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Microbes and Infection 16 (2014) 310e319

www.elsevier.com/locate/micinf

Original article

Characterisation and optimisation of organotypic oral mucosal models to


study Porphyromonas gingivalis invasion
Abigail Pinnock, Craig Murdoch, Keyvan Moharamzadeh, Simon Whawell, C.W. Ian Douglas*
Integrated Biosciences, University of Sheffield, School of Clinical Dentistry, 19 Claremont Crescent, Sheffield S10 2TA, UK
Received 28 October 2013; accepted 22 January 2014
Available online 31 January 2014

Abstract
Porphyromonas gingivalis is a Gram-negative, keystone pathogen in periodontitis that leads to tissue destruction and ultimately tooth loss.
The organism is able to infect oral epithelial cells and two-dimensional (monolayer) cultures have been used to investigate this process.
However, recently there has been interest in the use of three-dimensional, organotypic mucosal models to analyse infection. These models are
composed of collagen-embedded fibroblasts overlain with multilayers of oral epithelial cells. In this study we report for the first time significant
differences in the response of oral mucosal models to P. gingivalis infection when compared to monolayer cultures of oral epithelial cells.
Intracellular survival (3-fold) and bacterial release (4-fold) of P. gingivalis was significantly increased in mucosal models compared with
monolayer cultures, which may be due to the multi-layered nature and exfoliation of epithelial cells in these organotypic models. Furthermore,
marked differences in the cytokine profile between infected organotypic models and monolayer cultures were observed, particularly for CXCL8
and IL6, which suggested that degradation of cytokines by P. gingivalis may be less pronounced in organotypic compared to monolayer cultures.
These data suggest that use of oral mucosal models may provide a greater understanding of the host responses to P. gingivalis invasion than
simple monolayer cultures.
2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Porphyromonas gingivalis; Periodontitis; Epithelial cells; Oral mucosa; Chemokines; Innate immunity

1. Introduction
Porphyromonas gingivalis is a Gram-negative anaerobic
bacterium that significantly contributes to the pathogenesis of
periodontitis. This inflammatory disease affects the tissues that
surround and support the teeth and is the leading cause of
tooth-loss worldwide [1]. P. gingivalis can invade oral
epithelial cells in vivo [2] and in vitro [3e5], and has been
detected in a number of locations within the oral cavity,
including the buccal [6] and gingival mucosa [2]. Bacterial
internalisation by epithelial cells is thought to promote its
persistence within oral tissues by evasion of host immune

* Corresponding author. Academic Unit of Oral & Maxillofacial Pathology,


School of Clinical Dentistry, University of Sheffield, Claremont Crescent,
Sheffield S10 2TA, UK. Tel.: 44 114 2717957.
E-mail address: i.douglas@sheffield.ac.uk (C.W.I. Douglas).

responses and therapeutic agents, so prolonging the host


challenge and contributing to the chronic nature of this
disease.
There has been recent and continued interest in the utilisation of in vitro three-dimensional (3D) cultures of fullthickness epithelium as a more anatomically representative
model than two-dimensional, monolayer cultures [7]. The
culture of organotypic mucosal models (OMMs) has been
widely reported in the literature although they have been little
used for the study of host interactions with periodontal pathogens [8e10] and monolayer cultures have remained the main
epithelial model of choice [11,12].
The oral mucosa may provide a protective role to the effects
of internalised periodontal bacteria [13]. Therefore, such differences as the multi-cellular complexity and level of
differentiation of the oral mucosa between OMMs and monolayer cultures of oral epithelial cells may influence our understanding of the hostepathogen interaction. The main

1286-4579/$ - see front matter 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.micinf.2014.01.004

