Enterovirus 71 (EV-A71) receptors that have been identified to date cannot fully explain the pathogenesis of EV-A71, which is an important global cause of hand-foot-and-mouth disease and life-threatening encephalitis. We identified an interferon-gamma (IFNγ)-inducible EV-A71 cellular entry factor, human tryptophanyl-tRNA synthetase (hWARS), using genome-wide RNAi library screening. The importance of hWARS in mediating virus entry and infectivity was confirmed by virus attachment, in vitro pull-down, antibody/antigen blocking, and CRISPR/Cas9. Upon IFNγ treatment, induced hyperexpression and plasma membrane translocation of hWARS were observed, which sensitized semi-permissive (human neuronal NT2)/non-permissive (mouse fibroblast L929) cells to EV-A71 infection. Our hWARS-transduced mouse infection model showed pathological changes similar to patients with severe EV-A71 infection. The expression of hWARS is also required for productive infection by other human enteroviruses, including the clinically important CV-A16 and EV-D68. This is the first report on the discovery of an entry factor, hWARS, which can be induced by IFNγ for EV-A71. Given that a high level of IFNγ was observed in patients with severe EV-A71 infection, our findings extend the knowledge of the pathogenicity of EV-A71 in relation to the expression of entry factor upon IFNγ stimulation and the therapeutic options for treating severe EV-A71-associated complications.
Man Lung Yeung, Lilong Jia, Cyril C.Y. Yip, Jasper F.W. Chan, Jade L.L. Teng, Kwok-Hung Chan, Jian-Piao Cai, Chaoyu Zhang, Anna J. Zhang, Wan-Man Wong, Kin-Hang Kok, Susanna K.P. Lau, Patrick C.Y. Woo, Janice Y.C. Lo, Dong-Yan Jin, Shin-Ru Shih, Kwok-Yung Yuen
Cardiac two pore domain potassium channels (K2P) exist in organisms from Drosophila to humans, however their role in cardiac function is not known. We identified a K2P gene, CG8713 (sandman), in a Drosophila genetic screen and show that sandman is critical to cardiac function. Mice lacking an ortholog of sandman, TWIK related potassium channel (TREK-1 or Kcnk2), exhibit exaggerated pressure overload induced concentric hypertrophy and alterations in fetal gene expression, yet retain preserved systolic and diastolic cardiac function. While cardiomyocyte specific deletion of TREK-1 in response to in vivo pressure overload resulted in cardiac dysfunction, TREK-1 deletion in fibroblasts prevented deterioration in cardiac function. The absence of pressure overload induced dysfunction in TREK-1 KO mice was associated with diminished cardiac fibrosis and reduced activation of c-Jun N-terminal kinase activity (JNK) in cardiomyocytes and fibroblasts. These findings indicate a central role for cardiac fibroblast TREK-1 in the pathogenesis of pressure overload-induced cardiac dysfunction and serve as a conceptual basis for its inhibition for as a potential therapy.
Dennis M. Abraham, Teresa E. Lee, Lewis J. Watson, Lan Mao, Gurangad S. Chandok, Hong-Gang Wang, Stephan Frangakis, Geoffrey S. Pitt, Svati H. Shah, Matthew J. Wolf, Howard A. Rockman
Chronic allergic inflammatory diseases are a major cause of morbidity, allergic asthma alone affecting over 300 million people worldwide. Epidemiological studies demonstrate that environmental stimuli are associated with either promotion or prevention of disease. Major reductions in asthma prevalence are documented in European and US farming communities. Protection is associated with exposure of mothers during pregnancy to microbial breakdown products present in farm dusts and unprocessed foods, and enhancement of innate immune competence in the children. We sought to develop a scientific rationale for progressing these findings towards clinical application for primary disease prevention. Treatment of pregnant mice with a defined clinically-approved immune-modulator was shown to markedly reduce susceptibility of their offspring to development of the hallmark clinical features of allergic airway inflammatory disease. Mechanistically, offspring displayed enhanced dendritic cell-dependent airway mucosal immune surveillance function, which resulted in more efficient generation of mucosal-homing T-regulatory cells in response to local inflammatory challenge. We provide evidence that the principal target for maternal treatment effects was the fetal dendritic cell progenitor compartment, equipping the offspring for accelerated functional maturation of the airway mucosal dendritic cell network following birth. These data provide proof-of-concept supporting the rationale for development of transplacental immune reprogramming approaches for primary disease prevention.
