Dysregulation of circulating protease activity in Covid-19-associated superinfection

Infection by SARS-CoV-2 and subsequent COVID-19 can cause viral sepsis and septic shock. Several complications have been observed in patients admitted to the intensive care unit (ICU) with COVID-19, one of those being bacterial superinfection. Based on prior evidence that dysregulated systemwide proteolysis is associated with death in bacterial septic shock, we investigated whether protease activity and proteolysis could be elevated in COVID-19-induced sepsis with bacterial superinfection. In particular, we sought to assess the possible implications on the regulation of protein systems, such as for instance the proteins and enzymes involved in the clotting cascade. Blood samples collected at multiple time points during the ICU stay of four COVID-19 patients were analyzed to quantify: a) the circulating proteome and peptidome by mass spectrometry; b) plasma enzymatic activity of trypsin-like substrates and five clotting factors (plasmin, thrombin, factor VII, factor IX, factor X) by a fluorogenic assay. Of the four patients, one was diagnosed with bacterial superinfection on day 7 after beginning of the study and later died. The other three patients all survived (ICU length-of-stay 11.25{+/-}6.55 days, hospital stay of 15.25{+/-}7.18 days). Spikes in protease activity (factor VII, trypsin-like activity) were detected on day 7 for the patient who died. Corresponding increases in the total intensity of peptides derived by hydrolysis of plasma proteins, especially of fibrinogen degradation products, and a general reduction of coagulation proteins, were measured as well. A downregulation of endogenous enzymatic inhibitors, in particular trypsin inhibitors, characterized the non-surviving patient throughout her ICU stay. Enzymatic activity was stable in the patients who survived. Our study highlights the potential of multiomics approaches, combined with quantitative analysis of enzymatic activity, to i) shed light on proteolysis as a possible pathological mechanism in sepsis and septic shock, including COVID-19-induced sepsis; ii) provide additional insight into malfunctioning protease-mediated systems, such as the coagulation cascade; and iii) describe the progression of COVID-19 with bacterial superinfection.

Profiling Transcription Initiation in Peripheral Leukocytes Reveals Severity-Associated Cis-Regulatory Elements in Critical COVID-19

The contribution of transcription factors (TFs) and gene regulatory programs in the immune response to COVID-19 and their relationship to disease outcome is not fully understood. Analysis of genome-wide changes in transcription at both promoter-proximal and distal cis-regulatory DNA elements, collectively termed the active cistrome, offers an unbiased assessment of TF activity identifying key pathways regulated in homeostasis or disease. Here, we profiled the active cistrome from peripheral leukocytes of critically ill COVID-19 patients to identify major regulatory programs and their dynamics during SARS-CoV-2 associated acute respiratory distress syndrome (ARDS). We identified TF motifs that track the severity of COVID- 19 lung injury, disease resolution, and outcome. We used unbiased clustering to reveal distinct cistrome subsets delineating the regulation of pathways, cell types, and the combinatorial activity of TFs. We found critical roles for regulatory networks driven by stimulus and lineage determining TFs, showing that STAT and E2F/MYB regulatory programs targeting myeloid cells are activated in patients with poor disease outcomes and associated with single nucleotide genetic variants implicated in COVID-19 susceptibility. Integration with single-cell RNA-seq found that STAT and E2F/MYB activation converged in specific neutrophils subset found in patients with severe disease. Collectively we demonstrate that cistrome analysis facilitates insight into disease mechanisms and provides an unbiased approach to evaluate global changes in transcription factor activity and stratify patient disease severity.

Automated, miniaturized, and scalable screening of healthcare workers, first responders, and students for SARS-CoV-2 in San Diego County

BackgroundSuccessful containment strategies for SARS-CoV-2, the causative virus of the COVID-19 pandemic, have involved widespread population testing that identifies infections early and enables rapid contact tracing. In this study, we developed a rapid and inexpensive RT- qPCR testing pipeline for population-level SARS-CoV-2 detection, and used this pipeline to establish a clinical laboratory dedicated to COVID-19 testing at the University of California San Diego (UCSD) with a processing capacity of 6,000 samples per day and next-day result turnaround times. Methods and findingsUsing this pipeline, we screened 6,786 healthcare workers and first responders, and 21,220 students, faculty, and staff from UCSD. Additionally, we screened 6,031 preschool-grade 12 students and staff from public and private schools across San Diego County that remained fully or partially open for in-person teaching during the pandemic. Between April 17, 2020 and February 5, 2021, participants provided 161,582 nasal swabs that were tested for the presence of SARS-CoV-2. Overall, 752 positive tests were obtained, yielding a test positivity rate of 0.47%. While the presence of symptoms was significantly correlated with higher viral load, most of the COVID-19 positive participants who participated in symptom surveys were asymptomatic at the time of testing. The positivity rate among preschool-grade 12 schools that remained open for in-person teaching was similar to the positivity rate at UCSD and lower than that of San Diego County, with the children in private schools being less likely to test positive than the adults at these schools. ConclusionsMost schools across the United States have been closed for in-person learning for much of the 2020-2021 school year, and their safe reopening is a national priority. However, as there are no vaccines against SARS-CoV-2 currently available to the majority of school-aged children, the traditional strategies of mandatory masking, physical distancing, and repeated viral testing of students and staff remain key components of risk mitigation in these settings. The data presented here suggest that the safety measures and repeated testing actions taken by participating healthcare and educational facilities were effective in preventing outbreaks, and that a similar combination of risk-mitigation strategies and repeated testing may be successfully adopted by other healthcare and educational systems.

Increased peripheral blood neutrophil activation phenotypes and NETosis in critically ill COVID-19 patients

BackgroundIncreased inflammation is a hallmark of COVID-19, with pulmonary and systemic inflammation identified in multiple cohorts of patients. Definitive cellular and molecular pathways driving severe forms of this disease remain uncertain. Neutrophils, the most numerous leukocytes in blood circulation, can contribute to immunopathology in infections, inflammatory diseases and acute respiratory distress syndrome (ARDS), a primary cause of morbidity and mortality in COVID-19. Changes in multiple neutrophil functions and circulating cytokine levels over time during COVID-19 may help define disease severity and guide care and decision making. MethodsBlood was obtained serially from critically ill COVID-19 patients for 11 days. Neutrophil oxidative burst, neutrophil extracellular trap formation (NETosis), phagocytosis and cytokine levels were assessed ex vivo. Lung tissue was obtained immediately post-mortem for immunostaining. ResultsElevations in neutrophil-associated cytokines IL-8 and IL-6, and general inflammatory cytokines IP-10, GM-CSF, IL-1b, IL-10 and TNF, were identified in COVID-19 plasma both at the first measurement and at multiple timepoints across hospitalization (p < 0.0001). Neutrophils had exaggerated oxidative burst (p < 0.0001), NETosis (p < 0.0001) and phagocytosis (p < 0.0001) relative to controls. Increased NETosis correlated with both leukocytosis and neutrophilia. Neutrophils and NETs were identified within airways and alveoli in the lung parenchyma of 40% of SARS-CoV-2 infected lungs. While elevations in IL-8 and ANC correlated to COVID-19 disease severity, plasma IL-8 levels alone correlated with death. ConclusionsCirculating neutrophils in COVID-19 exhibit an activated phenotype with increased oxidative burst, NETosis and phagocytosis. Readily accessible and dynamic, plasma IL-8 and circulating neutrophil function may be potential COVID-19 disease biomarkers.