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Principal Investigator: Dan Jane-Wit
Organization: YALE UNIVERSITY
Fiscal Year: 2023
Award: $251,250
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY
During severe COVID-19, immune hyperactivation contributes to tissue injury and worsened patient
outcomes. Complement (C’) proteins are believed to centrally mediate COVID-19-related immune
hyperactivation. C’ are a conserved set of immune proteins involved in host defense that become activated on
various cell types in the lung during severe COVID-19. Upon terminal activation, C’ proteins assemble to form
pore-like membrane attack complexes (MACs) that insert into target cell surfaces as transmembrane
structures. The presence of MACs in pulmonary tissues strongly correlates with immune hyperactivation, but
underlying mechanisms are unknown.
The immune effects of MACs have been widely attributed to their cytolytic properties. However,
widespread MAC deposition occurs on alveolar endothelial cells (ECs) during severe COVID-19 in the absence
of significant EC death or vessel rarefaction. Instead, MAC-bound ECs show signs of EC dysfunction with
dysregulated NF-B activation and elaboration of pro-inflammatory cytokines. These patient-level observations
suggest immune effects of MACs that are separable from their cytolytic properties.
In this application we examine a novel role for intracellular MAC proteins as alarmins. We used a
SARS-CoV-2-derived antigen, S protein, to induce non-cytolytic MAC assembly on human ECs. Following
assembly on ECs, surface-bound MACs became rapidly internalized and transferred to Rab5+ endosomes.
The pool of intracellular, but not extracellular or surface-bound MACs, activated NF-B to induce elaboration of
inflammatory cytokines. The intraluminal milieu of Rab5+ endosomes caused a MAC protein, C9, to form
insoluble aggregates that stimulated aggrephagy, a specialized form of selective macroautophagy, to activate
NF-B. Via proteomic profiling of solubilized MAC complexes (Sc5b-9) in COVID-19 sera, we identified
guanylate binding protein 4 (GBP4) as a C9-binding protein regulating aggrephagy and NF-B activity.
Based on these exciting preliminary data we propose 2 Specific Aims to explore the hypothesis that
intracellular MAC proteins act as alarmins to initiate inflammatory signaling. We will consolidate role(s) for
GBP4 as an immune sensor by examining binding interactions with C9 using COVID-19 patient sera.
Secondly, we will identify mouse orthologs for human GBP4 that mediate C9 sensing and MAC-induced
immune hyperactivation in vivo in response to S protein-induced MAC and following infection by replication
competent SARS-CoV-2. Our studies introduce a new paradigm for understanding MAC-related immunity and
contribute to our long-term aim of understanding C’-induced inflammation. By doing this, druggable targets
ameliorating MAC-induced immune complications of severe COVID-19 may emerge.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><Alveolar><Antigens><Assay><Autoregulation><Binding><Binding Proteins><Bioassay><Biologic Assays><Biological Assay><Blood capillaries><Body Tissues><C 5b-9><C5b-9><COVID infected patient><COVID patient><COVID positive patient><COVID-19><COVID-19 S protein><COVID-19 antigen><COVID-19 infected patient><COVID-19 infection><COVID-19 patient><COVID-19 positive patient><COVID-19 spike glycoprotein><COVID-19 spike protein><COVID-19 virus><COVID-19 virus infection><COVID19><COVID19 S protein><COVID19 infection><COVID19 patient><COVID19 positive patient><COVID19 spike glycoprotein><COVID19 spike protein><COVID19 virus><CV-19><CV19><Cell Body><Cell Communication and Signaling><Cell Death><Cell Signaling><Cell surface><Cells><Clinical><CoV-2><CoV2><Complement><Complement Activation><Complement Complex C5b-9><Complement Membrane Attack Complex><Complement Proteins><Complex><Complication><Cytolysis><Cytolytic Terminal Complement Complex><Data><Deposit><Deposition><Diabetes Mellitus><Dropsy><Dysfunction><Edema><Endosomes><Endothelial Cells><Family><Functional disorder><GBP1><GBP1 gene><Guanylate-Binding Protein 1, Interferon Inducible, 67-KD><Homeostasis><Host Defense><Human><Hydrops><Hypertension><Immune><Immunes><Immunity><In Vitro><Infection><Inflammation><Inflammation Mediators><Inflammatory><Innate Immunity><Intracellular Communication