Kyle T. Mincham, Naomi M. Scott, Jean-Francois Lauzon-Joset, Jonatan Leffler, Alexander N. Larcombe, Philip A. Stumbles, Sarah A. Robertson, Christian Pasquali, Patrick G. Holt, Deborah H. Strickland
BACKGROUND. Evidence from rodent studies indicates that the sympathetic nervous system (SNS) regulates bone metabolism, principally via β2-adrenergic receptors (β2-ARs). Given conflicting human data, we used multiple approaches to evaluate the role of the SNS in regulating human bone metabolism. METHODS. (1) Bone biopsies were obtained from 19 young and 19 old women for assessment of ADRB1, ADRB2, and ADRB3 mRNA expression; (2) the relationship of β-blocker use to bone microarchitecture was assessed by high resolution-peripheral quantitative computed tomography in a population sample of 248 subjects; and (3) 155 postmenopausal women were randomized to one of five treatment groups for 20 weeks: placebo; propranolol, 20 mg twice a day (BID); propranolol, 40 mg BID; atenolol, 50 mg/d; and nebivolol, 5 mg/d. We took advantage of the β1-AR selectivity gradient of these drugs (propranolol [non-selective] << atenolol [relatively β1-AR selective] < nebivolol [highly β1-AR selective]) to define the β-AR selectivity for SNS effects on bone. RESULTS. (1) ADRB1and ADRB2, but not ADRB3, were expressed in human bone; (2) patients treated clinically with β1-AR selective blockers had better bone microarchitecture than non-users; and (3) relative to placebo, atenolol and nebivolol, but not propranolol, reduced the bone resorption marker serum C-telopeptide of type I collagen (by 19.5% and 20.6%, respectively; P < 0.01) and increased ultra-distal radius BMD (by 3.6% and 2.9%; P < 0.01 and P < 0.05, respectively). CONCLUSIONS. These three independent lines of evidence strongly support a role for adrenergic signaling in regulating bone metabolism in humans, principally via β1-ARs. TRIAL REGISTRATION. ClinicalTrials.gov NCT02467400. FUNDING. This research was supported by NIH grants AG004875, AR027065, and the Mayo Clinic CTSA (UL1 TR002377).
Sundeep Khosla, Matthew T. Drake, Tammie L. Volkman, Brianne S. Thicke, Sara J. Achenbach, Elizabeth J. Atkinson, Michael J. Joyner, Clifford J. Rosen, David G. Monroe, Joshua N. Farr
It is suggested that subtyping of complex inflammatory diseases can be based on genetic susceptibility and relevant environmental exposure (G+E). We propose that using matched cellular phenotypes in human subjects and corresponding pre-clinical models with the same G+E combinations are useful to this end. As an example, defective Paneth cells can subtype Crohn's disease (CD) subjects; Paneth cell defects have been linked to multiple CD susceptibility genes and are associated with poor outcome. We hypothesized that CD susceptibility genes interact with cigarette smoking, a major CD environmental risk factor, to trigger Paneth cell defects. We found that both CD subjects and mice with ATG16L1T300A (T300A; a prevalent CD susceptibility allele) developed Paneth cell defects triggered by tobacco smoke. Transcriptional analysis of full thickness ileum and Paneth cell-enriched crypt base cells showed the T300A-smoking combination altered distinct pathways, including pro-apoptosis, metabolic dysregulation, and selective down-regulation of the PPARγ pathway. Pharmacologic intervention by either apoptosis inhibitor or PPARγ agonist rosiglitazone prevented smoking-induced crypt apoptosis and Paneth cell defects in T300A mice and mice with conditional Paneth cell-specific knockout of Atg16l1. This study demonstrates how explicit G+E can drive disease relevant phenotype, and provides rational strategies to identify actionable targets.
