Porphyromonas Gingivalis Modulates TIR-domain-containing Adaptors Proteins Expression in Epithelial and Endothelial Cells

Objectives: The overpassing of epithelial and endothelium barriers and the host-bacterial interactions are crucial in the onset and development of periodontitis. Inflammatory cascades are initiated by the recognition of periodontal pathogens such as Porphyromonas gingivalis(Pg) by Toll-Like receptors (TLRs). TLR related pathways are activated after recruitment of TIR-domain-containing adaptors (TIRs). The aim of this study was to evaluate the effects of Pginfection on the expression of TIRs in gingival epithelial cells and endothelial cells.
Methods: TERT-2/OKF6 epithelial cells (GECs) and HUVEC endothelial cells (ECs) were cultured and then infected with PgATCC 33277 (MOI= 100) for 24 hours. TNF-αsecretion in supernatants has been evaluated with ELISA. After cell lysis, mRNA expression of the five TIRs (Myd88, Mal, Trif, Tram-1 and Sarm), TLR2 and 4 and the suppressor of cytokine signaling protein-1 (SOCS-1),was evaluated by RTqPCR. Immunofluorescence was performed for the TIRs on both cell types. Finally, the TLRs and TIRs protein-protein interaction and its inhibitor SOCS-1, were evaluated by a Pull-Down assay, co-immunoprecipitation and WB.
Results: In both cell types, Pginfection increases significantly TNF-αsecretionand mRNA expression of Mal, Myd88, Trif and Tram. The expression of TLR-2 and 4 was also increased at both mRNA and protein level following infection. Interestingly, Pg infection was mainly associated to an increased TLR-4/Mal/MyD88 interactions while interactions with the Trif/Tram complex were less affected as observed after co-immunoprecipitation in both cell types. Moreover, Pginfection reduced inhibitor SOCS-1 and increased the TIR SARM protein expression.
Conclusions: This study showed that Pgmodulates the expression of TIRs and their interactions with TLR-4. Mal-Myd88 protein-protein interaction associated with TLR4 was the main pathway activated during Pginfection, while infection was also able to decrease expression of inhibitor SOCS-1, simultaneously increasing SARM expression, implicated in the regulation of the inflammatory host response.Therefore, it is of interest to understand more precisely the role of TIRs proteins interactions in host-immune response in the context of periodontitis and to consider targeting some of them as therapeutic targets.