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
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Abstract
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 1β, 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.
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SciScore for 10.1101/2021.01.14.21249831: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Study design and oversight: The research protocol was approved by the UCSD, VASDHS and Rady Children’s Hospital institutional review boards (IRBs) and all participants or designated family member gave written informed consent.
Consent: After informed consent, blood was drawn on days 1, 3, 5, 7, 9, 1, most commonly between 8-10 am, and immediately prior to discharge.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when …
SciScore for 10.1101/2021.01.14.21249831: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Study design and oversight: The research protocol was approved by the UCSD, VASDHS and Rady Children’s Hospital institutional review boards (IRBs) and all participants or designated family member gave written informed consent.
Consent: After informed consent, blood was drawn on days 1, 3, 5, 7, 9, 1, most commonly between 8-10 am, and immediately prior to discharge.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
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:Our study has limitations. Our control subjects were younger, had higher numbers of Asian ancestry and fewer of Hispanic heritage than our COVID-19 patients. This situation is primarily due to the limitation to conduct human subjects research during the COVID-19 pandemic, such that we were limited to recruiting from employees at the VA San Diego Healthcare System. These control subjects were primarily to define the range of normal for neutrophil functional assays, run on different days, as there is significant day to day variability in these assays. Studying neutrophils from these control subjects on multiple days proved that it is feasible to define the activation state of neutrophils at a single timepoint as well as over time. It is unknown whether hypertension, cardiac disease, and ancestry impact neutrophil function. Diabetes and increasing age are associated with diminished neutrophil function [35-37]; thus, we do not believe that the increased neutrophil function in the COVID cohort is due to higher levels of these comorbidities. Another limitation is the size of our cohort, but by obtaining neutrophils, plasma and clinical data at multiple points over time, this approach increased the power of our study to detect clinically significant changes. The role of neutrophils in COVID-19 immunopathology is gaining interest rapidly. In aggregate, ours is the first study to report broad functional assessments of circulating neutrophils isolated from critically ill COVID-19 patie...
Results from TrialIdentifier: No clinical trial numbers were referenced.
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.
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SciScore for 10.1101/2021.01.14.21249831: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Methods Study design and oversight The research protocol was approved by the UCSD, VASDHS and Rady Children’s Hospital institutional review boards (IRBs) and all participants or designated family member gave written informed consent. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Lung tissue was obtained within 2 h postmortem from a 67 year-old male COVID-19 patient, intubated for 17 days prior to death in July 2020. Table 2: Resources
Antibodies Sentences Resources Primary antibodies used for immunofluorescence were: CD66b (1:100, 392902, Biolegend) CD66b…SciScore for 10.1101/2021.01.14.21249831: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement Methods Study design and oversight The research protocol was approved by the UCSD, VASDHS and Rady Children’s Hospital institutional review boards (IRBs) and all participants or designated family member gave written informed consent. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Lung tissue was obtained within 2 h postmortem from a 67 year-old male COVID-19 patient, intubated for 17 days prior to death in July 2020. Table 2: Resources
Antibodies Sentences Resources Primary antibodies used for immunofluorescence were: CD66b (1:100, 392902, Biolegend) CD66bsuggested: (BioLegend Cat# 392902, RRID:AB_2728422)Software and Algorithms Sentences Resources Neutrophils were isolated using PolymorphprepTM (PROGEN) per manufacturer’s instructions as previously described [21]. PolymorphprepTMsuggested: NoneStatistical analysis Statistical analyses were performed using GraphPad Prism Version 8.4.3 (San Diego). GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)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:
Our study has limitations. Our control subjects were younger, had higher numbers of Asian ancestry and fewer of Hispanic heritage than our COVID-19 patients. This situation is primarily due to the limitation to conduct human subjects research during the COVID-19 pandemic, such that we were limited to recruiting from employees at the VA San Diego Healthcare System. These control subjects were primarily to define the range of normal for neutrophil functional assays, run on different days, as there is significant day to day variability in these assays. Studying neutrophils from these control subjects on multiple days proved that it is feasible to define the activation state of neutrophils at a single timepoint as well as over time. It is unknown whether hypertension, cardiac disease, and ancestry impact neutrophil function. Diabetes and increasing age are associated with diminished neutrophil function [35-37]; thus, we do not believe that the increased neutrophil function in the COVID cohort is due to higher levels of these comorbidities. Another limitation is the size of our cohort, but by obtaining neutrophils, plasma and clinical data at multiple points over time, this approach increased the power of our study to detect clinically significant changes. The role of neutrophils in COVID-19 immunopathology is gaining interest rapidly. In aggregate, ours is the first study to report broad functional assessments of circulating neutrophils isolated from critically ill COVID-19 patie...
Results from TrialIdentifier: No clinical trial numbers were referenced.
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.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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