The Ability of Resveratrol to Attenuate Ovalbumin-Mediated Allergic Asthma Is Associated With Changes in Microbiota Involving the Gut-Lung Axis, Enhanced Barrier Function and Decreased Inflammation in the Lungs

Abstract

Asthma is a chronic respiratory disease highly prevalent worldwide. Recent studies have suggested a role for microbiome-associated gut-lung axis in asthma development. In the current study, we investigated if Resveratrol (RES), a plant-based polyphenol, can attenuate ovalbumin (OVA)-induced murine allergic asthma, and if so, the role of microbiome in the gut-lung axis in this process. We found that RES attenuated allergic asthma with significant improvements in pulmonary functions in OVA-exposed mice when tested using plethysmography for frequency (F), mean volume (MV), specific airway resistance (sRaw), and delay time(dT). RES treatment also suppressed inflammatory cytokines in the lungs. RES modulated lung microbiota and caused an abundance of Akkermansia muciniphila accompanied by a reduction of LPS biosynthesis in OVA-treated mice. Furthermore, RES also altered gut microbiota and induced enrichment of Bacteroides acidifaciens significantly in the colon accompanied by an increase in butyric acid concentration in the colonic contents from OVA-treated mice. Additionally, RES caused significant increases in tight junction proteins and decreased mucin (Muc5ac) in the pulmonary epithelium of OVA-treated mice. Our results demonstrated that RES may attenuate asthma by inducing beneficial microbiota in the gut-lung axis and through the promotion of normal barrier functions of the lung.

Keywords: asthma; gut microbiome; lung microbiome; ovalbumin; resveratrol.

Figure 1

Effect of resveratrol on physiological functions of the lungs in OVA-administered mice. BALB/c mice were initially sensitized by intraperitoneal injection of ovalbumin followed by an intranasal challenge on day 7. These mice received vehicle (Ova-veh) or RES (Ova-res) on days 1-14. On day 15, the mice were chosen randomly and placed in one of 4 chambers of Buxco instrument for pulmonary function analysis or sacrificed for other examination. (A) Pulmonary function tests included changing patterns of frequency (F), mean volume (MV), specific airway resistance (sRaw) and delay time (dT) among naïve, Ova-veh and Ova-res group as baseline response (BL, green arrow), PBS exposure response (red arrow), and 25 mg/ml methacholine response (Mch, red arrow). Bar graphs present the statistical comparison of frequency, MV, sRaw and dT among naïve, Ova-veh and Ova-res group at the baseline response (B) and after challenge with 25 mg/mL methacholine (C). (D) Immunofluorescent staining showing the expression of MUC5ac protein in lung sections of naïve, Ova-veh and Ova-res group mice observed under Leica microscope. Statistical comparison of MUC5ac protein expression among naïve, Ova-veh and Ova-res group (E). RT-qPCR data to measure the expression of tight junction proteins, occludin, claudin-18, E-cadherin and ZO-1 in purified epithelial cells from mice (F). Vertical bars in all panels represent Mean+/-SEM data from groups of 5 mice. *p<0.05, **p<0.01, ***p<0.001, ^#p<0.0001.

Figure 2

Effect of resveratrol on lung microbiota. OVA and RES treatments were performed as described in Figure 1 legend. The lung microbiota profile was conducted as described in Methods. (A) Principal coordinator analysis showing the specific distribution of lung microbiota in naïve, Ova-veh and Ova-res groups. (B) Pie charts showing the percentages of common bacterial phyla in the lung microbiota among naïve, Ova-veh and Ova-res groups. (C) cladogram showing the least significant discriminative changes of lung microbiota between Ova-veh and Ova-res group with at least 2-fold changes. (D) qPCR showing the abundance of Akkermansia muciniphila in mouse lung microbiota among naïve, Ova-veh and Ova-res groups. Vertical bars in panel D represent Mean+/-SEM data from groups of 5 mice. In panel (A), 2 mice out of 5 were excluded by Nephele setting). ^#p<0.0001.

Figure 3

Effect of resveratrol on gut microbiota. The gut microbiota profile was conducted as described in Methods. (A) Principal coordinator analysis shows the specific distribution of gut microbiota in the mice belonging to the naïve, Ova-veh and Ova-res groups. (B) Pie charts showing the percentages of common bacterial phyla in the gut microbiota among naïve, Ova-veh and Ova-res groups. (C) Cladogram showing the least significant discriminative changes of gut microbiota between Ova-veh and Ova-res group with at least 2-fold changes. (D) qPCR was used to detect the abundance of Bacteroides acidifaciens in mouse gut microbiota among naïve, Ova-veh and Ova-res groups. Mass spectrometry–gas chromatography analysis showing the differences of butyric acid (E), and acetic acid, and propionic acids (F) concentrations in mouse colonic materials among naïve, Ova-veh and Ova-res groups. Vertical bars represent Mean+/-SEM data from groups of 5 mice. *p<0.05, **p<0.01, ^#p<0.0001.

Figure 4

Effect of resveratrol on LPS metabolism and cytokine production. OVA and RES treatments were performed as described in Figure 1 legend. (A) Heat map showing the predicted metagenomic functions of lung microbiota between Ova-veh and Ova-res group. The predicted biological functions were normalized to the mean OTUs of naïve group. (B) LPS concentrations in bronchoalveolar lavage fluid (BALF) were measured in Ova-veh and Ova-res groups. (C) RT-qPCR assay to measure gene expression of inflammatory and anti-inflammatory cytokines in the isolated mononuclear cells from mouse lungs of Ova-veh and Ova-res groups. ELISA measurements of different cytokine levels in the serum (D) and BALF (E) of OVA-exposed mice treated either with VEH or RES. Vertical bars in all panels represent Mean+/-SEM data from groups of 5 mice. In panel (A), 1 mouse out of 5 in Ova-veh and Ova-res groups were excluded by Nephele setting). ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ^#p<0.0001.

Figure 5

Effect of RES on the integrity and functions of mouse epithelial cells. MLE-15 mouse epithelial cell layer was exposed to LPS and treated with either RES or VEH. (A) Transepithelial electrical resistance (TEER) measurement system was used in the analysis of the integrity and permeability of cultured MLE-15 mouse epithelial cell layer exposed to LPS and treated with either RES or VEH. RT-qPCR was used to detect the gene expression in the LPS-treated MLE-15 cells incubated with RES or VEH. The genes studied included: (B) MyD88, (C) Tight junction proteins (claudin, E-Cad, occuludin, ZO-1, ZO-2 and ZO-3), (D) Cytokines and chemokines (CCR2, IL-10, IL-13, IL-1β, IL-2, IL-4, IL-4, IL-6, TGF-β and TNF-α). (E) ELISA was used in the measurement of cytokines (IL-1β, IL-4, IL-6, IL-10, IL-13, TGF-β, IFN-γ and TNF-α) in the supernatants of MLE-15 cell cultures in the presence of LPS and RES or VEH (E). ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ^#p<0.0001.