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

Citation:

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.

Abstract:

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.