P. gingivalis Modulates Keratinocytes through FOXO Transcription Factors

Denis F Kinane,Manjunatha R Benakanakere,Anastasios Moschidis,Guangyu Dong,Shuai Li,Dana T Graves,Javier Ortiz

doi:10.1371/journal.pone.0078541

Denis F Kinane, Manjunatha R Benakanakere + Show 5 more

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https://doi.org/10.1371/journal.pone.0078541

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Abstract

P. gingivalis is a prominent periodontal pathogen that has potent effects on host cells. In this study we challenged gingival epithelial cells with P. gingivalis with the aim of assessing how mRNA levels of key target genes were modulated by P. gingivalis via the transcription factors FOXO1 and FOXO3. Primary mono- and multi-layer cultures of gingival epithelial cells were challenged and barrier function was examined by fluorescent dextran and apoptosis was measured by cytoplasmic histone associated DNA. Gene expression levels were measured by real-time PCR with and without FOXO1 and FOXO3 siRNA compared to scrambled siRNA. P. gingivalis induced a loss of barrier function and stimulated gingival epithelial cell apoptosis in multilayer cultures that was in part gingipain dependent. P. gingivalis stimulated an increase in FOXO1 and FOXO3 mRNA, enhanced mRNA levels of genes associated with differentiated keratinocyte function (keratin-1, -10, -14, and involucrin), increased mRNA levels of apoptotic genes (BID and TRADD), reduced mRNA levels of genes that regulate inflammation (TLR-2 and -4) and reduced those associated with barrier function (integrin beta-1, -3 and -6). The ability of P. gingivalis to modulate these genes was predominantly FOXO1 and FOXO3 dependent. The results indicate that P. gingivalis has pronounced effects on gingival keratinocytes and modulates mRNA levels of genes that affect host response, differentiation, apoptosis and barrier function. Moreover, this modulation is dependent upon the transcription factors FOXO1 or FOXO3. In addition, a new function for FOXO1 was identified, that of suppressing TLR-2 and TLR-4 and maintaining integrin beta -1, beta -3 and beta -6 basal mRNA levels in keratinocytes.

Highlights

Human gingival epithelial cells function as an important part of the innate host defense to limit invasion by microorganisms [1]
F. nucleatum and S. gordonii had no effect on epithelial barrier function nor was it affected by leupeptin in the absence of P. gingivalis (P.0.05) (Figure 1B)
We examined the impact of P. gingivalis on differentiated gingival epithelial cells and on key genes that regulate key aspects of keratinocyte function

Summary

Introduction

Human gingival epithelial cells function as an important part of the innate host defense to limit invasion by microorganisms [1]. This is true for the gingiva due to the high bacterial load that is present as a biofilm on adjacent tooth surfaces or that adheres directly to gingival epithelium. Gingival epithelial cells come into contact with a variety of bacteria. These include well-defined periodontal pathogens such as P. gingivalis as well as commensal organisms such as S. gordonii [4]. Gingival epithelial cells respond to bacteria in a number of different ways and the response in part depends upon the particular bacteria present

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