A. Pinnock et al. / Microbes and Infection 16 (2014) 310e319

consequences of P. gingivalis interaction with the epithelium


include intracellular bacterial survival, release of chemokines
and cytokines by epithelial cells, and host cell death [20] all of
which are features likely to contribute to the initiation and/or
propagation of the disease. However, much of the literature
regarding these host cell responses is conflicting. For example,
the time of intracellular survival of P. gingivalis varies
considerably in the literature from 6 h [14] to 4e8 days
[15,16,29]. In addition, some studies have shown that P. gingivalis-infected epithelial cells display increased production of
chemokines and cytokines [17,19], whereas others show chemokine/cytokine paralysis or degradation [18]. These conflicting data may be due to the use of different strains of P. gingivalis
or differences in the epithelial cells employed in these studies.
This study investigates these aspects further and reports significant differences between host cell responses to P. gingivalis
infection when oral epithelial cells are grown as monolayers or
as OMMs. Our data provides further evidence to support the
importance of using OMMs to study bacterial internalisation
and subsequent end-point analyses (e.g. chemokine profiling).
2. Methods and materials
2.1. Materials
All materials were purchased from Sigma, Poole, UK unless otherwise stated. The murine monoclonal P. gingivalis
antibody (clone 1B5) was a kind gift from Prof. M. Curtis,
Barts and The London School of Medicine, London, UK [21].
2.2. Bacterial strains and culture conditions
P. gingivalis strains NCTC 11834 (National Collection of
Type Cultures) and W50 [22], were cultured on fastidious
anaerobe agar (FA; LabM Limited, Lancashire, UK) supplemented with 10% horse blood (Oxoid, Fisher Scientific,
Loughborough, UK) at 37  C in an anaerobic atmosphere
(80% N2, 10% H2, 10% CO2; MiniMACS Anaerobic Workstation, Don Whitley Scientific, UK).
2.3. Epithelial cell culture conditions
Normal oral keratinocytes (NOK) were isolated from
buccal biopsies as previously described [23]. Briefly, biopsies
were obtained with written, informed consent from patients
attending the Charles Clifford Dental Hospital, Sheffield,
under ethical approval (reference number 09/H1308/66).
Biopsies were washed and incubated overnight at 4  C with
0.1% (w/v) trypsin. The epithelium and the connective
tissue layers were separated by gentle scraping. Primary
epithelial cells were seeded into a tissue culture flask along
with 5  105 lethally irradiated murine 3T3 fibroblasts
(XCELLentis, Gent, Belgium). Primary fibroblasts were isolated from the connective tissue layer by incubation with
0.05% (w/v) type I collagenase (GibcoBRL, Paisley, Scotland)
and then cultured in Dulbeccos Modified Eagles Medium
(DMEM) GlutaMAX (Gibco, UK) supplemented with

311

10% foetal calf serum (v/v, FCS), 625 ng ml1 amphotericin


B, 50 U ml1 penicillin and 50 U ml1 streptomycin (complete medium (CDMEM)). Keratinocytes were cultured in
CDMEM supplemented additionally with 0.1 mM adenine,
5 mg ml1 insulin, 5 mg ml1 transferrin, 5 mg ml1 triiodothyronine, 0.4 mM hydrocortisone and 10 ng ml1 epidermal
growth factor. Primary keratinocytes were passaged up to 3
times, each time seeding with irradiated 3T3 fibroblasts, and
primary fibroblasts were passaged up to 9 times. The H357
epithelial cell line, originally isolated from a squamous cell
carcinoma of the tongue (a kind gift from Prof. Stephen Prime,
University of Bristol, UK), was also cultured in supplemented
CDMEM.
2.4. Oral mucosal models (OMMs)
Rat-tail type I collagen was isolated from the tails of Wistar
rats at the end of a licenced study (by Mrs C Freeman, University of Sheffield) as previously described [24]. Extracted
collagen was dissolved in 0.1 M acetic acid at 4  C, freezedried and re-dissolved in 0.1 M acetic acid to a stock
concentration of 8 mg ml1, and stored at 4  C. Keeping all
reagents on ice, models were cultured using a protocol adapted
from Ref. [25]. Human primary buccal fibroblasts at a concentration of 1  106 per model, in CDMEM, were added to a
solution of 13.8 mg ml1 DMEM, 2.25 mg ml1 sodium bicarbonate, 2 mM HEPES, 6.3 mM NaOH, 8.5% (v/v) FCS,
2.1 mM L-glutamine and 5.28 mg ml1 rat-tail type I collagen.
The resultant fibroblast-containing suspension was incubated
at 37  C for 1e2 h until solidified. Once solid, OMMs were
completely submerged in supplemented CDMEM for 2 days
following which either NOK or the H357 cell line were seeded
at a density of 1  106 cells/model in supplemented CDMEM
for 1e2 days to allow keratinocyte adhesion. OMMs were
raised to the air-to-liquid interface for approximately 7 days or
left completely submerged in supplemented CDMEM for 2e3
days.
2.5. Antibiotic protection assay
The number of viable intracellular P. gingivalis cells was
assessed in monolayers and OMMs using an antibiotic protection assay as previously described [26], or a modified
version of this method for analysis of OMMs. Monolayers and
OMMs were incubated in DMEM GlutaMAX and Hams
F-12 (3:1) for 1 h at 37  C and then blocked with 2% (w/v)
bovine serum albumin (BSA) in DMEM GlutaMAX for
1 h at 37  C. P. gingivalis suspended in the same
DMEM GlutaMAX and Hams F-12 medium (3:1) was
applied at a multiplicity of infection (MOI) of 100 (monolayer) or 2  107 cells/300 ml (OMM; determined to be an
equivalent MOI 100 for an identical surface area of monolayer
cultured cells), were incubated at 37  C with monolayers and
OMMs for 1.5 or 4 h respectively in 5% CO2 (preliminary
assays indicated that the viability of P. gingivalis in this atmosphere was unaffected; post-invasion viability: anaerobic
3.77  0.58%, aerobic 4.46  0.33%, p 0.15, n 3).

