Murine Alveolar Macrophages Rapidly Accumulate Intranasally Administered SARS-CoV-2 Spike Protein leading to Neutrophil Recruitment and Damage

Reviewer #1 (Public Review):

The manuscript by Park et. al. examines the interaction of macrophages with SARS-CoV-2 spike protein and subsequent inflammatory reactions. The authors demonstrate that following intranasal delivery of spike it rapidly accumulates in alveolar macrophages. Inflammation associated with internalized spike recruits neutrophils to the lung, where they undergo a cell death process consistent with NETosis. The authors demonstrate that modifications spike to contain high mannose reduces uptake of spike protein and limits the inflammation induced. This finding could have implications on vaccine development, as vaccines containing modified spike could be safer and better tolerated.

The authors use a number of different techniques, including in vivo modeling, imaging, human and murine systems to interrogate their hypotheses. These systems provide robust supporting information for their conclusions. There are two key aspects from the current manuscript which would add key evidence. The authors suggest that neutrophils exposed to spike protein undergo a process of NETosis. To confirm this hypothesis inhibitors of NETosis should be used to demonstrate that the cell death is prevented. Additionally, vaccination of a murine model with the modified spike protein would add additional support to the conclusion that modified spike protein would be less inflammatory while maintaining its utility as a vaccine antigen.

Reviewer #2 (Public Review):

The paper describes the various types of immune cells interacting with SARS-CoV-2 spike protein and undergoing pathological changes upon different routes of administration into mice mainly in the absence of human ACE-2. Multiple murine cell types in the lungs, the cremaster muscle and surrounding tissues, and the liver were studied. The spike interactions with various cells from the human peripheral blood ex vivo and in cultures were also examined. This study focused on hACE-2-independent effects of the spike protein in vivo in mice and in vitro on human leukocytes and touched upon the potential involvement of sialic-acid-binding lectins (Siglec) as non-hACE-2 receptors for spike. Hence, a multitude of aspects about spike-cell interactions was studied, although each was covered without significant depths and the key findings are difficult to parse through. Many inconsistencies are not explained and the critical experimental parameters and controls are missing. Ultimately, the main message of the study is buried among supporting vs confounding data.

Reviewer #3 (Public Review):

The study focuses on in vivo and in vitro cellular responses intranasal instillation of glycoforms and mutants of SARS-CoV2 spike trimer or spike bearing VLP. Collectively, the experiments suggest that SARS-CoV2 spike has pro-inflammatory roles through increase M1 macrophage associated cytokines and induction of neutrophil netosis, a proinflammatory cell death mechanisms. These effects seem largely independent of hACE2 interaction and partly depend upon interactions with scavenger receptors on macrophages and neutrophils. A strength of the study is that a number sophisticated methods are used, including intravital microscopy in the cramaster and liver as well as acute lung slice models, to look at uptake of the spike proteins and immune cell dynamics. The weakness is that some of the reagents maybe contaminated with uncharacterized glycoforms and some important controls, such as control spike protein and control VLP are unevenly applied or not included. Given the breadth of the studies, it would be ideal for the authors to prioritise strengthening the most important in vivo results in the best animal models with the strongest controls to be able to realise the full impact of the results.