Multi-species Microbial Modulation of the RANK/RANKL (Receptor Activator of Nuclear Factor κB/Receptor Activator of Nuclear Factor κB Ligand) Pathway Induces Osteoclastogenic Priming in Apical Periodontitis

Background: Apical periodontitis (AP) is a prevalent inflammatory condition affecting the periapical tissues surrounding the tooth root, often initiated by microbial invasion. Objective: The objective of this study is to evaluate the osteoclastogenic priming effects of microbial species associated with primary and secondary AP on human macrophages and to determine their capacity to modulate the RANK/RANKL-OPG (receptor activator of nuclear factor kappa-B, receptor activator of nuclear factor kappa-B ligand, osteoprotegerin) pathway​. Materials and methods: THP-1 monocytes/macrophages were stimulated with live bacteria, with or without RANKL, and analyzed for RANK/RANKL/OPG expression by flow cytometry. Cytokine levels (interleukin-1β (IL-1β), tumoral necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70)) were measured using cytometric bead arrays. Results: Macrophages challenged with Enterococcus, Streptococcus, Actinomyces, and Candida exhibited an increase in RANK⁺, RANKL⁺, and RANK⁺RANKL⁺ cellular populations. Enterococcus and Candida promoted sustained RANK/RANKL expression across conditions, while Streptococcus and Actinomyces elicited significant double-positive cell expansion. Early microbial stimulation triggered robust TNF-α, IL-1β, and IL-6 production, peaking with Streptococcus intermedius, Enterococcus faecalis, and Actinomyces, followed by a decrease at 96 hours. In contrast, IL-8 secretion remained high among species and time points, with Actinomyces inducing the strongest response. Conclusion: Our data suggest that AP-associated bacteria can induce osteoclastogenic priming and promote the release of pro-inflammatory cytokines via RANK/RANKL activation, indicating a potential role in the early processes leading to periapical bone resorption.