologic neoplasia. The calcium channel blockers nifedipine and SKF-96365, either alone or in combination with metformin, MCC950, or anakinra (IL-1 receptor antagonist), suppressed the growth of mutant CHIP cells and partially restored normal hematopoiesis. Targeting CHIP-mutant cells with these drugs could be a potential therapeutic approach to treat CH and its associated abnormalities in individuals with obesity.
Authors
Santhosh Kumar Pasupuleti, Baskar Ramdas, Sarah S. Burns, Lakshmi Reddy Palam, Rahul Kanumuri, Ramesh Kumar, Taruni Reddy Pandhiri, Utpal P. Dave, Nanda Kumar Yellapu, Xinyu Zhou, Chi Zhang, George E. Sandusky, Zhi Yu, Michael C. Honigberg, Alexander G. Bick, Gabriel K. Griffin, Abhishek Niroula, Benjamin L. Ebert, Sophie Paczesny, Pradeep Natarajan, Reuben Kapur
Abstract
Many patients with hepatocellular carcinoma (HCC) do not respond to the first-line immune checkpoint inhibitor treatment. Immunization with effective cancer vaccines is an attractive alternative approach to immunotherapy. However, its efficacy remains insufficiently evaluated in preclinical studies. Here, we investigated HCC-associated self/tumor antigen, α-fetoprotein–based (AFP-based) vaccine immunization for treating AFP (+) HCC mouse models. We found that AFP immunization effectively induced AFP-specific CD8+ T cells in vivo. However, these CD8+ T cells expressed exhaustion markers, including PD1, LAG3, and Tim3. Furthermore, the AFP vaccine effectively prevented c-MYC/Mcl1 HCC initiation when administered before tumor formation, while it was ineffective against full-blown c-MYC/Mcl1 tumors. Similarly, anti-PD1 and anti–PD-L1 monotherapy showed no efficacy in this murine HCC model. In striking contrast, AFP immunization combined with anti–PD-L1 treatment triggered significant inhibition of HCC progression in most liver tumor nodules, while in combination with anti-PD1, it induced slower tumor progression. Mechanistically, we demonstrated that HCC-intrinsic PD-L1 expression was the primary target of anti–PD-L1 in this combination therapy. Notably, the combination therapy had a similar therapeutic effect in the cMet/β-catenin mouse HCC model. These findings suggest that combining the AFP vaccine and immune checkpoint inhibitors may be effective for AFP (+) HCC treatment.
Authors
Xinjun Lu, Shanshan Deng, Jiejie Xu, Benjamin L. Green, Honghua Zhang, Guofei Cui, Yi Zhou, Yi Zhang, Hongwei Xu, Fapeng Zhang, Rui Mao, Sheng Zhong, Thorsten Cramer, Matthias Evert, Diego F. Calvisi, Yukai He, Chao Liu, Xin Chen
Abstract
Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) in response to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs (CVOs) of the brain focus on mechanosensitive membrane proteins. The present study demonstrated that intracellular protein kinase WNK1 was involved. Focusing on vascular-organ-of-lamina-terminalis (OVLT) nuclei, we showed that WNK1 kinase was activated by water restriction. Neuron-specific conditional KO (cKO) of Wnk1 caused polyuria with decreased urine osmolality that persisted in water restriction and blunted water restriction–induced AVP release. Wnk1 cKO also blunted mannitol-induced AVP release but had no effect on osmotic thirst response. The role of WNK1 in the osmosensory neurons in CVOs was supported by neuronal pathway tracing. Hyperosmolality-induced increases in action potential firing in OVLT neurons was blunted by Wnk1 deletion or pharmacological WNK inhibitors. Knockdown of Kv3.1 channel in OVLT by shRNA reproduced the phenotypes. Thus, WNK1 in osmosensory neurons in CVOs detects extracellular hypertonicity and mediates the increase in AVP release by activating Kv3.1 and increasing action potential firing from osmosensory neurons.
Authors
Xin Jin, Jian Xie, Chia-Wei Yeh, Jen-Chi Chen, Chih-Jen Cheng, Cheng-Chang Lien, Chou-Long Huang
Abstract
Programmed cell death ligand 1 (PD-L1) is an immune checkpoint protein frequently expressed in human cancers that contributes to immune evasion through its binding to PD-1 on activated T cells. Unveiling the mechanisms underlying PD-L1 expression is essential for understanding the impact of the immunosuppressive microenvironment and is also crucial for the purpose of reboosting antitumor immunity. However, how PD-L1 is regulated, particularly at translational levels, remains largely unknown. Here, we discovered that a long noncoding RNA (lncRNA), HIF-1α inhibitor at translation level (HITT), was transactivated by E2F transcription factor 1 (E2F1) under IFN-γ stimulation. It coordinated with regulator of G protein signaling 2 (RGS2) in binding to the 5′ UTR of PD-L1, resulting in reduced PD-L1 translation. HITT expression enhanced T cell–mediated cytotoxicity both in vitro and in vivo in a PD-L1–dependent manner. The clinical correlation between HITT/PD-L1 and RGS2/PD-L1 expression was also detected in breast cancer tissues. Together, these findings demonstrate the role of HITT in antitumor T cell immunity, highlighting activation of HITT as a potential therapeutic strategy for enhancing cancer immunotherapy.