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A. Pinnock et al. / Microbes and Infection 16 (2014) 310e319

Following incubation, epithelial cells were washed three times


with PBS to remove non-adherent bacteria and external,
adherent bacteria were killed using 200 mg ml1 metronidazole
for 1 h. After washing the metronidazole from the cells, sterile
distilled water was used to osmotically lyse the epithelial cells.
OMMs were mechanically lysed by homogenisation (Tissue
Ruptor, Qiagen, West Sussex, UK). The number of exfoliated
epithelial cells in the culture medium was determined using a
haemocytometer prior to homogenisation.
2.6. Immunohistochemistry
OMMs were paraffin embedded, sectioned (4 mM) and
mounted onto glass slides (VWR International, Lutterworth,
UK). Sections were deparaffinised, rehydrated, peroxidase
activity quenched with 3% hydrogen peroxide and sections
subjected to high temperature antigen retrieval in citrate
buffer. Sections were then blocked with 100% horse serum for
30 min at room temperature. The murine monoclonal P. gingivalis antibody (clone 1B5) was diluted in horse serum (1:50)
and incubated with the sections overnight at 4  C in a humidified atmosphere. Slides were washed in PBS and incubated with biotinylated secondary antibody for 30 min
(VECTASTAIN Elite ABC-Peroxidase Kits; Vector Laboratories, Peterborough, UK) prepared according to the manufacturers instructions. After washing, slides were incubated
with avidin biotinylated enzyme complex (ABC) reagent
(Vector Laboratories) for 30 min. Finally, slides were washed
and 3,30 -diaminobenzidine tetrahydrochloride (DAB) (Vector
Laboratories) substrate was added. Slides were counterstained
with haematoxylin, using a Leica ST4020 Small Linear
Stainer (Leica Microsystems, Milton Keynes, UK) and
mounted using DPX non-aqueous mounting medium (Merck,
Nottingham, UK). An isotype mouse IgG1 control (Dako,
Copenhagen, Denmark) antibody (1:50) was used to confirm
the specificity of the primary antibody.

ProlongGold anti-fade reagent (Invitrogen, Paisley, UK).


Staining was visualised using the Zeiss Axiovert 200 inverted
fluorescence microscope with Axiovision imaging software
(Zeiss, Ltd).
2.8. Chemokine array
Previous studies assessing the detection of chemokine
release from epithelial cells treated with P. gingivalis have
described a local chemokine paralysis effect [18]. Therefore,
NOK monolayers and NOK-OMM were pre-stimulated with
25 ng ml1 TNF-a (Peprotec, UK) for 4 h to increase the
levels of chemokine release prior to the addition of P. gingivalis, allowing for easier assessment of changes in chemokine
detection. Following pre-stimulation, both monolayers and
OMMs were incubated with P. gingivalis NCTC 11834 for
1.5 h and 4 h respectively. Models containing intracellular P.
gingivalis were incubated in serum-free medium with
200 mg ml1 metronidazole (to kill the external adherent
bacteria) for 4 h and the conditioned medium assessed for the
presence of chemokines using the RayBio Human Inflammation Antibody Array 3 (Insight Biotechnology Ltd, Middlesex, UK) according to the manufacturers instructions. The
chemiluminescence signal was detected using a Compact X4
Automatic X-Ray Film Processor (Xograph Healthcare,
Gloucester, UK). Signal intensities were analysed using
Quantity One software (Bio-Rad, UK) and calculated relative
to their internal positive controls after subtraction of the
negative background intensity.
2.9. Statistical analysis
Statistical analyses were performed using a Students unpaired two-tailed t test.
3. Results

