TNF$\alpha$ expression by Porphyromonas gingivalis-stimulated macrophages relies on Sirt1 cleavage


Sai R.K. Meka, Tahsin Younis, Eli Reich, Jinan Elayyan, Ashok Kumar, Emmanuelle Merquiol, Galia Blum, Shira Kalmus, Yonathan H. Maatuf, George Batshon, Gabriel Nussbaum, Yael Houri-Haddad, and Mona Dvir-Ginzberg. 2021. “TNF$\alpha$ expression by Porphyromonas gingivalis-stimulated macrophages relies on Sirt1 cleavage.” Journal of Periodontal Research, 56, 3, Pp. 535–546.


Objective: Periodontitis is one the most common chronic inflammatory conditions, resulting in destruction of tooth-supporting tissues and leading to tooth loss. Porphyromonas gingivalis activates host macrophages to secrete pro-inflammatory cytokines and elicit tissue damage, in part by inducing NF-kappa-B transactivation. Since NF$ąppa$B transactivation is negatively regulated by the Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase enzyme Sirt1, we sought to assess if RAW264.7 macrophages exposed to P. gingivalis demonstrate impaired Sirt1 activity, to ultimately induce a pro-inflammatory response. Methods: RAW264.7 macrophages were incubated with heat- killed P. gingivalis for 2, 4, 8, and 24 h. Stimulated RAW264.7 were assessed for TNF$\alpha$ expression via PCR, ELISA, and ChIP analysis. Following the activation of RAW264.7 macrophages, immunoblot analysis was executed to detect modifications in Sirt1 and the NF$ąppa$B subunit RelA that is essential for NF$ąppa$B transcriptional activity. Results: TNF$\alpha$ expression was elevated 4 h after exposure to P. gingivalis. ChIP confirmed that RelA was enriched in the mouse TNF$\alpha$ promoter 4 h following stimulation, which correlated with the increased TNF$\alpha$ mRNA levels. Preceding TNF$\alpha$ expression, we detected Phosphoserine 536 and acetylated lysine 310 of RelA after 2 hours exposure with P. gingivalis. Moreover, reduced Sirt1 activity was associated with its cleavage in RAW264.7 protein extracts, after 2 hours of P. gingivalis exposure. Blocking TLR2/4 signaling prevented Sirt1 cleavage, loss of deacetylase activity, and TNF$\alpha$ secretion, while co-administering CA074Me (a cathepsin B inhibitor) with P. gingivalis reduced RelA promoter enrichment, resulting in impaired TNF$\alpha$ expression. Conclusions: Together, the results suggest that P. gingivalis induces TNF$\alpha$ expression, at least in part, by enhancing cleavage of Sirt1 via a TLR-dependent signaling circuit.