Elsevier

Archives of Oral Biology

Volume 54, Issue 8, August 2009, Pages 749-756
Archives of Oral Biology

Trehalose inhibits inflammatory cytokine production by protecting IκB-α reduction in mouse peritoneal macrophages

https://doi.org/10.1016/j.archoralbio.2009.05.003Get rights and content

Abstract

Objective

The aim of this study was to examine whether trehalose, a disaccharide, could inhibit Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS)-enhanced production of inflammatory cytokines in mouse peritoneal macrophages.

Design

Mouse peritoneal macrophages were treated with trehalose and stimulated with P. gingivalis LPS. Interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) levels in the culture supernatant were measured by ELISA. The mRNA levels of the cytokines in macrophages were analysed by semi-quantitative RT-PCR. DNA and protein synthesis were measured by incorporation of [3H] thymidine or [14C] praline into mouse peritoneal macrophages. IκB-α reductions were assessed by western blot.

Results

Treatment with trehalose suppressed LPS-induced IL-1β and TNF-α production and downregulated transcription of these cytokines. Furthermore, trehalose inhibited LPS-induced reduction of IκB-α. In addition, we also observed expression of the trehalose receptor (T1R3) in mouse peritoneal macrophages.

Conclusion

These results may suggest that trehalose inhibits LPS-induced production of IL-1β and TNF-α in mouse peritoneal macrophages by inhibiting degradation of IκB-αvia the trehalose receptor T1R3.

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

References (61)

  • G. Nelson et al.

    Mammalian sweet taste receptors

    Cell

    (2001)
  • G.Q. Zhao et al.

    The receptors for mammalian sweet and umami taste

    Cell

    (2003)
  • A.D. Haffajee et al.

    Microbial etiological agents of destructive periodontal diseases

    Periodontol 2000

    (1994)
  • J. Slots et al.

    The occurrence of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius in destructive periodontal disease in adults

    J Clin Periodontol

    (1986)
  • J.L. Dzink et al.

    The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases

    J Clin Periodontol

    (1988)
  • H. Birkedal-Hansen

    Role of cytokines and inflammatory mediator in tissue destruction

    J Periodontal Res

    (1993)
  • B. Henderson et al.

    Bacterial modulins: a novel class of virulence factor which cause host tissue pathology by inducing cytokine synthesis

    Microbial Rev

    (1996)
  • P.S. Tobias et al.

    Lipopolysaccharide dependent cellular activation

    J Periodontal Res

    (1997)
  • U. Zappa et al.

    Cell populations and episodic periodontal attachment loss in humans

    J Clin Periodontol

    (1991)
  • K.S. Kornman et al.

    The host response to the microbial challenge in periodontitis: assembling the players

    Periodontol 2000

    (1997)
  • C.G. McFarlane et al.

    The release of interleukin-1 beta, tumor necrosis factor-alpha and interferon-gamma by cultured peripheral blood mononuclear cells from patients with periodontitis

    J Periodontal Res

    (1990)
  • M. Wilson et al.

    Cytokine-induced components of periodontopathogenic bacteria

    J Periodontal Res

    (1996)
  • R.A. Lindemann et al.

    Production of interleukin-1 and tumor necrosis factor by human peripheral monocytes activated by periodontal bacteria and extracted lipopolysaccharides

    J Dent Res

    (1988)
  • C.Y. Chiang et al.

    Interleukin-1 and tumor necrosis factor activities partially account for calvarial bone resorption induced by local injection of lipopolysaccharide

    Infect Immun

    (1999)
  • M. Miyauchi et al.

    Cytokine expression in rat molar gingival periodontal tissues after topical application of lipopolysaccharide

    Histochem Cell Biol

    (2001)
  • H. Okada et al.

    Cytokine expression in periodontal health and disease

    Crit Rev Oral Biol Med

    (1998)
  • K. Kobayashi et al.

    Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction

    J Exp Med

    (2000)
  • A.S. Baldwin

    The NF-κB and IκB proteins: new discoveries and insights

    Annu Rev Immunol

    (1996)
  • F. Chen et al.

    New insights into the role of nuclear factor-κB, a ubiquitous transcription factor in the initiation of disease

    Clin Chem

    (1999)
  • M.S. Hayden et al.

    Signaling to NF-κB

    Genes Dev

    (2004)
  • Cited by (0)

    View full text