Review 2: "Daytime variation in SARS-CoV-2 infection and cytokine production"

Stephan Holtkamp

doi:doi:10.1162/2e3983f5.ad0f7a30

This study provides novel insight into time of day of SARS-CoV-2 infection and macrophage response, which could have therapeutic implications. However, reviewers suggest that claims could be misleading and were not adequately supported by their analytic methods.

by Stephan Holtkamp

Published onOct 23, 2020

Daytime variation in SARS-CoV-2 infection and cytokine production

by Aïssatou Bailo Diallo, Laetitia Gay, Benjamin Coiffard, Marc Leone, Soraya Mezouar, and Jean-Louis Mege

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Published on Sep 12, 2020
www.biorxiv.org

Description

Abstract S. Ray and A. Reddy recently anticipated the implication of circadian rhythm in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of the coronavirus disease (Covid-19). In addition to its key role in the regulation of biological functions, the circadian rhythm has been suggested as a regulator of viral infections. Specifically, the time of day of infection was found critical for illness progression, as has been reported for influenza, respiratory syncytial and parainfluenza type 3 viruses. We analyzed circadian rhythm implication in SARS-CoV-2 virus infection of isolated human monocytes, key actor cells in Covid-19 disease, from healthy subjects. The circadian gene expression of Bmal1 and Clock genes was investigated with q-RTPCR. Monocytes were infected with SARS-CoV-2 virus strain and viral infection was investigated by One-Step qRT-PCR and immunofluorescence. Interleukin (IL)-6, IL-1β and IL-10 levels were also measured in supernatants of infected monocytes. Using Cosinor analysis, we showed that Bmal1 and Clock transcripts exhibited circadian rhythm in monocytes with an acrophase and a bathyphase at Zeitgeber Time (ZT)6 and ZT17. After forty-eight hours, the amount of SARS-CoV-2 virus increased in the monocyte infected at ZT6 compared to ZT17. The high virus amount at ZT6 was associated with significant increased release in IL-6, IL-1β and IL-10 compared to ZT17. Our results suggest that time day of SARS-CoV-2 infection affects viral infection and host immune response. They support consideration of circadian rhythm in SARS-CoV-2 disease progression and we propose circadian rhythm as a novel target for managing viral progression.Importance The implication of circadian rhythm (CR) in pathogenesis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been recently anticipated. The time of day of infection is critical for illness progression as reported for influenza, respiratory syncytial and parainfluenza type 3 viruses. In this study, we wondered if SARS-CoV-2 infection and cytokine production by human monocytes, innate immune cells affected by Covid-19, were regulated by CR. Our results suggest that time day of SARS-CoV-2 infection affects viral infection and host immune response. They support consideration of circadian rhythm in SARS-CoV-2 disease progression and we propose circadian rhythm as a novel target for managing viral progression.

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 presented manuscript has given scientific evidence that time-of-day-dependent infection with SARS-CoV-2 yields a temporal response with respect to chemokine production in human primary macrophages.

Claims are not strongly supported, but may yield some insight by the data and methods used. Decision-makers should consider the claims in this study not actionable (except to prompt further research), unless the weaknesses are clearly understood and there is other theory and evidence to further support them based on the methods and data.

The manuscript supports previous findings by e.g. Ray and Reddy [1] and is positioned well in currently existing literature.

The clarity regarding recommended actions that results from the findings is given, as for example time-of-day dependent administration of medication against a SARS-CoV-2 infection is suggested.

The work is presented in a clear and accurate manner.

The data provided gives a well-designed first insight into how the circadian clock might influence the infection with SARS-CoV-2 in macrophages. However, there is data lacking to confirm the link between the molecular clock and the infection and subsequent cytokine production.

Questions to consider:

a. Is the oscillation in Bmal1 and Clock still persistent after 48 hours in cell culture?

b. How does a lack of Bmal1 lead to a stronger infection? (Use of Bmal1 KO cells?)

c. What about Per and Cry?

d. The analysed cytokines show time-of-day variations in macrophages tested in other publications. Was that taken into consideration? qPCR of cytokines?

e. What about time-of-day dependent administration of antiviral products in this model?

Authors are asked to provide more evidence for a molecular link and to exacerbate the time-of-day immuno-reactivity of macrophages. With this rather major revision, the manuscript should be accepted to the RR:C19 Journal.

Review 2: "Daytime variation in SARS-CoV-2 infection and cytokine production"

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