Resolvin E1 Reverses Experimental Periodontitis and Dysbiosis

Abstract

Periodontitis is a biofilm-induced inflammatory disease characterized by dysbiosis of the commensal periodontal microbiota. It is unclear how natural regulation of inflammation affects the periodontal biofilm. Promoters of active resolution of inflammation, including resolvin E1 (RvE1), effectively treat inflammatory periodontitis in animal models. The goals of this study were 1) to compare periodontal tissue gene expression in different clinical conditions, 2) to determine the impact of local inflammation on the composition of subgingival bacteria, and 3) to understand how inflammation impacts these changes. Two clinically relevant experiments were performed in rats: prevention and treatment of ligature-induced periodontitis with RvE1 topical treatment. The gingival transcriptome was evaluated by RNA sequencing of mRNA. The composition of the subgingival microbiota was characterized by 16S rDNA sequencing. Periodontitis was assessed by bone morphometric measurements and histomorphometry of block sections. H&E and tartrate-resistant acid phosphatase staining were used to characterize and quantify inflammatory changes. RvE1 treatment prevented bone loss in ligature-induced periodontitis. Osteoclast density and inflammatory cell infiltration in the RvE1 groups were lower than those in the placebo group. RvE1 treatment reduced expression of inflammation-related genes, returning the expression profile to one more similar to health. Treatment of established periodontitis with RvE1 reversed bone loss, reversed inflammatory gene expression, and reduced osteoclast density. Assessment of the rat subgingival microbiota after RvE1 treatment revealed marked changes in both prevention and treatment experiments. The data suggest that modulation of local inflammation has a major role in shaping the composition of the subgingival microbiota.

Figure 1. RvE1 treatment prevents and reverses alveolar bone loss

(A) Prevention of periodontitis: alveolar bone loss in the disease (vehicle + ligature) group prevented by RvE1 (0.1μg/μl). The area of exposed root surface and the distance between CEJ and alveolar bone level are outlined. (B) The area of bone loss in mm² and linear bone loss in distance from the CEJ to bone in mm are significantly reduced (~30–40%) in both the RvE1 groups (0.1μg/μl) and (0.5μg/μl). (n=4 in the no ligature group, the ligature + vehicle group, and the ligature+ RvE1 (0.1μg/μl) group; n=3 in the ligature+RvE1 (0.5μg/μl) group). (C) Regeneration of alveolar bone: established periodontitis was treated by topical application of RvE1 as a monotherapy. ~30–40% of lost bone was restored with RvE1 therapy. (D) The area of bone loss in mm² and linear bone loss in distance from the CEJ to bone in mm are significantly reduced (~30–40%) with RvE1 treatment, but alveolar bone loss in the vehicle group worsened. (n=6 in the no ligature group and the ligature alone group; n=3 in the ligature + vehicle group and the ligature + RvE1 group).

Figure 2. RvE1 treatment controls osteoclast activity

(A) Osteoclast density was defined as active osteoclast count (TRAP positive multinucleated osteoclasts) around the interproximal bone divided by the total area of the interproximal bone. Prevention of periodontitis: the osteoclast density in the RvE1 groups (0.1μg/μl) and (0.5μg/μl) is significantly lower than the ligature + vehicle group. (n=4 in the no ligature group, the ligature + vehicle group, and the ligature+ RvE1 (0.1μg/μl) group; n=3 in the ligature+RvE1 (0.5μg/μl) group). (B) Periodontal bone regeneration: RvE1 treatment reduced the osteoclast density by 60% compared to the vehicle group and the ligature alone group. (n=6 in the no ligature group and the ligature alone group; n=3 in the ligature + Vehicle group and the ligature + RvE1 group).

Figure 3. Impact of RvE1 on Periodontal Gene Expression

(A) A heat map of top 200 differentially expressed genes (absolute log2 fold change) compares periodontitis vs. health, Low dose RvE1 (0.1μg/μl) vs. periodontitis, and High dose RvE1 (0.5μg/μl) vs. periodontitis. The color intensity reflects the log 2 fold change levels for each comparison with red for up- and blue for down-regulation. Genes (in rows) were ordered based on the dendrogram derived from hierarchical clustering of log2 values of all rows, therefore, genes with similar differential expression patterns were grouped together. (B) Genes differentially expressed (mean fold change ≥ 2^0.6, adjusted p-value <0.1) in the comparison between disease (ligature + vehicle) and health (no ligature) and between RvE1 (0.5μg/μl) and disease (ligature+ vehicle). The genes are listed by the order of adjusted p-value in the comparison between disease and health. The p-values are adjusted using the Benjamini–Hochberg procedure. (C) Relative mRNA expression (2^−ΔΔCT) was used to analyze the gene expression levels of inflammation related genes in different groups. In the treatment experiment, gene expression of Cxcl1 in the RvE1 group is significantly lower than that in the vehicle group. Gene expression of Ptgs2 in the RvE1 group is significantly lower than that in the ligature alone group.

Figure 4. Shifts in subgingival microbiota associated with periodontal regeneration following RvE1 treatment

(A) Mean relative abundance difference between day 0 and day 20 is calculated by subtracting mean relative abundance at baseline (day 0) from mean relative abundance at 20 days post ligature. Positive values indicate the mean relative abundance on day 20 is larger than the mean relative abundance on day 0. OTUs that do not match any known genera in the reference data base are named at the family level. The most notable changes are the loss of Rothia spp. And the overgrowth of Streptococcus spp. as disease develops. (B)(C) Microbial shifts associated with the periodontal bone regeneration induced by RvE1 (0.25 μg/ml) treatment. Day 32-Panel B and day 40-Panel C are shown. Positive values indicate the mean relative abundance of the RvE1 group is larger than the mean relative abundance of the Vehicle group. Notably, most of the genera with increased relative abundance after disease induction exhibit a trend to return to level more associate with a healthy periodontium. (n=6 in the no ligature group and the ligature alone group; n=3 in the ligature + Vehicle group and the ligature + RvE1 group)”.

Figure 5. Shifts in subgingival microbiota composition during periodontal disease initiation and development

Prevention of periodontitis: shifts in the composition of the microbiota assessed by 16S rDNA sequencing were assessed during development of ligature induced periodontitis and compared to the low and high doses treatment with RvE1 during disease induction. (A)(B) Mean relative abundance difference between the 0.1μg/μl- Panel A, or 0.5μg/μl- Panel B. RvE1 treatment and vehicle treated control is calculated by subtracting mean relative abundance of the vehicle treatment from mean relative abundance of the RvE1treated doses at 14 days post ligature. Positive values indicate the mean relative abundance of the RvE1 group is larger than the mean relative abundance of the vehicle group. The family name of the bacterium is listed for the OTUs that are not assigned to any known genus. (C)(D) Mean relative abundance differences on day 28 for the two doses of RvE1 compared to vehicle. RvE1 treatment induces major shifts in the subgingival microbiota. Over time, RvE1 treatment tends to shift the microbiota toward a composition more associated with periodontal health. (n=4 in the no ligature group, the ligature + vehicle group, and the ligature+ RvE1 (0.1μg/μl) group; n=3 in the ligature+RvE1 (0.5μg/μl) group).