Interleukin-35 inhibits alveolar bone resorption during periodontitis by modulating the Treg/Th17 imbalance.

Objectives: Periodontitis is a chronic inflammatory disease, in which the locally deployed immune response against a dysbiotic subgingival microbiome results in alveolar bone resorption and tooth loss. In this context, the Th17 pattern of immune response has been associated to the increase of RANKL:OPG levels and consequently osteoclasts over-activation during periodontitis; otherwise, the T regulatory (Treg) response, has been associated to the regulation of the Th17 response and inhibition of periodontitis progression. Interleukin (IL)-35 is a recently described cytokine mostly produced by Tregs, with exclusive immunoregulatory properties, and with the ability to induce its own expression and production in Tregs; nevertheless, its role during periodontitis remains undetermined. Therefore, this work aimed to elucidate the potential protective role of IL-35 during experimental periodontitis.
Methods: Experimental periodontitis was induced in C57BL/6 mice using silk ligatures. Then, ligated mice received palatal or intraperitoneal injections of IL-35 every other day for 15 days. Sham-injected and non-ligated mice were used as controls. Alveolar bone resorption was analyzed using micro-CT and scanning electron microscopy (SEM), and differentiated osteoclasts were detected by TRAP staining. RANKL:OPG levels were detected by ELISA in crevicular fluid, and their expression levels were quantified by qPCR and immunofluorescence of periodontal lesions. The proportion of Tregs and Th17 lymphocytes in draining lymph nodes and periodontal lesions was determined by flow cytometry, and the expression of Treg- or Th17-related cytokines was quantified by qPCR.
Results: IL-35 inoculations diminished alveolar bone resorption, TRAP⁺osteoclast detection, RANKL:OPG expression and production, Th17 lymphocytes detection, Th17-associated cytokines, and receptors expression, and further increased Tregs detection and Treg-related cytokines expression in comparison with the sham-injected group.
Conclusions: IL-35 was able to arrest experimental periodontitis by inhibiting alveolar bone resorption and modulating the Treg/Th17 imbalance and could be potentially used as a novel therapy for periodontitis.