In vitromodeling of host-parasite interactions: the 'subgingival' biofilm challenge of primary human epithelial cells

Abstract

BackgroundMicrobial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria.ResultsWe describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes.ConclusionWe developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases.

The RANKL–OPG system is differentially regulated by supragingival and subgingival biofilm supernatants

“…Supernatants were collected after 64.5 h of supragingival or subgingival biofilm culture. The composition of the biofilm-associated species on the hydroxyapatite discs was determined by bacterial culture analysis as previously described [16,17]. The data represents the bacterial mean counts ± SD from triplicate biofilm cultures.…”

“…An in vitro subgingival biofilm model has so far been used to study apoptotic and pro-inflammatory cytokine responses in gingival epithelial cells [17].…”

“…After a total 64.5 h of incubation, at an advanced stage of biofilm maturation, the culture supernatants were collected, filtered and stored at -80°C. The composition of biofilms on the hydroxyapatite discs at the time of supernatant collection was performed by bacterial culture analysis, as previously described [16,17], and the respective bacterial counts are provided in Table 1. It is anticipated that supernatants from earlier stages of biofilm culture would be less virulent, or at least less representative an established biofilm stage.…”

“…The bacterial protein concentration in these supernatants was determined by the BCA Protein Assay (Pierce). For the experiments, these biofilm supernatant preparations were diluted into the final cell culture medium and maintained in the cell culture for up to 24 h [17]. Their concentration is expressed as total protein (µg/ml) present in the cell cultures.…”

Involvement of the TREM-1/DAP12 pathway in the innate immune responses to Porphyromonas gingivalis

“…For this analysis, two oligonucleotide probes purchased from Microsynth (Balgach, Switzerland), were used simultaneously, one specific for P. gingivalis (L-Pging1006-2 labelled at the 5'-end with Cy3), and one universal bacterial probe (EUB338 labelled at the 5'-end with Cy5). The sequences of these probes as well as the standard hybridization conditions are provided elsewhere (Guggenheim et al, 2009;Zijnge et al, 2010). For nuclear staining, the samples were further incubated with DAPI (Invitrogen, Life Technologies, Basel, Switzerland), to counter-stain DNA.…”