Evaluation of off-label rapamycin use on oral health.

Rapamycin (sirolimus) is an FDA approved drug with immune modulating properties that is being prescribed off-label in adults as a preventative therapy to maintain healthspan. We recently published one of the first reports on 333 adults with a history of off-label rapamycin use. Along with presenting evidence that rapamycin can be used safely in adults of normal health status, we discovered that about 26% of rapamycin users also reported oral health changes. Given the recent evidence highlighting the potential benefits of rapamycin and its derivatives in enhancing oral health, we conducted a secondary data analysis to profile the oral health of off-label rapamycin users, the true incidence of mouth sores, and present specific case studies of periodontal bone loss quantification using an FDA-approved artificial intelligence platform. Contrary to expected findings and previous literature, dimensions of rapamycin usage (such as length of use, dosage, and interval) were not found to be related to the incidence of mouth ulcers in rapamycin users. Notably, among rapamycin users, the most deleterious forms of ulcers were found to be infrequent and not statistically linked to rapamycin usage, with most rapamycin users having a common transient form of mouth ulcers. Additionally, we describe the general oral health outcomes of off-label rapamycin users and provide recommendations for individuals engaging in off-label rapamycin to be regularly checked by a dentist or an oral health care provider. This report was limited by being a secondary data analysis taken from survey data that focused on a more holistic health model. Future studies will use a focused survey that collects data on more dimensions of oral health outcomes while including questions on oral health for non-rapamycin-using participants.

Evaluation of off-label rapamycin use to promote healthspan in 333 adults.

Rapamycin (sirolimus) is an FDA-approved drug with immune-modulating and growth-inhibitory properties. Preclinical studies have shown that rapamycin extends lifespan and healthspan metrics in yeast, invertebrates, and rodents. Several physicians are now prescribing rapamycin off-label as a preventative therapy to maintain healthspan. Thus far, however, there is limited data available on side effects or efficacy associated with use of rapamycin in this context. To begin to address this gap in knowledge, we collected data from 333 adults with a history of off-label use of rapamycin by survey. Similar data were also collected from 172 adults who had never used rapamycin. Here, we describe the general characteristics of a patient cohort using off-label rapamycin and present initial evidence that rapamycin can be used safely in adults of normal health status.

Engineered osteoclasts resorb necrotic alveolar bone in anti-RANKL antibody-treated mice.

Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of antiresorptive medications such as denosumab (humanized anti-RANKL antibody), yet its pathophysiology remains elusive. It has been posited that inhibition of osteoclastic bone resorption leads to the pathological sequelae of dead bone accumulation, impaired new bone formation, and poor wound healing in MRONJ, but this hypothesis has not been definitively tested. We previously engineered myeloid precursors with a conditional receptor activator of nuclear factor kappa-Β intracellular domain (iRANK cells), which differentiate into osteoclasts in response to a chemical inducer of dimerization (CID) independently of RANKL. In this study, we showed that CID-treated iRANK cells differentiated into osteoclasts and robustly resorbed mineralized surfaces even in the presence of anti-RANKL antibody in vitro. We then developed a tooth extraction-triggered MRONJ model in nude mice using anti-RANKL antibody to deplete osteoclasts. This model was used to determine whether reconstitution of engineered osteoclasts within sockets could prevent specific pathological features of MRONJ. Locally delivered iRANK cells successfully differentiated into multinucleated osteoclasts in response to CID treatment in vivo as measured by green fluorescent protein (GFP), tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, matrix metallopeptidase 9 (MMP-9), and cathepsin K staining. Sockets treated with iRANK cells + CID had significantly more osteoclasts and less necrotic bone than those receiving iRANK cells alone. These data support the hypothesis that osteoclast deficiency leads to accumulation of necrotic bone in MRONJ.

Rapamycin rejuvenates oral health in aging mice.

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.

Age is the single greatest risk factor for many human diseases, including cancer, heart disease, and dementia. This is because, as the body ages, it becomes less able to repair itself. One way to prevent age-related disease and extend lifespan, at least in laboratory animals, is to use a drug called rapamycin. Mice treated with rapamycin live longer, have stronger hearts, and respond better to vaccination. But, despite these promising observations, the use of rapamycin as an anti-aging treatment is still under investigation. One open question is what age-related diseases rapamycin can help to prevent or treat. In the United States, more than 60% of adults over the age of 65 have gum disease. These people are also more likely to have other age-related diseases, like heart disease or Alzheimer's. This association between gum problems and other age-related diseases prompted An et al. to ask whether it might be possible to treat gum disease by targeting aging. To find out whether rapamycin could improve gum health, An et al. performed three-dimensional CT scans on mice as they aged to measure the bone around the teeth. Some of mice were treated with rapamycin, while the rest received a placebo. The mice that received the placebo started to show signs of gum disease as they aged, including inflammation and loss of bone around the teeth. The types of bacteria in their mouths also changed as they aged. Treating mice with rapamycin not only delayed the onset of these symptoms, but actually reversed them. After eight-weeks of the drug, the older mice had lost less bone and showed fewer signs of inflammation. There was also a shift in their mouth bacteria, restoring the balance of species back to those found in younger mice. Rapamycin is already approved for use in people, so a clinical trial could reveal whether it has the same effects on gum health in humans as it does in mice. But there are still unanswered questions about how rapamycin affects the mouth as it ages. These include how the drug works at a molecular level, and how long the changes to gum health persist after treatment stops.

