Quercetin inhibits rhinovirus replication in vitro and in vivo

Abstract

Rhinovirus (RV), which is responsible for the majority of common colds, also causes exacerbations in patients with asthma and chronic obstructive pulmonary disease. So far, there are no drugs available for treatment of rhinovirus infection. We examined the effect of quercetin, a plant flavanol on RV infection in vitro and in vivo. Pretreatment of airway epithelial cells with quercetin decreased Akt phosphosphorylation, viral endocytosis and IL-8 responses. Addition of quercetin 6h after RV infection (after viral endocytosis) reduced viral load, IL-8 and IFN responses in airway epithelial cells. This was associated with decreased levels of negative and positive strand viral RNA, and RV capsid protein, abrogation of RV-induced eIF4GI cleavage and increased phosphorylation of eIF2α. In mice infected with RV, quercetin treatment decreased viral replication as well as expression of chemokines and cytokines. Quercetin treatment also attenuated RV-induced airway cholinergic hyperresponsiveness. Together, our results suggest that quercetin inhibits RV endocytosis and replication in airway epithelial cells at multiple stages of the RV life cycle. Quercetin also decreases expression of pro-inflammatory cytokines and improves lung function in RV-infected mice. Based on these observations, further studies examining the potential benefits of quercetin in the prevention and treatment of RV infection are warranted.

Copyright © 2012 Elsevier B.V. All rights reserved.

Figures

Fig. 1

Cells pretreated with quercetin show attenuated RV-stimulated IL-8 responses. BEAS-2B cells were pretreated with DMSO or quercetin for 1 h. Cells were infected with sham, UV-RV1B, RV1B, UV-RV39 or RV39 in the presence of DMSO or quercetin and incubated for 24 h. IL-8 in the media was measured by ELISA. Data represent average and SD calculated from three independent experiments (∗p ⩽ 0.05, different from sham; †p ⩽ 0.05, different from media or DMSO treated cells).

Fig. 2

Quercetin pretreatment decreases RV endocytosis and RV-induced Akt phosphorylation. BEAS-2B cells were pretreated with 10 μM quercetin or DMSO and incubated with sham, UV-RV1B, RV1B, UV-RV39 or RV39 for 30 min at 4 °C, washed to remove unbound virus and cells were further incubated for 30 min at 37 °C. Endocytosed virus was detected using antibody to RV followed by flow cytometry (A and B). Data represent average and SD calculated from three independent experiments (∗p ⩽ 0.05, different from sham; †p ⩽ 0.05, different from media or DMSO treated cells). (C) Cells were pretreated with DMSO or quercetin and incubated with RV1B for 30 min. Cells were washed and incubated with antibody to RV capsid protein (green) and EEA1 antibody (red). Nuclei were counter-stained with DAPI (blue) and cells were visualized by confocal microscopy. Arrows represent co-localization of RV1B with EEA1. Cells pretreated with quercetin were infected with sham or RV1B and incubated for 30 min. Total cell lysates were subjected to Western blot analysis with antibodies to p-Akt or total Akt (D). Images and blot are representative of three independent experiments.

Fig. 3

Addition of quercetin 6 h post RV infection attenuates virus-stimulated IFN responses in airway epithelial cells. BEAS-2B cells were infected with sham, UV-RV1B, RV1B, UV-RV39 or RV1B, and incubated for 90 min. Infection medium was replaced with fresh medium and incubated for another 4.5 h. DMSO or 10 μM quercetin was added to the cells and incubation continued for another 18 h. Levels of IFN mRNA (panels A–C) and IL-8 protein (D) were determined by qPCR and ELISA, respectively. Expression of IFN genes were normalized to G3PDH and then expressed as fold change over sham-infected cells. Data represent average and SD calculated from three independent experiments (∗p ⩽ 0.05, different from UV-RV in (A–C); and different from sham in (D); †p ⩽ 0.05, different from media or DMSO treated cells).

Fig. 4

Quercetin added 6 h after RV infection decreases viral load and replication. Cells were infected and treated with quercetin as described in Fig. 3. Cells along with media was collected at 6 (just before adding quercetin) or 24 h (18 h after addition of quercetin or DMSO) after RV infection and infectious viral load was determined (A). Total RNA extracted from cells incubated for a total of 24 h post RV infection was used for determination of vRNA (B). The amplified qPCR product of negative strand RNA was subjected to agarose gel electrophoresis to show the amplified negative-strand vRNA (C). This result was representative of three experiments (∗p ⩽ 0.05, different from UV-RV in (A and B); and different from sham in (D); †p ⩽ 0.05, different from media or DMSO treated cells).

