SARS-CoV-2 infection in the lungs of human ACE2 transgenic mice causes severe inflammation, immune cell infiltration, and compromised respiratory function

ABSTRACTSevere Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) emerged in late 2019 and has spread worldwide resulting in the Coronavirus Disease 2019 (COVID-19) pandemic. Although animal models have been evaluated for SARS-CoV-2 infection, none have recapitulated the severe lung disease phenotypes seen in hospitalized human cases. Here, we evaluate heterozygous transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice results in high levels of viral infection in lung tissues with additional spread to other organs. Remarkably, a decline in pulmonary function, as measured by static and dynamic tests of respiratory capacity, occurs 4 days after peak viral titer and correlates with an inflammatory response marked by infiltration into the lung of monocytes, neutrophils, and activated T cells resulting in pneumonia. Cytokine profiling and RNA sequencing analysis of SARS-CoV-2-infected lung tissues show a massively upregulated innate immune response with prominent signatures of NF-kB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection recapitulates many features of severe COVID-19 infection in humans and can be used to define the mechanistic basis of lung disease and test immune and antiviral-based countermeasures.

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Abstract 1414: Pim-1 Stimulates Cardiac Progenitor Cell Proliferation And Asymmetric Division

Circulation ◽

10.1161/circ.118.suppl_18.s_321-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Christopher T Cottage ◽

Savilla Tuck ◽

Kimberlee Fischer ◽

Natalie Gude ◽

John Muraski ◽

...

Keyword(s):

Transgenic Mice ◽

Cell Fate ◽

Cellular Proliferation ◽

Gene Promoter ◽

Asymmetric Division ◽

Postnatal Growth ◽

Chain Gene ◽

Ki 67 ◽

Population Number ◽

Mechanistic Basis

Cardiac progenitor cells (CPCs) blunt cardiomyopathic damage and increase survival following adoptive transfer into hearts subjected to myocardial infarction (MI), but the initial survival, persistence, and long term engraftment of the donated cell population remains problematic. Previous studies from our group have demonstrated that transgenes driven by the α -myosin heavy chain gene promoter are expressed in the CPC population allowing for enhanced proliferation and survival. This study details a genetic engineering strategy to augment the salutary effects of CPCs through the use of a serine/threonine kinase named Pim-1 that promotes cellular proliferation and survival. Transgenic mice created with cardiac-specific Pim-1 overexpression (Pim-wt) exhibit enhanced Pim-1 activity in both cardiomyocytes and CPCs, both of which show increased proliferative activity assessed using BrdU or Ki-67 markers relative to non-transgenic (NTG) controls. However, CPC population number was not increased in the Pim-wt hearts during normal postnatal growth or after infarction challenge, suggesting that Pim-1 expression promotes asymmetric division resulting in maintenance of the CPC pool as well as expansion of the cardiomyocyte population. Localization and quantitation of cell fate determinants Numb and α -adaptin by confocal microscopy were employed to assess levels of asymmetric division in the CPC population. Polarization of Numb in mitotic phospho-histone positive cells demonstrates asymmetric division in 65% of the CPC population in hearts of Pim-wt mice versus 26% in NTG hearts after infarction challenge. Similarly, Pim-wt hearts had fewer cells with uniform α -adaptin staining indicative of symmetrically dividing CPCs, with in 36% of the CPCs versus vs. 73% in NTG sections. These findings define a mechanistic basis for enhanced myocardial regeneration in transgenic mice overexpressing Pim-1 kinase in the myocardial lineage cells.

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Disparate Interferon Signaling and Shared Aberrant Basaloid Cells in Single-Cell Profiling of Idiopathic Pulmonary Fibrosis and Systemic Sclerosis-Associated Interstitial Lung Disease

Frontiers in Immunology ◽

10.3389/fimmu.2021.595811 ◽

2021 ◽

Vol 12 ◽

Author(s):

Eleanor Valenzi ◽

Tracy Tabib ◽

Anna Papazoglou ◽

John Sembrat ◽

Humberto E. Trejo Bittar ◽

...