2.7. Immunofluorescence

3.1. Invasion of monolayer and OMM by P. gingivalis

Plate-cultured P. gingivalis NCTC 11834 were washed 3


times in PBS and labelled with 5-(6)-carboxyfluorescin succinylester (Invitrogen) in PBS (0.4 mg ml1) for 30 min in the
dark at 4  C. After washing, fluorescently-labelled P. gingivalis NCTC 11834 (MOI 100 in serum-free medium) was
added to air-exposed H357-OMMs and incubated overnight at
5% CO2, 37  C. Preliminary data revealed that fluorescently
labelling the P. gingivalis cells did not significantly decrease
their viability or cell invasion capability (post-invasion viable
count: unlabelled bacteria 4.22  0.96%; FITC-labelled
3.27  0.32%, p 0.18). OMMs were then fixed in 10%
(v/v) PBS-buffered formalin and embedded in optimum cutting temperature (OCT) (FisherScientific), at approximately
43  C. Sections (10 mm) were prepared using a Microm
HM560 cryostat (ThermoScientific), at 20 to 30  C, and
mounted on microscope slides. Slides were flooded with
1 mg ml1 Hoechst 33342 (ThermoScientific, Northumberland, UK) for 2 min, washed and then mounted using

The OMMs used in this study displayed a well-organised


architecture consisting of a multi-layered epithelium and a
fibroblast-populated connective tissue layer (Fig. 1). OMMs
generated from NOK exhibited a more stratified epithelium
than those generated with H357 cells. In addition, the thickness of the epithelium could be successfully controlled by
exposing the surface of the OMM to air or keeping it submerged with medium (Fig. 1). Both NOK and H357 OMMs
showed similar patterns of expression of the epithelial markers
cytokeratin 13, 14 and E-cadherin when compared to sections
of normal healthy oral mucosa (data not shown). Invasion of P.
gingivalis into air-exposed and submerged H357 and NOK
OMM was assessed visually by immunohistochemistry and
immunofluorescence (Fig. 2). Staining indicated that P. gingivalis was able to penetrate the epithelium in both H357 and
NOK OMM. Staining of air-exposed H357 OMM sections
(Fig. 2A) showed a greater level of P. gingivalis penetration
than in NOK OMM (Fig. 2B). P. gingivalis is more likely to

A. Pinnock et al. / Microbes and Infection 16 (2014) 310e319

313

Fig. 1. Haematoxylin and eosin staining of air-exposed and submerged OMM cultured using H357 and NOK. The H357 cell line and NOK were cultured on a
fibroblast-populated collagen matrix at the air-to-liquid interface or completely submerged in culture medium.

invade junctional epithelium where pocket deepening is


occurring and since this epithelium is only a few cells thick,
the opportunity to penetrate through the epithelium is likely to
be greater. We found that P. gingivalis was able to penetrate
completely through the thin epithelium and was localised in
the connective tissue in submerged OMM that are only a few
layers thick (Fig. 2C). Whereas, bacteria were observed only
in the upper layers of the thicker epithelium in the OMM
grown at an air-to-liquid interface (Fig. 2D).
Comparing monolayer cultures of H357 cells with monolayers of NOK, the percentage invasion by P. gingivalis (using
an antibiotic protection assay) was slightly higher in the H357
cultures than was seen with the NOK monolayer cultures but
this was not statistically significant (3.19  1.68% and
2.15  0.90% respectively, Fig. 3A). While some work has
been done looking at P. gingivalis invasion of OMM [8e10],
there are no reports comparing P. gingivalis invasion in this
with that which occurs in monolayer cultures. Consequently,
we undertook such a comparison and found that the percentage invasion of H357 cell OMMs by P. gingivalis strain NCTC
11834 was 3.38  0.45% which was not significantly different
from that obtained with H357 monolayer cultures
(3.19  1.68%; Fig. 3A). However, invasion of NOK OMM by
P. gingivalis NCTC 11834 was significantly lower than invasion into H357 OMM (0.98  1.02% versus 3.38  0.45%
respectively; p < 0.05; Fig. 3A). These data confirm the observations of the immunostaining studies (Fig. 2) and suggests
that there may be differences between the two cell types in
their expression of cell surface proteins/receptors important

for invasion of this bacterium or the rate of epithelial cell


desquamation at the OMM surface. It must be noted that
because the invasion experiments of monolayer and OMM
were performed over different periods of time (e.g. 1.5 h for
monolayer and 4 h for OMM), only the total percentage invasion was assessed. This is because invasion of monolayers
was greatest after 1.5 h, whereas invasion of OMM by
P. gingivalis was greatest after 4 h (data not shown). This
allowed for comparison of maximal percentage invasion
values between the two culture systems.
The levels of P. gingivalis invading the thinner-layered,
submerged H357 OMM was reduced but not statistically
different from the invasion of H357 OMM grown at an air-toliquid interface (1.28  0.96% and 2.30  1.74% respectively;
Fig. 3B).
3.2. Intracellular survival and bacterial release of P.
gingivalis from H357 monolayer and OMM cultures
To compare the intracellular survival of P. gingivalis over
time in both H357 monolayer and OMM cultures, an antibiotic protection assay was performed and the epithelial
cultures were further incubated and assessed for intracellular
survival by releasing bacteria from lysed epithelial cells.
H357 cells were used for this assay because monolayer and
OMM cultures using these cells were shown to have the same
or higher percentage P. gingivalis invasion than NOK cultures (Fig. 3A). Consequently, H357 cells were deemed a
better model to use because with the greater number of

