The miR-1260b-NFAT5 axis regulates inflammasome priming and M1 macrophage polarization in periodontitis

Macrophage-driven inflammatory programs and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays a critical role for periodontal tissue destruction; however, the transcriptional regulators that mediate these responses remain unclear. This study therefore examined the role of the microRNA-1260b (miR-1260b)-nuclear factor of activated T-cells 5 (NFAT5) axis in inflammatory macrophage responses relevant to periodontal disease.

NFAT5 was identified as a candidate miR-1260b target using integrated bioinformatic screening and investigated through gain- and loss-of-function approaches in RAW264.7 macrophages. Inflammasome-related experiments were performed in J774A.1 macrophages using an LPS/ATP two-signal stimulation protocol. Further analyses included a ligature-induced mouse experimental periodontitis model with local NFAT5 small interfering RNA delivery, macrophage polarization assays in murine bone marrow-derived macrophages stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ), and validation experiments in human peripheral blood mononuclear cell (PBMC)-derived macrophages.

Transfection with miR-1260b reduced NFAT5 expression and attenuated LPS-induced nuclear accumulation in macrophages. In vivo analyses showed increased NFAT5 expression in inflamed gingival tissues, whereas local NFAT5 knockdown was associated with reduced NLRP3 and interleukin-1 beta (IL-1β) expression. In J774A.1 macrophages, NFAT5 silencing under LPS/ATP stimulation reduced Nlrp3 and Il1b mRNA expression, together with lower NLRP3 and pro-IL-1β protein levels and reduced IL-1β levels in culture supernatants. Additional in vitro experiments demonstrated reduced M1-associated inflammatory gene expression following NFAT5 silencing. Flow cytometric analysis further showed a reduced proportion of CD86⁺ macrophage subsets after NFAT5 knockdown, accompanied by decreased pro-inflammatory cytokine production in both murine and human macrophage systems.

These findings identify NFAT5 as a key regulator of inflammasome priming that contributes to inflammatory macrophage polarization in periodontal inflammation and suggest the miR-1260b-NFAT5 axis as a potential target for host-modulatory approaches.