Fig. 5

Quercetin inhibits cleavage of eIF4GI, increases phosphorylation of eIF2α and decreases levels of VP2. BEAS-2B cells were infected with RV and incubated for 90 min. Infection medium was replaced with fresh media, incubated for 4.5 h and then quercetin (10 μM) (or DMSO) were added to the cells. After a total of 24 h, total protein was isolated and subjected to Western blot analysis using antibody to eIF4GI (A), eIF2α (B) or VP2 (C). Blots are representative of three independent experiments.

Fig. 6

Withdrawal of quercetin several hours before RV infection does not limit rhinovirus infectivity in vitro. BEAS-2B cells were treated with DMSO or 10 μM quercetin overnight. Cells were shifted to fresh media without quercetin or DMSO and incubated for 8 h. Cells were then infected in the absence of quercetin, and incubated for 18 h. Infectious viral load was determined as described in Fig. 4 (A). vRNA and IFN responses (B–E) were determined by qPCR. IL-8 protein levels were determined by ELISA (F). Data represent average and SD values calculated from three independent experiments (∗p ⩽ 0.05, different from sham infected cells).

Fig. 7

Quercetin decreases viral load in vivo. C57BL/6 mice were infected with RV1B or sham by the intranasal route. Two hours after RV infection, mice were orally gavaged with quercetin (0.2 mg/mouse) or 50% propylene glycol (PG) and then once a day up to 4 days. Mice were sacrificed and infectious viral load (A) was determined by measuring CCID50/ml. Positive strand vRNA (B) was determined by qPCR and negative strand vRNA (C) was detected by qPCR followed by agarose gel electrophoresis. (A and B) Data represent geomean and range of data from two independent experiments carried out in triplicate (†p ⩽ 0.05, different from mice infected with RV1B infected and treated with PG). (C) PCR product from two representative animals from PG and quercetin group showing negative-strand vRNA and 18S RNA.

Fig. 8

Quercetin decreases RV-stimulated chemokine and cytokine responses in vivo. Mice were infected with RV1B and treated with quercetin or vehicle as described in the Fig. 5 legend. After sacrifice, mouse lungs were excised, homogenized in PBS, centrifuged and supernatant examined by ELISA (A–D) or total RNA was extracted from the lungs and mRNA expression of IFN-α and IFN-λ1 was measured by qPCR (E and F). Data represent mean ± SD calculated from triplicate experiments (∗p ⩽ 0.05, different from sham; †p ⩽ 0.05, different from mice infected with RV and treated with vehicle).

Fig. 9

Compared to vehicle-treated animals, RV-infected mice treated with quercetin show decreased airways responsiveness to methacholine. Mice were infected with RV1B and then treated with DMSO or quercetin as described above. After 1 day, mice were anesthetized and airways responsiveness to methacholine challenge was measured (n = 3; ∗p ⩽ 0.05, different from sham infected mice; †p ⩽ 0.05, different from mice infected with RV1B and treated with vehicle, two-way ANOVA).

Fig. 10

Treatment of quercetin during RV infection is necessary to limit RV-stimulated cytokine responses. Mice were treated with quercetin or vehicle as described in Fig. 7 for 10 days. Forty hours after the last quercetin or vehicle treatment, mice were infected with RV1B or sham and sacrificed 24 h post-infection and lungs harvested for determination of cytokine proteins by ELISA (A–D) or IFN mRNA expression by qPCR (E–G). Data represent mean ± SD calculated from triplicate experiments (n = 5, ∗p ⩽ 0.05, different from sham, ANOVA).

Fig. 11

Treatment of quercetin during RV infection is necessary to reduce viral load and improve lung function. Mice were treated with quercetin or vehicle as described in Fig. 7 for 10 days. Forty hours after the last quercetin or vehicle treatment, mice were infected with RV1B or sham. After 1 day, some mice sacrificed, lungs harvested for determination of vRNA (A) by qPCR and infectious viral load (B). Rest of the mice were anesthetized and airways responsiveness to methacholine challenge was measured (C) (n = 3–5, data in (A and B) represents range and median; ∗p ⩽ 0.05, different from sham infected mice, two-way ANOVA).