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A. Pinnock et al. / Microbes and Infection 16 (2014) 310e319

Fig. 2. Detection of P. gingivalis cells within cultured OMMs. P. gingivalis cells (unlabelled (AeC) or FITC-labelled (D)) were incubated with air-exposed H357OMM (A&D), air-exposed NOK-OMM (B) or submerged H357-OMM (C) for 4 h. OMMs were then stained using a P. gingivalis-specific antibody (clone 1B5)
and counter-stained with haematoxylin (AeC) or OMMs were cryosectioned and Hoechst used to stain the epithelial cell nuclei (D). Staining was detected in the
superficial layers of air-exposed H357-OMM (A) and deeper within the OMMs (arrows in C&D). Penetration of P. gingivalis into the connective tissue of
submerged H357-OMM (C). Staining of P. gingivalis within NOK-OMM (B), arrows indicate staining of intracellular bacteria. Images are representative of at least
three independent experiments.

internalised bacteria the possibility of detecting any changes


in intracellular survival over time would be higher. Using this
model, the intracellular viability of P. gingivalis was found to
decrease in a time-dependent manner in both monolayer and
OMM to reach zero after 18 h for monolayer cultures but was
more prolonged with the OMM (still 0.001% at 48 h;
Fig. 4A). This prolonged, though low, viability of P. gingivalis within organotypic cultures suggests that features of
OMM structure or physiology may contribute to the sustained survival of this bacterium.
Periodontitis is a cyclical disease and so patients may
exhibit active disease followed by periods of quiescence. It has
been proposed that P. gingivalis may escape or be released
from epithelial cells to cause re-infection of the periodontal
tissues [27]. To assess this using H357 monolayer and OMM
cultures, cells were briefly treated with metronidazole
following bacterial invasion, to kill external P. gingivalis and
then cultured for a further 3, 6, 18 and 24 h. Viable P. gingivalis were looked for extracellularly in the culture medium
with maximal values being detected at 6 h for monolayer and
24 h for OMM cultures (Fig. 4B). At these time points the
levels of P. gingivalis within the extracellular medium were
similar for both monolayer and OMM with 23.34  8.52% and
20.82  2.14% of the total intracellular extracellular bacteria respectively. Therefore, the release of intracellular
P. gingivalis into the culture medium appeared to be occurring

in both monolayer and OMM systems. This may not be due to


active release by epithelial cells alone but also to exfoliation of
the infected epithelial cells into the culture medium. Indeed,
exfoliated cells were detectable in the medium of both
monolayer and OMM at similar levels (10.0  2.50% and
10.0  2.80% respectively, of the original epithelial count
from monolayer and on the surface of OMM) over a 48 h
period (Fig. 4C). It should be noted that P. gingivalis could not
grow in the culture medium used for the monolayer or OMM
culture systems (data not shown).
3.3. Chemokine/cytokine release from monolayer and
OMM in response to intracellular P. gingivalis
Periodontitis is an inflammatory disease and the release of
pro-inflammatory chemokines and cytokines by the oral
epithelium has previously been demonstrated [28]. However,
the literature is ambiguous concerning the contribution of
P. gingivalis to the chemokine load, and in particular what
effect intracellular P. gingivalis have on the detection of these
chemokines/cytokines [8,29]. Here, the contribution of intracellular P. gingivalis (following killing of external bacteria
with metronidazole) on chemokine/cytokine release was
determined using a semi-quantitative antibody array. NOKs
were chosen for this assay, rather than H357, because the
response of NOK in terms of the chemokine/cytokine release

A. Pinnock et al. / Microbes and Infection 16 (2014) 310e319

Monolayer
P<0.05

5
Invasion (%)

315

OMM

4
3
2
1
0

H357

NOK

Invasion (%)

5
4
3
2
1
0

Air-exposed

Submerged

Fig. 3. Invasion of monolayer and OMM cultured using H357 or NOK by P.


gingivalis NCTC 11834. Monolayers and OMMs were cultured using H357
(A) or NOK and OMMs were cultured using H357 at the air-to-liquid interface
or submerged (B). P. gingivalis NCTC 11834 were added at MOI 100 for 1.5 h
(monolayer) or 4 h (OMM), after which metronidazole was added to kill the
external adherent P. gingivalis. Intracellular bacteria were released by osmotic
lysis using sterile distilled water and scraping. Bacterial colonies were
numerated by serial dilutions and plating onto blood-FA plates. The number of
intracellular bacteria recovered is expressed as a percentage of the infecting
inoculum. Graphs show mean  SD of three independent experiments performed in triplicate, p < 0.05.

was likely to be more representative of the in vivo response.


