Characterization of low-density granulocytes in COVID-19

Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.

1. You agree on the fact that LDG may not comprise of a separate phenotype of neutrophils, yet you persist with implying that LDG are such a population in the Abstract and Discussion. You also neglect to cite studies that do not support the main hypothesis that neutrophils are important in the initiation of disease up to the moment patients have to go to the ICU. These points need to be addressed.
• LDGs are no longer called a distinct subset of circulating neutrophils in this manuscript. We deemed useful to refer to them as granulocytes of lower density on some occasions, to avoid the misconception of LDGs being a different type of neutrophils. Their isolation from PBMC has been highlighted more than in the previous version, and their presence in this fraction continues to be explained by their mere lower density, which can arise from an immature mononuclear or hyposegmented nucleus and/or by a loss of density of some mature PMNs, which we hypothesize could arise from activation.
2. Discussion about cause and consequence. Neutrophils might still be guilty by association. This is not carefully addressed in the Discussion.
• We have rewritten the parts of the discussion that were misleading by suggesting a role of LDGs in the initiation of the disease (or pathogenesis). This idea has been redirected towards neutrophils being actors during the general pathophysiology of COVID-19, contributing to disease progression. This goes in line with their proposed role as modulators and suppressors of the adaptive immune response, given their demonstrated suppressive effect in T lymphocytes.
3. A left shift during inflammatory conditions associated with the occurrence of progenitors in the peripheral blood is expected. This is normal physiology and not necessarily pathophysiology and accordingly should be acknowledged.
• The 'guilty by association' issue has been addressed by detailing the emergency myelopoiesis as a response of inflammation, which is why we support the idea that LDGs in infectious processes are indeed a product of a 'left-shift', instead of being exclusive for COVID-19. Spijkerman et al. (2021) has been added as a reference in this matter, and their findings on a lack of activation of neutrophils at hospital presentation was discussed.
4. The complex multidimensional data supporting the presence of progenitors in the peripheral blood is not much better than the 2D plots on the expression of CD11b and CD16. You do not acknowledge Hidalgo's paper in Trends in Immunology (ref 26) that already used this gating strategy. This acknowledgement should be clearly made. Responses to reviewer's comments: Reviewer: although the authors "fully agree" on the issue that LDG'S are not a specific subset but either young and/or activated neutrophils, they persist throughout the article with the suggestion that LDG comprise of separate subsets of neutrophils (1-4) (2019) and Pedersen et al (2016). Both studies have now been acknowledged in the discussion.
Reviewer: the issue of the difficulties obtaining relevant disease controls is well taken, but should be better discussed in terms of left shifts in other acute infectious diseases.

o We understand and have broaden the discussion concerning emergency myelopoiesis as a physiological response to inflammation and/or infection. This left shift has been acknowledged and the study by Spijkerman et al. (2021) comparing COVID-19 neutrophil compartment response to other bacterial and viral infections has been made. We believe this goes in line with demonstrating that during acute inflammatory processes such as infections the increase in neutrophils of lower density comes from the premature release of granulocyte precursors, which should also be taken into consideration during chronic pathological processes where LDGs are better defined and are still considered a different subset of cells compared to their normal-density counterparts.
Reviewer: the issue here was that the alternative pathogenesis of COVID19 might drive the disease including causing tissue damage through thrombo-embolisms and reperfusion injury. More severe disease would then lead to more release of DAMP's leading to more activation of the neutrophil compartment. So the left shift can be the consequence of disease rather than the cause. In fact, Spijkerman et al. (JLB 2021) supports this hypothesis by not finding much indications of an activated neutrophil compartment in COVID19 patients during hospital admission. This study is ignored but at least the concept should be discussed. We hope these modifications are in line with the requests made, as well as the answers to the reviewer's questions. If further changes are needed, do not hesitate to inform us and we will make sure to comply with the demands.