Co-Culture of P. gingivalis and F. nucleatum Synergistically Elevates IL-6 Expression via TLR4 Signaling in Oral Keratinocytes.

Periodontitis, characterized by persistent inflammation in the periodontium, is intricately connected to systemic diseases, including oral cancer. Bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, play a pivotal role in periodontitis development because they contribute to dysbiosis and tissue destruction. Thus, comprehending the interplay between these bacteria and their impacts on inflammation holds significant relevance in clinical understanding and treatment advancement. In the present work, we explored, for the first time, their impacts on the expressions of pro-inflammatory mediators after infecting oral keratinocytes (OKs) with a co-culture of pre-incubated P. gingivalis and F. nucleatum. Our results show that the co-culture increases IL-1ß, IL-8, and TNF-α expressions, synergistically augments IL-6, and translocates NF-kB to the cell nucleus. These changes in pro-inflammatory mediators-associated with chronic inflammation and cancer-correlate with an increase in cell migration following infection with the co-cultured bacteria or P. gingivalis alone. This effect depends on TLR4 because TLR4 knockdown notably impacts IL-6 expression and cell migration. Our study unveils, for the first time, crucial insights into the outcomes of their co-culture on virulence, unraveling the role of bacterial interactions in polymicrobial diseases and potential links to oral cancer.

Porphyromonas gingivalis-Helicobacter pylori co-incubation enhances Porphyromonas gingivalis virulence and increases migration of infected human oral keratinocytes.

Background: Porphyromonas gingivalis is part of the subgingival biofilm and a keystone species in the development of periodontitis. Interactions between P.gingivalis and other bacteria in biofilms have been shown to affect bacterial virulence. Helicobacter pylori also inhabits the subgingival biofilm, but the consequences of interactions there with P.gingivalis remain unknown. Here, we investigated how the pre-incubation of P.gingivalis with H.pylori affects P.gingivalis virulence. Methods: We assayed P.gingivalis internalization by oral keratinocytes (OKs), hemagglutination and biofilm formation to identify alterations in virulence after pre-incubation with H. pylori. Also, we evaluated viability and migration of OKs infected with P. gingivalis, as well as the role of toll-like receptor 4 (TLR4).   In addition, we quantified the mRNA of genes associated with P.gingivalis virulence. Results: Pre-incubation of P.gingivalis with H.pylori enhanced P.gingivalis biofilm formation, bacterial internalization into OKs and hemagglutination. Infection with pre-incubated P.gingivalis increased OK migration in a manner dependent on the O-antigen and linked to  increased expression of the gingipain RgpB. Also, OK TLR4 participates in these events, because upon TLR4 knock-down, pre-incubated P.gingivalis no longer stimulated OK migration. Discussion: We provide here for the first time insight to the consequences of direct interaction between P.gingivalis and H.pylori. In doing so, we shed light on the mechanism by which H. pylori presence in the oral cavity increases the severity or progression of periodontitis.