The results of the antibody array, for both NOK-monolayer
and NOK-OMM cultures, indicated that there was a marked
decrease in the production of the majority of chemokines/cytokines detectable when P. gingivalis was present compared
with the un-treated control (Fig. 5). For monolayer cultures,
the presence of intracellular P. gingivalis resulted in the
reduction of all tested cytokines, including exogenously added
TNF-a, apart from IL-1a. The semi-quantitative array indicated that IL-a increased 2.3-fold in the presence of intracellular P. gingivalis (Fig. 5C), however, this was not the case
for the OMM cultures which showed a decrease in IL-1a (1
fold), but an increase in CXCL8 and IL-6 by approximately
0.5 and 0.3 fold respectively when compared to un-infected
models and this was in contrast to monolayer cultures
(Fig. 5C). From the qualitative analysis of the cytokine arrays
there appears to be more cytokines constitutively produced by
OMM, compared with monolayer (e.g. dots for TGF-b1, IL3,
MIP-1b and ICAM-1) are more pronounced on the OMM
arrays (Fig. 5A). In addition, following infection, the degradation of cytokines was greater in monolayer than OMM, with

Fig. 4. Line charts to show the percentage invasion, CFU recovered from
supernatant and number of exfoliated epithelial cells after invasion of H357
monolayers and H357-OMM by P. gingivalis. H357 monolayers and H357OMM were cultured and exposed to P. gingivalis NCTC 11834 for 90 min
or H357-OMM was exposed to P. gingivalis NCTC 11834 for 4 h. Following
which H357 were washed and incubated with metronidazole for 1 h to kill the
external adherent bacteria. Invasion was calculated as the number of intracellular bacteria as a percentage of the original inoculum. Time 0 h refers to
measurements made immediately after metronidazole treatment. Representative wells were then washed and incubated at 37  C/5% CO2 and measurements made at the time points indicated in the graphs. Measurements included
percentage invasion (A) and the number of bacteria released from the
epithelial cells which is presented as the number of colony forming units
(CFU) counted by viable counting on blood agar plates, as a percentage of the
intracellular extracellular CFU (B). The number of exfoliated cells was also
counted at each time point (C). In all cases error bars indicate means  SEM
of three (monolayer) or two (OMM) independent experiments, all repeated in
triplicate.

CXCL8, IL-6, MCP-1 and CCL5 remaining after invasion of


OMM (Fig. 5A).
4. Discussion
As OMMs show similar immunohistochemical and
functional characteristics to normal oral mucosa than

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A. Pinnock et al. / Microbes and Infection 16 (2014) 310e319

- P. gingivalis

+ P. gingivalis

A B C D E F G H I J K L

A B C D E F G H I J

A B C D E F G H

A B C D E F G H

J K

Monolayer

J K L

OMM

A
B
1
Pos
Pos
2
Pos
Pos
3
IL-1
IL-2
4
IL-1
IL-2
5
IL-13
IL-15
6
IL-13
IL-15
7 RANTES TGF-1
8 RANTES TGF-1

C
D
E
Neg
Neg
Eotaxin
Neg
Neg
Eotaxin
IL-3
IL-4
IL-6
IL-3
IL-4
IL-6
IL-16 IL-17
IP-10
IL-16 IL-17
IP-10
TNF- TNF- s TNF R1
TNF- TNF- s TNF R1

Chemokine/
Cytokine

F
G
H
I
Eotaxin 2 GCSF GM-CSF ICAM-1
Eotaxin 2 GCSF GM-CSF ICAM-1
IL-6sR
IL-7
IL-8
IL-10
IL-6sR
IL-7
IL-8
IL-10
MCP-1
MCP-2
M-CSF
MIG
MCP-1
MCP-2
M-CSF
MIG
s TNF RII PDGF-BB TIMP-2
Blank
s TNF RII PDGF-BB TIMP-2
Blank

Also known as

J
K
L
IFN-
I-309
IL-1
IFN-
I-309
IL-1
IL-11 IL-12 p40 IL-12 p70
IL-11 IL-12 p40 IL-12 p70
MIP-1 MIP-1
MIP-1
MIP-1 MIP-1
MIP-1
Blank
Neg
Pos
Blank
Neg
Pos

