Micronutrient modulation of NF-κB in oral keratinocytes exposed to periodontal bacteria

http://ini.sagepub.com/Innate Immunity

http://ini.sagepub.com/content/early/2012/08/02/1753425912454761The online version of this article can be found at:

DOI: 10.1177/1753425912454761

published online 13 August 2012Innate ImmunityMichael R Milward, Iain LC Chapple, Kevin Carter, John B Matthews and Paul R Cooper

B in oral keratinocytes exposed to periodontal bacteriaκMicronutrient modulation of NF-

Published by:

http://www.sagepublications.com

On behalf of:

International Endotoxin & Innate Immunity Society

can be found at:Innate ImmunityAdditional services and information for

http://ini.sagepub.com/cgi/alertsEmail Alerts:

http://ini.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

What is This?

- Aug 13, 2012OnlineFirst Version of Record >>

at UNIV OF BIRMINGHAM on September 7, 2012ini.sagepub.comDownloaded from

http://ini.sagepub.com/

http://ini.sagepub.com/content/early/2012/08/02/1753425912454761

http://www.sagepublications.com

http://www.ieiis.org

http://ini.sagepub.com/cgi/alerts

http://ini.sagepub.com/subscriptions

http://www.sagepub.com/journalsReprints.nav

http://www.sagepub.com/journalsPermissions.nav

http://ini.sagepub.com/content/early/2012/08/02/1753425912454761.full.pdf

http://online.sagepub.com/site/sphelp/vorhelp.xhtml


XML Template (2012) [1.8.2012–9:51am] [1–12]K:/INI/INI 454761.3d (INI) [PREPRINTER stage]

Original Article

Micronutrient modulation of NF-iBin oral keratinocytes exposed toperiodontal bacteria

Michael R Milward, Iain LC Chapple, Kevin Carter,John B Matthews and Paul R Cooper

Abstract

Chronic periodontal diseases are characterised by a dysregulated and exaggerated inflammatory/immune response to

plaque bacteria. We have demonstrated previously that oral keratinocytes up-regulate key molecular markers of inflam-

mation, including NF-kB and cytokine signalling, when exposed to the periodontal bacteria Porphyromonas gingivalis and

Fusobacterium nucleatum in vitro. The purpose of the current study was to investigate whether a-lipoic acid was able to

abrogate bacterially-induced pro-inflammatory changes in the H400 oral epithelial cell line. Initial studies indicated that a-

lipoic acid supplementation (1–4 mM) significantly reduced cell attachment; lower concentrations (<0.5 mM) enabled

>85% cell adhesion at 24 h. While a pro-inflammatory response, demonstrable by NF-kB translocation, gene expression

and protein production was evident in H400 cells following exposure to P. gingivalis and F. nucleatum, pre-incubation of

cells with 0.5 mM a-lipoic acid modulated this response. a-Lipoic acid pre-treatment significantly decreased levels of

bacterially-induced NF-kB activation and IL-8 protein production, and differentially modulated transcript levels for IL-8,

IL-1b, TNF-a and GM-CSF, TLR2, 4, 9, S100A8, S100A9, lysyl oxidase, NF-kB1, HMOX, and SOD2. Overall, the data

indicate that a-lipoic acid exerts an anti-inflammatory effect on oral epithelial cells exposed to periodontal bacteria and

thus may provide a novel adjunctive treatment for periodontal diseases.

Keywords

a-Lipoic acid, epithelium, Fusobacterium nucleatum, periodontitis, Porphyromonas gingivalis

Date received: 16 January 2012; revised: 1 May 2012; 25 May 2012; accepted: 18 June 2012

Introduction

Periodontitis is a ubiquitous chronic inflammatory dis-ease that destroys the supporting structures of teeth; themost common form is chronic periodontitis which, ifuntreated, results in the breakdown of soft tissues andbone, leading, ultimately, to tooth loss. The disease-initiating factor is plaque bacteria present in a biofilmat, and below, the gingival margin;1 however, the sub-sequent tissue damage that characterises this disease isthe result of an aberrant and exaggerated host responsewithin susceptible individuals.2 The plaque biofilm isa complex ecosystem containing a wide diversity of bac-teria, two Gram-negative anaerobes Porphyromonasgingivalis and Fusobacterium nucleatum, which arewidely regarded as key to disease progression.3

Fusobacterium nucleatum interacts with other earlycolonising bacteria enabling a ‘bridge’ for the develop-ment of a more mature and pathogenic biofilm4 that is

colonised subsequently by strict anaerobes, includingP. gingivalis, which expresses a range of virulence fac-tors and is strongly associated with periodontal diseasepathogenesis.5

An aberrant host immune response has beenreported in periodontitis, with neutrophils frompatients demonstrating hyperactivity/reactivity2 andtheir excessive production of reactive oxygen species(ROS) is believed to be partly responsible for thelocal tissue damage, exacerbated by reduced localtissue antioxidant defences.6 The mechanisms whereby

School of Dentistry, College of Medical & Dental Sciences, University

of Birmingham, UK

Corresponding author:

Michael R Milward, School of Dentistry, College of Medical and

Dental Sciences, University of Birmingham, St Chads Queensway,

Birmingham B4 6NN, UK.

