| Abstract: | The hallmark of periodontal disease is the progressive destruction of gingival soft tissue and alveolar bone, which is initiated by inflammation in response to an invasive and persistent bacterial insult. In recent years, it has become apparent that this tissue destruction is associated with a decrease in local regulatory processes, including a decrease of forkhead box P3-expressing regulatory lymphocytes. Accordingly, we developed a controlled release system capable of generating a steady release of a known chemoattractant for regulatory lymphocytes, C-C motif chemokine ligand 22 (CCL22), composed of a degradable polymer with a proven track record of clinical translation, poly(lactic-co-glycolic) acid. We have previously shown that this sustained presentation of CCL22 from a point source effectively recruits regulatory T cells (Tregs) to the site of injection. Following administration of the Treg-recruiting formulation to the gingivae in murine experimental periodontitis, we observed increases in hallmark Treg-associated anti-inflammatory molecules, a decrease of proinflammatory cytokines, and a marked reduction in alveolar bone resorption. Furthermore, application of the Treg-recruiting formulation (fabricated with human CCL22) in ligature-induced periodontitis in beagle dogs leads to reduced clinical measures of inflammation and less alveolar bone loss under severe inflammatory conditions in the presence of a diverse periodontopathogen milieu.Periodontitis is characterized by destructive inflammation of the periodontal tissue including the gingiva, periodontal ligament, and alveolar bone, and it is considered the most pressing oral health concern today, affecting more than 78 million individuals in the United States alone (1). Importantly, this disease affects not only tooth loss, but also may impact the incidence of diabetes; cardiovascular, kidney, rheumatologic, and respiratory diseases; and even premature childbirth (2). To date, clinical approaches have been focused on abrogation of invasive bacterial species that trigger local and systemic inflammatory and immune responses (3). Specifically, the current standard of care involves debridement and is sometimes accompanied by local or systemic administration of broad-spectrum antibiotic agents (4). However, given that reestablishment of periodontal lesions is common, patients must repetitively undergo these procedures. In addition, treatment is ineffective in as much as 30% of the population (5, 6).A large body of literature now suggests that bacterial species (albeit protagonists) are secondary to the host immune response in regard to the etiology of periodontal disease progression (7–9). Specifically, various lymphocyte subsets can accumulate in the periodontium, leading to the local expression of soft tissue-destroying matrix metalloproteinases (10) (MMPs) and receptor activator of NF-κB (RANK) ligand (RANKL) (11) (the primary activation factor for osteoclasts), initiating alveolar bone resorption. Several recent reports have also shown that another lymphocyte subset called regulatory T cells (Tregs) can accumulate in the gingival tissues during periodontal disease in humans and in experimental models (12–15), and helps protect the host from harmful inflammation. However, it appears that, when Tregs are present in insufficient numbers, progression of the disease is accelerated (15).Accordingly, we sought to develop a strategy for increasing local numbers of regulatory lymphocytes through the recruitment of endogenous Tregs (16) [mimicking a mechanism tumors use to evade immune responses (17)]. Specifically, a natural gradient of a known chemoattractant for regulatory lymphocytes, C-C motif chemokine ligand 22 (CCL22) (16, 18), could be artificially reproduced by using controlled release from a local site. Recently, we developed polymer microspheres capable of steadily releasing CCL22 by using a model-aided design process that specifies the requisite formulation properties (such as porosity) and polymer composition (16). Importantly, this process permits the tuning of release behavior using degradable polymers such as poly(lactic-co-glycolic) acid (PLGA) that are already known to be safe and biocompatible and also exhibit a proven track record of clinical translation (19, 20). This CCL22-releasing formulation has been shown to be effective at recruiting Tregs in vitro and in vivo (16). These recruited Tregs have the potential to influence the local immunological milieu, shifting it toward homeostasis (16). Based on these observations, we hypothesized that this biodegradable, controlled-release formulation of CCL22 administered locally in the periodontium, may recruit Tregs and effectively abrogate periodontal disease symptoms without necessarily reducing local bacterial numbers. Furthermore, the presence of Tregs may actually help to balance the proinflammatory response and generate an environment that is conducive to periodontal tissue regeneration as well as bone regeneration, possibly through expression of IL-10 and osteocalcin (15).By using Actinobacillus actinomycetemcomitans (Aa)-induced murine and ligature-induced canine models, we demonstrate that the Treg recruiting formulation significantly halts the progression of periodontitis as determined by significant decreases in alveolar bone resorption (mice and dogs), as well as clinical scores of disease progression (dogs). Furthermore, in mice, our Treg recruiting formulation leads to a significant decrease in the production of proinflammatory cytokines in the periodontal tissues (along with an increase in anti-inflammatory cytokines) as well as a decrease in markers of soft and hard tissue destruction (along with an increase in markers of soft and hard tissue regeneration). Overall, the Treg-recruiting formulations described herein may serve as a tool for the study of the role of Treg in periodontal disease, and even suggest a unique treatment modality that intends to harness the body’s own sophisticated immune regulatory mechanisms through the recruitment of cells. |
