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F U L L T E X T S O U R C E : bioRxv
Abstract
Periodontal disease is an age-associated disorder clinically defined by periodontal bone loss, inflammation of the specialized tissues that surround and support the tooth, and microbiome dysbiosis. Currently, there is no therapy for reversing periodontal disease, and treatment is generally restricted to preventive measures or tooth extraction. The FDA-approved drug rapamycin slows aging and extends lifespan in multiple organisms, including mice. Here we demonstrate that short-term treatment with rapamycin rejuvenates the aged oral cavity of elderly mice, including regeneration of periodontal bone, attenuation of gingival and periodontal bone inflammation, and revertive shift of the oral microbiome toward a more youthful composition. This provides a geroscience strategy to potentially rejuvenate oral health and reverse periodontal disease in the elderly.
Single Sentence Summary Short-term treatment with rapamycin reverses periodontal bone loss, attenuates inflammation, and remodels the oral microbiome toward a more youthful state.
Main Text
Old age is associated with failure to maintain homeostasis resulting in degradation of cellular maintenance and repair processes (1) and is the single greatest risk factor for many human diseases including cardiovascular disorders, dementias, diabetes, and most cancers (2, 3). Interventions that target specific aging hallmarks have been shown to delay or prevent age-related disorders and extend lifespan in model organisms (4). Rapamycin, an FDA approved drug which directly inhibits the mechanistic target of rapamycin complex I (mTORC1), is one such intervention that extends lifespan and ameliorates a variety of age-related phenotypes (5). In mice, rapamycin extends lifespan when administered beginning at 9 or 20 months of age (6), and short-term treatments ranging from 6-12 weeks during adulthood have been shown to increase lifespan (7), improve cardiac function (8, 9) and restore immune function as measured by vaccine response (10). Initial indications suggest that mTORC1 inhibition can also reverse declines in age-related heart function in companion dogs and age-related immune function (11, 12) and skin aging (13) in humans.
The American Academy of Periodontology (AAP) defines periodontitis as inflammation of the periodontium, the specialized tissue surrounding and supporting the tooth structure, resulting in clinical attachment loss, alveolar (periodontal) bone loss and periodontal pocketing, and associated variability in the oral microbial (14). Most recent epidemiologic data in the U.S. population suggests that more than 60% of adults aged 65 years and older have periodontitis (15, 16), and diagnosis with periodontal disease is associated with increased risk for other age-related conditions including heart disease, diabetes, and Alzheimer’s disease (17-19). We have previously observed that aged mice treated with rapamycin have greater levels of periodontal bone than control animals (20), suggesting that inhibition of mTOR may delay age-related periodontal disease.
In order to understand potential mechanisms by which aging and mTOR activity influence oral health, we assessed whether transient rapamycin treatment is sufficient to regrow periodontal bone in aged animals. Two cohorts of animals were used for these studies: NIA-UW mice housed at the University of Washington and JAX mice housed at The Jackson Laboratory (see Methods). We used microCT (μCT) imaging to measure the amount of periodontal bone present in the maxilla and mandible of young (6 month), adult (13 month), and old (20 month) mice from the NIA-UW cohort (fig. S1). The amount of periodontal bone for the maxilla and mandible of each animal was calculated as the distance from the cementoenamel junction (CEJ) to alveolar bone crest (ABC) for 16 landmarked sites each on the buccal aspect of the maxillary and mandibular periodontium (fig. S2). Thus, larger values represent greater bone loss. As expected, there was a significant loss of periodontal bone with age in the NIA-UW cohort (Fig. 1, A and B). Mice treated with rapamycin for 8 weeks had significantly more bone at the end of the treatment period compared to mice that received the control diet (eudragit). To determine whether the increase in periodontal bone upon rapamycin treatment reflects attenuation of bone loss or growth of new bone, we performed μCT imaging on mice before and after treatment in the JAX cohort. Old mice randomized into either the eudragit control or rapamycin treatment groups had significantly less periodontal bone than young mice prior to the treatment period (Fig. 1F). After 8 weeks, the rapamycin treated mice had significantly more periodontal bone compared to eudragit controls and also compared to the pre-treatment levels for the same animals (Fig. 1, D to F). The presence of new bone following rapamycin treatment can be observed by comparison of μCT images from the same animals before and after treatment (Fig. 1, D and E).
Fig. S1.