Keyword(s):

Systemic Sclerosis ◽

Interstitial Lung Disease ◽

Idiopathic Pulmonary Fibrosis ◽

Pulmonary Fibrosis ◽

Lung Disease ◽

Single Cell ◽

Alveolar Type ◽

Type I ◽

Interferon Signaling ◽

Mesenchymal Transition

Idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) differ in the predominant demographics and identified genetic risk alleles of effected patients, however both diseases frequently progress to respiratory failure and death. Contrasting advanced SSc-ILD to IPF provides insight to the role dysregulated immunity may play in pulmonary fibrosis. To analyze cell-type specific transcriptome commonalities and differences between IPF and SSc-ILD, we compared single-cell RNA-sequencing (scRNA-seq) of 21 explanted lung tissue specimens from patients with advanced IPF, SSc-ILD, and organ donor controls. Comparison of IPF and SSc-ILD tissue identified divergent patterns of interferon signaling, with interferon-gamma signaling upregulated in the SPP1hi and FABP4hi macrophages, cytotoxic T cells, and natural kill cells of IPF, while type I interferon signaling and production was upregulated in the corresponding SSc-ILD populations. Plasmacytoid dendritic cells were found in diseased lungs only, and exhibited upregulated cellular stress pathways in SSc-ILD compared to IPF. Alveolar type I cells were dramatically decreased in both IPF and SSc-ILD, with a distinct transcriptome signature separating these cells by disease. KRT5-/KRT17+ aberrant basaloid cells exhibiting markers of cellular senescence and epithelial-mesenchymal transition were identified in SSc-ILD for the first time. In summary, our study utilizes the enriched capabilities of scRNA-seq to identify key divergent cell types and pathways between IPF and SSc-ILD, providing new insights into the shared and distinct mechanisms between idiopathic and autoimmune interstitial lung diseases.

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219.Analysis of ovarian macrophage populations using macrophage-specific green fluorescent protein (GFP) transgenic mice

Reproduction Fertility and Development ◽

10.1071/srb04abs219 ◽

2004 ◽

Vol 16 (9) ◽

pp. 219

Author(s):

C. E. Haynes ◽

R. J. Norman ◽

R. L. Robker

Keyword(s):

Transgenic Mice ◽

Hormone Receptors ◽

Ovarian Function ◽

Fluorescent Protein ◽

Immune Cell ◽

Ovarian Tissue ◽

Gene Promoter ◽

Peritoneal Macrophages ◽

Reproductive Tissues ◽

Green Fluorescent

Macrophages represent a major immune cell type in reproductive tissues and are thought to regulate multiple aspects of reproduction, including ovarian function. We have previously shown distinctive phenotypes and functions of ovarian macrophages such that many immunological mediators, such as cytokines and hormone receptors, are uniquely regulated within these cells across the oestrus cycle. In order to isolate macrophages from ovarian tissue by fluorescence activated cell sorting (FACS), we acquired transgenic mice (from DA Hume, Institute for Molecular Bioscience, University of Queensland) which express GFP exclusively in macrophages (1). In these mice GFP is expressed under direction of the c-fms gene promoter, which encodes the receptor for colony-stimulating factor-1 (CSF-1R), a major macrophage growth factor. Using flow cytometry we confirmed that 95% of peritoneal macrophages express GFP and 88% co-express GFP and the classical macrophage marker F4/80. The distribution of GFP+ macrophages in tissues was co-localized with macrophage markers F4/80 and major histocompatibility complex class II (MHCII) by immunohistochemistry using phycoerythrin (PE)-labelled antibodies. The liver, uterus and oviduct exhibited many GFP+ cells in characteristic macrophage distributions. Furthermore, GFP fluorescence was tightly co-localized with PE fluorescence of either F4/80 or MHCII, indicating that CSF-1R is expressed in the macrophages of these tissues. In contrast, macrophages in the ovary were positive for F4/80 and MHCII, but rarely expressed GFP. Thus unlike macrophages of other reproductive tissues, ovarian macrophages do not consistently express CSF-1R. In ovaries from gonadotrophin-primed immature females, GFP was not expressed in macrophages (F4/80+/MHCII+) surrounding follicles but was detected in macrophages within the regressing corpus luteum. Thus CSF-1R is a hormonally regulated gene, expressed only in specific subsets of ovarian macrophages suggesting that CSF-1 controls functional activities of ovarian macrophages at specific stages of the ovarian cycle. (1) Sasmono, R. T., et al. (2003) Blood 101, 1155–1163.