Authors
Qingyu Lin, Tong Liu, Xingwen Wang, Guixue Hou, Zhiyuan Xiang, Wenxin Zhang, Shanliang Zheng, Dong Zhao, Qibin Leng, Xiaoshi Zhang, Minqiao Lu, Tianqi Guan, Hao Liu, Ying Hu
Abstract
The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intratumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single-cell transcriptomic and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stress-responsive DTC cells, inflammatory ATC cells (iATCs), and mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs). Furthermore, our analysis identified 2 important milestones: (a) a diploid stage, in which iATC cells were diploids with inflammatory phenotypes and (b) an aneuploid stage, in which mATCs gained aneuploid genomes and mesenchymal phenotypes, producing excessive amounts of collagen and collagen-interacting receptors. In parallel, cancer-associated fibroblasts showed strong interactions among mesenchymal cell types, macrophages shifted from M1 to M2 states, and T cells reprogrammed from cytotoxic to exhausted states, highlighting new therapeutic opportunities for the treatment of ATC.
Authors
Lina Lu, Jennifer Rui Wang, Ying C. Henderson, Shanshan Bai, Jie Yang, Min Hu, Cheng-Kai Shiau, Timothy Pan, Yuanqing Yan, Tuan M. Tran, Jianzhuo Li, Rachel Kieser, Xiao Zhao, Jiping Wang, Roza Nurieva, Michelle D. Williams, Maria E. Cabanillas, Ramona Dadu, Naifa Lamki Busaidy, Mark Zafereo, Nicholas Navin, Stephen Y. Lai, Ruli Gao
Abstract
Plasma IL-6 is elevated after myocardial infarction (MI) and is associated with increased morbidity and mortality. Which cardiac cell type preferentially contributes to IL-6 expression and how its production is regulated are largely unknown. Here, we studied the cellular source and purinergic regulation of IL-6 formation in a murine MI model. We found that IL-6, measured in various cell types in post-MI hearts at the protein level and by quantitative PCR and RNAscope, was preferentially formed by cardiac fibroblasts (CFs). Single-cell RNA-Seq (scRNA-Seq) in infarcted mouse and human hearts confirmed this finding. We found that adenosine stimulated fibroblast IL-6 formation via the adenosine receptor A2bR in a Gq-dependent manner. CFs highly expressed Adora2b and rapidly degraded extracellular ATP to AMP but lacked CD73. In mice and humans, scRNA-Seq revealed that Adora2B was also mainly expressed by fibroblasts. We assessed global IL-6 production in isolated hearts from mice lacking CD73 on T cells (CD4-CD73–/–), a condition known to be associated with adverse cardiac remodeling. The ischemia-induced release of IL-6 was strongly attenuated in CD4-CD73–/– mice, suggesting adenosine-mediated modulation. Together, these findings demonstrate that post-MI IL-6 was mainly derived from activated CFs and was controlled by T cell–derived adenosine. We show that purinergic metabolic cooperation between CFs and T cells is a mechanism that modulates IL-6 formation by the heart and has therapeutic potential.
Authors
Christina Alter, Anne-Sophie Henseler, Christoph Owenier, Julia Hesse, Zhaoping Ding, Tobias Lautwein, Jasmin Bahr, Sikander Hayat, Rafael Kramann, Eva Kostenis, Jürgen Scheller, Jürgen Schrader
Abstract
Renal osteodystrophy (ROD) is a disorder of bone metabolism that affects virtually all patients with chronic kidney disease (CKD) and is associated with adverse clinical outcomes including fractures, cardiovascular events, and death. In this study, we showed that hepatocyte nuclear factor 4α (HNF4α), a transcription factor mostly expressed in the liver, is also expressed in bone, and that osseous HNF4α expression was dramatically reduced in patients and mice with ROD. Osteoblast-specific deletion of Hnf4α resulted in impaired osteogenesis in cells and mice. Using multi-omics analyses of bones and cells lacking or overexpressing Hnf4α1 and Hnf4α2, we showed that HNF4α2 is the main osseous Hnf4α isoform that regulates osteogenesis, cell metabolism, and cell death. As a result, osteoblast-specific overexpression of Hnf4α2 prevented bone loss in mice with CKD. Our results showed that HNF4α2 is a transcriptional regulator of osteogenesis, implicated in the development of ROD.
Authors
Marta Martinez-Calle, Guillaume Courbon, Bridget Hunt-Tobey, Connor Francis, Jadeah Spindler, Xueyan Wang, Luciene M. dos Reis, Carolina S.W. Martins, Isidro B. Salusky, Hartmut Malluche, Thomas L. Nickolas, Rosa M.A. Moyses, Aline Martin, Valentin David
Abstract
Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid “scramblases,” such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall–dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.
Authors
Alec A. Schmaier, Papa F. Anderson, Siyu M. Chen, Emale El-Darzi, Ivan Aivasovsky, Milan P. Kaushik, Kelsey D. Sack, H. Criss Hartzell, Samir M. Parikh, Robert Flaumenhaft, Sol Schulman
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Copyright © 2023 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)