Email: [email protected]

Innate Immunity

0(0) 1–12

! The Author(s) 2012

Reprints and permissions:

sagepub.co.uk/journalsPermissions.nav

DOI: 10.1177/1753425912454761

ini.sagepub.com




neutrophils are recruited to, or cleared from, the peri-odontal tissues may also have an important bearing onthe aetiology of this disease. The sulcular and junc-tional epithelia, in common with similar epitheliaat other sites in the body, were originally thoughtto simply provide a passive physical barrier, protectingthe host from the external environment. More recently,however, periodontal epithelium has been shown tobe a key orchestrator of the inflammatory response toa colonising biofilm and certain bacteria presentwithin.7 Indeed, recent work has demonstrated thatperiodontitis-associated bacteria, such as P. gingivalisand F. nucleatum, can initiate keratinocyte pro-inflammatory responses resulting in elevations in cyto-kine transcript and protein production in vitro.8 Centralto the pro-inflammatory response is NF-kB, a keyREDOX-sensitive transcription factor activated byincreases in intracellular oxidative stress caused by awide range of factors, including bacterial stimulation,8,9

resulting in downstream changes in gene expression andcytokine production.

a-Lipoic acid (ALA) (also known as a-lipoate orthiocytic acid) is a naturally-occurring di-thiol presentin a wide range of food substances, including meats,such as liver, kidney and heart, as well as vegetables,such as spinach and broccoli. Recently, ALA hasattracted considerable attention owing to its variedantioxidant actions. ALA is able to reduce levels ofoxidative stress by (i) direct free radical scavengingand (ii) regeneration of other intracellular antioxi-dants,10,11 most notably glutathione—a key antioxidantin maintaining cellular REDOX status.12

Owing to its ability to boost levels of intracellu-lar glutathione, ALA is able to regulate NF-kB activa-tion. 13 ALA has undergone extensive clinical trials andhas been shown to have efficacy in the treatment ofseveral chronic inflammatory diseases, for examplediabetes and cardiovascular disease.14,15 ALA thereforerepresents a potential candidate for modulation of NF-kB and the associated pro-inflammatory activity ofperiodontal epithelium. Thus, the aim of this studywas to investigate the effect of ALA on the pro-inflammatory response of oral keratinocytes exposedto periodontal bacteria by analysing NF-kB activation,gene expression and cytokine production.

Materials and methods

Bacterial culture and suspensions

Escherichia coli LPS (serotype 026:B6; Sigma, Poole,UK) was reconstituted with cell culture growth media(DMEM; Invitrogen, Paisley, UK) to produce a stocksolution (250mg/ml) which was stored at �20�C priorto use. Porphyromonas gingivalis (ATCC33277, isolatedfrom the human gingival sulcus) and F. nucleatum(ATCC10953, isolated from inflamed human gingivae)

were grown in broth culture at 37�C anaerobically, asdescribed previously.8 Cell suspensions were centri-fuged, and the bacterial pellet washed three timesusing PBS, heat-inactivated (100�C for 10min) and sus-pended in PBS to give a final concentration of 4� 108

cells/ml. Heat inactivation was confirmed by platingand culturing experiments. Bacterial suspensions werestored at �20�C prior to use for keratinocyte exposure.

Cell culture

Details of cell culture methods were as described pre-vioulsy.8,16 Cells were grown in a range of plastic-ware,including flasks, 96-well plates and Petri dishes(including some containing multi-well glass slides).H400 cells (an immortal cell line derived from an oralsquamous cell carcinoma of the alveolar process in a55-year-old patient) were cultured in DMEM(Invitrogen) supplemented with 10% FCS (Labtech,Ringmer, UK), 4mM glutamine (Sigma) and 0.5mg/mlhydrocortisone (Sigma). All cells were grown in anatmosphere of 5% CO2 at 37�C. Once cells wereseeded they were cultured for 4 d prior to mediachange and subsequently passaged on d 7. All experi-ments were performed between passages 4 and 20 usingsub-confluent cell monolayers and standard DMEMmedia supplemented with growth factors (FCS, glutam-ine and hydrocortisone), which are used routinelyfor H400 cell culture to promote viability and prolifer-ation.8,16 While hydrocortisone is known to haveanti-inflammatory actions, under the conditions andconcentrations we have employed this action is notparticularly evident, and its inclusion is rather toenhance cell viability and proliferation in our culturesystem.8,16 In addition, media is supplemented fre-quently with antibiotics which we consider a potentialconfounding factor of H400 cell bacterial stimulationand not representative of the in vivo situation. Thus,all experiments reported in this article were performedin the absence of antibiotic supplementation.

ALA

A 0.06M ALA (Sigma) stock solution was prepared in0.029M sodium hydroxide. The stock solution wasstored at �20�C in aliquots prior to use.

Effect of ALA on cell adherence

Cells were pre-incubated with ALA (0.0625–4mM) orvehicle control for 24 h to determine effects on celladherence. Following incubation culture media wasremoved, cell monolayers were washed with 10mlPBS and incubated with trypsin-EDTA for 10minwith regular agitation to ensure complete breakdownof the monolayer. The reaction was subsequentlystopped by using fresh cell culture media.

2 Innate Immunity 0(0)




Resulting cell suspensions were centrifuged and the cellpellet re-suspended in fresh culture media beforecell counts were performed using a haemocytometer(modified Neubauer).

Effect of ALA on NF-�B nuclear translocation in LPSstimulated keratinocytes

Escherichia coli LPS, a validated activator of NF-kBnuclear translocation in various cell types, was usedas a known positive control prior to testing relevantperiodontal pathogens in our test system. H400 cellscultured on glass slides within Petri dishes were pre-incubated with ALA (0.25–2mM) or vehicle controlfor 24 h prior to exposure to E. coli LPS (10 mg/ml;1 h) or vehicle control. Slides were then washed brieflyin PBS, shaken to remove excess PBS and processed forimmunocytochemical staining for NF-kB and manualassessment of percentage translocation, as described inthe following sections.

