Interleukin-8 Responses of Multi-Layer Gingival Epithelia to Subgingival Biofilms: Role of the “Red Complex” Species

Georgios N Belibasakis,Nagihan Bostanci,Thomas Thurnheer,Stefan Bereswill

doi:10.1371/journal.pone.0081581

Abstract

Periodontitis is an infectious inflammatory disease that results in the destruction of the tooth-supporting (periodontal) tissues. The Gram-negative anaerobic species Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, (also known as the “red complex” species) are highly associated with subgingival biofilms at periodontitis-affected sites. A major chemokine produced by the gingival epithelium in response to biofilm challenge, is interleukin (IL)-8. The aim of this in vitro study was to investigate the relative effect of the “red complex” species as constituents of subgingival biofilms, on the regulation of IL-8 by gingival epithelia. Multi-layered organotypic human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its 7-species variant, excluding the “red complex”. IL-8 gene expression and secretion analyses were performed by qPCR and ELISA, respectively. After 3 h, both biofilms up-regulated IL-8 gene expression, but the presence of the “red complex” resulted in 3-fold greater response. IL-8 secretion was also up-regulated by both biofilms, with no differences between them. After 24 h, the 10-species biofilm reduced IL-8 secretion to 50% of the control, but this was not affected when the “red complex” was absent. In conclusion, as part of biofilms, “red complex” species differentially regulate IL-8 in gingival epithelia, potentially affecting the chemotactic responses of the tissue.

Highlights

Periodontal diseases are infectious inflammatory diseases, caused by endogenous oral bacteria colonizing the tooth surfaces as polymicrobial biofilm communities
The multi-layered gingival epithelial cultures were challenged for 3 h and 24 h with either the 10-species subgingival biofilm, or its 7-species variant, which excluded the ‘‘red complex’’ species
After 3 h, the 10-species biofilm caused a significant enhancement of IL-8 expression, compared to the control group (6.3-fold), which was more potent than the effect caused by the 7-species biofilm lacking the ‘‘red complex’’ species (2.2-fold) (Figure 1)

Summary

Introduction

Periodontal diseases are infectious inflammatory diseases, caused by endogenous oral bacteria colonizing the tooth surfaces as polymicrobial biofilm communities. Interaction of biofilms with the juxtaposing periodontal tissues triggers an inflammatory response, aiming to prevent bacterial colonization and establishment [1]. IL-8 expression and production is induced in a number of cell types by T. denticola [6,7], T. forsythia [8,9], or P. gingivalis [9,10], as well as a mixed infection of all three species [11]. A 9-species biofilm including P. gingivalis and T. forsythia induced IL-8 production in human gingival epithelial cell cultures [18]. A number of studies are contrasting this trend, by demonstrating lack of effect or even inhibition of IL-8 production by epithelial cells in response to T. denticola [19,20,21], or P. gingivalis [19,22,23]. The capacity of P. gingivalis in planktonic state to inhibit IL-8 gene expression by gingival epithelial cells has been designated as ‘‘chemokine paralysis’’ [22]

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