The upper respiratory tract is compromised in the early period of COVID-19, but SARS-CoV-2 tropism at the cellular level is not fully defined. Unlike recent single cell RNA-sequencing analyses indicating uniformly low mRNA expression of SARS-CoV-2 entry-related host molecules in all nasal epithelial cells, we show that the protein levels are relatively high and their localizations are restricted to the apical side of multiciliated epithelial cells. In addition, we provide evidence in COVID-19 patients that SARS-CoV-2 is massively detected and replicated within the multiciliated cells. We observed these findings during the early stage of COVID-19, when infected ciliated cells are rapidly replaced by differentiating precursor cells. Moreover, our analyses reveal that SARS-CoV-2 cellular tropism is restricted to the nasal ciliated versus oral squamous epithelium. These results imply that targeting ciliated cells of the nasal epithelium during the early stage of COVID-19 could be an ideal strategy to prevent SARS-CoV-2 propagation.
Ji Hoon Ahn, JungMo Kim, Seon Pyo Hong, Sung Yong Choi, Myung Jin Yang, Young Seok Ju, Young Tae Kim, Ho Min Kim, MD Tazikur Rahman, Man Ki Chung, Sang Duk Hong, Hosung Bae, Chang-Seop Lee, Gou Young Koh
Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to GLP-1 receptor (GLP-1R) agonism in patients with type 2 diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine if GIPR agonism contributes, we compared the effect of tirzepatide in obese wild-type and Glp-1r null mice. In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino (BCAAs) and keto-acids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent and -independent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.
Ricardo J. Samms, Michael E. Christe, Kyla A. Collins, Valentina Pirro, Brian A. Droz, Adrienne K. Holland, Jessica L. Friedrich, Samantha Wojnicki, Debra L. Konkol, Richard Cosgrove, Ellen P.S. Conceição Furber, Xiaoping Ruan, Libbey S. O'Farrell, Annie M. Long, Mridula Dogra, Jill A. Willency, Yanzhu Lin, Liyun Ding, Christine C. Cheng, Over Cabrera, Daniel A. Briere, Jorge Alsina-Fernandez, Ruth E. Gimeno, Julie S. Moyers, Tamer Coskun, Matthew P. Coghlan, Kyle W. Sloop, William C. Roell
Synovial sarcoma is an aggressive malignancy with no effective treatments for patients with metastasis. The synovial sarcoma fusion, SS18-SSX, which recruits the SWI/SNF-BAF chromatin remodeling and polycomb repressive complexes, results in epigenetic activation of FGFR signaling. In genetic FGFR knockout models, culture, and xenograft synovial sarcoma models treated with the FGFR inhibitor BGJ398, we show that FGFR1, FGFR2, and FGFR3 were crucial for tumor growth. Transcriptome analyses of BGJ398-treated cells, histological and expression analyses of mouse and human synovial sarcoma tumors revealed prevalent expression of two ETS factors and FGFR targets, ETV4 and ETV5. We further demonstrate that ETV4 and ETV5 acted as drivers of synovial sarcoma growth, most likely through control of the cell cycle. Upon ETV4 and ETV5 knockdown, we observed a striking upregulation of DUX4 and its transcriptional targets that activate the zygotic genome and drive the atrophy program in facioscapulohumeral dystrophy (FSHD) patients. In addition to demonstrating the importance of inhibiting all three FGFR receptors, the current findings reveal potential nodes of attack for the cancer with the discovery of ETV4 and ETV5 as appropriate biomarkers and molecular targets, and activation of the embryonic DUX4 pathway as a promising approach to block synovial sarcoma tumors.
Joanna DeSalvo, Yuguang Ban, Luyuan Li, Xiaodian Sun, Zhijie Jiang, Darcy A. Kerr, Mahsa Khanlari, Maria Boulina, Mario R. Capecchi, Juha M. Partanen, Lin Chen, Tadashi Kondo, David M. Ornitz, Jonathan C. Trent, Josiane E. Eid
B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. As predicated by its prenatal origin, infant B-ALL (iB-ALL) shows an exceptionally silent DNA mutational landscape, suggesting that alternative epigenetic mechanisms may substantially contribute to its leukemogenesis. Here, we have integrated genome-wide DNA methylome and transcriptome data from 69 patients with de novo MLL-rearranged (MLLr) and non-MLLr iB-ALL leukemias uniformly treated according to Interfant-99/06 protocol. iB-ALL methylome signatures display a plethora of common and specific alterations associated with chromatin states related to enhancer and transcriptional control in normal hematopoietic cells. DNA methylation, gene expression and gene co-expression network analyses segregated MLLr away from non-MLLr iB-ALL and identified a coordinated and enriched expression of the AP-1 complex members FOS and JUN and RUNX factors in MLLr iB-ALL, consistent with the significant enrichment of hypomethylated CpGs in these genes. Integrative methylome-transcriptome analysis identified consistent cancer-cell vulnerabilities, revealed a robust iB-ALL-specific gene expression-correlating dmCpG signature and confirmed an epigenetic control of AP-1 and RUNX members in reshaping the molecular network of MLLr iB-ALL. Finally, pharmacological inhibition or functional ablation of AP-1 dramatically impaired MLLr-leukemic growth in vitro and in vivo using MLLr-iB-ALL patient-derived xenografts, providing rationale for new therapeutic avenues in MLLr-iB-ALL.