Effect of ALA on keratinocytes stimulated withperiodontal bacteria

H400 cells were pre-incubated for 24 h in 0.5mM ALAprior to exposure to P. gingivalis (ATCC33277) orF. nucleatum (ATCC10953). Periodontal bacteria wereincubated with H400 cells at a concentration of 1� 109

ml (equivalent to approximately 100 bacteria per epi-thelial cell) for 1 h. This ratio of bacteria (1 : 100, epi-thelial cell to bacteria) was comparable with previousstudies which indicate the maximum number of bac-teria associated with epithelial cells in the periodontalpocket, thereby providing clinical relevance.17 Cells orsupernatants were analysed subsequently for NF-kBnuclear translocation by high content analysis, geneexpression by sq-RTPCR and IL-8 protein by ELISA.

Immunocytochemistry of NF-�B translocation

Cell monolayers were fixed using dry acetone for 10minat room temperature (20–25�C), air dried and stainedby immunohistochemistry. The staining protocolemployed a MAb to the NF-kB p65 subunit (cloneF-6, diluted 1 : 100; Santa Cruz Biotechnology, SantaCruz, CA, USA) and followed a biotin–streptavidinimmunoperoxidase technique (StrAviGen, Biogenex,San Ramon, CA, USA) as reported previously.8 Theresulting bound peroxidase was visualised using3,30-diaminobenzidine reagent for 5min and counter-stained with Meyer’s haematoxylin before mountingin Xam. Slide-washing and dilution of reagents wereperformed using 0.01M PBS, pH 7.6. Positive- andnegative-staining controls were included, which con-sisted of replacement of the NF-kB monoclonal withKi67 (clone MM1, diluted 1 : 100; NovacastraTM,Vision Biosystems, Newcastle, UK) as a positive

control and replacement of NF-kB Ab with PBS or aMAb with irrelevant specificity, but the same immuno-globulin isotype as a negative control.

Quantification of NF-kB translocation was per-formed on cell monolayers using a microscope (Leica,Wetzlar, Germany) fitted with an eyepiece graticule(10� 10 0.01mm2 squares) and viewed at 100�magni-fication. Analysis using Hunting curves indicated thatcounting of 30 randomly selected fields provided repre-sentative cell counts. For cells to be classified as demon-strating NF-kB activation (positive), nuclear stainingfor p65 was required with no residual cytoplasmicstaining evident. The total and mean numbers of posi-tive cells were determined to calculate the percentage ofcells demonstrating NF-kB activation.

High content analysis of NF-�B nuclear translocation

H400 cells were cultured in 96-well plates (Corning,Amsterdam, The Netherlands) to semi-confluenceand either pre-incubated with 0.5mM ALA for 24 hor vehicle (negative control) prior to exposure toF. nucleatum, P. gingivalis or PBS (negative control).Cultures were then washed with PBS and fixed using1% formaldehyde solution for 20min, washed againwith PBS before filling wells with PBS. Plates werestored at 4�C prior to staining. Cells were incubatedwith the MAb NF-kB p65 subunit (clone F-6 diluted1 : 100; Santa Cruz) for 1 h at room temperature,washed and incubated with secondary Ab [anti-STAT1 rabbit polyclonal immunoglobulin (SantaCruz)]–conjugated to a fluorescent label (Santa Cruz)for 1 h. Detergent buffer (Cellomics, Reading, UK;200 ml) was added to the wells for 15min. Wellswere then washed with PBS before further detergentbuffer was added prior to analysis. Stained cultureswere analysed using an ArrayScan imaging cytometer(Cellomics) with ArrayScan II data acquisition soft-ware (Cellomics) used for automated quantification oflevels of cytoplasmic/nuclear NF-kB staining.

Semi-quantitative RT-PCR analysis

RNA was extracted from H400 cell cultures using acommercially-available kit (RNeasy�, Qiagen,Crawley, UK) and reverse transcription was performedusing the Omniscript RT kit (Qiagen) to generatesingle-stranded cDNA as described previously.8 Semi-quantitative RT-PCR assays were performed using theRedTaq PCR system (Sigma). The reaction mixtureconsisted of 50 ng of single-stranded cDNA, 12.5mlRedTaq ready reaction mix, 10.5ml dH2O, 1 ml of25 mM forward and reverse primer. Table 1 providesprimer sequences and reaction conditions. The resultingPCR mixture was amplified in a thermal cycler (Mastercycler Gradient; Eppendorf, Stevenage, UK). The ini-tial denaturing step was for 5min at 94�C which was

Milward et al. 3




Tab

le1.

Deta

ilsof

pri

mer

sequence

and

sem

i-quan

tita

tive

RT-

PC

Rco

nditio

ns.

All

DN

Ase

quence

sar

esh

ow

nin

the

5’to

3’ori

enta

tion.

Gene

Sym

bol

Acc

ess

ion

Num

ber

Sequence

Tm

(�C

)Pro

duct

(bp)

Cyc

leno.

