- Email: [email protected]
Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species
Accepted Manuscript Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species Dareen Fteita, Eija Könönen, Mervi Gürsoy, Xiaochu Ma, Herman O. Sintim, Ulvi Kahraman Gürsoy PII:
S1075-9964(18)30156-2
DOI:
10.1016/j.anaerobe.2018.09.001
Reference:
YANAE 1942
To appear in:
Anaerobe
Received Date: 25 April 2018 Revised Date:
28 August 2018
Accepted Date: 1 September 2018
Please cite this article as: Fteita D, Könönen E, Gürsoy M, Ma X, Sintim HO, Gürsoy UK, Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species, Anaerobe (2018), doi: 10.1016/j.anaerobe.2018.09.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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QUORUM SENSING MOLECULES REGULATE EPITHELIAL CYTOKINE
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RESPONSE AND BIOFILM-RELATED VIRULENCE OF THREE PREVOTELLA
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SPECIES
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Dareen Fteitaa,*, Eija Könönena, b, Mervi Gürsoya, Xiaochu Mac, Herman O. Sintimc, Ulvi
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Kahraman Gürsoya
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a
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Finland
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Department of Periodontology, Institute of Dentistry, University of Turku, FI-20520, Turku,
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Welfare Division, Oral Health Care, City of Turku, Turku, Finland
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c
Department of Chemistry and Purdue Institute for Drug Discovery and Purdue Institute of
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Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette,
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Indiana, USA
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Correspondence:
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Department of Periodontology, Institute of Dentistry, University of Turku,
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20520 Turku, Finland.
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Tel: +358 40 9650024
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E-mail addresses:
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[email protected] (D. Fteita), [email protected] (E. Könönen), [email protected] (M.
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Gürsoy), [email protected] (X. Ma), [email protected] (H. O. Sintim),
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[email protected] (U.K. Gürsoy).
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Key words:
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autoinducer-2; estradiol; gingival keratinocytes; inflammatory response; pathogenicity;
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Prevotella
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ACCEPTED MANUSCRIPT ABSTRACT
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Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of
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bacteria, and at the same time activates immune response in eukaryotic organisms. We
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recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its
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analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the
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four species in the Prevotella intermedia group. Here, we examined the combined effects of
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estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK)
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against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella
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pallens, and 2) biofilm formation of these three Prevotella species. All experiments were
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performed in the presence or absence of estradiol, and with different QS molecules: DPD and
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its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-
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1β, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was
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quantitatively evaluated by measuring protein concentration via the Bradford method, and the
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microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging.
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Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with
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estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with
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estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced
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regulation of HMK interleukin expression and, at the same time, inhibited the biofilm
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formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in
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biofilm mass after application of butyl-DPD, which was most detectable among the P.
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intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In
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conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine
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response and, at the same time, to inhibit the biofilm formation of P. intermedia and P.
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nigrescens.
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1.
INTRODUCTION
In bacteria, cell-to-cell communication is coordinated and gene expression is regulated in
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synchronization with a cell density-dependent signaling pathway, known as quorum sensing
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(QS) (Bassler, 1999; Shao and Demuth, 2010; Galloway et al., 2011). QS is a molecular
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process by which a variety of bacteria maintain a diverse array of physiological activities,
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including motility, biofilm formation, and virulence (Atkinson et al., 1999; Shiner et al.,
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2006; Sintim et al., 2010). QS relies on the production, release, and detection of signaling
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molecules, termed autoinducers (AIs). AIs are classified into three major classes: N- acyl
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homoserine lactone (AHL) or AI-1 for Gram-negative bacteria, AI-2 for both Gram-negative
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and Gram-positive bacteria, and cyclic oligopeptides for Gram-positive bacteria (Sintim et al.,
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2010). In addition, evidence on the use of the AI-3 system by Escherichia coli and Salmonella
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exists in literature (Walters and Sperandio, 2006). Efforts have been made at inhibiting
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bacteria-to-bacteria crosstalk by quenching native QS responses with synthetic small
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molecules (Roy et al., 2010).
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Beside their impact on bacterial gene expression and virulence, QS molecules have a
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regulatory effect on the host immune response (Zargar et al., 2015). The gingival epithelium
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and underlying lamina propria constitute a unique physical barrier to protect periodontal
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tissues against microbial invasion, mechanical stress, and environmental hazards (Chung et
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al., 2007; Dawson et al., 2013). Furthermore, as an active player in the human innate
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immunity, gingival keratinocytes express proinflammatory and, to a lesser extent, anti-
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inflammatory cytokines and chemokines (Chung et al., 2007; Suchett-Kaye et al., 1998). AI-1
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molecules have the capability to induce alterations in epithelial barrier functions, leading to
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disruption of the epithelial integrity (Vikström et al., 2006; Eum et al., 2014). Moreover,
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different AHLs have been found to modulate epithelial homeostasis, cell migration, and
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apoptosis (Cooley et al., 2008; Karlsson et al., 2012; Schwarzer et al., 2012). Our group
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human gingival keratinocytes (Elmanfi et al., 2018).
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Prevotella intermedia, Prevotella nigrescens, Prevotella pallens, and Prevotella aurantiaca
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are the four phylogenetically close species within the P. intermedia group (Shah and Gharbia,
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1992; Könönen et al., 1998; Sakamoto et al., 2010). With their known ability to utilize
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maternal steroids (estrogen and progesterone), the increase in numbers of P. intermedia and
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P. nigrescens has been linked to gingival inflammation and pregnancy-related gingivitis
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(Kornman and Loesche, 1982; Gürsoy et al., 2009). Furthermore, Prevotella species are able
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to stimulate gingival epithelial cells to express interleukin (IL)-6, IL-8, and tumour necrosis
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factor (TNF)-α (Ji et al., 2007; Schincaglia et al., 2017).
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Recently, we demonstrated that a chemically synthesized QS molecule, dihydroxy-2, 3-
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pentanedione (DPD), and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD) inhibit
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estradiol-induced biofilm-related virulence of P. aurantiaca (Fteita et al., 2017). In the
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present study, we hypothesized that DPD (the universal precursor of the QS molecule AI-2)
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and its analogs modulate human gingival keratinocyte cytokine response against bacterial cell
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extracts and, at the same time, inhibit biofilm formation by competing with or inhibiting the
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native AI-2 signaling pathway of these P. intermedia group species. Therefore, we examined
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the protein expression profiles of IL-1β, IL-6, and IL-8 of human gingival keratinocytes,
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challenged by the whole cell extracts (WCE) of P. intermedia, P. nigrescens, and P. pallens
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in the presence and absence of QS molecules and estradiol. In addition, the role of QS
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molecules and estradiol in the biofilm formation was assessed.
