S100A9-induced release of interleukin (IL)-6 and IL-8 through toll-like receptor 4 (TLR4) in human periodontal ligament cells
Introduction
Periodontitis is a chronic inflammatory disease of the tooth-supporting tissues that is characterized by alveolar bone loss (Cochran, 2008, Di Benedetto et al., 2013). The role of bacterial infection in the etiology of periodontitis is well supported (Hernandez et al., 2011), but the role of the host immune response is not fully understood. It is possible that the activation of the host inflammatory and immune responses may also contribute to the destruction of periodontal tissues (Cochran, 2008, Di Benedetto et al., 2013). Periodontal ligament (PDL) links teeth to alveolar bone and participates in alveolar bone remodeling. PDL cells reside in PDL space, characterized by the production of collagen fibers and mineralized tissue (Jonsson et al., 2011). Pattern recognition receptors (PRRs) on the resident cells of the periodontium play an important role in the innate immune response by recognizing bacteria-derived factors and promoting the production of cytokines and chemokines, which are key mediators of inflammation and bone loss (Cochran, 2008, Di Benedetto et al., 2013).
S100A8 and S100A9 are calcium-binding proteins that have been identified as important endogenous damage-associated molecular patterns (DAMP) or alarmins (Schiopu and Cotoi, 2013). S100A8 and S100A9 are recognized by PRRs and promote extracellular pro-inflammatory functions (Schiopu and Cotoi, 2013). S100A8 and S100A9 exist as non-covalently bonded homodimers, but S100A8 and S100A9 form the non-covalent heterodimer calprotectin (or the S100A8/A9 complex) in the presence of calcium (Simard et al., 2013).
Due to their pro-inflammatory functions, S100A8, S100A9, and calprotectin are associated with numerous autoimmune and inflammatory diseases, including arthritis (Foell et al., 2007), systemic lupus erythematous (Bjork et al., 2009), Crohn's disease (Lugering et al., 1995), periodontitis (Meng et al., 2002, Kojima et al., 2000), as well as other various diseases (Kallberg et al., 2012, Cesaro et al., 2012, Ichikawa et al., 2011, Boyd et al., 2008). The levels of calprotectin in gingival crevicular fluid and serum are increased in patients with periodontitis and decreased following periodontal treatment; therefore, calprotectin may be used as a biomarker to diagnose periodontitis and to monitor the response to periodontal treatment (Kido et al., 1999, Andersen et al., 2010).
S100A8, S100A9, and calprotectin are clearly related to periodontal disease but the pathological roles of these proteins have not been investigated thoroughly. Certain periodontal pathogens, such as porphyromonas gingivalis lipopolysaccharide, may promote the release of calprotectin from neutrophils and monocytes (Kido et al., 2004, Suryono et al., 2003, Kido et al., 2005), but the mechanisms of calprotectin release from neutrophils and monocytes have yet to be elucidated. Furthermore, S100A8 and S100A9 may also play distinct roles in the onset of periodontal disease. For instance, Nishii et al. (2013) reported the expression of S100A9 was significantly increased at the presence of microflora, while S100A8 protein expression was not affected by bacterial infection.
The main receptors for S100A8, S100A9, and calprotectin are toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE), but the specific receptors and pathways for S100A8, S100A9, and calprotectin depend on different cell types. For instance, S100A9 triggers nuclear factor (NF)-κB responses via TLR4 in THP-1 cells (Riva et al., 2012) and triggers NF-κB and extracellular-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) responses via RAGE in lung fibroblast cells (Xu et al., 2013), whereas S100A8, S100A9, and calprotectin triggers MAPKs responses via TLR4 and RAGE in endothelial cells (Wang et al., 2014).
Our previous study reported that calprotectin promotes pro-inflammatory responses in PDL cells via the S100A9 subunit (Zheng et al., 2014), but no published studies have investigated the potential receptor and signaling pathways engaged by S100A9 in human PDL cells. We hypothesized that S100A9 induces the production of IL-6 and IL-8 from human PDL cells through NF-κB and MAPKs signaling pathways mediated by TLR4.
Section snippets
Ethics statement
This study was approved by the Ethics Committee of Peking University School of Stomatology (Approval number PKUSSIRB-2011007). Participants and/or their guardians provided written informed consent before participating in this study.
Reagents
Human recombinant S100A9 were produced as previously described by Ryckman et al. (2003) and were found to contain less than 1 pg endotoxin/μg of protein measured by Limulus amebocyte assay (Sigma–Aldrich, St. Louis, MO). The RAGE blocking antibody was from R&D systems
S100A9 promoted secretion of IL-6 and IL-8 from human PDL cells in a dose- and time-dependent manner
We previously reported that S100A9 but not S100A8 promoted the release of IL-6, IL-8 from PDL cells (Zheng et al., 2014). To confirm the effect of S100A9 on the production of IL-6 and IL-8, human PDL cells were treated by S100A9 with concentrations of 1, 10, and 100 μg/mL. As expected, the mRNA levels of IL-6 and IL-8 were increased in a dose-dependent manner (Fig. 1A and B). ELISA results further confirmed the trend in up-regulation of supernatant IL-6 and IL-8 levels, which were comparable to
Discussion
This is the first study to identify the mechanisms of S100A9-induced release of IL-6 and IL-8 from human PDL cells in vitro. Our results demonstrated that: (1) the functional receptor of S100A9 in human PDL cells is TLR4, but not RAGE; (2) S100A9 promotes the secretion of IL-6 through the NF-κB and p38 MAPK pathways; (3) S100A9 promotes the release of IL-8 via the NF-κB, ERK1/2, JNK1/2 and p38 MAPK pathways; and (4) ROS is involved in S100A9-mediated secretion of IL-6 and IL-8 from PDL cells.
Conclusion
Our study revealed that S100A9 promotes inflammatory cytokines expression of PDL cells through TLR4-mediated activation, but not RAGE. Additionally, our results revealed that the secretion of IL-6 and IL-8 from human PDL cells is through different signaling pathways and that the release of both cytokines depends on ROS production. Our findings build on the proposed mechanisms linking pro-inflammatoy S100A9 to autoimmune and inflammatory diseases.
Author contributions
HM and JH conceived and designed the experiments. HG, YZ, XZ, and LP performed the experiments. HG analyzed the data and wrote the paper.
Conflict of interests
The authors declare no conflicts of interests.
Acknowledgements
The authors appreciate Dr. Philippe A. Tessier (centre de recherche en Infectiologie, Centre de recherche du Centre Hospitalier Universitaire de Quebec, and Faculty of Medecine, Universite Laval, Quebec, Canada) for his kind gift of human S100A9 plasmids. This work was supported by the National Natural Science Foundation of China (30271411, 30471882, 30973319, 81271149).
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