Toll-

like

rece

pto

r2

TLR

2N

M_003264.3

F-G

AT

GC

CTA

CT

GG

GT

GG

AG

AA

61

392

31

R-C

GC

AG

CT

CT

CA

GA

TT

TA

CC

C

Toll-

like

rece

pto

r4

TLR

4N

M_138444-2

F-A

AC

CA

TC

CT

GG

TC

AT

TC

TC

G61

315

36

R-C

GG

AA

AT

TT

TC

TT

CC

CG

TT

T

Toll-

like

rece

pto

r9

TLR

9A

B045180.1

F-C

TG

CG

TC

TC

CG

TG

AC

AA

TTA

61

443

36

R-G

TC

CT

GT

GC

AA

AG

AT

GC

TG

A

NF-kB

1N

F-�B

1N

M_003998

F-C

CT

GG

AT

GA

CT

CT

TG

GG

AA

A61

366

26

R-C

TT

CG

GT

GT

AG

CC

CA

TT

TG

T

Tum

our

necr

osi

sfa

ctor-a

TN

F-�

NM

_000594.2

F-A

AG

AA

TT

CA

AA

CT

GG

GG

CC

T60

402

38

R-G

GC

TA

CA

TG

GG

AA

CA

GC

CTA

Inte

rleukin

-1beta

IL-1

BN

M_000576.2

F-T

CC

AG

GG

AC

AG

GA

TA

TG

GA

G60

292

28

R-T

TC

TG

CT

TG

AG

AG

GT

GC

TG

A

Inte

rleukin

-8IL

-8N

M_000584.2

F-TA

GC

AA

AA

TT

GA

GG

CC

AA

GG

60

204

30

R-G

GA

CT

TG

TG

GA

TC

CT

GG

CTA

Gra

nulo

cyte

mac

rophag

eco

lony

stim

ula

ting

fact

or

GM

CSF

M11220.1

F-A

CT

AC

AA

GC

AG

CA

CT

GC

CC

T60

303

31

R-C

TT

CT

GC

CA

TG

CC

TG

TA

TC

A

Monocy

teC

hem

oat

trac

tant

Pro

tein

-1/C

CL2

MCP-

1/C

CL2

NM

_002982.3

F-G

CC

TC

CA

GC

AT

GA

AA

GT

CT

C60

339

35

R-G

CT

GC

AG

AT

TC

TT

GG

GT

TG

T

Hem

eoxyg

enas

e1

HM

0X

1N

M_0002133.1

F-A

AC

CT

CC

AA

AA

GC

CC

TG

AG

T61

207

32

R-C

AC

CC

CA

AC

CC

TG

CT

ATA

AA

S100

calc

ium

bin

din

gpro

tein

A8

MRP8

/S100A8

BC

005928

F-A

TT

TC

CA

TG

CC

GT

CT

AC

AG

G61

232

30

R-C

AG

CC

TC

TG

GG

CC

CA

GTA

AC

TC

S100

calc

ium

bin

din

gpro

tein

A9

MRP1

4/S

100A9

NM

_002965.2

F-C

AG

CT

GG

AA

CG

CA

AC

ATA

GA

61

228

23

R-T

CA

GC

AT

GA

TG

AA

CT

CC

TC

G

CC

L20

CCL2

0/M

IP-3a

NM

_004591.1

F-G

CA

AG

CA

AC

TT

TG

AC

TG

CT

G60

341

27

R-C

AA

GT

CC

AG

TG

AG

GC

AC

AA

A

Lysy

loxid

ase

LOX

NM

_002317

F-A

CA

GG

GT

GC

TG

CT

CA

GA

TT

T61

381

24

R-C

CA

GG

TA

GC

TG

GG

GT

TTA

CA

Supero

xid

edis

muta

se2

SOD

2S7

7127

F-T

GG

AG

GC

AT

CTA

GT

GG

AA

AA

A61

305

34

R-C

CC

AG

TC

TC

TC

CC

CA

TTA

CA

Tm

,an

neal

ing

tem

pera

ture

(�C

);bp,bas

epai

rs;(F

),fo

rwar

dpri

mer;

(R),

reve

rse

pri

mer.





then followed by amplification cycles comprising 94�Cfor 20 s, 60–61�C for 20 s and 72�C for 20 s, (see Table 1for cycle number and annealing temperature per assay)with a final 10min extension at 72�C. Six microlitreswere removed from amplified reactions and separatedon a 1.5% agarose gel (containing 0.5 mg/ml ethidiumbromide; Sigma). The resulting gel was visualised underultraviolet illumination using the EDAS 120 system(Kodak, Hemel Hempstead, UK) and the capturedimage was imported into AIDA image analysis soft-ware (Fuji, Bedford, UK). The volume density ofamplified products was normalised against GAPDHhousekeeping expression levels. All semi-quantitativeRT-PCR experiments were performed in duplicateand representative graphs plotted.

ELISA

A commercially-available sandwich ELISA kit (IDSLtd, Boldon, UK) enabling sensitive and specific detec-tion/quantification was used to determine levels of IL-8present in cell culture media. Culture media was filteredusing a 0.2 micron filter (Millipore, Watford, UK) toremove cell debris prior to analysis. One hundredmicrolitres of standard or sample was added to wellsand processed following the manufacturer’s protocol.Absorbance was read at 450 nM (Spectrophotometer,Jenway, Stone, UK). Known IL-8 concentrations wereused to generate a standard curve from which levels ofIL-8 from the experimental samples (pg/ml) were deter-mined. The detection limit for the assay was 25 pg/ml.

Results

Determination of ALA concentrations to be used inexperiments using periodontal bacteria

As ALA culture supplementation has been reportedpreviously to modulate NF-kB activation18 and initialexperimentation indicated an ability to disrupt celladhesion, dose–response experiments were performedto identify suitable concentrations for use with theH400 cell line. Data demonstrated that with increasingconcentrations of ALA, adherent cell numbersdecreased dose-dependently and ALA concentrationsgreater the 0.5mM resulted in less than 50% cell adher-ence at 24 h (Figure 1). Reports in the literature indicatethe ability of ALA to reduce expression of adhesionmolecules,19 which may explain our adhesion data.Further investigation of H400 cells exposed to ALAindicated that non-adhered cells remained viable(Trypan blue exclusion staining) and after washing (toremove ALA), were able to re-adhere to culture plastic-ware and proliferate normally (data not shown).Combined data indicated that the use of � 0.5mM

ALA was the most appropriate for subsequentexperimentation.

