Trehalose inhibits inflammatory cytokine production by protecting IκB-α reduction in mouse peritoneal macrophages
Introduction
Periodontal diseases are a group of inflammatory disorders that lead to the destruction of tooth-supporting tissues and are caused by a specific group of Gram-negative anaerobic bacteria, including Porphyromonas gingivalis (P. gingivalis).1 The host response to these bacteria and their products is a critical determinant in the initiation and progression of periodontitis.2, 3 More specifically, lipopolysaccharides (LPS) of Gram-negative bacteria are potent inducers of pro-inflammatory cytokines and lead to periodontal tissue destruction.4, 5 LPS is a major component of the outer membrane of Gram-negative bacteria.6 Monocytes and macrophages, which are found in higher numbers in active periodontal lesions than in inactive sites,7 play an important role in the host inflammatory response to periodontopathogens.8 LPS induces inflammatory cytokines, such as interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), in the macrophages and neutrophilic leucocytes that infiltrate areas infected with bacteria9, 10, 11 and in mice calvaria12 and rat molar gingival periodontal tissues.13 The continuous, high secretion of various cytokines – including IL-1β and TNF-α in host cells with subsequent stimulation by periodontopathogens modulates periodontal tissue destruction.14 IL-1β and TNF-α induce bone resorption by acting both directly and indirectly on osteoclasts.15
Nuclear transcription factor kappa-B (NF-κB) is one of the most important transcription factors, and is found in cell types that express cytokines, chemokines, growth factors, cell adhesion molecules, and some acute phase proteins in healthy and disease states.16, 17 The activation of NF-κB involves the phosphorylation of the IκBs at two critical serine residues (Ser32, Ser36) via the IκB kinase (IKK) complex. Once the IκBs have been phosphorylated, they are ubiquitinated and reduced by the 26S proteasome. The resulting free NF-κB is then translocated into the nucleus, where it binds to NF-κB binding sites in the promoter regions of target genes, and induces the transcription of pro-inflammatory mediators, e.g., TNF-α, IL-1β and IL-6.18, 19 Furthermore, LPS have been implicated in the inducible expression of many genes in macrophages, including pro-inflammatory cytokines such as TNF-α, IL-1α and -β, and IL-6 via specific NF-κB translocation.20
Trehalose is a non-reducing disaccharide which consists of two molecules of glucose bound to glucose by an α, α-1, 1 linkage.21, 22 Since trehalose stabilises and protects the cell membrane, it has been used as a preservative for tissues in organ transplantation.23 In dental research, it has been reported that exposure of intra-oral and tooth surfaces to trehalose exhibits a slower pH response than exposure to sucrose.24 In addition, trehalose undergoes less acid fermentation and production by streptococci mutants, and rat dental caries are reduced by the presence of dietary trehalose.24 Besides these properties, we have recently shown that trehalose prevents reduction of bone mineral density in ovariectomized (OVX) mice25 and rats26. Moreover, Nishizaki et al.27 demonstrated that trehalose attenuates bone loss and osteoclast formation in OVX mice. Finally, trehalose inhibits excessive osteoclastogenesis in the LPS-injected mouse model of osteoporosis, suppresses LPS-induced TNF-α production by bone marrow cells in LPS-injected mice,28 and IL-6 secretion by bone marrow cells in OVX mice.29 However, there have been no reports on its anti-inflammatory effect in periodontopathogen-activated macrophages.
Therefore, we hypothesised that trehalose may have a beneficial effect in periodontitis by exerting an anti-inflammatory effect. Thus, we investigated the protein production and mRNA expression of inflammatory cytokines in macrophages stimulated with P. gingivalis LPS. In addition, we investigated whether trehalose influences the LPS-induced reduction of IκB-α.
Section snippets
Animals
All animal experiments were performed in accordance with the Guideline on Animal Care and Use established by Ohu University. Specific pathogen-free, inbred ICR male mice 5 weeks of age were purchased from CLEA Japan, Inc. (Tokyo, Japan). They were maintained under standard laboratory conditions (at room temperature of 22–24 °C with relative humidity of 50–60% and a 14/10 h light/dark cycle).
Preparation of mouse peritoneal macrophages
Preparation of mouse peritoneal macrophages was performed by a modification of a previously described
Inhibitory effect of trehalose on LPS-induced IL-1β and TNF-α production
To analyse the potential anti-inflammatory properties of trehalose, we used mouse peritoneal macrophages, which can produce IL-1β and TNF-α upon stimulation with P. gingivalis LPS. Cells were treated with various doses of trehalose and stimulated with LPS for 12 h. IL-1β and TNF-α production in the culture supernatants were increased upon LPS stimulation. It was found that trehalose leads to reduced LPS-induced IL-1β production in a dose-dependent manner (Fig. 1A). High doses of trehalose (0.1,
Discussion
Periodontitis is a chronic inflammatory disorder and a bone disease affecting oral tissues. The host inflammatory response to periodontopathogens is considered a major factor causing the local tissue destruction observed in periodontitis. P. gingivalis has been predominantly isolated from subgingival plaques and is thought to be a major etiologic agent in adult periodontitis. Macrophages participate in the host response induced by periodontopathogens and are the principal target for LPS.34
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