J. Ramon Tejedor, Clara Bueno, Meritxell Vinyoles, Paolo Petazzi, Antonio Agraz-Doblas, Isabel Cobo, Raúl Torres-Ruiz, Gustavo F. Bayón, Raúl F. Pérez, Sara López-Tamargo, Francisco Gutierrez-Agüera, Pablo Santamarina-Ojeda, Manuel Ramírez-Orellana, Michela Bardini, Giovanni Cazzaniga, Paola Ballerini, Pauline Schneider, Ronald W. Stam, Ignacio Varela, Mario F. Fraga, Agustín F. Fernández, Pablo Menéndez
BACKGROUND. Although convalescent plasma has been widely used to treat severe coronavirus disease 2019 (COVID-19), data from randomized controlled trials that support its efficacy are limited. METHODS. We conducted a randomized, double-blind, placebo-controlled trial among adults hospitalized with severe and critical COVID-19 at five sites in New York City (USA) and Rio de Janeiro (Brazil). Patients were randomized in a 2:1 ratio to receive a single transfusion of either convalescent plasma or placebo (normal control plasma). The primary outcome was clinical status at 28 days following randomization, measured using an ordinal scale and analyzed using a proportional odds model in the intention-to-treat population. RESULTS. Of 223 participants enrolled, 150 were randomized to receive convalescent plasma and 73 to normal control plasma. At 28 days, no significant improvement in clinical status was observed in participants randomized to convalescent plasma (OR 1.50, 95% confidence interval (CI) 0.83-2.68, p=0.180). However, 28-day mortality was significantly lower in participants randomized to convalescent plasma versus control plasma (19/150 [12.6%] versus 18/73 [24.6%], OR 0.44, 95% CI 0.22-0.91, p=0.034). The median titer of anti-SARS-CoV-2 neutralizing antibody in infused convalescent plasma units was 1:160 (IQR 1:80-1:320). In a subset of nasopharyngeal swab samples from Brazil that underwent genomic sequencing, no evidence of neutralization-escape mutants was detected. CONCLUSIONS. In adults hospitalized with severe COVID-19, use of convalescent plasma was not associated with significant improvement in clinical status at day 28. However, a significant improvement in mortality was observed, which warrants further evaluation. TRIAL REGISTRATION. ClinicalTrials.gov, NCT04359810 FUNDING. Amazon Foundation. Skoll Foundation.
Max R. O'Donnell, Beatriz Grinsztejn, Matthew J. Cummings, Jessica E. Justman, Matthew R. Lamb, Christina M. Eckhardt, Neena M. Philip, Ying Kuen Cheung, Vinay Gupta, Esau João, Jose H. Pilotto, Maria Pia Diniz, Sandra Wagner Cardoso, Darryl Abrams, Kartik N. Rajagopalan, Sarah E. Borden, Allison Wolf, Leon Claude Sidi, Alexandre Vizzoni, Valdilea G. Veloso, Zachary C. Bitan, Dawn E. Scotto, Benjamin J. Meyer, Samuel D. Jacobson, Alex Kantor, Nischay Mishra, Lokendra V. Chauhan, Elizabeth F. Stone, Flavia Dei Zotti, Francesca La Carpia, Krystalyn E. Hudson, Stephen A. Ferrara, Joseph Schwartz, Brie A. Stotler, Wen-Hsuan W. Lin, Sandeep N. Wontakal, Beth Shaz, Thomas Briese, Eldad A. Hod, Steven L. Spitalnik, Andrew Eisenberger, Walter I. Lipkin
Sepsis survivors exhibit impaired responsiveness to antigen (Ag) challenge associated with increased mortality from infection. The contribution of follicular dendritic cells (FDCs) in the impaired humoral response in sepsis-surviving mice is investigated in this study. We demonstrated that mice subjected to sepsis from cecal ligation and puncture (CLP) have reduced NP-specific high-affinity class-switched Ig antibodies compared to sham control mice following immunization with the T-dependent Ag, NP-CGG. NP-specific germinal center (GC) B cells in CLP mice exhibited reduced TNFα and AID mRNA expression compared to sham mice. CLP mice showed a reduction in FDC clusters, a reduced binding of immune complexes on FDCs, and reduced mRNA expression of CR2, ICAM-1, VCAM-1, FcγRIIB, TNFR1, IKK2 and LTbR compared to sham mice. Adoptive transfer studies showed there was no B cell-intrinsic defect. In summary, our data suggest that the reduced Ag-specific antibody response in CLP mice is secondary to a disruption in FDC and GC B cell function.