To determine the effect of ALA on NF-kB transloca-tion under simulated pro-inflammatory conditions inH400 cells, cultures were pre-incubated with a rangeof ALA concentrations (0–2mM) for 24 h and thenexposed to E. coli LPS (10 mg/ml) for 1 h. In these initialexperiments E. coli LPS was used as this represents awell-characterised activator of the NF-kB pathway20

and has been used previously to explore the modulatoryeffects of ALA in other cellular systems.21 Cell mono-layers were stained immunocytochemically and manualcounts were performed to determine the percentage ofcells exhibiting nuclear translocation of NF-kB. Datademonstrated a dose-dependent inhibition of levels ofNF-kB nuclear translocation with increasing levels ofALA, with 0.5mM and 0.25mM ALA supplementationresulting in a 78% and 59% reduction in NF-kB acti-vation respectively (Figure 2). Based on these celladherence and NF-kB activation studies further experi-ments investigating the effects of ALA on keratinocyteresponses to periodontal bacteria were performed with0.5mM ALA.

NF-�B nuclear translocation and expressionregulation by periodontal bacteria and ALA

To determine the kinetics of NF-kB nuclear transloca-tion in H400 cells, using the periodontal bacterial sti-muli P. gingivalis and F. nucleatum, high contentanalysis was performed. Initial experiments aimed toidentify a suitable time-point for assay of NF-kB activ-ity in H400 cells exposed to periodontal bacteria, whichthat demonstrated NF-kB translocation appearedto peak at different time-points for P. gingivalis(�60min) and F. nucleatum (�100min) (Figure 3).Subsequent experiments utilised 60min of exposurefor both P. gingivalis (�100% stimulation) and F.nucleatum (�84% stimulation) to enable comparisonbetween bacteria and consistency with work publishedpreviously.8 These experiments demonstrated that NF-kB translocation was stimulated significantly by bothP. gingivalis (P¼ 0.0016) and F. nucleatum (P¼ 0.02;Figure 4) compared with un-stimulated controls.Furthermore, cells pre-incubated with ALA (0.5mM

for 24 h) prior to bacterial exposure demonstrated sig-nificantly reduced levels of NF-kB activation whencompared with stimulated cells that had not been pre-treated with ALA (P. gingivalis, P¼ 0.0026; F. nuclea-tum, P¼ 0.008) (Figure 4).

Semi-quantitative RT-PCR was performed subse-quently to assess transcript level regulation (Figure 5)in genes identified previously as being associated withthe inflammatory response, bacterial recognition(TLR), epithelial protection, antioxidant defence andtissue repair, as well as transcripts which we have iden-tified previously as being regulated differentially by P.gingivalis and F. nucleatum.8 IL-8, GM-CSF, TNF-aand IL-1b demonstrated increased levels of gene

Milward et al. 5




expression in H400 cells when exposed to bothP. gingivalis and F. nucleatum when compared withun-stimulated controls. Pre-incubation with ALA(0.5mM, 24 h) prior to bacterial exposure reducedthese bacterially-induced transcript levels. In contrast,ALA pre-treatment of H400 cell cultures resulted insignificant increases in TLR4 and TLR9 expression,while TLR2 transcript levels were decreased minimally.Exposure to F. nucleatum stimulated increases in bothS100A8 and S100A9 transcript levels in H400 cellscompared with un-stimulated controls, while P. gingi-valis exposure resulted in minimal transcript levelchange compared with un-stimulated controls. Pre-incubation with ALA substantially reduced these tran-scripts in both stimulated and un-stimulated conditionsfor both these genes. Superoxide dismutase-2 (SOD-2)and haemoxygenase 1 (HMOX) are two transcriptsimportant in antioxidant cellular defence. SOD-2demonstrated increased expression following H400exposure to P. gingivalis and F. nucleatum, whileALA pre-incubation affected these induced levels

minimally. HMOX gene expression was not increasedfollowing F. nucleatum stimulation of H400 cells; how-ever, a clear increase in transcript level was evident fol-lowing P. gingivalis stimulation, which was reduced toun-stimulated levels following H400 cell pre-incubationwith ALA.Previous microarray analysis demonstrateddown-regulation of lysyl oxidase (LOX) expression fol-lowing H400 cell stimulation with P. gingivalis andF. nucleatum.8 In agreement with this finding datahere demonstrated that H400 cell stimulation with F.nucleatum resulted in a minimal reduction in LOXexpression while P. gingivalis stimulation resulted in agreater down-regulation of expression. However, ALAsupplementation alone reduced LOX transcript levelssignificantly compared with both un-stimulated andF. nucleatum-stimulated cultures. A further minimaldecrease was also detected in P. gingivalis-stimulatedcultures. Pro-inflammatory related transcript,NF-kB1, which we have demonstrated previously tobe increased in H400 cells in response to P. gingivalisor F. nucleatum,8 were further characterised in this

120.0

(A) (B) (C)

(D)

100.0

80.0

60.0

40.0

20.0

0.00 0.0625 0.125 0.25

Concentration ALA (mM)

% c

ontr

ol v

alue

0.5 1 2 4

Figure 1. H400 cells incubated with (A) control solution (DMEM only; negative control) and (B) 0.5 mM a-lipoic acid for 24 h;

(C) 4 mM a-lipoic acid for 24 h; (D) H400 cells incubated with a range of concentrations of a-lipoic acid for 24 h and counts of attached

cells determined (mean value� standard deviation).





model system. Concomitantly, increased expressionwas detected for NF-kB1 following cellular stimulationwith both P. gingivalis and F. nucleatum. Bacterially-induced levels of this transcript were decreased follow-ing ALA pre-treatment of cultures.

