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.
Physiological and cellular functions operate in a 24-hour cyclical pattern orchestrated by an endogenous process known as the circadian rhythm. Circadian rhythms represent intrinsic oscillations of biological functions that allow for adaptation to cyclic environmental changes. Key clock genes that affect the persistence and periodicity of circadian rhythms include BMAL1/CLOCK, Period 1, Period 2, and Cryptochrome. Remarkable progress has been made in our understanding of circadian rhythms and their role in common medical conditions. A critical review of the literature supports the association between circadian misalignment and adverse health consequences in sepsis, obstructive lung disease, obstructive sleep apnea, and malignancy. Circadian misalignment plays an important role in these disease processes and can affect disease severity, treatment response, and survivorship. Normal inflammatory response to acute infections, airway resistance, upper airway collapsibility, and mitosis regulation follows a robust circadian pattern. Disruption of normal circadian rhythm at the molecular level affects severity of inflammation in sepsis, contributes to inflammatory responses in obstructive lung diseases, affects apnea length in obstructive sleep apnea, and increases risk for cancer. Chronotherapy is an underused practice of delivering therapy at optimal times to maximize efficacy and minimize toxicity. This approach has been shown to be advantageous in asthma and cancer management. In asthma, appropriate timing of medication administration improves treatment effectiveness. Properly timed chemotherapy may reduce treatment toxicities and maximize efficacy. Future research should focus on circadian rhythm disorders, role of circadian rhythm in other diseases, and modalities to restore and prevent circadian disruption.
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.
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.
Background: Neutrophils are key players in the immune and aid in the defense against microorganisms. Neutrophil extracellular traps (NETs) are extracellular DNA complexes, which are released during NETosis, a programmed form of cell death. Although NETs are crucial in the fight against infectious agents, an overabundance of neutrophils has been implicated in many inflammatory lung conditions. Our aim is to determine whether an overabundance of NETosis is associated with clinical deterioration of patients with COVID-19. Methods: Circulating polymorphonuclear cells (neutrophils) were isolated from human peripheral blood of 20 human subjects with COVID-19. Neutrophils were seeded in 96-well plates and treated with 0, 2.5 nM, 25 nM, and 250 nM of phorbol 12-myrisate 13-acetate (PMA) or 12 uM nigericin for 2 hours to stimulate NET production via canonical and noncanonical pathways, respectively. Following incubation, wells were treated with micrococcal nuclease, supernatants were collected from each well, and extracellular DNA content to quantify NETosis was detected by fluorescent plate reader. We calculated acute physiology and chronic health evaluation (APACHE-II) scores for every human subject. These were calculated at the same time point at which the neutrophils were collected. They were then compared to the degree of NETosis and absolute neutrophil count (ANC). These were analyzed using a simple linear regression model. We also categorized participants based on APACHE-II scores (APACHE-II 15) and compared them to rates of NETosis using a bar graph. Results: APACHE II is a widely used ICU mortality prediction score that is used to risk-stratify patients. We found that participants with higher APACHE-II scores had higher rates of NETosis, both at 0 nM PMA and when stimulated with nigericin (figure 1a-b). This suggests that higher rates of NETosis correlate with increased disease severity. Additionally, we found a positive correlation between ANC and NETosis (Figure 1c-1d), suggesting that ANC itself is a reliable marker of NETosis and disease severity. Conclusion: NETosis is an important player in immune system defense but has also been implicated in various inflammatory lung conditions. We found that in patients with COVID-19, there was a positive correlation between worsening disease state, measure by APACHE II scores, and increased NETosis. This suggests that over-activation of neutrophils may play a role in disease progression. We also found a positive correlation between NETosis and ANC, indicating that the degree of circulating neutrophils is a reliable marker of the functional state of neutrophils, as well as disease severity.
