Antiviral and Anti-Inflammatory Activities of Fluoxetine in a SARS-CoV-2 Infection Mouse Model

Abstract

The coronavirus disease 2019 (COVID-19) pandemic continues to cause significant morbidity and mortality worldwide. Since a large portion of the world's population is currently unvaccinated or incompletely vaccinated and has limited access to approved treatments against COVID-19, there is an urgent need to continue research on treatment options, especially those at low cost and which are immediately available to patients, particularly in low- and middle-income countries. Prior in vitro and observational studies have shown that fluoxetine, possibly through its inhibitory effect on the acid sphingomyelinase/ceramide system, could be a promising antiviral and anti-inflammatory treatment against COVID-19. In this report, we evaluated the potential antiviral and anti-inflammatory activities of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and against variants of concern in vitro, i.e., SARS-CoV-2 ancestral strain, Alpha B.1.1.7, Gamma P1, Delta B1.617 and Omicron BA.5. Fluoxetine, administrated after SARS-CoV-2 infection, significantly reduced lung tissue viral titres and expression of several inflammatory markers (i.e., IL-6, TNFα, CCL2 and CXCL10). It also inhibited the replication of all variants of concern in vitro. A modulation of the ceramide system in the lung tissues, as reflected by the increase in the ratio HexCer 16:0/Cer 16:0 in fluoxetine-treated mice, may contribute to explain these effects. Our findings demonstrate the antiviral and anti-inflammatory properties of fluoxetine in a K18-hACE2 mouse model of SARS-CoV-2 infection, and its in vitro antiviral activity against variants of concern, establishing fluoxetine as a very promising candidate for the prevention and treatment of SARS-CoV-2 infection and disease pathogenesis.

Keywords: COVID-19; SARS-CoV-2; anti-depressant; fluoxetine; inflammation; mouse model.

Figure 1

Fluoxetine reduced viral load and inflammatory response in K18-hACE2 mice infected with SARS-CoV-2. (A) Experimental design of the study; (B) infectious viral loads in the lungs of vehicle- and fluoxetine-treated mice infected with SARS-CoV-2 at day 2 and 6 post-infection expressed as log10 TCID50/mL; (C) viral RNA levels in the lungs of vehicle- and fluoxetine-treated mice infected with SARS-CoV-2 at day 2 and 6 post-infection expressed as Lung SARS-CoV-2 RNA load (RNA copies/µL). (D) Cytokine and chemokine transcripts in lung tissue at day 2 and 6 post-infection of SARS-CoV-2-infected mice, treated with vehicle or with fluoxetine. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 2

Fluoxetine effect on lung levels of (A) ceramide 16:0, (B) hexosylceramide 16:0, (C) hexosylceramide 16:0 / ceramide 16:0 ratio, and (D) sphingomyelin 16:0, at day 2 post-infection. *, p < 0.05.

Figure 3

Fluoxetine activity on SARS-CoV-2 variants in vitro. (A) Activity of fluoxetine (12.5 µM) or mock (water) in Vero E6 cells infected with SARS-CoV-2 (Pasteur, B.1), B.1.1.7 (UK), P1, B.1.617 (Delta) and BA.5 (Omicron) variants measured by assessing cell viability and viral load. (B) Antiviral activity of fluoxetine (12.5 µM) measured in A549 cells expressing hACE2-TMPRSS2 infected with SARS-CoV-2 (Pasteur, B.1), B.1.1.7 (UK), P1, B.1.617 (Delta) and BA.5 (Omicron) variants measured by cell viability and viral load. Representative of two to seven independent experiments, data are expressed as mean + SEM. *, p < 0.05, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 4

Fluoxetine inhibits SARS-CoV-2 infection of 2D human airway cells. (A) Representative images of immunofluorescent stainings of mNeon Green SARS-CoV-2 infected airway cultures. Acetylated tubulin (AcTUB, red) stains cell ciliates, phalloidin-coupled Alexa Fluor 647 stains F-actin (purple) and DAPI stains nuclei (blue). Scale bar = 10 µm. (B) Live virus titre measured on apical washes 72 h after infection with mNeon Green SARS-Cov-2. Data from three independent experiments on two independent donors, expressed as mean ± SEM, **** p < 0.0001.