Ta-Chiang Liu, Justin T. Kern, Kelli L. VanDussen, Shanshan Xiong, Gerard E. Kaiko, Craig B. Wilen, Michael W. Rajala, Roberta Caruso, Michael J. Holtzman, Feng Gao, Dermot P.B. McGovern, Gabriel Nunez, Richard D. Head, Thaddeus S. Stappenbeck
Inflammation occurs in all tissues in response to injury or stress and is the key process underlying hepatic fibrogenesis. Targeting chronic and uncontrolled inflammation is one strategy to prevent liver injury and fibrosis progression. Here, we demonstrate that triggering receptor expressed on myeloid cells-1 (TREM-1), an amplifier of inflammation, promotes liver disease by intensifying hepatic inflammation and fibrosis. In the liver, TREM-1 expression is limited to liver macrophages and monocytes and is highly upregulated on Kupffer cells, circulating monocytes, and monocyte-derived macrophages in a mouse model of chronic liver injury and fibrosis induced by carbon tetrachloride (CCl4) administration. TREM-1 signaling promotes pro-inflammatory cytokine production and mobilization of inflammatory cells to the site of injury. Deletion of Trem1 reduced liver injury, inflammatory cell infiltration, and fibrogenesis. Reconstitution of Trem1-deficient mice with Trem1-sufficient Kupffer cells restored recruitment of inflammatory monocytes and severity of liver injury. Markedly increased infiltration of liver fibrotic areas with TREM-1-positive Kupffer cells and monocytes/macrophages was found in patients with hepatic fibrosis. Our data support a role of TREM-1 in liver injury and hepatic fibrogenesis and suggests that TREM-1 is a master regulator of Kupffer cell activation, which escalates chronic liver inflammatory responses, activates hepatic stellate cells, and reveals a novel mechanism of promotion of liver fibrosis.
Anh Thu Nguyen-Lefebvre, Ashwin Ajith, Vera Portik-Dobos, Daniel David Horuzsko, Ali Syed Arbab, Amiran Dzutsev, Ramses Sadek, Giorgio Trinchieri, Anatolij Horuzsko
Human endogenous retroviruses (hERVs) are remnants of exogenous retroviruses that have integrated into the genome throughout evolution. We developed a computational workflow, hervQuant, which identified over 3,000 transcriptionally active hERVs within The Cancer Genome Atlas (TCGA) pan-cancer RNA-seq database. hERV expression was associated with clinical prognosis in several tumor types, most significantly clear cell renal cell carcinoma (ccRCC). We explored two mechanisms by which hERV expression may influence the tumor-immune microenvironment in ccRCC: through 1) RIG-I-like signaling, and 2) retroviral antigen activation of adaptive immunity. We demonstrated the ability of hERV signatures associated with these immune mechanisms to predict patient survival in ccRCC, independent of clinical staging and molecular subtyping. We identified potential tumor-specific hERV epitopes with evidence of translational activity through the use of a ccRCC Ribo-seq dataset, validated their ability to bind HLA in vitro, and identified presence of MHC tetramer-positive T cells against predicted epitopes. hERV sequences identified through this screening approach were significantly more highly expressed in ccRCC tumors responsive to treatment with programmed death receptor-1 (PD-1) inhibition. hervQuant provides new insights into the role of hERVs within the tumor-immune microenvironment as well as evidence for hERV expression-based biomarkers for patient prognosis and response to immunotherapy.
