Macrophage activation by RelA following P. gingivalis challenge: possible regulation by SIRT1/RelA axis.

Objectives:
Porphyromonas gingivalis is the major pathogen in the development of adult periodontitis. SIRT1 is a highly conserved NAD+ dependent protein deacetylase that is involved in diverse cellular processes. In inflammation, SIRT1 was shown to inhibit NF-kappaB signaling via p65/RelA deacetylation, thereby protecting from inflammatory damage. Here we examine the mechanism by which SIRT1 may regulate macrophage activation in P.gingivalis challenge.
Methods:
RAW 264.7 macrophage cell lines activated with 10 μL of 10⁹ CFU/mL heat killed P.gingivalis 33277 ATCC for different time incubations: 2, 4, 8, and 24h . Following treatment, cells harvested and processed for immunoblot analysis of SIRT1, Rel-A and acetylated-RelA. Further, protein extracts and supernatants analyzed for TNFα levels by Enzyme-linked immunosorbent assay (ELISA).
Results:
We observed an increase in TNFα secretion levels after challenging raw cells with P. gingivalis (Pg), which is also confirmed by mRNA expression of TNFα. SIRT1 levels increased after 4 hours from the challenge time. We additionally observed decreased levels of acetyl-P65 after the challenge with P. gingivalis, possibly due to the rising levels of SIRT1.
Conclusions:
The results support that Pg. induces TNFα expression and protein secretion which is accompanied with increased SIRT1 levels and decreased p65 acetylation. In contrast to several reports indicating that the deacetylation of p65 inhibits its transcriptional activity, RAW cells show enhanced TNFα expression with reduced acetylation of p65, supporting it may possess enhanced transcriptional activity under these conditions. Future endeavors will focus on Chromatin immunoprecipitation and immunofluorescence to detect enrichment of p65 in the nucleus under P. gingivalis challenge.