Enhanced neutrophil extracellular trap formation in COVID-19 is inhibited by the protein kinase C inhibitor ruboxistaurin
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Abstract
Neutrophil extracellular traps (NETs) are web-like DNA and protein lattices which are expelled by neutrophils to trap and kill pathogens, but which cause significant damage to the host tissue. NETs have emerged as critical mediators of lung damage, inflammation and thrombosis in coronavirus disease 2019 (COVID-19) and other diseases, but there are no therapeutics to prevent or reduce NETs that are available to patients.
Methods
Neutrophils were isolated from healthy volunteers (n=9) and hospitalised patients with COVID-19 at the acute stage (n=39) and again at 3–4 months post-acute sampling (n=7). NETosis was measured by SYTOX green assays.
Results
Here, we show that neutrophils isolated from hospitalised patients with COVID-19 produce significantly more NETs in response to lipopolysaccharide (LPS) compared to cells from healthy control subjects. A subset of patients was captured at follow-up clinics (3–4 months post-acute sampling), and while LPS-induced NET formation is significantly lower at this time point, it remains elevated compared to healthy controls. LPS- and phorbol myristate acetate (PMA)-induced NETs were significantly inhibited by the protein kinase C (PKC) inhibitor ruboxistaurin. Ruboxistaurin-mediated inhibition of NETs in healthy neutrophils reduces NET-induced epithelial cell death.
Conclusion
Our findings suggest ruboxistaurin could reduce proinflammatory and tissue-damaging consequences of neutrophils during disease, and since it has completed phase III trials for other indications without safety concerns, it is a promising and novel therapeutic strategy for COVID-19.
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SciScore for 10.1101/2021.08.24.21262336: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: Human samples: Hospitalised patients with COVID-19 (n=39) admitted to the Royal Hallamshire Hospital, Sheffield, UK were recruited to the study and provided fully informed consent via The Sheffield Teaching Hospitals Observational Study of Patients with Pulmonary Hypertension, Cardiovascular and other Respiratory Diseases (
IRB: Ethical approval was given by the Yorkshire & The Humber - Sheffield Research Ethics Committee.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources Wells were blocked and permeabilized with buffer containing 5% bovine serum albumin (BSA), 0.1% saponin … SciScore for 10.1101/2021.08.24.21262336: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: Human samples: Hospitalised patients with COVID-19 (n=39) admitted to the Royal Hallamshire Hospital, Sheffield, UK were recruited to the study and provided fully informed consent via The Sheffield Teaching Hospitals Observational Study of Patients with Pulmonary Hypertension, Cardiovascular and other Respiratory Diseases (
IRB: Ethical approval was given by the Yorkshire & The Humber - Sheffield Research Ethics Committee.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources Wells were blocked and permeabilized with buffer containing 5% bovine serum albumin (BSA), 0.1% saponin and 5% normal goat serum, and then stained with a rabbit anti-myeloperoxidase (MPO) (A0398) primary antibody for 90 minutes at 37°C. anti-myeloperoxidase ( MPOsuggested: (Antibodies-Online Cat# ABIN235893, RRID:AB_10764910)A secondary goat anti-rabbit AlexaFluor® 594 antibody (ab150088) was added for 45 minutes at 37°C. anti-rabbitsuggested: (Abcam Cat# ab150088, RRID:AB_2890663)Software and Algorithms Sentences Resources Images were constructed using FIJI image analysis software and the background was subtracted for the DAPI channel using FiJI FiJIsuggested: (Fiji, RRID:SCR_002285)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:A limitation of our study is around the demographics of the healthy control subjects compared to the patient cohort, the latter of whom are older, have more comorbidities and are receiving medications that may impact on neutrophil function (including dexamethasone). However, studying patients at approximately three months post-hospitalisation means participants serve as appropriate age- and comorbidity-matched controls and allow up to understand differences in neutrophil function at the acute stage of the disease. Vaccination weakens the link between infection and critical illness, but vaccine breakthroughs are seen, particularly in the case of viral variants which will continue to emerge (28). It is therefore critical that we develop alternative and complementary strategies to prevent severe COVID-19, and the innate immune response is an ideal target for this. Since NETs are known drivers of pathology in a number of diseases including but not limited to COVID-19, targeting NETosis is a logical therapeutic strategy for the future. A growing number of studies describe increased levels of NETosis in COVID-19 as well as the deleterious role of NETs in driving inflammation, thrombosis and disease severity, but few have offered a solution. Our study indicates that ruboxistaurin could reduce NET formation and ultimately diminish airway inflammation and other events including microvascular thrombosis, and is a novel and promising therapeutic strategy for COVID-19 (6, 8). Furthermore...
Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04817332 Completed STOP-COVID19: Superiority Trial Of Protease Inhibition in CO… NCT04359654 Recruiting Nebulised Dornase Alfa for Treatment of COVID-19 Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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