Dynamic activity in cis-regulatory elements of leukocytes identifies transcription factor activation and stratifies COVID-19 severity in ICU patients.

Transcription factor programs mediating the immune response to coronavirus disease 2019 (COVID-19) are not fully understood. Capturing active transcription initiation from cis-regulatory elements such as enhancers and promoters by capped small RNA sequencing (csRNA-seq), in contrast to capturing steady-state transcripts by conventional RNA-seq, allows unbiased identification of the underlying transcription factor activity and regulatory pathways. Here, we profile transcription initiation in critically ill COVID-19 patients, identifying transcription factor motifs that correlate with clinical lung injury and disease severity. Unbiased clustering reveals distinct subsets of cis-regulatory elements that delineate the cell type, pathway-specific, and combinatorial transcription factor activity. We find evidence of critical roles of regulatory networks, showing that STAT/BCL6 and E2F/MYB regulatory programs from myeloid cell populations are activated in patients with poor disease outcomes and associated with COVID-19 susceptibility genetic variants. More broadly, we demonstrate how capturing acute, disease-mediated changes in transcription initiation can provide insight into the underlying molecular mechanisms and stratify patient disease severity.

Absolute quantification of plasma mitochondrial DNA by droplet digital PCR marks COVID-19 severity over time during intensive care unit admissions.

Increased plasma mitochondrial DNA concentrations are associated with poor outcomes in multiple critical illnesses, including COVID-19. However, current methods of cell-free mitochondrial DNA quantification in plasma are time-consuming and lack reproducibility. Here, we used next-generation sequencing to characterize the size and genome location of circulating mitochondrial DNA in critically ill subjects with COVID-19 to develop a facile and optimal method of quantification by droplet digital PCR. Sequencing revealed a large percentage of small mitochondrial DNA fragments in plasma with wide variability in coverage by genome location. We identified probes for the mitochondrial DNA genes, cytochrome B and NADH dehydrogenase 1, in regions of relatively high coverage that target small sequences potentially missed by other methods. Serial assessments of absolute mitochondrial DNA concentrations were then determined in plasma from 20 critically ill subjects with COVID-19 without a DNA isolation step. Mitochondrial DNA concentrations on the day of enrollment were increased significantly in patients with moderate or severe acute respiratory distress syndrome (ARDS) compared with those with no or mild ARDS. Comparisons of mitochondrial DNA concentrations over time between patients with no/mild ARDS who survived, patients with moderate/severe ARDS who survived, and nonsurvivors showed the highest concentrations in patients with more severe disease. Absolute mitochondrial DNA quantification by droplet digital PCR is time-efficient and reproducible; thus, we provide a valuable tool and rationale for future studies evaluating mitochondrial DNA as a real-time biomarker to guide clinical decision-making in critically ill subjects with COVID-19.

Increased Peripheral Blood Neutrophil Activation Phenotypes and Neutrophil Extracellular Trap Formation in Critically Ill Coronavirus Disease 2019 (COVID-19) Patients: A Case Series and Review of the Literature.

BACKGROUND: Increased inflammation has been well defined in coronavirus disease 2019 (COVID-19), while definitive pathways driving severe forms of this disease remain uncertain. Neutrophils are known to contribute to immunopathology in infections, inflammatory diseases, and acute respiratory distress syndrome, a primary cause of morbidity and mortality in COVID-19. Changes in neutrophil function in COVID-19 may give insight into disease pathogenesis and identify therapeutic targets. METHODS: Blood was obtained serially from critically ill COVID-19 patients for 11 days. Neutrophil extracellular trap formation (NETosis), oxidative burst, phagocytosis, and cytokine levels were assessed. Lung tissue was obtained immediately postmortem for immunostaining. PubMed searches for neutrophils, lung, and COVID-19 yielded 10 peer-reviewed research articles in English. RESULTS: Elevations in neutrophil-associated cytokines interleukin 8 (IL-8) and interleukin 6, and general inflammatory cytokines IFN-inducible protien-19, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 1ß, interleukin 10, and tumor necrosis factor, were identified both at first measurement and across hospitalization (P < .0001). COVID-19 neutrophils had exaggerated oxidative burst (P < .0001), NETosis (P < .0001), and phagocytosis (P < .0001) relative to controls. Increased NETosis correlated with leukocytosis and neutrophilia, and neutrophils and NETs were identified within airways and alveoli in lung parenchyma of 40% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected lungs available for examination (2 of 5). While elevations in IL-8 and absolute neutrophil count correlated with disease severity, plasma IL-8 levels alone correlated with death. CONCLUSIONS: Literature to date demonstrates compelling evidence of increased neutrophils in the circulation and lungs of COVID-19 patients. Importantly, neutrophil quantity and activation correlates with severity of disease. Similarly, our data show that circulating neutrophils in COVID-19 exhibit an activated phenotype with enhanced NETosis and oxidative burst.