Pro-inflammatory Roles of NOD2 in Human Gingival Fibroblasts

Objectives: Since human gingival fibroblasts (HGFs) are the major cells in periodontal tissues, we hypothesized that HGFs are expressed the nucleotide-binding oligomerization domain (NOD) 2 for peptidoglycan components, which induce innate immune responses against invading bacteria. Methods: We used HGFs isolated from three clinically healthy gingiva during routine distal wedge surgical procedures. The HGFs that grew from the gingiva were cultured primarily on plastic dishes in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and antibiotics at 37°C in humidified air with 5% CO2. Confluent cells were transferred and cultured for use in the present study. We examined NOD2 expression in HGFs by RT-PCR and flow cytometry analysis. HGFs were stimulated with NOD2 agonist, Muramyldipeptide (MDP), and IFN-gamma for 24h. The supernatants from HGFs were collected, and IL-6, IL-8, CCL2, CXCL10, and CXCL11 concentration of the culture supernatants was measured in triplicate with enzyme-linked immunosorbent assay (ELISA). In some experiments, we pre-incubated HGFs with pharmacologic NF-kappaB inhibitor prior to the stimulation. We used RT-PCR and immunohistochemistry to examine the NOD2 expression in periodontal tissues. Results: NOD2 is constitutively expressed in HGFs. MDP stimulation enhanced IL-6, IL-8 and CCL2 in a dose-dependent manner. IL-6, IL-8 and CCL2 productions induced by MDP were significantly inhibited by NF-kappaB inhibitor. Moreover, MDP enhanced CXCL10 and CXCL11 productions by IFN-gamma-stimulated HGFs, though single stimuli of MDP did not induce CXCL10 and CXCL11 productions. Furthermore, we elucidated IFN-gamma enhanced NOD2 expression in HGFs. Additionally, RT-PCR and immunohistochemistry revealed that NOD2 were expressed in periodontally diseased tissues. Conclusion: These findings indicate that NOD2 in HGFs is the functional receptor involved in inflammatory reactions in periodontal tissues, which might be responsible for periodontal pathogenesis. This work was supported by a Grant-in-Aid for Scientific Research (B) (19791616) from the Japan Society for the Promotion of Science.