Fold Change
Monolayer

OMM

CXCL8

Interleukin 8

-0.7

0.5

IL-1

Interleukin 1

2.3

-0.1

IL-6

Interleukin 6

-0.1

0.3

CCL2

Monocyte chemotactic protein 1 (MCP-1)

-0.1

-0.6

CXCL10

Interferon-gamma-inducible protein 10 (IP-10)

-0.1

-0.1

CCL5

Regulated upon activation, normal T cell expressed and


secreted (RANTES)

-0.1

-0.6

TIMP-2

Tissue inhibitor of metalloprotease 2

-0.1

-0.1

TNF-

Tumour necrosis factor

-0.1

-0.1

Fig. 5. Cytokine immunoblot of NOK-monolayer and NOK-OMM stimulated with TNF-a and P. gingivalis NCTC 11834. NOK monolayer and NOK OMM were
pre-stimulated with TNF-a for 4 h. Monolayers and OMMs were exposed to TNF-a with or without P. gingivalis for 1.5 h and 4 h respectively. Monolayers and
OMMs were washed and incubated with metronidazole for 4 h during which time cytokines were released. A cytokine array was performed and the density of each
dot analysed. Using the internal positive control (Pos), the relative density was calculated between eP. gingivalis and P. gingivalis for monolayer and OMM.
Immunoblots (A), location of cytokine antibodies on the array membranes (B), fold changes between infected and non-infected blots (C). Results shown are
representative of duplicate experiments.

monolayers [8,13], it was hypothesised that organotypic


cultures of the oral epithelium could provide more insight
into bacterial invasion and host responses of oral epithelial
cells. In this study we have shown that there are marked
differences in the total percentage invasion, intracellular
viability, bacterial release and cytokine profile following

epithelial cell invasion of monolayer and OMM by


P. gingivalis.
Calculating the percentage invasion of bacteria into
epithelial cells is commonly used to investigate trends in
experimental data; for example, to investigate which strains of
bacteria invade cells at a greater level than others and the

A. Pinnock et al. / Microbes and Infection 16 (2014) 310e319

effect of therapeutic agents on bacterial survival. In the last


15e20 years, epithelial cell invasion by P. gingivalis has been
shown by many research groups [3,4]. The most commonly
used epithelial cell in these studies has been KB (now shown
to be a HeLa contaminated cell line) or NOK. NOK cultured
from the oral mucosa retain some in vivo characteristics
compared with immortalised oral epithelial cells and cancer
cell lines, indicating that the use of NOK may be a more
representative in vitro model [3]. The levels of bacterial invasion shown in the present study for NOK and H357 cells are
similar to those previously reported in the literature [12] but
they differed when grown as OMM. Indeed, in terms of
modelling the oral mucosa more closely, organotypic models
have recently been championed [8,9]. This is because of their
multi-layered nature and the presence of a fibroblastcontaining scaffold that is comparable with the oral mucosa.
In this study, we found that the total percentage bacterial invasion of H357 monolayer and OMM cultures were similar,
suggesting that either H357 monolayer or H357 OMM may be
suitable to study experimental trends in invasion data. Despite
this, significant differences were shown for bacterial survival
and chemokine/cytokine release between monolayer and
OMM, suggesting that while monolayer cultures may be
suitable to investigate trends in experimental data, OMM may
be important for elucidating experimental end-points, such as
protein release, receptor expression and cellular viability.
NOK OMM did not support as high a level of P. gingivalis
invasion as H357 OMM, which might be due to differences in
the expression, activation and/or polarity of epithelial cell
surface receptors, cellular differentiation and/or intracellular
signalling pathways of these two cell types.
Immunohistochemistry of OMM tissue sections indicated
that P. gingivalis has the capacity to infiltrate the upper most
layers of the epithelium of air-exposed NOK, and in particular,
H357 OMM. This is in agreement with previously reported
literature that shows the cellular invasion of P. gingivalis
through multiple epithelial layers [8e10,30]. In addition, we
observed that P. gingivalis could enter the connective
component of OMM but only in models containing a thin layer
(4e5 cells thick) of epithelium, which is similar in thickness
to junctional epithelium at the base of the gingival pocket. The
movement of P. gingivalis through the epithelium and into the
connective tissue layer is particularly important because
in vivo the blood vessels are situated close to the epithelial
layers, presenting a risk of low level bacteraemia, which may
play a role in atherosclerosis, endocarditis or other systemic
diseases [31]. Therefore, it appears that the fewer epithelial
cell layers the bacterium is required to move through, the
greater the opportunity for the pathogen to breach host defences and to cause damage.
Intracellular survival is important for the continuation of
disease; for example, intracellular pathogens may be expelled
by cells allowing them to re-infect neighbouring cells. Current
data is conflicting as to the length of time P. gingivalis can
survive within human cells. Intracellular persistence has been
reported for anything up to 4e8 days [15,16,29]. However, we
show that for both monolayer and OMM the intracellular