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Psoriasis-like skin disorder in transgenic mice expressing a RIG-I Singleton–Merten syndrome variant

International Immunology ◽

10.1093/intimm/dxaa071 ◽

2020 ◽

Author(s):

Ahmed Abu Tayeh ◽

Masahide Funabiki ◽

Shota Shimizu ◽

Saya Satoh ◽

Lee Sumin ◽

...

Keyword(s):

Transgenic Mice ◽

Histological Analysis ◽

Skin Lesions ◽

Type I ◽

Jak Inhibitor ◽

Interferon Signaling ◽

Epidermal Hyperplasia ◽

Cytoplasmic Rna ◽

Proliferation And Differentiation ◽

Inducible Gene

Abstract Mutations in DDX58 (DExD/H-box helicase 58), which encodes the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I), were recently identified in the rare autoimmune disease Singleton–Merten syndrome (SMS). We report the spontaneous development of psoriasis-like skin lesions as an SMS-like symptom in transgenic mice harboring one of the RIG-I SMS variants, E373A. Histological analysis revealed typical characteristics of psoriasis, including the abnormal proliferation and differentiation of keratinocytes leading to epidermal hyperplasia, and infiltrates consisting of neutrophils, dendritic cells and T cells. Levels of the IL-23/IL-17 immune axis cytokines were high in the skin lesions. Rag2−/− transgenic mice showed partial amelioration of the phenotype, with down-regulation of inflammatory cytokines, including IL-17A, suggesting the importance of lymphocytes for the pathogenesis similar to that of human psoriasis. Of note, IL-17A deficiency abolished the skin phenotype, and treatment using the JAK inhibitor tofacitinib not only prevented onset, but also improved the skin manifestations even after onset. Our study provides further evidence for the involvement of RIG-I activation in the onset and progression of psoriasis via type I interferon signaling and the IL-23/IL-17 axis.

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Cancer stemness, intratumoral heterogeneity, and immune response across cancers

10.1101/352559 ◽

2018 ◽

Author(s):

Alex Miranda ◽

Phineas T Hamilton ◽

Allen W Zhang ◽

Etienne Becht ◽

Artur Mezheyeuski ◽

...

Keyword(s):

Cancer Progression ◽

Immune Cell ◽

Antigen Expression ◽

Therapy Resistance ◽

Intratumoral Heterogeneity ◽

Type I ◽

Interferon Signaling ◽

Cancer Stemness ◽

Tumor Phenotype ◽

The Impact

SummaryRegulatory programs that control the function of stem cells are active in cancer and confer properties that promote progression and therapy resistance. However, the impact of a stem cell-like tumor phenotype (“sternness”) on the immunological properties of cancer has not been systematically explored. Using gene expression-based metrics, we evaluate the association of stemness with immune cell infiltration and genomic, transcriptomic, and clinical parameters across 21 solid cancers. We find pervasive negative associations between cancer stemness and anticancer immunity. This occurs despite high stemness cancers exhibiting increased mutation load, cancer-testis antigen expression, and intratumoral heterogeneity. Stemness was also strongly associated with cell-intrinsic suppression of endogenous retroviral expression and type I interferon signaling and increased expression of several therapeutically accessible signaling pathways. Thus, stemness is not only a fundamental process in cancer progression but may represent a unifying mechanism linking antigenicity, intratumoral heterogeneity, and immune suppression across cancers.

