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Quercetin Preserves Oral Cavity Health by Mitigating Inflammation and Microbial Dysbiosis - PubMed

  • ️Fri Jan 01 2021

Quercetin Preserves Oral Cavity Health by Mitigating Inflammation and Microbial Dysbiosis

Erin C Mooney et al. Front Immunol. 2021.

Abstract

Failure to attenuate inflammation coupled with consequent microbiota changes drives the development of bone-destructive periodontitis. Quercetin, a plant-derived polyphenolic flavonoid, has been linked with health benefits in both humans and animals. Using a systematic approach, we investigated the effect of orally delivered Quercetin on host inflammatory response, oral microbial composition and periodontal disease phenotype. In vivo, quercetin supplementation diminished gingival cytokine expression, inflammatory cell infiltrate and alveolar bone loss. Microbiome analyses revealed a healthier oral microbial composition in Quercetin-treated versus vehicle-treated group characterized by reduction in the number of pathogenic species including Enterococcus, Neisseria and Pseudomonas and increase in the number of non-pathogenic Streptococcus sp. and bacterial diversity. In vitro, Quercetin diminished inflammatory cytokine production through modulating NF-κB:A20 axis in human macrophages following challenge with oral bacteria and TLR agonists. Collectively, our findings reveal that Quercetin supplement instigates a balanced periodontal tissue homeostasis through limiting inflammation and fostering an oral cavity microenvironment conducive of symbiotic microbiota associated with health. This proof of concept study provides key evidence for translational studies to improve overall health.

Keywords: A20; IL-6; NF-κB; TNF; flavonoids; microbiome; periodontal diseases; toll-like receptor (TLR).

Copyright © 2021 Mooney, Holden, Xia, Li, Jiang, Banson, Zhu and Sahingur.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1

Quercetin Treatment Decreases Periodontal Inflammation and Bone Loss. (A) Schematic presentation demonstrating the experimental design system. In male C57BL/6J mice, silk 5-0 ligatures were placed on the left side of the maxilla interdentally between first and second molars, while the right sides were left unligated as controls. Mice were treated with Quercetin by oral gavage or vehicle matched control six days prior to ligature placement and maintained with the same treatment regimen one-week post-ligation. Mice were euthanized and periodontal tissues were examined. (B) Representative images of 3D micro-CT reconstructions of periodontal bone in Quercetin treated and vehicle treated mice. The distance between horizontal lines represents the distance between cemental-enamel junction and alveolar bone crest. (C) Representative two-dimensional images of sagittal slice views of Quercetin-treated and vehicle-treated mice. Linear measurements were taken of the alveolar bone loss (ABL) in the interdental space from the cemental-enamel junction to alveolar bone crest. (D) Distance between the cemental-enamel junction and alveolar bone crest was analyzed and plotted to determine periodontal bone loss (n=37 mice for Quercetin treatment, n=21 mice for vehicle treatment). The average of 4 CEJ-ABC measurements were calculated per mouse. Individual mouse datapoints are plotted. Averages and standard deviations are shown. ****p ≤ 0.0001. ####Ligated gingival tissues versus corresponding experimental controls (p ≤ 0.0001). (E) Representative images from H&E stained histological sections from gingival tissues derived from ligated and control periodontium in Quercetin-treated and vehicle-treated mice. (F) Quantitative analysis of inflammatory cell infiltrate in the periodontium. Positive cells were counted per µm2 gingival connective tissue. Data are representative of three independent experiments, with a minimum of seven mice analyzed in each treatment group per experiment. Averages and standard deviations are shown. **p ≤ 0.01, ****p ≤ 0.0001. (G, H) Gingival tissues derived from ligated and control periodontium in Quercetin treated and vehicle treated mice were digested and applied to RNA preparation. The mRNA levels of mTNF (G), mIL-6 (H) were determined with qRT-PCR and the relative mRNA expression levels were plotted (n=11 for vehicle treatment, n=26 for Quercetin treatment). Averages and standard deviations are shown. **p ≤ 0.01, ***p<0.001, ****p ≤ 0.0001. ####Ligated gingival tissues versus corresponding experimental controls (p ≤ 0.0001). ###Ligated gingival tissues versus corresponding experimental controls (p ≤ 0.001). ##Ligated gingival tissues versus corresponding experimental controls (p ≤ 0.01).

Figure 2
Figure 2

Quercetin Treatment Increases Bacterial Diversity in Mice with Experimental Periodontitis. (A) Schematic diagram illustrating the periodontal induction and treatment experimental design and timeline of oral swab collection for subsequent 16S rRNA sequencing microbiome analysis. (B, C) Alpha diversity was assessed in the oral microbiota of 5 vehicle- and 13 Quercetin-treated mice before and after the induction of periodontitis and the Evenness (B) and Shannon index (C) of vehicle- and Quercetin-treated mice on Day 1, 6 and 13 are shown. *p ≤ 0.05, **p ≤ 0.01. (D, E) Dissimilarity was assessed in the oral microbiota of 5 vehicle- and 13 Quercetin-treated mice before and after periodontitis induction and the ordination is shown by the t-Distributed Stochastic Neighbor Embedding (t-SNE) plot (D). The Bray-Curtis dissimilarity between vehicle- and Quercetin-treated mice in Days 1 (left), 6 (middle) and 13 (right) are shown (E). ***p ≤ 0.001.