and Signaling><Intracellular Membranes><Isoforms><Life><Ligand Binding Protein><Ligand Binding Protein Gene><Lung><Lung Respiratory System><Lung damage><Lysis><Mediating><Membrane><Membrane Attack Complex><Mice><Mice Mammals><Modern Man><Molecular><Molecular Interaction><Murine><Mus><Native Immunity><Natural Immunity><Non-Specific Immunity><Nonspecific Immunity><Ortholog><Orthologous Gene><Outcome><Patient outcome><Patient-Centered Outcomes><Patient-Focused Outcomes><Patients><Phenotype><Physiological Homeostasis><Physiopathology><Process><Property><Protein Binding><Protein Isoforms><Proteins><Proteomics><Receptosomes><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 S protein><SARS-CoV-2 antigen><SARS-CoV-2 infected patient><SARS-CoV-2 infection><SARS-CoV-2 patient><SARS-CoV-2 positive patient><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV2><SARS-CoV2 S protein><SARS-CoV2 antigen><SARS-CoV2 infection><SARS-CoV2 spike glycoprotein><SARS-CoV2 spike protein><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SC5b-9><SC5b-9 complement complex><Sequence Homology><Severe Acute Respiratory Coronavirus 2><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 coronavirus 2><Severe acute respiratory syndrome coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Signal Transduction><Signal Transduction Systems><Signaling><Surface><Symptoms><Terminal Complement Complex><Testing><Tissues><Vascular Hypertensive Disease><Vascular Hypertensive Disorder><Wuhan coronavirus><biological signal transduction><bound protein><capillary><cell type><complement pathway regulation><corona virus disease 2019><coronavirus disease 2019><coronavirus disease 2019 S protein><coronavirus disease 2019 antigen><coronavirus disease 2019 infected patient><coronavirus disease 2019 infection><coronavirus disease 2019 patient><coronavirus disease 2019 positive patient><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike protein><coronavirus disease 2019 virus><coronavirus disease infected patient><coronavirus disease patient><coronavirus disease positive patient><coronavirus disease-19><coronavirus disease-19 patient><coronavirus disease-19 virus><coronavirus infectious disease-19><coronavirus patient><cytokine><cytokine release syndrome><cytokine storm><diabetes><druggable target><guanylate><hCoV19><high blood pressure><hyperpiesia><hyperpiesis><hypertensive disease><hypertensive disorder><immunogen><improved><in vivo><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><inflammatory mediator><injury to tissue><insoluble aggregate><life-threatening COVID><life-threatening COVID-19><life-threatening SARS-CoV-2><life-threatening coronavirus disease><life-threatening coronavirus disease 2019><life-threatening severe acute respiratory syndrome coronavirus 2><lung injury><membrane assembly><membrane structure><nCoV2><necrocytosis><neo-antigen><neo-epitopes><neoantigens><neoepitopes><novel><pathophysiology><patient infected with COVID><patient infected with COVID-19><patient infected with SARS-CoV-2><patient infected with coronavirus disease><patient infected with coronavirus disease 2019><patient infected with severe acute respiratory syndrome coronavirus 2><patient oriented outcomes><patient with COVID><patient with COVID-19><patient with COVID19><patient with SARS-CoV-2><patient with coronavirus disease><patient with coronavirus disease 2019><patient with severe acute respiratory distress syndrome coronavirus 2><protein aggregate><protein aggregation><protein complex><pulmonary><pulmonary damage><pulmonary injury><pulmonary tissue damage><pulmonary tissue injury><response><sensor><serious COVID><serious COVID-19><serious SARS-CoV-2><serious coronavirus disease><serious coronavirus disease 2019><serious severe acute respiratory syndrome coronavirus 2><severe COVID><severe COVID-19><severe COVID19><severe SARS-CoV-2><severe acute respiratory syndrome coronavirus 2 antigen><severe acute respiratory syndrome coronavirus 2 infected patient><severe acute respiratory syndrome coronavirus 2 patient><severe acute respiratory syndrome coronavirus 2 positive patient><severe coronavirus disease><severe coronavirus disease 19><severe coronavirus disease 2019><severe severe acute respiratory syndrome coronavirus 2><social role><soluble C5b-9 complex><soluble complement C5b-9><stem><tissue injury><vascular inflammation><virtual>