A Pathomimetic Gingiva-on-a-Chip for Assessing Microbiota-Directed Anti-Inflammatory Therapeutics

/ABSTRACT Periodontitis, or severe gum disease, is one of most prevalent inflammatory conditions worldwide. Left untreated, it ultimately results in the loss of teeth, while also increasing risk for several comorbidities, including heart disease, diabetes, and rheumatoid arthritis. The etiology of periodontitis is strongly tied to an ecologically imbalanced oral microbiota, in which disease-promoting microbes (pathobionts) proliferate at the expense health-promoting microbes (symbionts). Despite this, current periodontitis treatments, such as the debridement of dental plaque, indiscriminately eliminate both pro-inflammatory pathobionts and beneficial symbionts. A major benefit of symbionts stems from their capacity to produce antimicrobials that suppress pathobionts. Selectively enhancing the production of such antimicrobials could therefore represent a more targeted therapeutic option for periodontitis. In preliminary studies, we found that that an abundant diet-derived metabolite in human saliva, nitrate, can be converted by symbionts into an antimicrobial (nitrite) that inhibits pathobionts. To identify additional metabolites with similar activity, we performed an initial screen using ex vivo human saliva cultures, revealing that the plant metabolite arbutin also inhibits pathobionts in a microbiota-dependent manner. In line with these findings, it has been consistently reported that nitrate-rich foods, such as leafy greens, can reduce microbiota imbalance and even gingival inflammation, while in other disease settings, arbutin has also been reported to dampen inflammation. However, whether these metabolites are anti-inflammatory because they induce symbionts to inhibit pathobionts has not been fully elucidated. Furthermore, numerous other symbiont- promoting metabolites that target pathobionts likely await discovery, necessitating accurate high-throughput modeling of oral microbiota-host crosstalk to probe downstream impacts on gingival inflammation. To this end, we propose leveraging our experience in microphysiological (Organ-on-a-Chip) systems to develop a novel Gingiva-on-a-Chip. This microfluidic device will recapitulate key physico-chemical aspects of the periodontal disease site (crevice between gum and tooth), while enabling stable co-culture of epithelial cells with human oral microbiota. Using this bioengineered model, we will pursue the following Aims to explore the anti-inflammatory potential of symbiont-promoting metabolites. In Aim 1, we will utilize Gingiva Chips to test whether nitrate reduces inflammation by inducing symbionts to inhibit pathobionts, while in Aim 2, we will expand our initial screen to identify additional novel microbiota-dependent antimicrobial metabolites, before again utilizing Gingiva Chips to assess their impacts on inflammation. The successful completion of this project will result in an improved technology for deciphering oral microbiota-host interactions, while reinforcing the novel concept that symbiont- promoting metabolites can be translated into targeted microbiota-directed therapies to ameliorate inflammation. Project Number: 1R21DE035249-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Apollo Stacy | Institution: CLEVELAND CLINIC LERNER COM-CWRU, CLEVELAND, OH | Award Amount: $442,750 | Activity Code: R21 | Study Section: Oral, Dental and Craniofacial Sciences Study Section[ODCS] View on NIH RePORTER: https://reporter.nih.gov/project-details/11216240