IADR Abstract Archives
Cytokine Responses of Human Gingival Epithelial Keratinocytes to Anaeroglobus geminatus
Objectives: Anaeroglobus geminatus is a Gram-negative anaerobic species recently identified by metagenomic approaches in subgingival biofilms, potentially associated with periodontal disease. The interactions of this newly discovered species with the host have barely been characterized. The aim of this study was to investigate the potential cytokine regulatory effects of A. geminatus on human gingival epithelial keratinocytes (HGEK).
Methods: HGEK were cultivated in defined keratinocyte serum-free medium. Isolated A. geminatus (type strain CCUG44773T) was grown under anaerobic conditions on blood agar culture plates. For the experiments, bacterial colonies were harvested, suspended in physiological saline and added to the HGEK cultures at multiplicity of infection (MOI) 0.1, 1 and 10, under 2% oxygen conditions, for 4 h and 24 h. Upon completion of the experiments, the secreted culture supernatants were collected and analyzed by a 10-plex cytokine immunoassay, in a Luminex200 device. One-way analysis of variance was used to compare differences between the control and experimental groups, within each time-point.
Results: After 4 h of challenge with A. geminatus, there were no changes in the secretion of any of the 10 cytokines studied by the HGEK. However, after 24 h, the secretion of IL-10, IL-6, IL-4 was significantly (p<0.05) increased in a concentration-dependent manner, by increasing MOI. The secretion of IL-1β, GM-CSF, IL-5, IL-2 and TNF-α was also increased significantly (p<0.05) reaching, however, a plateau at MOI 1. The secretion of IFN-γ and IL-8 were not affected by the A. geminatus-challenge. Among the studied cytokines, the highest regulatory responses to A. geminatus, compared to the unchallenged control, were observed on IL-1β (2.3-fold), IL-6 (2.7-fold), IL-10 (2.1-fold), IL-2 (2.1-fold), and IL-4 (2.3-fold).
Conclusions: Anaeroglobus geminatus appears to cause a late (24 h) inflammatory response by HGEK, with distinct inflammatory signatures regarding the type of cytokines affected, and the MOI-dependency.
Methods: HGEK were cultivated in defined keratinocyte serum-free medium. Isolated A. geminatus (type strain CCUG44773T) was grown under anaerobic conditions on blood agar culture plates. For the experiments, bacterial colonies were harvested, suspended in physiological saline and added to the HGEK cultures at multiplicity of infection (MOI) 0.1, 1 and 10, under 2% oxygen conditions, for 4 h and 24 h. Upon completion of the experiments, the secreted culture supernatants were collected and analyzed by a 10-plex cytokine immunoassay, in a Luminex200 device. One-way analysis of variance was used to compare differences between the control and experimental groups, within each time-point.
Results: After 4 h of challenge with A. geminatus, there were no changes in the secretion of any of the 10 cytokines studied by the HGEK. However, after 24 h, the secretion of IL-10, IL-6, IL-4 was significantly (p<0.05) increased in a concentration-dependent manner, by increasing MOI. The secretion of IL-1β, GM-CSF, IL-5, IL-2 and TNF-α was also increased significantly (p<0.05) reaching, however, a plateau at MOI 1. The secretion of IFN-γ and IL-8 were not affected by the A. geminatus-challenge. Among the studied cytokines, the highest regulatory responses to A. geminatus, compared to the unchallenged control, were observed on IL-1β (2.3-fold), IL-6 (2.7-fold), IL-10 (2.1-fold), IL-2 (2.1-fold), and IL-4 (2.3-fold).
Conclusions: Anaeroglobus geminatus appears to cause a late (24 h) inflammatory response by HGEK, with distinct inflammatory signatures regarding the type of cytokines affected, and the MOI-dependency.