Cross-Institution Experimental Design
The NIA-UW colonies were received directly from the NIA Aged Rodent Colony at 4, 11, and 18-months, then acclimated for two months within the UW facilities (ARCF) until they reached 6 (Young), 13 (Adult), and 20-months (Old). The Young and Adult cohorts were harvested for oral tissues and microbiome. The Old cohorts were randomized and either given Eudragit or 42ppm eRAPA within the food for 8 weeks. For the JAX colonies, an initial microCT image was taken prior to the 8-week treatment and then a final microCT before harvest. All animals were harvested at the end of 8 weeks, ∼22-months old.
Fig. S2.
Assay for Measuring Periodontal Bone Loss.
Representative image of a mandible is shown. Periodontal bone loss was measured as distance from the cementoenamel junction (CEJ, white arrows) to alveolar bone crest (ABC, orange arrows) on 16 predetermined landmarks on the buccal aspect of maxillary and mandibular periodontium. The CEJ-ABC distances were totaled for each mouse.
Fig. 1.
Rapamycin reverses age-associated periodontal bone loss (NIA-UW and JAX).
(A and B) Representative images of NIA-UW (A) maxillary and (B) mandibular teeth of Young, Old, and Old treated with 42ppm eRAPA (rapamycin) revealing age-associated periodontal bone loss. 8 weeks of rapamycin attenuated periodontal bone loss. © Box-and-whiskers plots shows median, 25th and 75th percentile with whiskers at the 5th and 95th percentile. Statistical analysis was completed using unpaired t-test, with p-values < 0.05 were considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.005 (D and E) Representative images of the (D) maxillary and (E) mandibular teeth from the same animal in the JAX cohort before treatment (labeled Old) and after 8 weeks of 42ppm eRAPA (labeled Old+Rapamycin). On both the maxilla and mandible, there is periodontal bone loss around and in-between the molars, but after 8 weeks of 42ppm eRAPA the bone loss is reversed. White arrowheads indicate areas of bone loss and bone loss reversal (F) Box-and-whiskers plots shows median, 25th and 75th percentile with whiskers at the 5th and 95th percentile. Longitudinal comparison was completed with the same animal at baseline or after 8 weeks with either eudragit (control) or 42ppm eRAPA (rapamycin). Statistical analysis was completed using paired t-test, with p-values < 0.05 were considered statistically significant. * p<0.05, ** p<0.01.
Normal bone homeostasis results from a balance between new bone growth and bone resorption, which is reflected by the ratio of RANKL (receptor-activator of nuclear factor-κB ligand) to OPG (osteoprotegerin), and dysregulation of this balance contributes to bone loss in periodontitis (21). Consistent with bone loss during aging, we detected significantly greater levels of RANKL in old animals of both cohorts compared to young animals (Fig. 2, A and B) OPG levels remained relatively stable, resulting in an increase in the RANKL:OPG ratio indicative of bone resorption exceeding bone formation (Fig. 2C). These age-associated defects in bone homeostasis were suppressed by eight weeks of rapamycin treatment (Fig. 3). In addition to increased RANKL:OPG ratio, a significant increase in TRAP+ cells was also observed in periodontal bone with age (Fig. 3, D and E). TRAP (tartrate-resistant acid phosphatase) is a histochemical marker of bone resorbing osteoclasts (22, 23). Rapamycin treatment for eight weeks also decreased TRAP+ cells. Together, our data indicate that rapamycin reverses periodontal bone loss in the aging murine oral cavity at least in part through inhibition of bone resorption.
Fig. 2.
Rapamycin attenuates age-associated increase in RANKL expression and TRAP+ cells in periodontal bone.
(A and B) RANKL and OPG expression was determined by Western blot analysis of total lysates from the periodontal bone of aged animals (Young, Adult, and Old) and Old animals treated for 8 weeks with 42ppm rapamycin (eRAPA). The periodontal bone within both the NIA-UW and JAX Colonies showed an increased expression of RANKL while 8 weeks of rapamycin treatment ameliorated the increased RANKL expression. Each lane represents individual periodontal bone samples. © Quantification of RANKL/OPG of the NIA-UW Western blot analysis. (D) Representative histological sections of the alveolar bone furcation that have undergone TRAP azo-dye staining with FastGreen counterstain. (E) Enumeration of TRAP+ cells within the periodontal bone from two-independent observers reveals an increase number of TRAP+ cells with age and diminishes with rapamycin treatment. Statistical analysis completed with unpaired t-test, with significance set to p<0.05. * p<0.05, ** p<0.01, *** p<0.005.
Fig. 3.
