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

Guggenheim B, Gmür R, Galicia JC, Stathopoulou PG, Benakanakere MR, Meier A, Thurnheer T, Kinane DF - BMC Microbiol. (2009)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Plattenstrasse 11, CH-8032 Zürich, Switzerland. bernie@zzmk.uzh.ch

ABSTRACT

Background: Microbial 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.

Results: We 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.

Conclusion: We 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.

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Biofilm structure visualized by CLSM and TEM. CLSM images of a 64.5 h 9-species biofilm stained by multiplex FISH for (A) V. dispar (purple; VEI217-ROX, 40% formamide), C. rectus (blue; Camp655-Cy5, 30% formamide), F. nucleatum (red; Fnuc133c-Cy3, 30% formamide), and P. intermedia (green; L-Pint649-2-FAM, 30% formamide), (B) V. dispar (purple; VEI217-ROX, 40% formamide), A. naeslundii (red; L-Act476-2-Cy3, 25% formamide), S. intermedius (green; L-Sco/int172-2-FAM, 25% formamide), and S. oralis (blue; MIT447-Cy5, 25% formamide), and (C) V. dispar (purple; VEI217-ROX, 40% formamide), T. forsythia (green; Tfor582-FAM, 40% formamide), P. gingivalis (red; L-Pgin1006-Cy3, 30% formamide), and C. rectus (blue; Camp655-Cy5, 30% formamide). Images are 1-μm-thick transverse (large images), sagittal (right) and coronal (bottom) slices at the positions indicated by the fine lines. The length of the bars indicates 20 μm. (D) TEM image of a 64.5 h multi-species biofilm demonstrating the predominance of varius cocci or very short rods (S. oralis, S. intermedius, V. dispar, P. intermedia) and of the fusiform F. nucleatum cells. Bar = 5 μm.
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Figure 2: Biofilm structure visualized by CLSM and TEM. CLSM images of a 64.5 h 9-species biofilm stained by multiplex FISH for (A) V. dispar (purple; VEI217-ROX, 40% formamide), C. rectus (blue; Camp655-Cy5, 30% formamide), F. nucleatum (red; Fnuc133c-Cy3, 30% formamide), and P. intermedia (green; L-Pint649-2-FAM, 30% formamide), (B) V. dispar (purple; VEI217-ROX, 40% formamide), A. naeslundii (red; L-Act476-2-Cy3, 25% formamide), S. intermedius (green; L-Sco/int172-2-FAM, 25% formamide), and S. oralis (blue; MIT447-Cy5, 25% formamide), and (C) V. dispar (purple; VEI217-ROX, 40% formamide), T. forsythia (green; Tfor582-FAM, 40% formamide), P. gingivalis (red; L-Pgin1006-Cy3, 30% formamide), and C. rectus (blue; Camp655-Cy5, 30% formamide). Images are 1-μm-thick transverse (large images), sagittal (right) and coronal (bottom) slices at the positions indicated by the fine lines. The length of the bars indicates 20 μm. (D) TEM image of a 64.5 h multi-species biofilm demonstrating the predominance of varius cocci or very short rods (S. oralis, S. intermedius, V. dispar, P. intermedia) and of the fusiform F. nucleatum cells. Bar = 5 μm.

Mentions: Bacteria were stained by multiplex FISH and assessed by confocal laser scanning microscopy (CLSM). Representative images are shown in Fig. 2. 64.5 h biofilms had a thickness of 40 to 60 μm with broad distribution of S. intermedius, P. intermedia and F. nucleatum. In contrast, T. forsythia, and P. gingivalis were restricted to microcolonies. C. rectus occurred dispersed, but here and there also in microcolonies. Images from transmission electron microscopy (TEM) show the predominance of various cocci and short rods (streptococci, P. intermedia, V. dispar) interspersed by prominent elongated fusiform cells (F. nucleatum subsp. vincentii) (Fig. 2D).


