Immune response characterization of primary gingival fibroblasts from Grade C periodontitis patients

Background: Grade C, Stage 3–4 Periodontitis (Perio4C) is a rapidly destructive disease caused by an unequilibrated immune response that starts after the primary contact of the periodontopathogens with the gingival tissue. However, it is still unclear how this imbalanced response initiates and what is the role of the connective tissue cells in the progression of this disease. Thus, this study aims to assess the local immune response of Perio4C patients through the exposure of primary gingival fibroblast cells (GFs) with Aggregatibacter actinomycetemcomitans protein extract (AaPE) and the quantification of the inflammatory cytokines interleukin (IL)-4, IL-17, tumor necrosis factor (TNF)-α, IL-1β, interferon (IFN)-γ, and IL-10 super-family members (IL-10, IL-19, and IL-24) secreted by them. Methods: Gingival biopsies from nine periodontally health (PH) and eight Perio4C patients were harvested, and the primary culture of GFs was obtained. The cells were exposed to AaPE (5 and 20 μg/ml) and 12-myristate 13-phorbol acetate and ionomycin – calcium salt (PMA). The supernatant was collected after 1.5 and 3 h, and a cytokine panel was evaluated. Results: Clustering analysis indicated dissimilar and stimuli-dependent cytokine production between Perio4C and PH subjects. Perio4C GFs presented lower production of IL-4, TNF-α, IFN-γ, IL-17, IL-10, IL-24, and IL-19, while IL-1β levels were similar to the PH group, leading to a disruption in the pro-/anti-inflammatory cytokine ratio (p < 0.05). IL-1β and IL-10 super-family were the most discriminative representants for PH and Perio4C, respectively. Conclusion: GFs from individuals with Perio4C tended to hypo-respond to stimulation with AaPE, producing lower concentrations of some pro- and anti-inflammatory molecules, trending to develop a pro-inflammatory extracellular environment.