TNFα 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*, Mona Dvir-Ginzberg*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

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 NFκB 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 TNFα 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 NFκB subunit RelA that is essential for NFκB transcriptional activity. Results: TNFα expression was elevated 4 h after exposure to P. gingivalis. ChIP confirmed that RelA was enriched in the mouse TNFα promoter 4 h following stimulation, which correlated with the increased TNFα mRNA levels. Preceding TNFα 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 TNFα secretion, while co-administering CA074Me (a cathepsin B inhibitor) with P. gingivalis reduced RelA promoter enrichment, resulting in impaired TNFα expression. Conclusions: Together, the results suggest that P. gingivalis induces TNFα expression, at least in part, by enhancing cleavage of Sirt1 via a TLR-dependent signaling circuit.

Original languageAmerican English
Pages (from-to)535-546
Number of pages12
JournalJournal of Periodontal Research
Volume56
Issue number3
DOIs
StatePublished - Jun 2021

Bibliographical note

Publisher Copyright:
© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Keywords

  • Porphyromonas gingivalis
  • RelA
  • Sirt1
  • TNFα

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