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2.
MATERIAL AND METHODS
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2.1.
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Prior to each experiment, immortalized human gingival keratinocytes (HMK) cell lines
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(Mäkelä et al., 1998) were cultured for at least 72 hours in a keratinocyte-SFM growth
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medium, containing human recombinant epidermal growth factor, bovine pituitary extract
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(cat 17005-075, Fisher Scientific, Paisley, Scotland), and antibiotics (penicillin 10.000 U/ml
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and streptomycin 100 µg/ml, Fisher Scientific, Bleiswijk, The Netherlands), and incubated at
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37°C and 5% CO2. Growth culture media were replaced every second day and the cells were
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passaged every 4-5 days or when the flask confluency reached 80-90%. The HMK cells used
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in the study were collected from passage numbers range from 22-24.
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Cell culture methods
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2.2.
Bacterial strains and culture methods
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The type and one clinical strain of P. intermedia (ATCC 25611T and AHN 8290), P.
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nigrescens (ATCC 33563T and AHN 8293), and P. pallens (NCTC 13042T and AHN 9283)
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were used in the present work. All strains were provided by the National Institute for Health
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and Welfare, Helsinki, Finland. The strain identification has been described in details
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previously (Könönen et al., 2000).
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In each experiment, the bacterial strains were revived from -70°C stored stocks and grown on
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Brucella blood agar plates, supplemented with hemin (5 mg L-1) and vitamin K1 (10 mg L-1),
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and cultured in an anaerobic chamber (Whitley A35 Anaerobic Workstation, Don Whitley
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Scientific Ltd., West Yorkshire, UK) with an atmosphere of 10% H2, 5% CO2, and 85% N2 at
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37ºC for 72 hours. Before each experiment, pure bacterial cultures were obtained by
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passaging clearly distinct colonies for a second growth cycle on new Brucella agar plates and
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with the same incubation conditions. For a liquid phase of growth, bacterial strains were
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grown in Todd-Hewitt broth (Becton, Difco™ and BBL™, USA), supplemented with 5 g L-1
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yeast extracts, 750 mg L-1 cysteine, 5 mg L-1 hemin, and 5 mg L-1 menadione (Sigma
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Chemical Co., St. Louis, USA) for 24 hours in anaerobic condition before running each
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protocol (Fteita et al., 2014).
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2.3.
Estradiol suspensions
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The estradiol concentration of 90 nmol L-1 was the only concentration used in the study,
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simulating the serum estradiol concentration equivalent during the second trimester of
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pregnancy (O’Leary et al., 1991). Besides, an estradiol concentration of 0 nmol L-1 served as a
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control.
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2.4.
4,5-dihydroxy-2,3-pentanedione (DPD) and its C1-alkyl analogs suspensions
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DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD) were synthesized according
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to a protocol by Roy et al. (2010). Stock solutions in dimethyl sulfoxide (DMSO) were
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diluted with culture media to a final molarity of 10 nM, 100 nM, 1 µM, and 10 µM. Handling
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of stock solutions has been described previously (Fteita et al., 2017).
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2.5.
Whole cell extract preparations
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Preparation of WCE was performed according to Itoh et al. (2009) and has been described in
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detail elsewhere (Fteita et al., 2017). Briefly, purely cultured bacterial colonies were grown
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on agar for 72 hours and then collected and suspended in 0.3% CHAPS detergent (Thermo
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Fisher Scientific, USA) to enhance cell membrane rupture. Bacterial suspensions were
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sonicated at power 80 for 20 seconds. The optical density (OD) of each bacterial suspension
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was spectrophotometrically adjusted to the lowest strain OD (3.0) at 490 nm.
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Co-culture of gingival keratinocytes with WCE and analogs
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When HMK cell confluency reached 80-90%, cells were trypsinized, centrifuged, and 3x105
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of HMK cells/well were incubated overnight in 12-well plates at 37°C and 5% CO2. The wells
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were rinsed twice with PBS and an aliquot of a 1 ml of cell culture growth media containing
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10 µM of DPD or its analogs was added in each test well in triplicates. Cells incubated with
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fresh media containing no DPD or its analogs served as control triplicates. In test groups, for
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each well seeded with 3x105/ml of HMK cells, a known volume of WCE (equivalent to
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3x103/ml of sonicated bacteria) was loaded to match the standard ratio accepted in cell culture
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studies (WCE:HMK cells is equal to 1:100).
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After overnight incubation, cells were first checked for over-confluency and then the medium
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was taken out and immediately replaced with PBS to prevent cell drying. The wells were
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rinsed twice and in the third rinse, PBS was replaced with 300 µL of lysis buffer (L2912,
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Sigma-Aldrich, St. Louis, USA) and kept on a medium speed shaker for 10 minutes. Finally,
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by using a sterile plastic scraper, the well bottoms were scraped with an adequate hand
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mechanical force to ensure the collection of all lysed cells. Cell lysates were collected and
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stored in -70ºC until further analysis. Immediately before running the cytokine analyses, the
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cell lysates were thawed and sonicated for 5 seconds each.
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2.7.
Cytokine response profiles
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The HMK lysate samples were allowed to thaw and were centrifuged at 10000 g for 5
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minutes. Lysate concentrations of IL-1β, IL-6, and IL-8 were detected by the Luminex®
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xMAP™ technique (Luminex Corporation, Austin, TX) and by using commercially provided
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Bio-Plex kits (cytokine group I assays; Bio-Rad, Santa Rosa, CA) based on the
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manufacturer’s instructions. The minimal detection limit of cytokine concentrations detected
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by the Luminex assay was 0.22 pg/mL for IL-1β, 2.19 pg/mL for IL-6, and 1.27 pg/mL for
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IL-8.
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2.8.
Biofilm mass quantification
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Biofilm mass, in the presence and absence of estradiol, was measured by determining the
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amount of protein present using the Bradford protein assay of biological samples (Hammond
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and Kruger, 1988) and incorporating the microwave enhancement protocol (Akins and Tuan,
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1995; Fteita et al., 2014; 2017). Briefly, after coating the 96-well plate with clarified saliva
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for 1 hour at 37 ºC, each Prevotella strain was incubated anaerobically with estradiol (0 and
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90 nmol L-1) in pentaplicates for 48 hours. The wells were blotted, rinsed twice with PBS, and
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loaded with 0.2N of NaOH and sonicated. Then the protein quantification using the
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microwave enhancement protocol and Bradford assay (Bio-Rad, USA) was undertaken (Fteita
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et al., 2015). Biofilm mass was detected using a colorimetric method at an absorbance of 595
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nm using a micro-plate reader. Actual protein levels were standardized using a scattering
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formula which included five different standard concentrations of bovine serum albumin
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(Sigma-Aldrich, USA) (Fteita et al., 2014; 2017).