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Activated FoxM1 Attenuates Streptozotocin-Mediated β-Cell Death

Molecular Endocrinology ◽

10.1210/me.2014-1024 ◽

2014 ◽

Vol 28 (9) ◽

pp. 1435-1447 ◽

Cited By ~ 12

Author(s):

Maria L. Golson ◽

Matthew F. Maulis ◽

Jennifer C. Dunn ◽

Greg Poffenberger ◽

Jonathan Schug ◽

...

Keyword(s):

Cell Death ◽

Transgenic Mice ◽

Cell Injury ◽

Immune Cell ◽

Cell Mass ◽

Sequencing Analysis ◽

Β Cell ◽

Partial Pancreatectomy ◽

Cell Ablation ◽

Mass Expansion

The forkhead box transcription factor FoxM1, a positive regulator of the cell cycle, is required for β-cell mass expansion postnatally, during pregnancy, and after partial pancreatectomy. Up-regulation of full-length FoxM1, however, is unable to stimulate increases in β-cell mass in unstressed mice or after partial pancreatectomy, probably due to the lack of posttranslational activation. We hypothesized that expression of an activated form of FoxM1 could aid in recovery after β-cell injury. We therefore derived transgenic mice that inducibly express an activated version of FoxM1 in β-cells (RIP-rtTA;TetO-hemagglutinin (HA)-Foxm1ΔNRD mice). This N-terminally truncated form of FoxM1 bypasses 2 posttranslational controls: exposure of the forkhead DNA binding domain and targeted proteasomal degradation. Transgenic mice were subjected to streptozotocin (STZ)-induced β-cell ablation to test whether activated FoxM1 can promote β-cell regeneration. Mice expressing HA-FoxM1ΔNRD displayed decreased ad libitum–fed blood glucose and increased β-cell mass. β-Cell proliferation was actually decreased in RIP-rtTA:TetO-HA-Foxm1NRD mice compared with that in RIP-rtTA mice 7 days after STZ treatment. Unexpectedly, β-cell death was decreased 2 days after STZ treatment. RNA sequencing analysis indicated that activated FoxM1 alters the expression of extracellular matrix and immune cell gene profiles, which may protect against STZ-mediated death. These studies highlight a previously underappreciated role for FoxM1 in promoting β-cell survival.

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Molecular Characterization of a Type I Quantitative Factor V Deficiency in a Thrombosis Patient that Is “Pseudo Homozygous” for Activated Protein C Resistance

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655948 ◽

1997 ◽

Vol 77 (02) ◽

pp. 252-257 ◽

Cited By ~ 32

Author(s):

Joan F Guasch ◽

Ruud P M Lensen ◽

Rogier M Bertina

Keyword(s):

Protein C ◽

Dna Analysis ◽

Activated Protein C ◽

Type I ◽

Factor V ◽

Sequencing Analysis ◽

Apc Resistance ◽

Quantitative Factor ◽

Factor V Deficiency ◽

Fv Leiden

SummaryResistance to activated protein C (APC), which is associated with the FV Leiden mutation in the large majority of the cases, is the most common genetic risk factor for thrombosis. Several laboratory tests have been developed to detect the APC-resistance phenotype. The result of the APC-resistance test (APC-sensitivity ratio, APC-SR) usually correlates well with the FV Leiden genotype, but recently some discrepancies have been reported. Some thrombosis patients that are heterozygous for FV Leiden show an APC-SR usually found only in homozygotes for the defect. Some of those patients proved to be compound heterozygotes for the FV Leiden mutation and for a type I quantitative factor V deficiency. We have investigated a thrombosis patient characterized by an APC-SR that would predict homozygosity for FV Leiden. DNA analysis showed that he was heterozygous for the mutation. Sequencing analysis of genomic DNA revealed that the patient also is heterozygous for a G5509→A substitution in exon 16 of the factor V gene. This mutation interferes with the correct splicing of intron 16 and leads to the presence of a null allele, which corresponds to the “non-FV Leiden” allele. The conjunction of these two defects in the patient apparently leads to the same phenotype as observed in homozygotes for the FV Leiden mutation.

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