key: cord-0986501-lfuxhlki
authors: Diallo, Aïssatou Bailo; Gay, Laetitia; Coiffard, Benjamin; Leone, Marc; Mezouar, Soraya; Mege, Jean-Louis
title: Daytime variation in SARS-CoV-2 infection and cytokine production
date: 2020-09-11
journal: bioRxiv
DOI: 10.1101/2020.09.09.290718
sha: d6fe8fbfd179e80f34d372bdbbe659dcd1f34407
doc_id: 986501
cord_uid: lfuxhlki

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.

The implication of circadian rhythm (CR) in pathogenesis of Severe Acute Respiratory 61

Syndrome Coronavirus 2 (SARS-CoV-2) has been recently anticipated (1, 2). The CR regulates 62 physiological processes in living organisms with a period of 24 hours (3). Rhythmicity depends on 63 central and peripheral oscillators whose activity relies on two main feedback loops managed by a 64 clock genes cascade under the regulation of the main clock gene Bmal1 (3). The host susceptibility to 65 microorganism is likely under control of biological clocks (4). The time of day of infection is critical 66 for illness progression as reported for influenza, respiratory syncytial and parainfluenza type 3 67 viruses (5-7). We previously reported that CR is a key actor at the interface between infection 68 susceptibility, clinical presentation and prognosis of infection (4, 8) . 69

There are some evidences that enable to anticipate the role of CR in SARS-CoV-2 infection. 70

The absence of Bmal1 has an impact on intracellular replication of coronaviruses, especially 71 vesicular trafficking, endoplasmic reticulum and protein biosynthesis (9) . Knock-out of Bmal1 72 markedly decreases the replication of several viruses such as Dengue or Zika (10). Finally, among 73 key proteins involved in SARS-CoV-2 interaction with the host recently published (11), it has been 74 identified 30% of them being associated with circadian pathway (1). Clearly, the evidences of an 75 implication of CR in SARS-CoV-2 infection of human cells are lacking. In this study, we wondered 76 if SARS-CoV-2 infection and cytokine production by human monocytes, innate immune cells 77 affected by Covid-19, were regulated by CR. 78

We first wondered if the infection of monocytes, innate immune cells affected by , obey to circadian oscillations. Every 3 hours during 24 hours, total RNA was extracted and 81 expression of Bmal1 and Clock genes was investigated in unstimulated monocytes as previously 82 described (8). Expression of investigating genes exhibited CR in monocytes with an acrophase (peak 83 of the rhythm) and a bathyphase (trough of the rhythm) at Zeitgeber (German name for synchronizer) 84

Time (ZT)6 and ZT17 (Fig.1A ). These two time points represent the beginning of the active and the 85 resting periods in humans (13). To assess the involvement of CR in infection of monocytes with 86 SARS-CoV-2, we incubated monocytes with SARS-CoV-2 during the bathyphase (ZT6) and 87 acrophase (ZT17) during 48 hours. Then, viral RNA was extracted to evaluate Covid-19 virus 88 amount and the phagocytosis index was calculated at ZT6 and ZT17 using immunofluorescence. The 89 amount of SARS-CoV-2 virus was higher in monocytes cultured at ZT6 than at ZT17, suggesting a Covid-19 disease is characterized by runaway immune system leading to a cytokine storm 97 consisting of high circulating levels of cytokines including IL-6, IL-1 and IL-10 (14). We wondered 98

if the interaction of SARS-CoV-2 with monocytes affected cytokine production at two points of the 99 CR. The amounts of IL-1, IL-6 and IL-10 were significantly increased at ZT6 (Fig.1D ) when the 100 amount of infection is highest. Hence, the interaction of SARS-CoV-2 with monocytes resulted in 101 distinct cytokine pattern according to daytime. 102

We demonstrate here that the time day of SARS-CoV-2 infection determines consistently 103 viral infection/replication and host immune response. It is likely that SARS-CoV-2 exploits clock 104 pathway for its own gain. Our findings support consideration of CR in SARS-CoV-2 disease 105 progression and suggest that CR represents a novel target for managing viral progression. This study 106 also highlights the importance of the time of treatment administration to Covid-19 patients since CR 107 was found regulating pharmacokinetics of several drugs (15). Several treatments are proposed to 108 prevent the occurrence of severe forms in Covid-19. They include passive immunization, cytokines, 109 anti-cytokine antibody or corticoids (16). All these candidates affect the immune response known to 110 oscillate during the day and their administration according to CR of SARS-CoV-2. Finally, the well-6 documented CR disturbance in intensive care units (17) with 4% fetal bovine serum (FBS), as previously described (18). 118

Human monocytes were isolated from peripheral blood mononuclear cells from healthy donors 119 (convention n°7828, Etablissement Français du Sang, Marseille, France) following CD14 selection 120 using MACS magnetic beads (Miltenyi Biotec, Bergisch, Germany) as previously described (19). 

Coronaviruses: An Overview of Their Replication and 200

Coronaviruses

The circadian clock components BMAL1 and REV-206

ERBα regulate flavivirus replication

ZT17