Periodontitis is an inflammatory disease that leads to the destruction of periodontal tissue, including alveolar bone [1]. In response to external stimuli, such as dental plaque and dental calculus (DC), various types of leukocytes infiltrate periodontal tissue, and release inflammatory mediators, such as prostaglandins, matrix metalloproteinases, and cytokines, which promote periodontal tissue destruction [2]. Among these mediators, interleukin (IL)-1β has the capacity to trigger potent bone resorption [3], and IL-1β has been detected in the periodontal tissue and gingival crevicular exudates of patients with periodontitis, suggesting its involvement in alveolar bone resorption ^[4][5][4,5].

The production of IL-1β is regulated both transcriptionally and post-transcriptionally [6]. The transcription of pro-IL-1β can be triggered by the binding of Toll-like receptors (TLRs), IL-1 receptors, and tumor necrosis factor (TNF) receptors with their ligands, leading to the nuclear translocation of nuclear factor (NF)-κB [7]. The transcriptional activation of the IL-1β promoter results in the synthesis of a 35 kD pro-IL-1β, which is biologically inactive and remains in the cytosol [8]. For the maturation of IL-1β, pro-IL-1β must be processed by caspase-1 [9]. Assembly of the inflammasome, which consists of nucleotide-binding oligomerization domain leucine-rich repeat containing protein (NLR), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and the cysteine protease pro-caspase-1, converts pro-caspase-1 to active caspase-1 by autocatalysis; caspase-1, in turn, cleaves pro-IL-1β into its mature IL-1β form [10].

IL-18 and IL-33 are also IL-1 family members that can be cleaved by caspase-1 ^[11][12][11,12]. IL-18 is known to be a proinflammatory cytokine that induces the production of interferon (IFN)-γ in T cells [13], and promotes osteoclastogenesis by upregulating receptor activator of nuclear factor-kappa B ligand (RANKL) production in T cells in synovitis in rheumatoid arthritis [14]. IL-33 binds to type 4 IL-1 receptors and is regarded as a Th2-promoting inflammatory cytokine [15]. The precursor form of IL-33 is already biologically active and is inactivated via cleavage by caspase-1 ^[11][16][11,16]. IL-33 has been reported to inhibit osteoclastogenesis ^[17][18][17,18]. DC is a deposit frequently found in periodontal pockets, and is 70–80% inorganic material [19]. The remaining organic component consists of proteins, leukocytes, and microorganisms [20]. The crystalline components of DC are mainly hydroxyapatite, brushite, tricalcium phosphate, and octacalcium phosphate ^[19][21][19,21]. Recent studies have shown that stimulation of neutrophils and macrophages with DC activates the nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome and produces IL-1β [22]. In addition, IL-18 and IL-33 can be produced by mouse macrophages stimulated with DC via the NLRP3 inflammasome. However, the effects of these cytokines stimulated with DC on osteoclastogenesis have not been investigated. It is difficult to distinguish the effects of DC from those of dental biofilms because DC is always covered with biofilms that contain highly viable bacteria [23]. However, accumulated evidence has shown that there is a clear association between DC deposition and periodontitis ^[23][24][23,24].

2. Effects of Culture Supernatants from Mouse Macrophages Stimulated with Dental Calculus on Osteoclast Formation

When bone marrow macrophages (BMMs) primed with M-CSF and RANKL were incubated with the supernatant from the NLRP3-deficient mouse macrophages stimulated with DC, osteoclast formation was promoted, suggesting the effects of pro-inflammatory cytokines in the supernatants (Figure 1a). The supernatant of WT mouse macrophages stimulated with DC induced higher numbers of osteoclasts than the supernatant from NLRP3-deficient mouse macrophages, suggesting the effect of another pro-inflammatory cytokine (Figure 1b). In contrast to the results of RANKL-primed BMMs, the supernatants from both WT and NLRP3-deficient mouse macrophages stimulated with DC suppressed osteoclast formation in RANKL-primed RAW-D cells (Figure 1a,b). Incubation of RANKL-primed RAW-D cells with the supernatant from unstimulated WT or NLRP3-deficient mouse macrophages did not affect the osteoclastogenesis.