The Flavonoid Cyanidin Shows Immunomodulatory and Broad-Spectrum Antiviral Properties, Including SARS-CoV-2

Abstract

New antiviral treatments are needed to deal with the unpredictable emergence of viruses. Furthermore, vaccines and antivirals are only available for just a few viral infections, and antiviral drug resistance is an increasing concern. Cyanidin (a natural product also called A18), a key flavonoid that is present in red berries and other fruits, attenuates the development of several diseases, through its anti-inflammatory effects. Regarding its mechanism of action, A18 was identified as an IL-17A inhibitor, resulting in the attenuation of IL-17A signaling and associated diseases in mice. Importantly, A18 also inhibits the NF-κB signaling pathway in different cell types and conditions in vitro and in vivo. In this study, we report that A18 restricts RSV, HSV-1, canine coronavirus, and SARS-CoV-2 multiplication, indicating a broad-spectrum antiviral activity. We also found that A18 can control cytokine and NF-κB induction in RSV-infected cells independently of its antiviral activity. Furthermore, in mice infected with RSV, A18 not only significantly reduces viral titers in the lungs, but also diminishes lung injury. Thus, these results provide evidence that A18 could be used as a broad-spectrum antiviral and may contribute to the development of novel therapeutic targets to control these viral infections and pathogenesis.

Keywords: A18; SARS-CoV-2; antiviral; broad-spectrum; cyanidin; immunomodulatory.

Figure 1

Effect of A18 on virus macromolecular synthesis. (A,B) Protein expression. IF staining was performed to detect the intracellular localization of (A) SARS-CoV-2 spike glycoprotein in calu-3 cells infected with SARS-CoV-2 (moi = 0.1) and (B) RSV gF in A549 cells infected with RSV A2 (moi = 1) and treated or not with A18 (25 mg/mL) at 24 h p.i. Magnification: 400X. (C) Viral RNA synthesis. Total cellular RSV RNA was extracted at the indicated times p.i. and viral RNA was quantified by qRT-PCR. The amounts of viral RNA in untreated and treated cells with A18 (25 mg/mL) in A549 cells were calculated in comparison to the content of viral RNA in untreated infected cells at 6 h p.i., defined as 1. Data represent mean ± SD for n = 3 independent experiments, performed in duplicate. * Significantly different from CV (p-value < 0.05).

Figure 2

Influence of the duration of treatment with A18 on RSV infectivity. Virus adsorption: A549 cells were infected with RSV A2 (moi = 1), HCLE cells were infected with HSV-1 KOS (moi = 1), and calu-3 cells with SARS-CoV-2 (moi = 1) together with 25 μg/mL of A18 and incubated for 1 h at 4 °C. The inoculum was discarded, and cells were washed, supplemented with fresh medium, and further incubated for 24 h at 37 °C. Virus yields were collected and titrated by plaque assay. Virus internalization: A549 cells were infected with RSV A2 (moi = 1), HCLE cells were infected with HSV-1 KOS (moi = 1), and calu-3 cells with SARS-CoV-2 (moi = 1) and incubated for 1 h at 4 °C. The inoculum was discarded, and cells were washed and treated with 25 μg/mL of A18 for 2 h at 37 °C. Afterwards, cells were washed, and the non-internalized virus was inactivated with citrate buffer (pH 3) for 1 min. Cells were further incubated with fresh medium for 24 h at 37 °C. Virus yields were collected and titrated by plaque assay. Data represent mean ± SD for n = 3 independent experiments, performed in duplicate. * Significantly different from CV (p-value < 0.05).

Figure 3

Effect of A18 on cytokine production in RSV infected cells. HEp-2, A549, and J774A.1 cells were infected with RSV A2 (moi = 1) and treated or not with A18 (25 mg/mL) and BAY 11-7085 (10 mg/mL) during 24 h. IL-6, IL-8, and TNF-α were determined by ELISA. CC: cell control (unstimulated cells). Data represent mean ± SD for n = 3 independent experiments, performed in triplicate. * Significantly different from RSV-infected cells; # Significantly different from CC (p-value < 0.05).

Figure 4

Effect of A18 on NF-κB activation in RSV infected cells. HEp-2, A549, and J774A.1 cells were transfected with 0.5 μg of NF-κB-LUC reporter vector and 0.5 μg of β-galactosidase control plasmid. After 24 h, cells were infected with RSV A2 (moi = 1) and treated or not with A18 (25 μg/mL) and BAY 11-7085 (10 mg/mL) during 6 h. Luciferase activity was measured in cell extracts, and each value was normalized to β-galactosidase activity in relative luciferase units (RLUs). CC: cell control (unstimulated cells). Data represent mean ± SD for n = 3 independent experiments, performed in duplicate. * Significantly different from RSV-infected cells; # Significantly different from CC (p-value < 0.05).

Figure 5

In vivo evaluation of A18 antiviral effect. Female Balb/c mice were infected with RSV line 19 and A2 (5 × 10⁶ PFU) by intranasal instillation, concomitant with 6 mg/kg of A18 i.p. or 3 mg/kg of A18 by i.n. injection on day 0. On days 1–4, all mice received further inoculations of A18 i.p. or i.n. (A,B) Weight was monitored and assessed as a percentage of starting weight. Day 0 refers to the time right before inoculation. * At day 2, values for A18-treated infected mice were significantly higher than infected mice (p-value < 0.05). (C) The animals were killed on day 4 and the lungs were used for titration of infectious virus. Data show mean ± SD from n = 6 mice/condition. * Significantly different from RSV-infected mice (p-value < 0.05).

Figure 6

Cytokines gene expression determined by real-time PCR. Pulmonary cytokines expression was assessed on day 8 p.i. and the data were analyzed using the 2^−ΔΔCt formula. Actin was used as an internal for determination of gene expression. Data show mean ± SD from n = 3 mice/condition. * Significantly different from RSV-infected mice (p-value < 0.05).

Figure 7

Lung histology. (A) Light micrographic images of pulmonary histology of H&E-stained lungs collected at day 8 p.i., shown at original magnification ×100, representative of n = 3/condition. (B) An index of pathologic changes in H&E slides was obtained by scoring the inflammatory infiltrate around the airways and vessels for the greatest severity. The Inflammation Index was calculated as the average of the airways’ index, and it was plotted as a percentage with respect to mock-treated infected mice, which was considered 100% of inflammation. * Significantly different from mock-treated infected mice (p-value < 0.05); One-way ANOVA with Tukey post-test.