While transcriptional regulation in H400 cells by ALAwas demonstrated further confirmation that these changes

translated to effects on functional protein levels was inves-tigated. Subsequently, analysis of the levels of pro-inflam-matory cytokine, interleukin (IL-8), a surrogate markerof NF-kB activation demonstrated that stimulation withP. gingivalis and F. nucleatum increased secreted proteinlevels of IL-8, while pre-incubation of cells with ALAsignificantly reduced these stimulated levels (Figure 6).

50

60

(A)

(B)

40

50

30

20

% tr

anns

loca

tion

0

10

0

Un-stimulated (No ALA)

Stimulated (No ALA)

0.25mM 0.5mM 1mM 2mM

(i) (iii) (v)

(ii) (iv) (vi)

Figure 2. Analysis of H400 cells pre-incubated with a range of a-lipoic acid concentrations for 24 h prior to stimulation with E. coli

LPS (1 h; 10 mg/ml) and immunocytochemically assayed for NF-kB translocation. (A) Images of stained cultures including (i) negative-

staining control (primary Ab replaced with PBS), (ii) vehicle (DMEM) containing no ALA, (iii) 0.25 mM ALA, (iv) 0.5 mM ALA, (v) 1.0 mM

ALA, and (vi) 2.0 mM ALA. Nuclear counterstaining has been used and scale bars are shown. Cytoplasmic localisation is progressively

more pronounced as a-lipoic acid concentrations increase. (B) Graphical analysis showing percentage of total cell numbers exhibiting

nuclear NF-kB localisation (mean� SD).

Milward et al. 7




Discussion

Current evidence regarding periodontal disease patho-genesis indicates that the substantive tissue damageseen in patients is a result of a dysregulated, non-resol-ving inflammation in response to the plaque biofilm22

associated with oxidative stress23 and elevated neutro-phil reactive oxygen species release2 characteristic ofthe periodontitis phenotype. Emerging data indicate

that, initially, the oral epithelium detects the presenceof periodontal bacteria7 and, plays an important rolein orchestrating the subsequent pro-inflammatoryresponse. The regulation of epithelial responses there-fore provides a point for potential therapeutic interven-tion and management of this disease. The NF-kBsignalling pathway is a key molecular regulator of thecellular inflammatory response and is REDOX sensi-tive; therefore, subsequent changes in intracellular

1.8

1.6

1.4

1.2

1

0.8

Nuc

lear

/cyt

opla

smic

Rat

io

0.6

0.4

0.2

0 30 60

Time (min)

F.nucleatum

P.gingivalis

Un-stimulated

90 120 150 180 210 240

Figure 3. NF-kB translocation kinetics for H400 cells exposed to P. gingivalis and F. nucleatum determined by high content analysis

over a 240-min time period. Levels of translocation are shown as nucelear:cytoplasmic ratios (mean� SD, n¼ 3).

1.6

1.4

1.2

1

0.8

0.6

0.4

0.2

0Un-stimulated Un-stimulated

+ALA PG stimulated PG+ALA FN stimulated FN + ALA

Nuc

lear

/cyt

opla

smic

ratio

Figure 4. High content immunocytochemical analysis of NF-kB in H400 cells exposed to P. gingivalis or F. nucleatum� a-lipoic acid

(n¼ 3, mean� SD). Cultures were pre-incubated with 0.5 mM ALA for 24 h prior to exposure to bacteria for 1 h. Symbols indicate

statistical significant differences between exposure groups (*P¼ 0.0016; +P¼ 0.0026; �P¼ 0.02; P¼ 0.008).





oxidative stress levels regulate pathway activation andresult in downstream sequelae, including cytokine pro-duction.24 This pathway therefore provides an idealtarget for modulation of the inflammatory responseand, as the natural di-thiol ALA is reported to boostintracellular antioxidant capacity, it has been identifiedas an ideal candidate for modulating epithelial-driveninflammation. This study investigated the effects ofALA on key stages in periodontal pathogen stimulatedepithelial cell activation, including bacterial componentrecognition via TLRs, NF-kB signalling, transcrip-tional responses and cytokine production. The expres-sion of three key TLRs were investigated and datademonstrated that ALA pre-treatment may result inincreases in keratinocyte expression of these moleculeswhich, hypothetically, may have translated to increasesin the cells’ sensitivity and ability to detect bacteria.Conversely, however, epithelial cell pre-incubationwith ALA with subsequent bacterial stimulationresulted in reduced levels of NF-kB activation, suggest-ing that increases in TLR transcript levels are not dir-ectly translated to increases in inflammation owing

to ALA’s ability to modulate activation of NF-kB.These findings are in agreement with data from previ-ous studies which have analysed a range of other celltypes and have shown that ALA pre-treatment reducesthe initiation and propagation of the inflammatoryresponse.25–28

ALA is important in regenerating intracellularantioxidants, including glutathione, which is centralto maintaining cellular REDOX status.10–12 Mechani-stically, it has been shown that ALA is internalisedinto the cell and converted to its more active antioxi-dant form, dihydrolipoic acid (DHLA).29 Preliminaryexperiments have demonstrated that incubation ofALA with H400 cells enhances its antioxidant capacity,as determined using an enhanced chemiluminescenceassay30 (data not shown), indicating that H400 cellsare able to metabolise ALA to its more active antioxi-dant form and suggesting that our observations arelikely explained by ALA boosting intracellular antioxi-dant levels in H400 cells.

