Chikako Hayashi, Takao Fukuda, Miyu Shida, Meng Xiao, Ziyu Wang, Naoaki Ryo, Masaaki Toyoda, Kentaro Kawakami, Takanori Shinjo, Tsukasa Aoki, Jinfeng Li, Mwannnes Ahmad, Takaharu Taketomi, Takeshi Uchiumi, Terukazu Sanui, Fusanori Nishimura
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.
INTRODUCTION: 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.
METHODS: 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.
RESULTS: 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.
DISCUSSION: 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.