Bronchoalveolar Lavage Single-Cell Transcriptomics Identifies Immune Cells Driving COVID-19 Severity in Patients

The continuous threats posed by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, including the emergence of potentially more infectious and deadly variants, necessitate ongoing studies to uncover novel and detailed mechanisms driving disease severity. Using single-cell transcriptomics, we conducted a secondary data analysis of bronchoalveolar lavage fluid (BALF) from COVID-19 patients of varying severities and healthy controls to comprehensively examine immune responses. We observed significant immune cell alterations correlating with disease severity. In severe cases, macrophages showed upregulation of pro-inflammatory genes TNF and IL1{beta}, contributing to severe inflammation and tissue damage. Neutrophils exhibited increased activation, marked by S100A8, CXCL8, and IL1{beta} expression, with extended viability and reduced phagocytosis. Genes such as MCL1 and HIF1 supported extended viability, while MSR1 and MRC1 indicated reduced phagocytosis. Enhanced formation of neutrophil extracellular traps (NETs) and reduced clearance, indicated by NET-associated markers, were linked to thrombo-inflammation and organ damage. Both macrophages and neutrophils in severe cases showed impaired efferocytosis, indicated by decreased expression of MSR1 and TREM2 in macrophages and downregulation of FCGR3B in neutrophils, leading to the accumulation of apoptotic cells and exacerbating inflammation. Severe cases were characterized by M1 macrophages with high TNF and IL1{beta}, while milder cases had M2 macrophages with elevated PPAR{gamma}. Low-density neutrophils (LDNs) increased significantly in severe cases, showing higher CXCR4 and CD274 and lower FCGR3B compared to high-density neutrophils (HDNs). NK and T cells in severe cases demonstrated altered receptor and gene expression, with increased activation markers IFN{gamma} and ISG15, suggesting a paradoxical state of activation and exhaustion. This imbalance suggests a potential mechanism for immune dysregulation and ineffective antiviral responses in severe COVID-19. This analysis highlights the critical role of dysregulated neutrophil, macrophage, NK, and T cell responses in severe COVID-19, identifying potential therapeutic targets and providing novel insights into the disease. Author SummarySevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, poses continuous health threats due to emerging, potentially more infectious and deadly variants. We used single-cell gene analysis from lung fluid samples, known as bronchoalveolar lavage (BAL), from COVID-19 patients to understand why some cases become more severe than others. In severe cases, immune cells called macrophages and neutrophils showed higher levels of genes that trigger inflammation and cause damage to the body. These cells were more active and lived longer but were less capable of clearing away dead cells and debris, leading to prolonged inflammation. Severe cases also had more neutrophils that were less effective in fighting infections. Another type of immune cell, NK and T cells, showed changes indicating an ineffective response to the virus, with signals that were not properly coordinated to fight the infection. This imbalance in the immune response can lead to severe inflammation and organ damage. Our findings highlight potential targets for treatments to help manage severe COVID-19 and improve patient outcomes. Understanding these immune cell behaviors through single-cell gene analysis could guide the development of new therapies and improve strategies for treating severe COVID-19.