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2.9.
Biofilm formation under SEM
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Each OD-adjusted Prevotella strain was grown on saliva-coated glass coverslips and
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anaerobically incubated with 10 µM of DPD and its analogs and with the functioning
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concentration of estradiol (90 nmol L-1) for 24 hours. The glass coverslips were rinsed twice
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with PBS followed by a chemical fixation with 5% glutaraldehyde in 0.16 mol 1-1 s-
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collidine–HCl buffer, pH 7.4 for 20 minutes, and followed by incubation in graded series of
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ethanol (50%, 70%, and 98%) with 5 minutes embedding intervals in each concentration.
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and carbon thread coated for 1 second. The specimen microtopography was examined by
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LEO 1530 Gemini scanning electron microscope (Carl Zeiss, Oberkochen, Germany, 2001).
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The image magnification was standardized to correspond to a Polaroid 545 print and an image
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size of 8.9x11.4 cm.
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2.10. Statistical analysis
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The results were presented as mean ± standard deviation of the data from the mean. Since all
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data were normally distributed, multiple comparisons between the different test groups were
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performed by using the single factor “one-way ANOVA” with a two-tailed probability level
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of statistical significance followed by a post hoc test for inter-group comparisons. All
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experiments were performed at least twice at different time points in pentaplicates. A P-value
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<0.05 was accepted as statistically significant.
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RESULTS
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3.
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3.1.
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After 24 hours of incubation, the WCE of P. intermedia (ATCC 25611and AHN 8290) and
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P. nigrescens (ATCC 33563 and AHN 8293) significantly increased (Fig. 1a-d), while those
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of P. pallens (NCTC 13042 and AHN 8293) decreased the expression of IL-6 and IL-8 of
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HMK cells (Fig. 1e and 1f). DPD enhanced and butyl-DPD neutralized the WCE activated
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IL-6 and IL-8 responses. No statistically significant difference was observed in IL-1β
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expression. Estradiol alone had a suppressive effect on the expression of IL-1β by HMK cells
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incubated with WCE of P. intermedia ATCC 25611 and AHN 8290 (Fig. 1a and 1b), and IL-
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Expression of IL-1β, IL-6, and IL-8 in cultured human gingival keratinocytes
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6 and IL-8 with WCE of P. nigrescens AHN 8293 (Fig. 1d). When the same cells incubated
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with WCE of P. pallens AHN 8392, estradiol enhanced the expression of IL-1β (Fig. 1f).
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3.2.
Biofilm mass
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In the absence of estradiol, the biofilm mass of three strains was significantly inhibited with
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DPD and its analogs and no inhibition was detected with the two strains of P. pallens (Fig.
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2a). In the presence of estradiol, butyl DPD inhibited the biofilm formation of all Prevotella
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strains except for the type strain of P. intermedia and the two strains of P. pallens (Fig. 2b).
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3.3.
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SEM micrographs revealed a general decrease in the cell density and distribution of the
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biofilm grown on the glass cover-slips incubated with 10 µM of DPD and analogs. This
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inhibition in biofilm formation was most detectable with P. intermedia ATCC 25611
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incubated with estradiol and DPD, butyl DPD or isobutyl DPD and with P. nigrescens
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(ATCC 33563 and AHN 8293) incubated with estradiol and DPD, ethyl DPD, or butyl DPD
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(Supplementary figures).
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SEM imaging analyses of biofilm formation
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4.
DISCUSSION
The present study is the first to demonstrate the neutralizing effect of a DPD analog, butyl-
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DPD, on the Prevotella WCE-induced IL-6 and IL-8 expressions of human gingival
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keratinocytes. Moreover, estradiol-induced biofilm formations of P. nigrescens ATCC 33563
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and AHN 8293 and P. pallens NCTC 13042 were prone to the presence of DPD and ethyl
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DPD, and, to a lesser extent, butyl DPD. These results indicate that DPD analogs can
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simultaneously function as a host response regulator and a bacterial virulence inhibitor.
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P. intermedia group organisms are potential periodontopathogens that may significantly
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associate with the health-disease status of the periodontium (Gharbia et al., 1994; Könönen et
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al., 2000; Gürsoy at al., 2009; Sakamoto et al., 2010) and their growth has been found to
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positively correlate with increased estradiol and progesterone concentrations (Kornman and
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Loesche, 1982; Muramatsu and Takaesu, 1994). Some Prevotella species are capable of
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activating cytokine response through Toll-like receptor 2 (TLR-2), resulting in the production
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of IL-23 and IL-1 (de Aquino et al., 2014; Schincaglia et al., 2017). Besides, Prevotella can
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stimulate epithelial cell response to express IL-8, IL-6, and the C-C class chemokine 20
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(CCL20), which foster the mucosal immune responses and neutrophil migration to the site of
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infection (Ji et al., 2007; Matsui et al., 2014). According to our results, WCEs of P.
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intermedia and P. nigrescens enhance, whereas WCE of P. pallens suppresses the expressions
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of IL-6 and IL-8 in human gingival keratinocytes. This finding is interesting, since both P.
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intermedia and P. nigrescens are commonly detected in samples from periodontitis patients,
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while P. pallens is associated with periodontal health (Mättö et al., 1996; Könönen et al.,
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2000). In our previous studies, estradiol was found to be an important regulator of biofilm-
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related virulence of P. intermedia group bacteria (Fteita et al., 2014; 2015; 2017). In the
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present study, estradiol significantly suppressed the cytokine response of HMK cells
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incubated with WCE of P. intermedia (IL-1β) and P. nigrescens (IL-6 and IL-8). Also
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IL-1β in human keratinocytes (Kanda and Watanabe, 2003) and suppresses the release of
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TNF-α and IL-8 by the estrogen receptor β (ERβ) in cystic fibrosis bronchial epithelial
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cultured cells (Chotirmall et al., 2010). However, in the current work, when HMK cells were
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incubated with WCE of P. pallens, estradiol enhanced the IL-1β expression.
