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Sci Immunol ; 7(75):eadd4906, 2022.
Article in English | PubMed | ID: covidwho-2019704


Lung-infiltrating macrophages create a marked inflammatory milieu in a subset of patients with COVID-19 by producing a cytokine storm, which correlates with increased lethality. However, these macrophages are largely not infected by SARS-CoV-2, so the mechanism underlying their activation in the lung is unclear. Type I interferons (IFN-I) contribute to protecting the host against SARS-CoV-2 but may also have some deleterious effect, and the source of IFN-I in the lungs of infected patients is not well defined. Plasmacytoid dendritic cells (pDCs), a key cell type involved in antiviral responses, can produce IFN-I in response to SARS-CoV-2. We observed the infiltration of pDCs in the lungs of SARS-CoV-2-infected patients, which correlated with strong IFN-I signaling in lung macrophages. In patients with severe COVID-19, lung macrophages expressed a robust inflammatory signature, which correlated with persistent IFN-I signaling at the single-cell level. Hence, we observed the uncoupling in the kinetics of the infiltration of pDCs in the lungs and the associated IFN-I signature, with the cytokine storm in macrophages. We observed that pDCs were the dominant IFN-α-producing cells in response to the virus in the blood, whereas macrophages produced IFN-α only when in physical contact with infected epithelial cells. We also showed that IFN-α produced by pDCs, after the sensing of SARS-CoV-2 by TLR7, mediated changes in macrophages at both transcriptional and epigenetic levels, which favored their hyperactivation by environmental stimuli. Together, these data indicate that the priming of macrophages can result from the response by pDCs to SARS-CoV-2, leading to macrophage activation in patients with severe COVID-19.

Blood ; 138:1, 2021.
Article in English | EMBASE | ID: covidwho-1582278


Severe SARS-CoV-2 infection is complicated by dysregulation of the blood coagulation system and high rates of thrombosis, but virus-intrinsic mechanisms underlying this phenomenon are poorly understood. Increased intracellular calcium concentrations promote externalization of phosphatidylserine (PS), the membrane anionic phospholipid required for assembly and activation of the tenase and prothrombinase complexes to drive blood coagulation. TMEM16F is a ubiquitous phospholipid scramblase that mediates externalization of PS in a calcium-dependent manner. As SARS-CoV-2 ORF3a encodes a presumed cation channel with the ability to transport calcium, we hypothesized that ORF3a expression by infected host cells perturbs the cellular calcium rheostat, driving TMEM16F-dependent externalization of PS and enhancing procoagulant activity. Using a doxycycline-inducible system, synchronized expression of ORF3a in A549 pulmonary epithelial cells resulted in a time-dependent augmentation of tissue factor (TF) procoagulant activity exceeding 9-fold by 48 hours (p < 0.0001), with no change in TF cell-surface expression. This enhancement was dependent upon PS as determined by inhibition with the PS-binding protein lactadherin. Over 2-fold enhancement of prothrombinase activity (p < 0.0001) was also observed by 48 hours. ORF3a increased intracellular calcium levels by 18-fold at 48 hours (p < 0.0001), as determined by the intracellular calcium indicator fluo-4. After 16 hours of ORF3a expression, more than 60% of cells had externalized PS (p < 0.001) without increased cell death, as quantified by flow cytometry following annexin V binding. Immunofluorescence microscopy staining for ORF3a, annexin V, and nuclei confirmed ORF3a expression within internal and cell surface membranes and increased PS externalization. PS externalization was insensitive to the pan-caspase inhibitor z-VAD-FMK, and there was no evidence of apoptotic activation as determined by caspase-3 cleavage. By contrast, ORF3a expression did not augment coagulation in cells deficient in the calcium-dependent phospholipid scramblase TMEM16F. Similarly, ORF3a-enhanced TF procoagulant activity (p < 0.01) and prothrombinase activity (p<0.05) was completely abrogated using TMEM16 inhibitors, including the uricosuric agent benzbromarone that has been registered for human use in over 20 countries. Live SARS-CoV-2 infection of A549-ACE2 cells increased cell surface factor Xa generation at MOI 0.1 (p < 0.01) but not MOI 0.01 or following heat inactivation of the virus, and RNA sequencing confirmed ORF3a induction without increased F3 expression. RNA sequencing of human SARS-CoV-2 infected lung autopsy and control tissue (n= 53) confirmed these findings in vivo. Immunofluorescence staining for ORF3a and KRT8/18 and CD31 in SARS-CoV-2 infected human lung autopsy specimens demonstrated ORF3a expression in pulmonary epithelium and endothelial cells, highlighting the potential pathologic relevance of this mechanism. Here we demonstrate that expression of the SARS-CoV-2 accessory protein ORF3a increases the intracellular calcium concentration and TMEM16F-dependent PS scrambling to augment procoagulant activity of the tenase and prothrombinase complexes. Our studies of human cells and tissues infected with SARS-CoV-2 support the pathologic relevance of this mechanism. We highlight the therapeutic potential to target the ORF3a-TMEM16F axis as with benzbromarone to mitigate dysregulation of coagulation and thrombosis during severe SARS-CoV-2 infection. Disclosures: Schwartz: Miromatrix Inc: Membership on an entity's Board of Directors or advisory committees;Alnylam Inc.: Consultancy, Speakers Bureau. Schulman: CSL Behring: Consultancy, Research Funding.

PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-296897


The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has infected over 115 million people and caused over 2.5 million deaths worldwide. Yet, the molecular mechanisms underlying the clinical manifestations of COVID-19, as well as what distinguishes them from common seasonal influenza virus and other lung injury states such as Acute Respiratory Distress Syndrome (ARDS), remains poorly understood. To address these challenges, we combined transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues, matched with spatial protein and expression profiling (GeoMx) across 357 tissue sections. These results define both body-wide and tissue-specific (heart, liver, lung, kidney, and lymph nodes) damage wrought by the SARS-CoV-2 infection, evident as a function of varying viral load (high vs. low) during the course of infection and specific, transcriptional dysregulation in splicing isoforms, T cell receptor expression, and cellular expression states. In particular, cardiac and lung tissues revealed the largest degree of splicing isoform switching and cell expression state loss. Overall, these findings reveal a systemic disruption of cellular and transcriptional pathways from COVID-19 across all tissues, which can inform subsequent studies to combat the mortality of COVID-19, as well to better understand the molecular dynamics of lethal SARS-CoV-2 infection and other viruses.

PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-8946


COVID-19 patients commonly present with neurological signs of central nervous system (CNS) and/or peripheral nervous system dysfunction. However, which neural cells are permissive to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been controversial. Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively permissive to SARS-CoV-2 infection both in vitro and upon transplantation in vivo, and that SARS-CoV-2 infection triggers a DA neuron inflammatory and cellular senescence response. A high-throughput screen in hPSC-derived DA neurons identified several FDA approved drugs, including riluzole, metformin, and imatinib, that can rescue the cellular senescence phenotype and prevent SARS-CoV-2 infection. RNA-seq analysis of human ventral midbrain tissue from COVID-19 patients, using formalin-fixed paraffin-embedded autopsy samples, confirmed the induction of an inflammatory and cellular senescence signature and identified low levels of SARS-CoV-2 transcripts. Our findings demonstrate that hPSC-derived DA neurons can serve as a disease model to study neuronal susceptibility to SARS-CoV-2 and to identify candidate neuroprotective drugs for COVID-19 patients. The susceptibility of hPSC-derived DA neurons to SARS-CoV-2 and the observed inflammatory and senescence transcriptional responses suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.