Oral health in geroscience: animal models and the aging oral cavity.

Age is the single greatest risk factor for many diseases, including oral diseases. Despite this, a majority of preclinical oral health research has not adequately considered the importance of aging in research aimed at the mechanistic understanding of oral disease. Here, we have attempted to provide insights from animal studies in the geroscience field and apply them in the context of oral health research. In particular, we discuss the relationship between the biology of aging and mechanisms of oral disease. We also present a framework for defining and utilizing age-appropriate rodents and present experimental design considerations, such as the number of age-points used and the importance of genetic background. While focused primarily on rodent models, alternative animal models that may be particularly useful for studies of oral health during aging, such as companion dogs and marmoset monkeys, are also discussed. We hope that such information will aid in the design of future preclinical studies of geriatric dental health, thus allowing more reliability for translation of such studies to age-associated oral disease in people.

Corrigendum: Hepatic S6K1 Partially Regulates Lifespan of Mice with Mitochondrial Complex I Deficiency.

[This corrects the article on p. 113 in vol. 8, PMID: 28919908.].

Hepatic S6K1 Partially Regulates Lifespan of Mice with Mitochondrial Complex I Deficiency.

The inactivation of ribosomal protein S6 kinase 1 (S6K1) recapitulates aspects of caloric restriction and mTORC1 inhibition to achieve prolonged longevity in invertebrate and mouse models. In addition to delaying normative aging, inhibition of mTORC1 extends the shortened lifespan of yeast, fly, and mouse models with severe mitochondrial disease. Here we tested whether disruption of S6K1 can recapitulate the beneficial effects of mTORC1 inhibition in the Ndufs4 knockout (NKO) mouse model of Leigh Syndrome caused by Complex I deficiency. These NKO mice develop profound neurodegeneration resulting in brain lesions and death around 50-60 days of age. Our results show that liver-specific, as well as whole body, S6K1 deletion modestly prolongs survival and delays onset of neurological symptoms in NKO mice. In contrast, we observed no survival benefit in NKO mice specifically disrupted for S6K1 in neurons or adipocytes. Body weight was reduced in WT mice upon disruption of S6K1 in adipocytes or whole body, but not altered when S6K1 was disrupted only in neurons or liver. Taken together, these data indicate that decreased S6K1 activity in liver is sufficient to delay the neurological and survival defects caused by deficiency of Complex I and suggest that mTOR signaling can modulate mitochondrial disease and metabolism via cell non-autonomous mechanisms.

Rapamycin treatment attenuates age-associated periodontitis in mice.

Interventions that target biological mechanisms of aging have great potential to enhance quality of life by delaying morbidity and mortality. The FDA-approved drug rapamycin is a compelling candidate for such an intervention. In a previous study, it was reported that 3 months of rapamycin treatment is sufficient to increase life expectancy and remodel the gut microbiome in aged mice. Transient treatment with rapamycin or a rapamycin derivative has also been shown to delay immune stem cell senescence and rejuvenate immune function in aged mice and elderly people. Periodontal disease is an important age-related disease involving altered immune function, pathological changes to the oral microbiome, and systemic inflammation. Periodontal disease is defined clinically by loss of alveolar bone and by connective tissue degeneration. Here, we describe significant alveolar bone loss during aging in two different mouse strain backgrounds and report that rapamycin treatment is sufficient to reverse age-associated periodontal disease in mice. Partial restoration of youthful levels of alveolar bone is observed in 22-month-old rapamycin-treated mice as rapidly as 8 weeks after initiation of treatment. To the best of our knowledge, this represents the first intervention shown to substantially prevent or reverse age-associated alveolar bone loss. These findings suggest the possibility that inhibition of mTOR with rapamycin or other pharmacological agents may be useful to treat a clinically relevant condition for which there is currently no effective treatment.

Peroxotitanate- and monosodium metal-titanate compounds as inhibitors of bacterial growth.

Sodium titanates are ion-exchange materials that effectively bind a variety of metal ions over a wide pH range. Sodium titanates alone have no known adverse biological effects but metal-exchanged titanates (or metal titanates) can deliver metal ions to mammalian cells to alter cell processes in vitro. In this work, we test a hypothesis that metal-titanate compounds inhibit bacterial growth; demonstration of this principle is one prerequisite to developing metal-based, titanate-delivered antibacterial agents. Focusing initially on oral diseases, we exposed five species of oral bacteria to titanates for 24 h, with or without loading of Au(III), Pd(II), Pt(II), and Pt(IV), and measuring bacterial growth in planktonic assays through increases in optical density. In each experiment, bacterial growth was compared with control cultures of titanates or bacteria alone. We observed no suppression of bacterial growth by the sodium titanates alone, but significant (p < 0.05, two-sided t-tests) suppression was observed with metal-titanate compounds, particularly Au(III)-titanates, but with other metal titanates as well. Growth inhibition ranged from 15 to 100% depending on the metal ion and bacterial species involved. Furthermore, in specific cases, the titanates inhibited bacterial growth 5- to 375-fold versus metal ions alone, suggesting that titanates enhanced metal-bacteria interactions. This work supports further development of metal titanates as a novel class of antibacterials.