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Potential periodontopathogens, such as P. intermedia, Fusobacterium nucleatum, and
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Porphyromonas gingivalis, have been found to produce AI-2 (Frias et al., 2001; Sintim and
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Gürsoy, 2016). Considering that AI-2 also regulates immune cells, we tested the combined
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effects of DPD or its analogs with WCEs of the examined Prevotella species on the immune
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response of gingival keratinocytes. Our results indicate a difference between their actions;
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while butyl-DPD neutralizes the elevated cytokine response of gingival keratinocytes
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challenged with Prevotella WCEs, DPD enhances this effect. It was recently demonstrated
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that AI-2 signaling plays a role in the cytokine production of oral fibroblasts (Scheres et al.,
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2015) and colon carcinoma cells (Zargar et al., 2015). To our knowledge, this phenomenon
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has not been described in regards to oral keratinocytes. Butyl-DPD is a C1-alkyl analog of
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DPD, which enhances the integrity of the epithelium by stimulating the formation of occludin
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(Elmanfi et al., 2018). Although the underlying mechanism of this latter finding remains
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unexplained, it indicates a potential use of butyl-DPD or a variant thereof as a host-response
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regulator of human oral epithelial cells.
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Our research group has previously demonstrated a significant role of estradiol in the
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regulation of growth properties, proteolytic enzyme activity, and biofilm-related virulence of
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the P. intermedia group organisms (Fteita et al., 2014; 2015). Here, the biofilm mass of the
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three P. intermedia group members examined significantly increased in the presence of
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estradiol. Interestingly, butyl DPD had an inhibitory effect on P. intermedia AHN 8290 and
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P. nigrescens ATCC 33563 and AHN 8293, whereas it failed to inhibit the biofilm formation
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Könönen et al., 2000). This finding may reflect a promising potential specificity of butyl DPD
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in targeting species with higher pathogenic properties. Furthermore, as we have recently
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presented evidence that the disruption in QS signaling with C1-alkyl analogs of DPD may
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modify the biofilm-related virulence of the newest species within the P. intermedia group, P.
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aurantiaca (Fteita et al., 2017), here also we present by SEM imaging that estradiol
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stimulated the biofilm formation of P. intermedia ATCC 25611 and P. nigrescens AHN 8293,
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but DPD, butyl-DPD, and isobutyl DPD could significantly suppress this effect. In our
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previous study (Fteita et al., 2017) as well as in this study, by using the Bradford protein
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determination assay, P. pallens proved to be a good biofilm producer. In the present study,
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however, SEM imaging revealed only weak biofilms produced by P. pallens. The discrepancy
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between the total protein determination of biofilm mass and SEM analysis could be due to the
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fact that SEM imaging analyses include harsh sample preparation procedures, such as
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repeated specimen rinsing, glutaraldehyde fixation, and alcohol dehydration, which may
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affect the binding of bacterial biofilm on the glass surfaces (Chang & Rittmann, 1986).
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As a conclusion, butyl-DPD is able to neutralize the Prevotella WCE-induced cytokine
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expression of human gingival keratinocytes and to inhibit the estradiol-induced biofilm
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formation of P. intermedia and P. nigrescens. Such dual or multiple acting compounds are
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desired for the treatment of chronic inflammatory processes induced by oral bacteria,
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including pregnancy-related gingivitis.
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This work was supported by the Finnish Doctoral Program in Oral Sciences (FINDOS) (D.F.),
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and Finnish Dental Society Apollonia (U.K.G.). XCM thanks China Scholarship Council
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(No.201406140119) for financial support.
Conflict of interests
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The authors declare no conflict of interest.
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ACCEPTED MANUSCRIPT Schwarzer C, Fu Z, Patanwala M, Hum L, Lopez-Guzman M, Illek B et al. (2012) Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia. Cell Microbiol 14:698-709. Shah HN & Gharbia SE (1992) Biochemical and chemical studies on strains designated
sp. nov. Int J Syst Bacteriol 42: 542-546.
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Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens
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expression by oral bacteria and periodontal pathogens. Periodontol 2000 52:53-67.
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Shiner EK, Terentyev D, Bryan A, Sennoune S, Martinez-Zaguilan R, Li G, et al. (2006) Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signal- ling. Cell Microbiol 8: 1601-1610.
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new opportunity for biomarkers, molecular targets, and bacterial eradication. OMICS
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Suchett-Kaye G, Morrier JJ, Barsotti O (1998) Interactions between non-immune host cells and the immune system during periodontal disease: role of the gingival keratinocyte. Crit Rev Oral Biol Med 9:292-305.
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ACCEPTED MANUSCRIPT Vikström E, Tafazoli F, Magnusson KE (2006) Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells. FEBS Lett 580:6921-6928. Walters M, Sperandio V (2006) Quorum sensing in Escherichia coli and Salmonella. Int J
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Zargar A, Quan DN, Carter KK, Guo M, Sintim HO, Payne GF et al. (2015) Bacterial
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secretions of nonpathogenic Escherichia coli elicit inflammatory pathways: a closer
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investigation of interkingdom signaling. MBio 6:e00025.
Figure legends
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Figure 1a-f: Expression of IL-1β, IL-6, and IL-8 in cultured human gingival keratinocyte (HMK) cells after 24 hours of incubation with whole cell extract (WCE) of two Prevotella intermedia, two Prevotella nigrescens, and two Prevotella pallens strains, 90 nmol L-1 of
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estradiol, and 10 µM of dihydroxy-2, 3-pentanedione (DPD), ethyl DPD (eDPD), butyl DPD (bDPD), or isobutyl DPD (ibDPD). The results are demonstrated as mean ± standard
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deviation. Cytokine levels are measured in pg/ml. Asterisks indicate significant differences with the control (*P<0.05, **P<0.01, and ***P<0.001, the same values of significance applied to all other symbols). φ: represents significant differences with HMK and WCE, α: represents significant differences with HMK and estradiol, ¤: represents significant differences with HMK, WCE, and estradiol, and †: represents significant differences within DPD and its analogs.
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ACCEPTED MANUSCRIPT Figure 2a-b: Biofilm mass represented by protein levels of two Prevotella intermedia, two Prevotella nigrescens, and two Prevotella pallens strains, in the presence and absence of 90 nmol L-1 of estradiol, and 10 µM of dihydroxy-2, 3-pentanedione (DPD), ethyl DPD (eDPD), butyl DPD (bDPD), or isobutyl DPD (ibDPD). Data are presented as mg mL-1. Control bar
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represents OD adjusted bacterial suspensions and control (+) bar represents OD adjusted
bacterial suspensions with estradiol. Asterisks indicate significant differences with the control (Fig. 2a) and with control (+) (Fig. 2b) (*P < 0.05).
