IADR Abstract Archives
Macrophage Activation Through SIRT1/RelA Axis
Objectives: Introduction: Periodontal infection is the most common chronic inflammatory disease in humans, which results in destruction of tooth-supporting tissues and eventually leading to tooth loss. This process is characterized by destruction of the periodontal ligament, formation of periodontal pockets, and alveolar bone resorption. Porphyromonas gingivalis (Pg) remains the major pathogen in the development of adult periodontitis. Pg is able to activate host macrophages to secrete pro-inflammatory cytokines and elicit tissue damage. Our work aims to biochemically delineate the sequence of event leading to this activation.
Objectives
To characterize the levels of SIRT1, Acetylated-RelA and secreted cytokines followingheat killed Pg challenge of macrophage cell line.
To examine if Resolvin D1 possesses a protective effect in RelA/SIRT1 axis, following Pg challenge.
Methods: RAW264.7 macrophages were activated with 10 μL of 109 CFU/mL heat killed Pg 33277 ATCC and 100nM Resolvin D1 /placebo for 2, 4, 8, and 24h. Following treatment, cells harvested and processed for immunoblot analysis and real-time quantitative PCR to detect TNFa mRNA. Supernatants were analyzed for TNFa levels by Enzyme-linked immunosorbent assay (ELISA). Chromatin immunoprecipitation (ChIP) analysis was carried out to assess the enrichment of RelA on TNFa promoter .
Results: TNFa secretion levels was elevated 8h after challenging Pg stimulation, which is also confirmed by mRNA expression. Four hours post Pg challenge acetyl-RelA and cleaved SIRT1 (75SIRT1) are significantly increased. ChIP results, confirm that RelA is enriched on mouse TNFa- promoter 4h post-Pg stimulation, which correlates with the increased TNFa mRNA. Notably, administering Resolvin D1 with Pg reduced RelA enrichment explaining the reduced TNFa secretion in this treatment.
Conclusions: The results support that Pg. induces TNFa by enhanced cleavage of SIRT1. Leading to hyperacetylated RelA and increased transactivation of TNFa. These effects were significantly attenuated when Resolvin D1 was co-administered with Pg.
Objectives
To characterize the levels of SIRT1, Acetylated-RelA and secreted cytokines followingheat killed Pg challenge of macrophage cell line.
To examine if Resolvin D1 possesses a protective effect in RelA/SIRT1 axis, following Pg challenge.
Methods: RAW264.7 macrophages were activated with 10 μL of 109 CFU/mL heat killed Pg 33277 ATCC and 100nM Resolvin D1 /placebo for 2, 4, 8, and 24h. Following treatment, cells harvested and processed for immunoblot analysis and real-time quantitative PCR to detect TNFa mRNA. Supernatants were analyzed for TNFa levels by Enzyme-linked immunosorbent assay (ELISA). Chromatin immunoprecipitation (ChIP) analysis was carried out to assess the enrichment of RelA on TNFa promoter .
Results: TNFa secretion levels was elevated 8h after challenging Pg stimulation, which is also confirmed by mRNA expression. Four hours post Pg challenge acetyl-RelA and cleaved SIRT1 (75SIRT1) are significantly increased. ChIP results, confirm that RelA is enriched on mouse TNFa- promoter 4h post-Pg stimulation, which correlates with the increased TNFa mRNA. Notably, administering Resolvin D1 with Pg reduced RelA enrichment explaining the reduced TNFa secretion in this treatment.
Conclusions: The results support that Pg. induces TNFa by enhanced cleavage of SIRT1. Leading to hyperacetylated RelA and increased transactivation of TNFa. These effects were significantly attenuated when Resolvin D1 was co-administered with Pg.