PAR1- and PAR2-induced innate immune markers are negatively regulated by PI3K/Akt signaling pathway in oral keratinocytes.

BACKGROUND: Protease-Activated Receptors (PARs), members of G-protein-coupled receptors, are activated by proteolytic activity of various proteases. Activation of PAR1 and PAR2 triggers innate immune responses in human oral keratinocytes (HOKs), but the signaling pathways downstream of PAR activation in HOKs have not been clearly defined. In this study, we aimed to determine if PAR1- and PAR2-mediated signaling differs in the induction of innate immune markers CXCL3, CXCL5 and CCL20 via ERK, p38 and PI3K/Akt. RESULTS: Our data show the induction of innate immunity by PAR1 requires both p38 and ERK MAP kinases, while PAR2 prominently signals via p38. However, inhibition of PI3K enhances expression of innate immune markers predominantly via suppressing p38 phosphorylation signaled by PAR activation. CONCLUSION: Our data indicate that proteases mediating PAR1 and PAR2 activation differentially signal via MAP kinase cascades. In addition, the production of chemokines induced by PAR1 and PAR2 is suppressed by PI3K/Akt, thus keeping the innate immune responses of HOK in balance. The results of our study provide a novel insight into signaling pathways involved in PAR activation.

Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria.

Protease-activated receptors (PARs), nucleotide-binding oligomerization domain (NOD) receptors and Toll-like receptors (TLRs) play a role in innate immunity, but little is known about interaction between these receptors. The goal of this study was to investigate how silencing one receptor affects the expression of other receptors and downstream innate immune markers in response to bacteria. Human gingival epithelial cells (GECs) were transfected with siRNA specific for PAR1 or PAR2, then stimulated with periopathogen Porphyromonas gingivalis, bridging organism between pathogens and non-pathogens Fusobacterium nucleatum, or non-pathogen Streptococcus gordonii. PAR1 or PAR2 knock-down resulted in up-regulated NOD1 and NOD2 expression with P. gingivalis or F. nucleatum stimulation (p<0.01), as well as enhanced TLR2 and TLR4 expression when cells were stimulated by bacteria that utilize TLR2 or TLR4, respectively. Involvement of PARs for induction of CC chemokine ligand 20 (CCL20), a cytokine with antimicrobial properties, was observed following stimulation of the three bacterial species. Furthermore, results from multiple cytokine ELISA array showed receptors utilized in the induction of various innate immune markers are tailored to individual bacterium tested. Our data suggest complex interplay of several receptors is required for appropriate innate immune responses to the different types of bacteria present within the oral cavity and that receptor expression itself is altered depending on which organism the cell encounters.

Differential effects of periopathogens on host protease inhibitors SLPI, elafin, SCCA1, and SCCA2.

OBJECTIVE: Secretory leukocyte peptidase inhibitors (SLPI), elafin, squamous cell carcinoma antigen 1 and 2 (SCCA1 and SCCA2) are specific endogenous serine protease inhibitors expressed by epithelial cells that prevent tissue damage from excessive proteolytic enzyme activity due to inflammation. To determine the effects of various periopathogens on these protease inhibitors, we utilized human gingival epithelial cells (GECs) challenged with cell-free bacteria supernatants of various periopathogens Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. DESIGN: The gene expression and secretion of SLPI, elafin, SCCA1, and SCCA2 were determined using real-time PCR and ELISA, respectively. The direct effects of periopathogens and P. gingivalis gingipain mutants on these inhibitors were examined in vitro by Western Blot. The effect on the innate immune response of GECs was measured by expression of antimicrobial peptides: human beta-defenisin-2 (hBD2) and chemokine (C-C motif) ligand 20 (CCL20). RESULTS: We found that SLPI, SCCA2, elafin, hBD2, and CCL20 gene expression levels were significantly induced (p<0.001) in response to P. gingivalis, whose virulence factors include cysteine proteases, but not in response to stimulation by other bacteria. P. gingivalis reduced the secretion of SLPI and elafin significantly in GECs, and degraded recombinant SLPI, elafin, SCCA1, and SCCA2. Differential degradation patterns of SLPI, elafin, SCCA1, and SCCA2 were observed with different bacteria as well as P. gingivalis mutants associated with the loss of specific gingipains secreted by P. gingivalis. In addition, pretreatment of GECs with SLPI, SCCA1, or SCCA2 partially blocked hBD2 and CCL20 mRNA expression in response to P. gingivalis, suggesting a protective effect. CONCLUSION: Our results suggest that different periopathogens affect the host protease inhibitors in a different manner, suggesting host susceptibility may differ in the presence of these pathogens. The balance between cellular protease inhibitors and their degradation may be an important factor in susceptibility to periodontal infection.