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Supplementary figure (a-f): SEM micrographs reveal the difference in biofilm mass
microtopography of two Prevotella intermedia, two Prevotella nigrescens, and two Prevotella
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pallens strains after 24 hours of incubation with 90 nmol L-1 of estradiol and 10 µM of dihydroxy-2, 3-pentanedione (DPD), ethyl DPD, butyl DPD, or isobutyl DPD. The differences were evaluated visually in comparison with the control (with and without
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estradiol). Scale bars indicate 10 µm.
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ACCEPTED MANUSCRIPT Highlights 1) Quorum sensing (QS) signaling modifies biofilm formation and host immune response.
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2) QS-analogs effect on estradiol-induced virulence of Prevotella species was evaluated. 3) QS-analogs were found to modify the host response and virulence of Prevotella species.
4) Such analogs deserve further research targeting pregnancy-related gingivitis
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treatment.
S1075-9964(18)30156-2
DOI:
10.1016/j.anaerobe.2018.09.001
Reference:
YANAE 1942
To appear in:
Anaerobe
Received Date: 25 April 2018 Revised Date:
28 August 2018
Accepted Date: 1 September 2018
Please cite this article as: Fteita D, Könönen E, Gürsoy M, Ma X, Sintim HO, Gürsoy UK, Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species, Anaerobe (2018), doi: 10.1016/j.anaerobe.2018.09.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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QUORUM SENSING MOLECULES REGULATE EPITHELIAL CYTOKINE
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RESPONSE AND BIOFILM-RELATED VIRULENCE OF THREE PREVOTELLA
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SPECIES
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Dareen Fteitaa,*, Eija Könönena, b, Mervi Gürsoya, Xiaochu Mac, Herman O. Sintimc, Ulvi
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Kahraman Gürsoya
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a
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Finland
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Department of Periodontology, Institute of Dentistry, University of Turku, FI-20520, Turku,
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Welfare Division, Oral Health Care, City of Turku, Turku, Finland
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c
Department of Chemistry and Purdue Institute for Drug Discovery and Purdue Institute of
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Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette,
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Indiana, USA
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*
Correspondence:
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Department of Periodontology, Institute of Dentistry, University of Turku,
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20520 Turku, Finland.
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Tel: +358 40 9650024
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E-mail addresses:
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[email protected] (D. Fteita), [email protected] (E. Könönen), [email protected] (M.
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Gürsoy), [email protected] (X. Ma), [email protected] (H. O. Sintim),
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[email protected] (U.K. Gürsoy).
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Key words:
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autoinducer-2; estradiol; gingival keratinocytes; inflammatory response; pathogenicity;
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Prevotella
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ACCEPTED MANUSCRIPT ABSTRACT
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Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of
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bacteria, and at the same time activates immune response in eukaryotic organisms. We
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recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its
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analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the
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four species in the Prevotella intermedia group. Here, we examined the combined effects of
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estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK)
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against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella
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pallens, and 2) biofilm formation of these three Prevotella species. All experiments were
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performed in the presence or absence of estradiol, and with different QS molecules: DPD and
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its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-
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1β, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was
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quantitatively evaluated by measuring protein concentration via the Bradford method, and the
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microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging.
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Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with
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estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with
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estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced
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regulation of HMK interleukin expression and, at the same time, inhibited the biofilm
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formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in
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biofilm mass after application of butyl-DPD, which was most detectable among the P.
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intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In
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conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine
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response and, at the same time, to inhibit the biofilm formation of P. intermedia and P.
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nigrescens.
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1.
INTRODUCTION
In bacteria, cell-to-cell communication is coordinated and gene expression is regulated in
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synchronization with a cell density-dependent signaling pathway, known as quorum sensing
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(QS) (Bassler, 1999; Shao and Demuth, 2010; Galloway et al., 2011). QS is a molecular
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process by which a variety of bacteria maintain a diverse array of physiological activities,
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including motility, biofilm formation, and virulence (Atkinson et al., 1999; Shiner et al.,
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2006; Sintim et al., 2010). QS relies on the production, release, and detection of signaling
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molecules, termed autoinducers (AIs). AIs are classified into three major classes: N- acyl
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homoserine lactone (AHL) or AI-1 for Gram-negative bacteria, AI-2 for both Gram-negative
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and Gram-positive bacteria, and cyclic oligopeptides for Gram-positive bacteria (Sintim et al.,
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2010). In addition, evidence on the use of the AI-3 system by Escherichia coli and Salmonella
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exists in literature (Walters and Sperandio, 2006). Efforts have been made at inhibiting
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bacteria-to-bacteria crosstalk by quenching native QS responses with synthetic small
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molecules (Roy et al., 2010).
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Beside their impact on bacterial gene expression and virulence, QS molecules have a
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regulatory effect on the host immune response (Zargar et al., 2015). The gingival epithelium
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and underlying lamina propria constitute a unique physical barrier to protect periodontal
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tissues against microbial invasion, mechanical stress, and environmental hazards (Chung et
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al., 2007; Dawson et al., 2013). Furthermore, as an active player in the human innate
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immunity, gingival keratinocytes express proinflammatory and, to a lesser extent, anti-
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inflammatory cytokines and chemokines (Chung et al., 2007; Suchett-Kaye et al., 1998). AI-1
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molecules have the capability to induce alterations in epithelial barrier functions, leading to
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disruption of the epithelial integrity (Vikström et al., 2006; Eum et al., 2014). Moreover,
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different AHLs have been found to modulate epithelial homeostasis, cell migration, and
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apoptosis (Cooley et al., 2008; Karlsson et al., 2012; Schwarzer et al., 2012). Our group
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human gingival keratinocytes (Elmanfi et al., 2018).
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Prevotella intermedia, Prevotella nigrescens, Prevotella pallens, and Prevotella aurantiaca
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are the four phylogenetically close species within the P. intermedia group (Shah and Gharbia,
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1992; Könönen et al., 1998; Sakamoto et al., 2010). With their known ability to utilize
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maternal steroids (estrogen and progesterone), the increase in numbers of P. intermedia and
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P. nigrescens has been linked to gingival inflammation and pregnancy-related gingivitis
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(Kornman and Loesche, 1982; Gürsoy et al., 2009). Furthermore, Prevotella species are able
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to stimulate gingival epithelial cells to express interleukin (IL)-6, IL-8, and tumour necrosis
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factor (TNF)-α (Ji et al., 2007; Schincaglia et al., 2017).