Minakshi Rana, Andrea La Bella, Rivka Lederman, Bruce T. Volpe, Barbara Sherry, Betty Diamond
Interstitial kidney inflammation is present in various nephritides in which serum IL-23 is elevated. Here we show that IL-23 receptor (IL-23R) expressing murine and human renal tubular epithelial cells (TEC) respond to IL-23 by inducing intracellular calcium flux, enhanced glycolysis, and the upregulation of calcium/calmodulin kinase IV (CaMK4) which results in suppression of the expression of the arginine degrading enzyme arginase 1 (ARG1) thus increasing in situ levels of free L-Arginine (Arg). Limited availability of Arg suppresses the ability of infiltrating T cells to proliferate and produce inflammatory cytokines. TEC from humans and mice with nephritis express increased levels of IL-23R and CaMK4 but reduced levels of ARG1. TEC-specific deletion of Il23r or Camk4 suppressed inflammation whereas deletion of Arg1 exacerbated inflammation in different murine disease models. Finally, TEC-specific delivery of a CaMK4 inhibitor specifically curbed the renal inflammation in lupus-prone mice without affecting systemic inflammation. Our data offer first evidence on the immunosuppressive capacity of TEC through a mechanism that involves competitive uptake of Arg and signify the importance of modulation of an inflammatory cytokine on the function of non-lymphoid cells, which leads to the establishment of an inflammatory microenvironment. New approaches to treat kidney inflammation should consider restoring the immunosuppressive capacity of TEC.
Hao Li, Maria G. Tsokos, Rhea Bhargava, Iannis E. Adamopoulos, Hanni Menn-Josephy, Isaac E. Stillman, Philip Rosenstiel, Jarrat Jordan, George C. Tsokos
Severe asthma remains challenging to manage with limited treatment options. We have previously shown that targeting smooth muscle integrin α5β1 interaction with fibronectin can mitigate the effects of airway hyperresponsiveness by impairing force transmission. In this paper we show that another member of the integrin superfamily, integrin α2β1, is present in airway smooth muscle and capable of regulating force transmission via cellular tethering to the matrix protein collagen I, and to a lesser degree, laminin-111. The addition of an inhibitor of integrin α2β1 impaired IL-13-enhanced contraction in mouse tracheal rings and human bronchial rings, and abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. We confirmed that this effect was not due to alterations in classic intracellular myosin light chain phosphorylation regulating muscle shortening. Although IL-13 did not affect surface expression of α2β1, it did increase α2β1-mediated adhesion and the level of expression of an activation-specific epitope on the β1 subunit. We developed a method to simultaneously quantify airway narrowing and muscle shortening using two-photon microscopy and demonstrated that inhibition of α2β1 mitigated IL-13-enhanced airway narrowing without altering muscle shortening by impairing the tethering of muscle to the surrounding matrix. Our data identify cell-matrix tethering as an attractive therapeutic target to mitigate the severity of airway contraction in asthma.
Sean Liu, Uyen Ngo, Xin-Zi Tang, Xin Ren, Wenli Qiu, Xiaozhu Huang, William DeGrado, Christopher D.C. Allen, Hyunil Jo, Dean Sheppard, Aparna B. Sundaram
Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington’s disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of HD patients and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex of HD patients. Of these targets, SG components were enriched, including the SG nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of HD patient brains. Intriguingly, we also observed that the SG-associated TAR DNA-Binding Protein-43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1-granule positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.
Isabella I. Sanchez, Thai B. Nguyen, Whitney E. England, Ryan G. Lim, Anthony Q. Vu, Ricardo Miramontes, Lauren M. Byrne, Sebastian Markmiller, Alice L. Lau, Iliana Orellana, Maurice A. Curtis, Richard Lewis Maxwell Faull, Gene W. Yeo, Christie D. Fowler, Jack C. Reidling, Edward J. Wild, Robert C. Spitale, Leslie M. Thompson
T cells are involved in control of COVID-19, but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we assessed the magnitude, function and phenotype of SARS-CoV-2-specific CD4 T cells in 95 hospitalized COVID-19 patients (38 of them being HIV-1 and/or tuberculosis (TB) co-infected) and 38 non-COVID-19 patients, using flow cytometry. We showed that SARS-CoV-2-specific CD4 T cell attributes, rather than magnitude, associates with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity and enhanced HLA-DR expression. Moreover, HIV-1 and TB co-infection skewed the SARS-CoV-2 T cell response. HIV-1 mediated CD4 T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2; and a decrease in the polyfunctional capacity of SARS-CoV-2-specific CD4 T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mtb-specific CD4 T cells, with possible implications for TB disease progression. There results corroborate the important role of SARS-CoV-2-specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.
Catherine Riou, Elsa Du Bruyn, Cari Stek, Remy Daroowala, Rene T. Goliath, Fatima Abrahams, Qonita Said-Hartley, Brian W. Allwood, Nei-Yuan Hsiao, Katalin A. Wilkinson, Cecilia S. Lindestam Arlehamn, Alessandro Sette, Sean Wasserman, Robert J. Wilkinson
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