PROJECT SUMMARY SARS-CoV2 pandemic has already taken a high toll with ~700,000 (~155,00 US) deaths and 18 million (~5 million US) infections globally with unabated spread in many countries and new hotspots reemerging on a regular basis. While frantic efforts are underway to develop vaccines and therapies and there is high hope that these will be available over the next 6-12 months, there are no answers to how much and how long these will be effective. A continued effort on understanding the disease etiology, particularly in the organ first infected, and identifying new avenues of intervention therefore is important. The major route of virus infection is via the respiratory tract and virus is reported to spread via lung to other organs by vascular leakage by directly (through infection) or indirectly (by impairing ACE2 activity) affecting the endothelial and immune cells. Cells expressing ACE2 enzyme and other viral coreceptors (TMPRSS2 or Cathepsin L) are the major targets of viral infection. ACE2 is a key player in regulation of the Renin-Angiotensin system (RAS) pathway. By converting the product of ACE activity, angiotensin II (ang II), to angiotensin 1-7 (ang 1-7), ACE2 diminishes Ang II mediated deleterious effects that can include promoting vascular wall inflammation, endothelial dysfunction, endothelial cell and vascular smooth muscle cell migration, growth, proliferation, and thrombosis. Disruption of ACE2 by viral binding may reduce this protective effect. The inhibition of nitric oxide production, activation of megakaryocytes, complement and platelets can also cause thrombosis and thrombolytic dysfunction leading to clot formation in lung arteries and other organs. Since the early unprecedented global effort to identify the cellular targets of SARS-CoV2 using single cell RNA sequencing (scRNAseq) data from multiple human and non-human single cell datasets, several in depth reports on individual organs infected and cells and cellular pathways affected by this virus have appeared. Most of these reports are based on transcriptomic analysis of homogenized or disaggregated samples although some singleplex immunofluorescence analysis have been reported. COVID-19 tissue histology shows a very heterogenous disease which may be a function of multiple factors including cellular composition, spatial organization and neighboring cell activation. To understand these factors, here we propose an in situ multiplex immunofluorescence study of SARS- CoV2 positive and negative patient samples to spatially profile the cell types affected in the upper and lower respiratory tract and the role of RAS pathway activation in endothelial and immune cell dysfunction related to COVID-19 morbidity and mortality.

PROJECT NARRATIVE We propose to measure cellular responses in COVID-19 diseased upper airways and lungs and the role of the renin-angiotensin system pathway, a pathway that regulates arterial blood pressure, in causing patient morbidity and death. This will lead to new understanding of how cells respond to SARS-CoV-2 infection and hopefully identify new avenues for exploration to treat COVID-19 disease. This is a collaborative project between two major consortia, Human Biomolecular Atlas Program (HuBMAP) and Lung Molecular Atlas Program (LungMAP), both funded by the National Institutes of Health.