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Recently, we demonstrated that a chemically synthesized QS molecule, dihydroxy-2, 3-
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pentanedione (DPD), and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD) inhibit
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estradiol-induced biofilm-related virulence of P. aurantiaca (Fteita et al., 2017). In the
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present study, we hypothesized that DPD (the universal precursor of the QS molecule AI-2)
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and its analogs modulate human gingival keratinocyte cytokine response against bacterial cell
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extracts and, at the same time, inhibit biofilm formation by competing with or inhibiting the
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native AI-2 signaling pathway of these P. intermedia group species. Therefore, we examined
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the protein expression profiles of IL-1β, IL-6, and IL-8 of human gingival keratinocytes,
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challenged by the whole cell extracts (WCE) of P. intermedia, P. nigrescens, and P. pallens
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in the presence and absence of QS molecules and estradiol. In addition, the role of QS
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molecules and estradiol in the biofilm formation was assessed.
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2.
MATERIAL AND METHODS
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2.1.
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Prior to each experiment, immortalized human gingival keratinocytes (HMK) cell lines
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(Mäkelä et al., 1998) were cultured for at least 72 hours in a keratinocyte-SFM growth
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medium, containing human recombinant epidermal growth factor, bovine pituitary extract
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(cat 17005-075, Fisher Scientific, Paisley, Scotland), and antibiotics (penicillin 10.000 U/ml
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and streptomycin 100 µg/ml, Fisher Scientific, Bleiswijk, The Netherlands), and incubated at
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37°C and 5% CO2. Growth culture media were replaced every second day and the cells were
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passaged every 4-5 days or when the flask confluency reached 80-90%. The HMK cells used
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in the study were collected from passage numbers range from 22-24.
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Cell culture methods
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2.2.
Bacterial strains and culture methods
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The type and one clinical strain of P. intermedia (ATCC 25611T and AHN 8290), P.
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nigrescens (ATCC 33563T and AHN 8293), and P. pallens (NCTC 13042T and AHN 9283)
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were used in the present work. All strains were provided by the National Institute for Health
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and Welfare, Helsinki, Finland. The strain identification has been described in details
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previously (Könönen et al., 2000).
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In each experiment, the bacterial strains were revived from -70°C stored stocks and grown on
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Brucella blood agar plates, supplemented with hemin (5 mg L-1) and vitamin K1 (10 mg L-1),
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and cultured in an anaerobic chamber (Whitley A35 Anaerobic Workstation, Don Whitley
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Scientific Ltd., West Yorkshire, UK) with an atmosphere of 10% H2, 5% CO2, and 85% N2 at
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37ºC for 72 hours. Before each experiment, pure bacterial cultures were obtained by
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passaging clearly distinct colonies for a second growth cycle on new Brucella agar plates and
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with the same incubation conditions. For a liquid phase of growth, bacterial strains were
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grown in Todd-Hewitt broth (Becton, Difco™ and BBL™, USA), supplemented with 5 g L-1
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yeast extracts, 750 mg L-1 cysteine, 5 mg L-1 hemin, and 5 mg L-1 menadione (Sigma
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Chemical Co., St. Louis, USA) for 24 hours in anaerobic condition before running each
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protocol (Fteita et al., 2014).
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2.3.
Estradiol suspensions
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The estradiol concentration of 90 nmol L-1 was the only concentration used in the study,
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simulating the serum estradiol concentration equivalent during the second trimester of
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pregnancy (O’Leary et al., 1991). Besides, an estradiol concentration of 0 nmol L-1 served as a
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control.
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2.4.
4,5-dihydroxy-2,3-pentanedione (DPD) and its C1-alkyl analogs suspensions
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DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD) were synthesized according
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to a protocol by Roy et al. (2010). Stock solutions in dimethyl sulfoxide (DMSO) were
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diluted with culture media to a final molarity of 10 nM, 100 nM, 1 µM, and 10 µM. Handling
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of stock solutions has been described previously (Fteita et al., 2017).
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2.5.
Whole cell extract preparations
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Preparation of WCE was performed according to Itoh et al. (2009) and has been described in
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detail elsewhere (Fteita et al., 2017). Briefly, purely cultured bacterial colonies were grown
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on agar for 72 hours and then collected and suspended in 0.3% CHAPS detergent (Thermo
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Fisher Scientific, USA) to enhance cell membrane rupture. Bacterial suspensions were
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sonicated at power 80 for 20 seconds. The optical density (OD) of each bacterial suspension
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was spectrophotometrically adjusted to the lowest strain OD (3.0) at 490 nm.
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Co-culture of gingival keratinocytes with WCE and analogs
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When HMK cell confluency reached 80-90%, cells were trypsinized, centrifuged, and 3x105
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of HMK cells/well were incubated overnight in 12-well plates at 37°C and 5% CO2. The wells
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were rinsed twice with PBS and an aliquot of a 1 ml of cell culture growth media containing
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10 µM of DPD or its analogs was added in each test well in triplicates. Cells incubated with
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fresh media containing no DPD or its analogs served as control triplicates. In test groups, for
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each well seeded with 3x105/ml of HMK cells, a known volume of WCE (equivalent to
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3x103/ml of sonicated bacteria) was loaded to match the standard ratio accepted in cell culture
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studies (WCE:HMK cells is equal to 1:100).
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After overnight incubation, cells were first checked for over-confluency and then the medium
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was taken out and immediately replaced with PBS to prevent cell drying. The wells were
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rinsed twice and in the third rinse, PBS was replaced with 300 µL of lysis buffer (L2912,
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Sigma-Aldrich, St. Louis, USA) and kept on a medium speed shaker for 10 minutes. Finally,
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by using a sterile plastic scraper, the well bottoms were scraped with an adequate hand
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mechanical force to ensure the collection of all lysed cells. Cell lysates were collected and
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stored in -70ºC until further analysis. Immediately before running the cytokine analyses, the
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cell lysates were thawed and sonicated for 5 seconds each.
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2.7.
Cytokine response profiles
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The HMK lysate samples were allowed to thaw and were centrifuged at 10000 g for 5
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minutes. Lysate concentrations of IL-1β, IL-6, and IL-8 were detected by the Luminex®
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xMAP™ technique (Luminex Corporation, Austin, TX) and by using commercially provided
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Bio-Plex kits (cytokine group I assays; Bio-Rad, Santa Rosa, CA) based on the
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manufacturer’s instructions. The minimal detection limit of cytokine concentrations detected
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by the Luminex assay was 0.22 pg/mL for IL-1β, 2.19 pg/mL for IL-6, and 1.27 pg/mL for
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IL-8.
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2.8.