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

Guggenheim B, Gmür R, Galicia JC, Stathopoulou PG, Benakanakere MR, Meier A, Thurnheer T, Kinane DF - BMC Microbiol. (2009)

Biofilm structure visualized by CLSM and TEM. CLSM images of a 64.5 h 9-species biofilm stained by multiplex FISH for (A) V. dispar (purple; VEI217-ROX, 40% formamide), C. rectus (blue; Camp655-Cy5, 30% formamide), F. nucleatum (red; Fnuc133c-Cy3, 30% formamide), and P. intermedia (green; L-Pint649-2-FAM, 30% formamide), (B) V. dispar (purple; VEI217-ROX, 40% formamide), A. naeslundii (red; L-Act476-2-Cy3, 25% formamide), S. intermedius (green; L-Sco/int172-2-FAM, 25% formamide), and S. oralis (blue; MIT447-Cy5, 25% formamide), and (C) V. dispar (purple; VEI217-ROX, 40% formamide), T. forsythia (green; Tfor582-FAM, 40% formamide), P. gingivalis (red; L-Pgin1006-Cy3, 30% formamide), and C. rectus (blue; Camp655-Cy5, 30% formamide). Images are 1-μm-thick transverse (large images), sagittal (right) and coronal (bottom) slices at the positions indicated by the fine lines. The length of the bars indicates 20 μm. (D) TEM image of a 64.5 h multi-species biofilm demonstrating the predominance of varius cocci or very short rods (S. oralis, S. intermedius, V. dispar, P. intermedia) and of the fusiform F. nucleatum cells. Bar = 5 μm.
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Figure 2: Biofilm structure visualized by CLSM and TEM. CLSM images of a 64.5 h 9-species biofilm stained by multiplex FISH for (A) V. dispar (purple; VEI217-ROX, 40% formamide), C. rectus (blue; Camp655-Cy5, 30% formamide), F. nucleatum (red; Fnuc133c-Cy3, 30% formamide), and P. intermedia (green; L-Pint649-2-FAM, 30% formamide), (B) V. dispar (purple; VEI217-ROX, 40% formamide), A. naeslundii (red; L-Act476-2-Cy3, 25% formamide), S. intermedius (green; L-Sco/int172-2-FAM, 25% formamide), and S. oralis (blue; MIT447-Cy5, 25% formamide), and (C) V. dispar (purple; VEI217-ROX, 40% formamide), T. forsythia (green; Tfor582-FAM, 40% formamide), P. gingivalis (red; L-Pgin1006-Cy3, 30% formamide), and C. rectus (blue; Camp655-Cy5, 30% formamide). Images are 1-μm-thick transverse (large images), sagittal (right) and coronal (bottom) slices at the positions indicated by the fine lines. The length of the bars indicates 20 μm. (D) TEM image of a 64.5 h multi-species biofilm demonstrating the predominance of varius cocci or very short rods (S. oralis, S. intermedius, V. dispar, P. intermedia) and of the fusiform F. nucleatum cells. Bar = 5 μm.
Mentions: Bacteria were stained by multiplex FISH and assessed by confocal laser scanning microscopy (CLSM). Representative images are shown in Fig. 2. 64.5 h biofilms had a thickness of 40 to 60 μm with broad distribution of S. intermedius, P. intermedia and F. nucleatum. In contrast, T. forsythia, and P. gingivalis were restricted to microcolonies. C. rectus occurred dispersed, but here and there also in microcolonies. Images from transmission electron microscopy (TEM) show the predominance of various cocci and short rods (streptococci, P. intermedia, V. dispar) interspersed by prominent elongated fusiform cells (F. nucleatum subsp. vincentii) (Fig. 2D).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Oral Biology, Section for Oral Microbiology and General Immunology, University of Zürich, Plattenstrasse 11, CH-8032 Zürich, Switzerland. bernie@zzmk.uzh.ch

ABSTRACT

Background: Microbial 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.

Results: We 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.

Conclusion: We 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.

Show MeSH
Related in: MedlinePlus