317

viability of P. gingivalis significantly declines after 6 h to


almost zero by 48 h. This is in accordance with the findings of
Li et al. [14], who reported similar levels of intracellular
P. gingivalis in KB (HeLa), endothelial and smooth muscle
cells. These authors showed a decline in intracellular bacteria
to almost zero after 24e48 h in the numbers of intracellular P.
gingivalis after 90 min incubation [14]. This rapid decline in
intracellular viability may be due to the experimental conditions employed (e.g. the culture medium or the aerobic atmosphere necessary to maintain viability of the human cells)
or it is possible that some P. gingivalis cells enter an uncultivable state after several hours of intracellular occupancy
[14]. Interestingly, we observed that the intracellular viability
and persistence of P. gingivalis in OMM was greater than in
monolayers. Dickinson et al. [9] recently showed that P.
gingivalis cells are able to spread from cell-to-cell. Since the
OMM is a multi-layered epithelium, the opportunity to contact fresh cells may be greater in this model compared to cells
grown as monolayers and so this may account for the apparent
increased survival in the OMM. As well as survival, we
observed release of P. gingivalis from both monolayers and
OMM cultures and although we observed epithelial exfoliation in both model systems, which is in accordance with
previously reported literature [32], it is uncertain as to whether
this is sufficient to explain the presence of P. gingivalis in the
culture medium over time or whether this represents active
release. P. gingivalis has recently been reported to be actively
released from epithelial cells [33] but by either mechanism,
bacteria would be able to seed either local or distant sites in
the oral cavity. Intracellular replication was not found using
the experimental techniques employed in this study.
The first line of host defence against bacterial challenge is
the release of pro-inflammatory chemokines and cytokines.
Here, we investigated whether there were similarities or differences in the pro-inflammatory chemokine/cytokine release
in the presence of intracellular P. gingivalis produced by
monolayer and OMM. In accordance with previous studies,
we observed a decrease in the majority of chemokines/cytokines detected from both the monolayer [34,35] and OMM
cultures in the presence of this organism [9]. This attenuation
of the host response has been suggested to be important for
the survival of P. gingivalis at the site of infection by
depressing the host immune response [18]. Chemokine and
cytokine release was measured in infected monolayer and
OMM after removal of extracellular P. gingivalis following
treatment with metronidazole. Protease inhibitors were not
employed because we found that these dramatically decreased
the epithelial cell viability over this time period. Notwithstanding these issues, not all chemokines and cytokines were
reduced upon prolonged exposure to P. gingivalis. Indeed, for
OMM, there was a slight increase in the detection of CXCL8
and IL-6 compared with the un-stimulated control and
monolayer cultures. This is in accordance with previously
reported data from an organotypic culture model using palatal
epithelial cells [8]. This lack of attenuation of these chemokines in OMM compared with monolayers may be due to the
accumulation of these molecules in the multi-cellular layers of

318

A. Pinnock et al. / Microbes and Infection 16 (2014) 310e319

the OMM and consequent relative protection against the action of bacterial proteases or perhaps due to intrinsic differences between the physiology of the two model systems.
While it is possible that the action of gingipains might be
reduced in the aerobic conditions of these tissue culture systems, previous workers have reported that most of the proteolytic activity of P. gingivalis cultures was retained in the
presence of oxygen [36] and that gingipains are active in
tissue culture systems incubated aerobically [8]. We cannot
rule out that some of the cytokines secreted may have originated from oral fibroblasts within the OMM. However, experiments comparing stimulated OMM containing fibroblasts
to those without fibroblasts show that these cells contribute
only a small increase in CXCL8 production (approximately
0.4-fold) but no apparent contribution to the secretion of IL-6
(data not shown). Therefore, these data suggest that the fibroblasts may only play a minor role in chemokine/cytokine
contribution in the OMM and their presence seems unlikely to
explain the lack of attenuation of these cytokines compared
with monolayer cultures. Additionally, the chemokine/cytokine contribution from fibroblasts, as part of OMM, is an
intrinsic feature that qualifies these models as more representative of the oral tissues.
In summary, we have shown that monolayer cultures may
be suitable to determine trends in experimental data on cell
invasion by P. gingivalis. However, when requiring absolute
end-point values, the use of organotypic models are a more
relevant in vitro model of the cellular microenvironment
encountered by P. gingivalis in vivo.
Acknowledgements
The authors would like to thank GlaxoSmithKline for
funding this work.
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