Christof C. Smith, Kathryn E. Beckermann, Dante S. Bortone, Aguirre A. de Cubas, Lisa M. Bixby, Samuel J. Lee, Anshuman Panda, Shridar Ganesan, Gyan Bhanot, Eric M. Wallen, Matthew I. Milowsky, William Y. Kim, W. Kimryn Rathmell, Ronald Swanstrom, Joel S. Parker, Jonathan S. Serody, Sara R. Selitsky, Benjamin G. Vincent
Renin cells are crucial for survival: they control fluid-electrolyte and blood pressure homeostasis, vascular development, regeneration, and oxygen delivery to tissues. During embryonic development, renin cells are progenitors for multiple cell types which retain the memory of the renin phenotype. When there is a threat to survival, those descendants are transformed and reenact the renin phenotype to restore homeostasis. We tested the hypothesis that the molecular memory of the renin phenotype resides in unique regions and states of these cells’ chromatin. Using renin cells at various stages of stimulation, we identified regions in the genome where the chromatin is open for transcription, mapped histone modifications characteristic of active enhancers such as H3K27ac and deposition of transcriptional activators such a Med1 whose deletion results in ablation of Renin expression and low blood pressure. Using the rank ordering of super-enhancers, epigenetic re-writing, and enhancer deletion analysis, we found that renin cells harbor a unique set of super-enhancers that determine their identity. The most prominent Renin super-enhancer may act as a chromatin sensor of signals that convey the physiologic status of the organism and is responsible for the transformation of renin cell descendants to the renin phenotype, a fundamental process to ensure homeostasis.
Maria Florencia Martinez, Silvia Medrano, Evan A. Brown, Turan Tufan, Stephen Shang, Nadia Bertoncello, Omar Guessoum, Mazhar Adli, Brian C. Belyea, Maria Luisa S. Sequeira Lopez, R. Ariel Gomez
Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we show in novel autochthonous mouse models that EMT accelerates KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrate that HBP is required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delays KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAcylation) post-translational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerates lung tumorigenesis. Conversely, loss of O-GlcNAcylation delays lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-Myc correlates with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrate that O-GlcNAcylation is sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which is reinforced by epithelial plasticity programs.
Kekoa Taparra, Hailun Wang, Reem Malek, Audrey Lafargue, Mustafa A. Barbhuiya, Xing Wang, Brian W. Simons, Matthew Ballew, Katriana Nugent, Jennifer Groves, Russell D. Williams, Takumi Shiraishi, James Verdone, Gokben Yildirir, Roger Henry, Bin Zhang, John Wong, Ken Kang-Hsin Wang, Barry D. Nelkin, Kenneth J. Pienta, Dean Felsher, Natasha E. Zachara, Phuoc T. Tran
First generation immune checkpoint inhibitors including anti-CTLA-4 and anti-PD-1 antibodies have led to major clinical progress, yet resistance frequently leads to treatment failure. Thus, new targets acting on T cells are needed. CD33-related Siglecs are pattern recognition immune receptors binding to a range of sialoglycan ligands, which appear to function as self-associated molecular patterns (SAMPs) that suppress autoimmune responses. Siglecs are expressed at very low levels on normal T cells, and these receptors were not yet considered as interesting targets on T cells for cancer immunotherapy. Here, we show an upregulation of Siglecs including Siglec-9 on tumor-infiltrating T cells from non-small cell lung (NSCLC), colorectal and ovarian cancer patients. Siglec-9 expressing T cells co-expressed several inhibitory receptors including PD-1. Targeting of the sialoglycan-SAMP/Siglec pathway in vitro and in vivo resulted in increased anti-cancer immunity. T cell expression of Siglec-9 in NSCLC patients correlated with a reduced survival, and Siglec-9 polymorphisms showed associations with the risk of developing lung and colorectal cancer. Our data identify the sialoglycan-SAMP/Siglec pathway as new potential target to improve T cell activation for immunotherapy.
Michal A. Stanczak, Shoib S. Siddiqui, Marcel P. Trefny, Daniela S. Thommen, Kayluz Frias Boligan, Stephan von Gunten, Alexandar Tzankov, Lothar Tietze, Didier Lardinois, Viola Heinzelmann-Schwarz, Michael S. von Bergwelt-Baildon, Wu Zhang, Heinz-Josef Lenz, Younghan Han, Christopher I. Amos, Mohammedyaseen Syedbasha, Adrian Egli, Frank Stenner, Daniel E. Speiser, Ajit Varki, Alfred Zippelius, Heinz Läubli
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