Biofilm mass quantification
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Biofilm mass, in the presence and absence of estradiol, was measured by determining the
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amount of protein present using the Bradford protein assay of biological samples (Hammond
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and Kruger, 1988) and incorporating the microwave enhancement protocol (Akins and Tuan,
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1995; Fteita et al., 2014; 2017). Briefly, after coating the 96-well plate with clarified saliva
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for 1 hour at 37 ºC, each Prevotella strain was incubated anaerobically with estradiol (0 and
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90 nmol L-1) in pentaplicates for 48 hours. The wells were blotted, rinsed twice with PBS, and
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loaded with 0.2N of NaOH and sonicated. Then the protein quantification using the
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microwave enhancement protocol and Bradford assay (Bio-Rad, USA) was undertaken (Fteita
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et al., 2015). Biofilm mass was detected using a colorimetric method at an absorbance of 595
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nm using a micro-plate reader. Actual protein levels were standardized using a scattering
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formula which included five different standard concentrations of bovine serum albumin
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(Sigma-Aldrich, USA) (Fteita et al., 2014; 2017).
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2.9.
Biofilm formation under SEM
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Each OD-adjusted Prevotella strain was grown on saliva-coated glass coverslips and
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anaerobically incubated with 10 µM of DPD and its analogs and with the functioning
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concentration of estradiol (90 nmol L-1) for 24 hours. The glass coverslips were rinsed twice
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with PBS followed by a chemical fixation with 5% glutaraldehyde in 0.16 mol 1-1 s-
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collidine–HCl buffer, pH 7.4 for 20 minutes, and followed by incubation in graded series of
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ethanol (50%, 70%, and 98%) with 5 minutes embedding intervals in each concentration.
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and carbon thread coated for 1 second. The specimen microtopography was examined by
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LEO 1530 Gemini scanning electron microscope (Carl Zeiss, Oberkochen, Germany, 2001).
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The image magnification was standardized to correspond to a Polaroid 545 print and an image
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size of 8.9x11.4 cm.
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2.10. Statistical analysis
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The results were presented as mean ± standard deviation of the data from the mean. Since all
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data were normally distributed, multiple comparisons between the different test groups were
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performed by using the single factor “one-way ANOVA” with a two-tailed probability level
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of statistical significance followed by a post hoc test for inter-group comparisons. All
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experiments were performed at least twice at different time points in pentaplicates. A P-value
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<0.05 was accepted as statistically significant.
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RESULTS
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3.
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3.1.
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After 24 hours of incubation, the WCE of P. intermedia (ATCC 25611and AHN 8290) and
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P. nigrescens (ATCC 33563 and AHN 8293) significantly increased (Fig. 1a-d), while those
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of P. pallens (NCTC 13042 and AHN 8293) decreased the expression of IL-6 and IL-8 of
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HMK cells (Fig. 1e and 1f). DPD enhanced and butyl-DPD neutralized the WCE activated
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IL-6 and IL-8 responses. No statistically significant difference was observed in IL-1β
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expression. Estradiol alone had a suppressive effect on the expression of IL-1β by HMK cells
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incubated with WCE of P. intermedia ATCC 25611 and AHN 8290 (Fig. 1a and 1b), and IL-
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Expression of IL-1β, IL-6, and IL-8 in cultured human gingival keratinocytes
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6 and IL-8 with WCE of P. nigrescens AHN 8293 (Fig. 1d). When the same cells incubated
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with WCE of P. pallens AHN 8392, estradiol enhanced the expression of IL-1β (Fig. 1f).
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3.2.
Biofilm mass
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In the absence of estradiol, the biofilm mass of three strains was significantly inhibited with
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DPD and its analogs and no inhibition was detected with the two strains of P. pallens (Fig.
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2a). In the presence of estradiol, butyl DPD inhibited the biofilm formation of all Prevotella
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strains except for the type strain of P. intermedia and the two strains of P. pallens (Fig. 2b).
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3.3.
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SEM micrographs revealed a general decrease in the cell density and distribution of the
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biofilm grown on the glass cover-slips incubated with 10 µM of DPD and analogs. This
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inhibition in biofilm formation was most detectable with P. intermedia ATCC 25611
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incubated with estradiol and DPD, butyl DPD or isobutyl DPD and with P. nigrescens
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(ATCC 33563 and AHN 8293) incubated with estradiol and DPD, ethyl DPD, or butyl DPD
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(Supplementary figures).
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SEM imaging analyses of biofilm formation
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4.
DISCUSSION
The present study is the first to demonstrate the neutralizing effect of a DPD analog, butyl-
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DPD, on the Prevotella WCE-induced IL-6 and IL-8 expressions of human gingival
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keratinocytes. Moreover, estradiol-induced biofilm formations of P. nigrescens ATCC 33563
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and AHN 8293 and P. pallens NCTC 13042 were prone to the presence of DPD and ethyl
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DPD, and, to a lesser extent, butyl DPD. These results indicate that DPD analogs can
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simultaneously function as a host response regulator and a bacterial virulence inhibitor.
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P. intermedia group organisms are potential periodontopathogens that may significantly
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associate with the health-disease status of the periodontium (Gharbia et al., 1994; Könönen et
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al., 2000; Gürsoy at al., 2009; Sakamoto et al., 2010) and their growth has been found to
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positively correlate with increased estradiol and progesterone concentrations (Kornman and
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Loesche, 1982; Muramatsu and Takaesu, 1994). Some Prevotella species are capable of
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activating cytokine response through Toll-like receptor 2 (TLR-2), resulting in the production
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of IL-23 and IL-1 (de Aquino et al., 2014; Schincaglia et al., 2017). Besides, Prevotella can
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stimulate epithelial cell response to express IL-8, IL-6, and the C-C class chemokine 20
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(CCL20), which foster the mucosal immune responses and neutrophil migration to the site of
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infection (Ji et al., 2007; Matsui et al., 2014). According to our results, WCEs of P.
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intermedia and P. nigrescens enhance, whereas WCE of P. pallens suppresses the expressions
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of IL-6 and IL-8 in human gingival keratinocytes. This finding is interesting, since both P.
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intermedia and P. nigrescens are commonly detected in samples from periodontitis patients,
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while P. pallens is associated with periodontal health (Mättö et al., 1996; Könönen et al.,
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2000). In our previous studies, estradiol was found to be an important regulator of biofilm-
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related virulence of P. intermedia group bacteria (Fteita et al., 2014; 2015; 2017). In the
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present study, estradiol significantly suppressed the cytokine response of HMK cells
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incubated with WCE of P. intermedia (IL-1β) and P. nigrescens (IL-6 and IL-8). Also
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IL-1β in human keratinocytes (Kanda and Watanabe, 2003) and suppresses the release of
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TNF-α and IL-8 by the estrogen receptor β (ERβ) in cystic fibrosis bronchial epithelial
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cultured cells (Chotirmall et al., 2010). However, in the current work, when HMK cells were
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incubated with WCE of P. pallens, estradiol enhanced the IL-1β expression.
