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Principal Investigator: ANTHONY W ORVEDAHL
Organization: WASHINGTON UNIVERSITY
Fiscal Year: 2021
Award: $1,410,500
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY/ABSTRACT
The host inflammatory response is a double-edged sword that must vigorously defend against pathogens,
but also requires restraint to prevent unintended injury to the host. The Cytokine Storm Syndrome (CSS)
represents a state of unbridled inflammation that can be triggered by infections, including Severe Acute
Respiratory Syndrome-associated Coronavirus 2 (SARS-CoV-2). While evidence for dysregulated cytokine
responses exists for the SARS-CoV-2-associated CSS (S-CSS), the precise cell types and viral factors that
precipitate this response remain incompletely understood. Autophagy is a cytosol-to-lysosome degradative
pathway that has important functions in host immunity. We have recently shown that autophagy genes in myeloid
cells, preliminarily alveolar macrophages (aMΦ), confer protection in a murine model of CSS induced by
intravenous TNF. We hypothesize that host autophagy may also have a pleiotropic role in limiting the S-CSS.
We are motivated in this hypothesis since coronaviruses (CoVs) manipulate host autophagy-associated
membranes for their own replication via the nonstructural protein 6 (nsp6). This proposal for the NIH Director's
New Innovator Award will test the role of host autophagy and a viral antagonist in the triggering of S-CSS using
both established and innovative methods. The project will utilize a model for SARS-CoV-2 infection in which the
human ACE2 receptor (encoded by hAce2) is delivered to mouse lungs via adenovirus (AdV) vector. Additionally,
we will determine the role of aMΦ-specific host pathways by utilizing mice deficient for GM-CSF signaling and
devoid of aMΦ (Csf2rb-/-), that are durably restored with aMΦ by a single intranasal instillation of progenitor cells
in neonates. The role of SARS-CoV-2 nsp6 in viral pathogenesis will be determined with recombinant viruses
deleted for this factor or with naturally occurring point mutations hypothesized to facilitate infection. Moreover,
we will develop an AdV-hAce2 vector system expressing sgRNAs to edit genes directly in susceptible respiratory
cells in Cas9-transgenic recipient mice. We will generate pooled AdV sgRNA libraries via this method for in vivo
screening approaches that may identify host pathways important for regulating infection not otherwise
recapitulated by in vitro approaches. Further, we will reconstitute Csf2rb-/- mice with aMΦ cell progenitors
containing pooled CRISPR libraries to identify host genes important for not only the aMΦ response to SARS-
CoV-2 but also for fundamental aspects of aMΦ niche development. These studies have the potential to identify
new areas for the development of host- and viral-directed therapies (e.g., the autophagy pathway and nsp6,
respectively). The robust and versatile in vivo platforms established for functional genomic studies of a tissue
site critical for the proximal response to SARS-CoV-2 have broader implications for the study of complex cell
populations in diverse biological processes.
Terms: <(TNF)-α><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><ACE2><Ad vector><Adenoviral Vector><Adenoviridae><Adenovirus Vector><Adenoviruses><Alveolar><Alveolar Cell><Alveolar Macrophages><Alveolus><Area><Autophagocytosis><Award><Biological Function><Biological Process><Body Tissues><Bronchial Alveolus><COVID-19 associated cytokine storm><COVID-19 cytokine storm><COVID-19 induced cytokine storm><COVID-19 related cytokine storm><COVID-19 virus><COVID19 virus><CRISPR library><CRISPR-based library><CRISPR/Cas9 library><Cachectin><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Clustered Regularly Interspaced Short Palindromic Repeats library><CoV-2><CoV2><Complex><Coronaviridae><Coronavirus><Cytosol><Degradation Pathway><Degradative Pathway><Development><Disease><Disorder><Dissection><Epithelial Cells><Event><GM-CSF><Genes><Granulocyte-Macrophage Colony-Stimulating Factor><Histamine-Producing Cell-Stimulating Factor><Human><Immune response><Immunity><Immunological response><In Vitro><Incubators><Infection><Inflammation><Inflammatory Response><Injury><Intracellular Communication and Signaling><Intravenous><Libraries><Lung><Lung Respiratory System><Lysosomes><Macrophage-Derived TNF><Membrane><Methods><Mice><Mice Mammals><Modeling><Modern Man><Molgramostin><Monocyte-Derived TNF><Murine><Mus><Myeloid Cells><NIH><National Institutes of Health><Non-structural Protein><Nonstructural Protein><Pathway interactions><Phenotype><Point Mutation><Population><Precipitating Factors><Progenitor Cells><Pulmonary Macrophages><Receptor Protein><Respiratory Epithelium><Role><SARS corona virus 2><SARS-CoV-2><SARS-CoV-2 associated cytokine storm><SARS-CoV-2 cytokine storm><SARS-CoV-2 induced cytokine storm><SARS-CoV-2 related cytokine storm><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Signal Transduction><Signal Transduction Systems><Signaling><Site><Structure of respiratory epithelium><Syndrome><System><TC-GM-CSF><TNF><TNF A><TNF Alpha><TNF gene><TNF-α><TNFA><TNFα><Testing><Tissues><Transgenic Organisms><Tumor Necrosis Factor><Tumor Necrosis Factor-alpha><Tumor-Cell Human GM Colony-Stimulating Factor><United States National Institutes of Health><Viral><Viral Pathogenesis><Wuhan coronavirus><adeno vector><adenovector><angiotensin converting enzyme 2><angiotensin converting enzyme II><autophagy><biological signal transduction><cell type><corona virus><coronavirus disease 2019 associated cytokine storm><coronavirus disease 2019 cytokine storm><coronavirus disease 2019 induced cytokine storm><coronavirus disease 2019 related cytokine storm><coronavirus disease 2019 virus><cytokine><cytokine release syndrome><cytokine storm><developmental><functional genomics><granulocyte macrophage colony stimulating factor><hCoV19><host response><immune system response><immunoresponse><in vivo><injuries><innovate><innovation><innovative><membrane structure><mouse model><murine model><nCoV2><neonate><pathogen><pathway><prevent><preventing><pulmonary><receptor><recombinant virus><reconstitute><reconstitution><respiratory><response><restraint><screening><severe acute respiratory syndrome coronavirus 2 associated cytokine storm><severe acute respiratory syndrome coronavirus 2 cytokine storm><severe acute respiratory syndrome coronavirus 2 induced cytokine storm><severe acute respiratory syndrome coronavirus 2 related cytokine storm><social role><stem cells><tool><transgenic><vector><virus pathogenesis>