Interestingly, LOX gene expression appeared tobe reduced following exposure to F. nucleatum and

Rel

ativ

e ex

pres

sion

(%

)

100

100

100

100

0 0

GA

DP

H

TLR

4

TLR

9

NF

- KB

1

IL-1

b

GM

-CS

F

TM

F-a IL-8

SO

D-2

S10

0A3

S10

0A9

CC

L2

CC

L 20

LOX

HM

OX

TLR

2

0

0 0

0

0

00

0 0 0

000

100

100 100

100

100

100 100

100

100

LOXCCL-20CCL-2

SOD-2 HMOX S100A8 S100A9

IL-8TNFaGM-CSFIL 1b

TLR-2 TLR-4 TLR-9 NF-KB1

100

100(A)

(B)

(C)

(D)

(E)

No ALA / un-stimulatedALA / un-stimulatedF. nucleatum stimulated

KEY

F. nucleatum stimulated + ALAP. gingivalis stimulatedP. gingivalis stimulated + ALA

Un-stimulatedUn-stimulated + ALA

PG stimulatedFN stimulated

PG stimulated+ ALAFN stimulated + ALA

Figure 5. Gene transcription changes as determined by sq-RT-PCR analysis in H400 cells exposed to P. gingivalis (1 h), F. nucleatum

(1 h) or media (negative control) and pre-incubated with 0.5 mM a-lipoic acid for 24 h. (A) Genes associated with bacterial recognition

and the NF-kB transcription pathway; (B) pro-inflammatory cytokines; (C) genes associated with epithelial protection; (D) cell

recruitment and tissue repair associated genes; (E) representative gel images. All data shown is representative of at least duplicate

analyses where comparable results were obtained.

Milward et al. 9




P. gingivalis. LOX is a copper-dependent enzymeimportant in epithelial function, development andrepair, and functions by cross-linking elastin and colla-gen.31 In periodontitis breakdown of these tissues isassociated with disease progression. Therefore, reduc-tion in expression of LOX post P. gingivalis and F.nucleatum stimulation may play a role in the pathogenicmechanism induced by these bacteria and conceivablylead to the periodontal tissue breakdown and frustratedtissue healing characteristic of this disease. LOX geneexpression was also reduced in the presence of ALA;this may be due to the copper-chelating properties ofALA, an essential co-factor in the action of thisenzyme.32 ALA has been used in a range of chronicinflammatory diseases and reports in other systems,including skin25 and gastric ulcers,33 have suggestedthat ALA enhances healing. Further studies are there-fore needed to investigate functional protein produc-tion in response to ALA supplementation.

Genes that have a role in cellular protection, neutro-phil recruitment and activation (MRP-8/S100A8 andMRP-14/S100A9) were potentially reduced in bacte-rially-stimulated cells owing to pre-incubation withALA. While these data indicate that ALA supplemen-tation may reduce cell protection mechanisms followingbacterial challenge, these reductions may also result inreduced neutrophil chemotaxis and activation subse-quently modulating the pro-inflammatory response. Inaddition, recent work34 has demonstrated that theincreased expression of these molecules associateswith ROS-mediated transcription pathway activation,therefore their down-regulation in response to ALA

further supports its anti-inflammatory action. Pre-incubation of the H400 cells with ALA resulted in apotential decrease in transcript levels of NF-kB whichmay equate to reduced cellular levels of the translatedprotein which potentially diminishes the activationpotential of this pathway. In further support ofthis, pre-incubation of H400 cells with ALA prior tostimulation with P. gingivalis and F. nucleatum poten-tially reduced protein transcription levels of the keyneutrophil recruiting and activating cytokine, IL-8. Asthis molecule is implicated in the induction and propa-gation of a variety of chronic inflammatory responsesthe ability of ALA to modulate IL-8 production mayhave important ramifications for periodontal diseasemanagement. Pre-incubation of oral epithelial cellswith ALA has now, for the first time, been shown tomodulate periodontal bacterial induced NF-kB activa-tion, pro-inflammatory gene expression and cytokineproduction. It is, however, interesting to note thatALA does not abrogate the pro-inflammatoryresponse, but rather attenuates it. Indeed, completeblocking of this key first-line host response to bacterialchallenge may be detrimental as it may render the hostsusceptible to overwhelming bacterial invasion.Therefore, this characteristic of ALA has potentiallyimportant clinical implications; the aim of any newanti-inflammatory therapeutic agent would be toappropriately reduce exaggerated levels of inflamma-tion in periodontitis patients and facilitate resolutionof inflammation with minimal collateral tissue damage.

Our immunocytochemical and high-throughput datasuggest increased levels of NF-kB translocatory

7000

6000

5000

4000

3000pg p

er m

l

2000

1000

0

Key:No a-lipoate/ un-stimulated

a-lipoate/ un-stimulated

F. nucleatum stimulated

F. nucleatum stimulated+a-lipoate

P. gingivalis stimulated

P. gingivalis stimulated+a-lopoate

Figure 6. Secreted IL-8 levels (pg/ml) as determined by ELISA in H400 cell cultures pre-incubated with 0.5 mM a-lipoic acid for 24 h and

exposed to P. gingivalis (1 h), F. nucleatum (1 h) or media alone. Combinations of culture exposure conditions are shown in the key. Mean� SD.





