Review 2: "Targeting Neutrophils Extracellular Traps (NETs) Reduces Multiple Organ Injury in a COVID-19 Mouse Model"

RR:C19 Evidence Scale rating by reviewer:

• Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review:

The authors use an intranasal inoculum of SARS-CoV-2 virus in the humanized ACE2 receptor transgenic mouse line to induce an experimental model of COVID-19 in mice. This model has been previously published and the lung pathology established, but NETs have not yet been examined in this model. The presence of NETs and potential therapeutic targeting of them using gasdermin D or PAD inhibition was previously reported in the Syrian hamster model.

This manuscript uses the approach of DNase I administration, which in theory should degrade released NETs to smaller fragments. This general approach has been demonstrated in numerous other in vivo studies in other disease models where NETs play a pathogenic role, including myocardial infarction, stroke, wound healing, and deep vein thrombosis. The current study uses subcutaneous administration daily for 5 days. The half-life of this drug via this route of administration is not clear, as well as its localization in the lungs/blood. Several clinical trials administering DNase as an inhaled nebulizer therapy in severe COVID-19 have been initiated. These have demonstrated safety and show promise for efficacy. Experimental studies investigating how this treatment works on a cellular level in disease pathogenesis are important to conduct, and this study does precisely this.

The representative pathology shows very severe inflammation in the vehicle treated animals which are ameliorated by DNase administration. NETs are also shown to be reduced using immunostaining to identify NETting neutrophil and released NETs. It would be helpful to better understand how heterogenous this response was in different parts of the lung with additional representative images and fields of view, and quantification that encompasses this.

The systemic protection from cardiac tissue damage and kidney injury is also intriguing and points toward a role of NETs within the circulation as well as the efficacy of DNase in a non-standard administration route reaching the bloodstream. The histology images could be more informative with higher magnification fields of view and more specific staining, as well as arrows to point to different pathological features. The paired measurements of CK-MB and creatinine in blood samples are necessary to support the conclusions that the heart and kidney are involved and also protected with Dnase I administration.

This model is known to have brain involvement with longer timepoints, and this would be something of importance to test in this experimental treatment study as well. Are mice protected from mortality or from brain involvement with DNase administration? This would be powerful for Long COVID sufferers.

The study would be strengthened by use of nonparametric statistical testing provided the low sample sizes in each experiment.

Minor:

• DNase/DNAse should be spelled consistently

• Several of the references do not appear correct (for example reference 46, to which the DOI refers to a 2012 Nature Medicine paper with another title)

  In summary, this provides the first preclinical evidence supporting DNase administration in the humanized mouse model of SARS-CoV-2 infection. This is shown in the lung and also potentially in other organs including the heart and kidney. Further exploration behind the mechanism by which this protects, and more in-depth analysis of the systemic effects beyond the lung are logical next steps to extend the study. This approach may have important implications for therapeutic use for COVID-19, as well as for other inflammatory diseases where DNase could be administered systemically in the future.