Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication

Abstract

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE₂, one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE₂ receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE₂ signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE₂ signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on ACE2 expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients.

Figure 1.. SARS-CoV-2 infection induces PTGS2 expression in human and mouse systems

(A) Calu-3 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.05. PTGS2 expression was measured at 2 days post infection (dpi), normalized to ACTB. (B-C) PTGS2 expression in Calu-3 (B) and ACE2-overexpressing A549 (A549-ACE2) (C) cells following SARS-CoV-2 infection. Data are from GSE147507 (Blanco-Melo et al., 2020). (D) Huh7.5 cells were infected with SARS-CoV-2 at a MOI of 0.05. PTGS2 expression was measured at 2 dpi, normalized to ACTB. (E) Human bronchial epithelial cells (HBECs) were cultured at an air-liquid interface and then infected at the apical surface with 10⁴ plaque-forming units (PFU) of SARS-CoV-2. Cells were collected at 1, 2, and 3 dpi for single-cell RNA sequencing (scRNA-seq) (16). Volcano plot of differentially expressed genes in infected versus bystander ciliated cells pooled from all time points. PTGS2 is highlighted. (F) K18-hACE2 mice were infected intranasally with 1.2 × 10⁶ PFU of SARS-CoV-2. Ptgs2 expression in the lung was measured at 0, 2, 4, and 7 dpi. (G) Ptgs2 expression in the lung of K18-hACE2 mice following intranasal SARS-CoV-2 infection. Data are from GSE154104 (Winkler et al., 2020). All data points in this figure are presented as mean ± SEM. Data were analyzed by Welch’s two-tailed, unpaired t-test (A, D, F); Student’s two-tailed, unpaired t-test (B, C, G); and two-sided Mann-Whitney U test with continuity and Benjamini-Hochberg correction (E). *P < 0.05, ***P < 0.001, ****P < 0.0001. Data in (A, D) are representative of two independent experiments with three replicates per condition.

Figure 2.. NSAID treatment does not affect ACE2 expression in human cell lines

(A-B) Calu-3 (A) and Huh7.5 (B) cells were treated with different concentrations of ibuprofen or meloxicam for 48 hours. Cell viability was measured and calculated as a percentage of no treatment. (C) Calu-3 cells were treated with DMSO, 50 μM ibuprofen, or 50 μM meloxicam for 48 hours. Levels of prostaglandin E₂ (PGE₂) were measured in the supernatant. Dotted line represents limit of detection. (D-E) Calu-3 (D) and Huh7.5 (E) cells were treated with DMSO, 50 μM ibuprofen, or 50 μM meloxicam for 24 hours. ACE2 expression was measured and normalized to ACTB. All data points in this figure are presented as mean ± SEM. Data were analyzed by Welch’s two-tailed, unpaired t-test (C-E). **P < 0.01. ns, not significant. Data in (A-E) are representative of two independent experiments with three replicates per condition.

Figure 3.. NSAID treatment does not affect Ace2 expression in mouse tissues

(A-B) C57BL/6 mice were treated intraperitoneally with DMSO, 30 mg/kg ibuprofen, or 1 mg/kg meloxicam daily for 4 days. Ace2 expression was measured in the lung (A), heart (B), kidney (C), and ileum (D), normalized to Actb. All data points in this figure are presented as mean ± SEM. Data were analyzed by Welch’s two-tailed, unpaired t-test (A-D). ns, not significant. Data in (A-D) are pooled from two independent experiments with four to six mice per condition.

Figure 4.. NSAID treatment does not affect SARS-CoV-2 entry or replication

(A-B) Calu-3 (A) and Huh7.5 (B) cells were pre-treated with DMSO, 50 μM ibuprofen, or 50 μM meloxicam for 24 hours and then infected with SARS2-VSVpp or G-VSVpp expressing Renilla luciferase. Luminescence was measured at 24 hours post infection (hpi) and normalized to DMSO for each infection. (C-D) Calu-3 (C) and Huh7.5 (D) cells were pre-treated with DMSO, 50 μM ibuprofen, or 50 μM meloxicam for 24 hours and then infected with mNeonGreen reporter replication-competent SARS-CoV-2 (icSARS-CoV-2-mNG) at a MOI of 1. Frequency of infected cells was measured by mNeonGreen expression at 1, 2, and 3 dpi. All data points in this figure are presented as mean ± SEM. Data were analyzed by Student’s two-tailed, unpaired t-test (A-B) and two-way ANOVA (C-D). ns, not significant. Data in (A-B) are representative of two independent experiments with four replicates per condition. Data in (C-D) are representative of two independent experiments with five replicates per condition.