Hepatology ; 72(1 SUPPL):278A, 2020.
Article in English | EMBASE | ID: covidwho-986075


Background: COVID-19 was declared a pandemic by the World Health Organization, caused by severe acute respiratory syndrome corona-virus 2 (SARS-CoV-2) Respiratory failure is the most common mortality outcome in COVID-19 patients, yet serious and even fatal manifestations are seen across multiple organ systems Emerging clinical data from our own hospital revealed a high prevalence of initial presentations with GI manifestations Almost one third of patients presenting to our hospital reported at least one gastrointestinal manifestation including nausea, vomiting, diarrhea, or abdominal pain 62% of patients presented with biochemical evidence of liver injury Moreover, the presence of liver injury on presentation was associated with a significantly higher risk of ICU admission and death As this is a new and novel clinical entity, robust in vitro models that phenocopy SARS-CoV-2 infection and human COVID-19 disease are limited Current in vitro (e g Vero cells) and in vivo models (mouse models engineered with ACE2) are so distinct from human infection that they may not capture key components of viral infection or virus-host interactions Therefore, the development of robust human models of COVID-19 infection will be essential for the study of SARS-Cov-2 viral infection and to identify robust SARS-CoV-2 therapeutics Methods: Human pluripotent stem cells (hPSCs), including human embryonic stem cells hESCs) and induced pluripotent stem cells (hiPSCs), can be used to derive functional human cells/tissues/organoids for modeling human disease and drug discovery, including for infectious diseases Here we leveraged several stem cell platforms (e g endodermal lineages including hepatocyte and cholangiocyte) along with primary human hepatocytes and cholangiocyte organoid systems to study SARS-CoV-2 infection SARS-CoV-2 pseudoparticles were used to study SARS-CoV-2 viral entry SARS-CoV-2 (USA-WA1/2020) was used to validate viral infection and to study cellular response Autopsy liver samples from COVID-19 patients were obtained and compared to SARS-CoV-2 infected liver models Results: Adult hepatocyte and cholangiocyte organoids along with PSC derived hepatocytes and cholangiocytes are Permissive to SARS-CoV-2 virus infection and show similar transcriptome changes and chemokine responses for SARSCoV- 2 infection as seen in autopsy samples from COVID-19 Patients Conclusion: We report here the development of robust models of SARS-CoV-2 infection in primary and PSC derived hepatocyte and cholangiocytes which phenocopy COVID-19 hepatic disease These disease-relevant human cell/organoid-based platforms can be directly applied for drug screening and the evaluation of prospective antiviral therapeutics as well be used to delineate molecular mechanisms underlying COVID-19 disease.

PubMed; 2020.
Preprint in English | PubMed | ID: ppcovidwho-2670


Heart injury has been reported in up to 20% of COVID-19 patients, yet the cause of myocardial histopathology remains unknown. In order to study the cause of myocardial pathology in COVID-19 patients, we used a hamster model to determine whether following infection SARS-CoV-2, the causative agent of COVID-19, can be detected in heart tissues. Here, we clearly demonstrate that viral RNA and nucleocapsid protein is present in cardiomyocytes in the hearts of infected hamsters. Interestingly, functional cardiomyocyte associated gene expression was decreased in infected hamster hearts, corresponding to an increase in reactive oxygen species (ROS). This data using an animal model was further validated using autopsy heart samples of COVID-19 patients. Moreover, we show that both human pluripotent stem cell-derived cardiomyocytes (hPSC-derived CMs) and adult cardiomyocytes (CMs) can be infected by SARS-CoV-2 and that CCL2 is secreted upon SARS-CoV-2 infection, leading to monocyte recruitment. Increased CCL2 expression and macrophage infiltration was also observed in the hearts of infected hamsters. Using single cell RNA-seq, we also show that macrophages are able to decrease SARS-CoV-2 infection of CMs. Overall, our study provides direct evidence that SARS-CoV-2 infects CMs in vivo and proposes a mechanism of immune-cell infiltration and pathology in heart tissue of COVID-19 patients.