Rapamycin alters increased NF- κB expression and inflammatory cytokine profiles in periodontium
(A and C) NF- κB p65 and IκBα expression was determined by Western blot analysis of total lysates from the gingiva and periodontal bone (B and D) of aged animals (Young, Adult, and Old) and Old animals treated for 8 weeks with rapamycin (42ppm eRAPA). GAPDH was used a loading control. Both in the aging gingiva and periodontal bone, there is an overall increased expression of NF- κ B p65 with corresponding alteration of IκBα or p- IκBα. 8 weeks of 42ppm eRAPA treatment attenuates the altered changes seen with the increased NF- κB p65 expression. For the gingiva, each lane represents gingiva from animals co-housed (n=1-2), and each lane for the periodontal bone western blot represents individual samples. (E and F) Protein expression levels of mouse cytokines and chemokines was determined by a spotted nitrocellulose membrane assay (Proteome Profiler Mouse, R&D Systems) by loading pooled samples from (E) gingiva and (F) periodontal bone of Young and Old (Control, Eudragit), and Old animals treated for 8 weeks with rapamycin (42ppm eRAPA). Data is shown per manufacture’s protocol, with fold-change relative to Young (Set to 1), expressed as mean ± SEM. Only data showing statistical significance set to p< 0.05, except CXCL16 and MCSF (E).
Along with bone loss, gingival inflammation is a defining feature of periodontal disease. Aging is also associated with chronic accumulation of pro-inflammatory factors, a collective term referred to as inflammaging (24-27). The nuclear factor-κB (NF-κB) is a hub of immune and inflammatory response activated both during normal aging and as a consequence of periodontal disease (28-31). We first evaluated the NF-κB hub through NF-κB p65 and IκBα expressions levels. The NF-κB heterodimer consists of RelA (or p65) and p50. IκBα functions as a negative regulator of NF-κB by sequestering it in the cytoplasm. Degradation of IκBα or phosphorylated-IκBα leads to nuclear localization of NF-κB subunits which induce expression of target inflammatory genes, such as TNF-α and IL-1β (31). In both the gingival tissue and periodontal bone, there was an increase in p65 expression with corresponding decrease of IκBα levels, indicating an age-associated increase in NF-κB inflammatory signaling in the periodontium (Fig. 3, A and B). Eight weeks of rapamycin treatment was sufficient to reverse these changes. We also examined the levels of inflammatory cytokines in the oral cavity associated with normative aging and rapamycin treatment in mice. Consistent with the increase in NF-κB signaling, we found elevated expression of several cytokines in both the gingival tissue and the periodontal bone (Fig. 3, E and F). Eight weeks of rapamycin treatment reversed most age-associated chemokine and cytokine changes in both the gingival tissue and periodontal bone. Thus, transient treatment with rapamycin during middle-age can largely restore a youthful inflammatory state in both the gingiva and periodontal bone of mice.
Dysbiotic shifts in the oral microbiome are thought to play a significant role in the progression of periodontal disease in humans. We and others have previously shown that rapamycin treatment can remodel the gut microbiome in mice (32-34); however, the effect of rapamycin on the oral microbiome has not been explored. Therefore, we sought to evaluate effects of rapamycin on the aged oral microbiome using 16S rRNA gene sequencing and Amplicon Sequence Variant (ASV) analysis approach. Examination of the alpha diversity of the oral cavity illustrated a significant increase in species richness during aging that rapamycin treatment impacted (Fig. 4A, fig. S3). Among the most notable alterations in taxonomic abundance between groups was the reduction of Bacteroidetes phylum that had increased with age but reduced in the rapamycin-treated old animals (Fig. 4B, fig. S4). The Bacteroidetes phylum consists of over 7000 different species (35) and includes bacteria associated with human periodontal disease such as Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus (36-38). Further, both the Firmicutes and Proteobacteria phyla also showed a significant difference that was age and treatment dependent (Fig. 4B). In order to assess whether rapamycin is shifting the composition back towards a youthful state, we evaluated the beta diversity using weighted UniFrac distances. We discovered a significant separation of the oral microbiome between old control and old rapamycin-treated animals, while no significant differences were observed between young mice and old rapamycin-treated mice (Fig. 4C). Overall, we observed no significant differences in alpha diversity, beta diversity, nor relative taxonomic abundance between young mice and old mice treated with rapamycin, suggesting an eight-week treatment with rapamycin reverted the old oral microbiome to a more youthful state. This observation is further supported when analysis of the samples is performed independently by facility (UW-NIA or JAX) (fig. S5).
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