Figure 3
Figure 3

Quercetin Treatment Alters Microbial Composition Induced by Periodontitis. (A–C) Prominent bacterial taxa in 5 vehicle- and 13 Quercetin-treated mice at Day 1 (A), Day 6 (B) and Day 13 (C). (D) Significantly altered taxa in Quercetin treated compared to vehicle treated mice after periodontitis onset (Day 13) as determined by linear discriminant analysis effect size (LDA) scores using Silva-132 classifier.

Figure 4
Figure 4

Quercetin diminishes cytokine production in human macrophages challenged with oral bacteria and toll-like receptor agonists. THP-1 macrophages treated with Quercetin (5µg/mL) or vehicle matched control were infected with oral bacteria or toll-like receptor agonists for up to 24 hours. (A–E) IL-6 and TNF levels in the supernatants were determined with ELISA. ELISA was conducted on (A) macrophages infected with P. gingivalis (100 MOI), (B) macrophages infected with F. nucleatum (50 MOI), (C) macrophages infected with P. gingivalis LPS (10µg/mL), (D) macrophages infected with Pam3CSK4 (10ng/mL) and (E) macrophages infected with ODN 2006 (100µg/mL). Each experiment was performed at minimum three times independently and averages and standard deviations are shown. ****p ≤ 0.0001. ####Unstimulated control cells versus corresponding stimulated cells (p ≤ 0.0001). ###Unstimulated control cells versus corresponding stimulated cells (p ≤ 0.001).

Figure 5
Figure 5

Quercetin decreases NF-κB nuclear translocation upon LPS-and oral bacteria-induced inflammation in human macrophages. THP-1 macrophages treated with Quercetin (5µg/mL) or vehicle matched control were infected with LPS or oral bacteria for indicated times. NF-κB was stained with NF-κB antibody followed by Alexa-Fluor488 (green) and the nucleus was stained with DAPI (blue). Images were captured with Nikon Confocal laser microscope. (A) Representative images of vehicle- and Quercetin-treated cells infected with F. nucleatum (25 MOI) (C) and P. gingivalis (50MOI) and the quantitative analysis of nuclear to cytosolic ratio of NF-κB was shown, respectively (B, D). Scale bars=10µM. Data are representative of three independent experiments and averages and standard deviations are shown. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001. ####Unstimulated control cells versus corresponding stimulated cells (p ≤ 0.0001).

Figure 6
Figure 6

Quercetin modulates A20 mRNA levels in pre-clinical periodontitis disease models. (A) THP-1 macrophages treated with Quercetin (5µg/mL) or vehicle matched control were infected with oral bacteria (F.nucleatum [10 MOI]) or toll-like receptor agonist (Pam3CSK4 [10ng/mL]) for up to 6 hours and applied to RNA preparation. The mRNA levels of mA20 were determined with qRT-PCR and the relative mRNA expression levels were plotted. Each experiment was performed three times independently and averages and standard deviations are shown. *p ≤ 0.05. (B) Gingival tissues derived from ligated and control periodontium in Quercetin treated and vehicle treated mice were digested and applied to RNA preparation. The mRNA levels of mA20 were determined with qRT-PCR and the relative mRNA expression levels were plotted (n=11 for vehicle treatment, n=26 for Quercetin treatment). Averages and standard deviations are shown. *p ≤ 0.05. #Ligated gingival tissues versus corresponding experimental controls.

Figure 7
Figure 7

Quercetin supplement alleviates the progression of periodontitis by aiding in the preservation of host- and microbiome-homeostasis. A self-perpetuating pathogenic cycle of deregulated inflammation, exacerbated cytokine release, and bacterial dysbiosis fuels tissue destruction and the subsequent development of periodontitis in response to ligature placement. Quercetin, a ubiquitous polyphenolic flavonoid, promotes health through various biological mechanisms. Upon periodontitis induction, Quercetin supplement reduced gingival inflammation, alveolar bone loss and limits bacterial dysbiosis. At the cellular level, periodontal tissue homeostasis requires the stringent regulation of inflammatory pathways mainly driven by the activation of innate sensors, such as TLRs. Downstream of TLR activation, Quercetin supplement decreased cytokine production by regulating NF-κB activation and possibly modulating A20 expression, in response to oral bacteria and TLR agonist challenge. Collectively, the regulation of inflammation with Quercetin supplement has a significant impact on gingival inflammatory gene expression and bacterial composition that is coincident with ameliorating disease progression.

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