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Potential periodontopathogens, such as P. intermedia, Fusobacterium nucleatum, and
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Porphyromonas gingivalis, have been found to produce AI-2 (Frias et al., 2001; Sintim and
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Gürsoy, 2016). Considering that AI-2 also regulates immune cells, we tested the combined
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effects of DPD or its analogs with WCEs of the examined Prevotella species on the immune
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response of gingival keratinocytes. Our results indicate a difference between their actions;
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while butyl-DPD neutralizes the elevated cytokine response of gingival keratinocytes
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challenged with Prevotella WCEs, DPD enhances this effect. It was recently demonstrated
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that AI-2 signaling plays a role in the cytokine production of oral fibroblasts (Scheres et al.,
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2015) and colon carcinoma cells (Zargar et al., 2015). To our knowledge, this phenomenon
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has not been described in regards to oral keratinocytes. Butyl-DPD is a C1-alkyl analog of
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DPD, which enhances the integrity of the epithelium by stimulating the formation of occludin
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(Elmanfi et al., 2018). Although the underlying mechanism of this latter finding remains
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unexplained, it indicates a potential use of butyl-DPD or a variant thereof as a host-response
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regulator of human oral epithelial cells.
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Our research group has previously demonstrated a significant role of estradiol in the
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regulation of growth properties, proteolytic enzyme activity, and biofilm-related virulence of
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the P. intermedia group organisms (Fteita et al., 2014; 2015). Here, the biofilm mass of the
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three P. intermedia group members examined significantly increased in the presence of
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estradiol. Interestingly, butyl DPD had an inhibitory effect on P. intermedia AHN 8290 and
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P. nigrescens ATCC 33563 and AHN 8293, whereas it failed to inhibit the biofilm formation
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Könönen et al., 2000). This finding may reflect a promising potential specificity of butyl DPD
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in targeting species with higher pathogenic properties. Furthermore, as we have recently
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presented evidence that the disruption in QS signaling with C1-alkyl analogs of DPD may
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modify the biofilm-related virulence of the newest species within the P. intermedia group, P.
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aurantiaca (Fteita et al., 2017), here also we present by SEM imaging that estradiol
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stimulated the biofilm formation of P. intermedia ATCC 25611 and P. nigrescens AHN 8293,
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but DPD, butyl-DPD, and isobutyl DPD could significantly suppress this effect. In our
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previous study (Fteita et al., 2017) as well as in this study, by using the Bradford protein
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determination assay, P. pallens proved to be a good biofilm producer. In the present study,
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however, SEM imaging revealed only weak biofilms produced by P. pallens. The discrepancy
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between the total protein determination of biofilm mass and SEM analysis could be due to the
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fact that SEM imaging analyses include harsh sample preparation procedures, such as
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repeated specimen rinsing, glutaraldehyde fixation, and alcohol dehydration, which may
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affect the binding of bacterial biofilm on the glass surfaces (Chang & Rittmann, 1986).
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As a conclusion, butyl-DPD is able to neutralize the Prevotella WCE-induced cytokine
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expression of human gingival keratinocytes and to inhibit the estradiol-induced biofilm
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formation of P. intermedia and P. nigrescens. Such dual or multiple acting compounds are
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desired for the treatment of chronic inflammatory processes induced by oral bacteria,
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including pregnancy-related gingivitis.
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This work was supported by the Finnish Doctoral Program in Oral Sciences (FINDOS) (D.F.),
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and Finnish Dental Society Apollonia (U.K.G.). XCM thanks China Scholarship Council
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(No.201406140119) for financial support.
Conflict of interests
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The authors declare no conflict of interest.
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Figure legends
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Figure 1a-f: Expression of IL-1β, IL-6, and IL-8 in cultured human gingival keratinocyte (HMK) cells after 24 hours of incubation with whole cell extract (WCE) of two Prevotella intermedia, two Prevotella nigrescens, and two Prevotella pallens strains, 90 nmol L-1 of
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estradiol, and 10 µM of dihydroxy-2, 3-pentanedione (DPD), ethyl DPD (eDPD), butyl DPD (bDPD), or isobutyl DPD (ibDPD). The results are demonstrated as mean ± standard
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deviation. Cytokine levels are measured in pg/ml. Asterisks indicate significant differences with the control (*P<0.05, **P<0.01, and ***P<0.001, the same values of significance applied to all other symbols). φ: represents significant differences with HMK and WCE, α: represents significant differences with HMK and estradiol, ¤: represents significant differences with HMK, WCE, and estradiol, and †: represents significant differences within DPD and its analogs.
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ACCEPTED MANUSCRIPT Figure 2a-b: Biofilm mass represented by protein levels of two Prevotella intermedia, two Prevotella nigrescens, and two Prevotella pallens strains, in the presence and absence of 90 nmol L-1 of estradiol, and 10 µM of dihydroxy-2, 3-pentanedione (DPD), ethyl DPD (eDPD), butyl DPD (bDPD), or isobutyl DPD (ibDPD). Data are presented as mg mL-1. Control bar
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represents OD adjusted bacterial suspensions and control (+) bar represents OD adjusted
bacterial suspensions with estradiol. Asterisks indicate significant differences with the control (Fig. 2a) and with control (+) (Fig. 2b) (*P < 0.05).
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Supplementary figure (a-f): SEM micrographs reveal the difference in biofilm mass
microtopography of two Prevotella intermedia, two Prevotella nigrescens, and two Prevotella
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pallens strains after 24 hours of incubation with 90 nmol L-1 of estradiol and 10 µM of dihydroxy-2, 3-pentanedione (DPD), ethyl DPD, butyl DPD, or isobutyl DPD. The differences were evaluated visually in comparison with the control (with and without
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estradiol). Scale bars indicate 10 µm.
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ACCEPTED MANUSCRIPT Highlights 1) Quorum sensing (QS) signaling modifies biofilm formation and host immune response.
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2) QS-analogs effect on estradiol-induced virulence of Prevotella species was evaluated. 3) QS-analogs were found to modify the host response and virulence of Prevotella species.
4) Such analogs deserve further research targeting pregnancy-related gingivitis
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treatment.