activation with P. gingivalis compared with F. nuclea-tum stimulation. Thus, the finding of significantlyhigher epithelial secretion of IL-8 after F. nucleatumstimulation compared with P. gingivalis stimulationappears contrary to the NF-kB data. The possible rea-sons for this disparity in data are unclear; however, it ispossible that: (i) NF-kB activation does not directlycorrelate with IL-8 levels, for example levels of acti-vated transcripts may be significantly induced, butthis may not translate to increased protein levels; and(ii) the kinetics of activation of NF-kB likely influenceIL-8 levels, which were measured 24 h after bacterialchallenge. There is little published data on the kineticsof induction of NF-kB over a 24 h time period. It isclear from our data that a different temporal activityprofile exists for NF-kB activity when stimulated by thetwo bacteria, with nuclear translocation being highestfor P. gingivalis at 60min but lower than F. nucleatumat 90min and 120min after. Such differences in tem-poral profile may explain why higher IL-8 levels aredetected following F. nucleatum stimulation. In add-ition, while our data and that of others indicate thatnuclear translocation of NF-kB peaks at about 1 h afterstimulation and is recycled by 4 h, it is not clear as towhen nuclear translocation can be re-stimulated. Itmay, therefore, be feasible that different NF-kB activa-tion profiles exist for F. nucleatum and P. gingivalisstimulation within the 24-h time period, which mayalso explain the lack of correlation between 60-minNF-kB nuclear translocation and IL-8 levels deter-mined at 24 h.

ALA has been used widely for managing a range ofchronic inflammatory diseases, including those whichassociate with periodontitis, such as diabetes;35 how-ever, its potential in managing periodontal disease hasnot been proposed previously. This study investigatedthe effects of this natural di-thiol on periodontal patho-gen-stimulated oral epithelium in terms of NF-kB acti-vation, gene expression changes and cytokineproduction, utilising a previously well-characterisedepithelial cell culture system.8,16 Data indicated theability of periodontal bacteria (F. nucleatum and P.gingivalis) to stimulate a pro-inflammatory phenotypewhich was subsequently modified by pre-incubation ofH400 cells with ALA. These data suggest that ALAsupplementation, either via local or systemic delivery,may provide utility in managing periodontitis patients.

Funding

This research was part-funded by the Oral and Dental

Research Trust.

Acknowledgements

The authors would like to thank Imagen Biotech,Manchester, UK, for the high content analysis and

Professor Stephen Prime (University of Bristol, UK) for hiskind donation of the H400 cell line.

References

1. Page RC and Kornman KS. The pathogenesis of human peri-

odontitis: an introduction. Periodontol 2000 1997; 14: 9–11.

2. Matthews JB, Wright HJ, Roberts A, Cooper PR and Chapple

ILC. Hyper-active and reactive peripheral blood neutrophils in

chronic periodontitis. Clin Exp Immunol 2006; 147: 255–264.

3. Socransky SS, Haffajee AD, Cugini MA, et al. Microbial com-

plexes in subgingival plaque. J Clin Periodontol 1998; 25:

134–144.

4. Kolenbrander PE, Parrish KD, Andersen RN and Greenberg EP.

Intergeneric coaggregation of oral Treponema spp. with

Fusobacterium spp. and intrageneric coaggregation among

Fusobacterium spp. Infection & Immunity 1995; 63: 4584–4588.

5. Singh A, Wyant T, Anaya-Bergman C, et al. The capsule of

Porphyromonas gingivalis leads to a reduction in the host inflam-

matory response, evasion of phagocytosis, and increase in viru-

lence. Infect Immun 2011; 79: 4533–4542.

6. Chapple IL, Brock GR, Milward MR, et al. Compromised GCF

total antioxidant capacity in periodontitis: cause or effect? J Clin

Periodontol 2007; 34: 103–110.

7. Kusumoto Y, Hirano H, Saitoh K, et al. Human gingival epithe-

lial cells produce chemotactic factors interleukin-8 and monocyte

chemoattractant protein-1 after stimulation with Porphyromonas

gingivalis via toll-like receptor 2. J Periodontol 2004; 75: 370–379.

8. Milward MR, Chapple IL, Wright HJ, et al. Differential activa-

tion of NF-kappaB and gene expression in oral epithelial cells by

periodontal pathogens. Clin Exp Immunol 2007; 148: 307–324.

9. Ambili R, Santhi WS and Janam P. Expression of activated tran-

scription factor nuclear factor-kappaB in periodontally diseased

tissues. J Periodontol 2005; 76: 1148–1153.

10. Packer L, Witt EH and Tritschler HJ. Alpha-lipoic acid as a

biological antioxidant. Free Radic Biol Med 1995; 19: 227–250.

11. Matsugo S, Konishi T and Matsuo D. Re-evaluation of super-

oxide scavenging activity of dihydrolipoic acid and its analogues

by chemiluminescent method using 2-methyl-6-[p-methoxyphe-

nyl]-3,7-dihydroimidazo-[1,2-a]pyrazine-3-one (MCLA) as a

superoxide probe. Biochem Biophys Res Commun 1996; 227:

216–220.

12. Forman HJ, Zhang H and Rinna A. Glutathione: overview of its

protective roles, measurement, and biosynthesis. Mol Aspects

Med 2009; 30: 1–12.

13. Seo JY, Kim H and Kim KH. Transcriptional regulation by thiol

compounds in Helicobacter pylori-induced interleukin-8 produc-

tion in human gastric epithelial cells. Ann NY Acad Sci 2002; 973:

541–545.

14. Midaoui AE, Elimadi A, Wu L, et al. Lipoic acid prevents hyper-

tension, hyperglycemia, and the increase in heart mitochondrial

superoxide production. Am J Hypertens 2003; 16: 173–179.

15. Packer L, Kraemer K and Rimbach G. Molecular aspects of

lipoic acid in the prevention of diabetes complications.

Nutrition 2001; 17: 888–895.

16. Prime SS, Nixon SVR, Crane LJ, et al. The behaviour of human

oral squamous cell carcinoma in cell culture. J Path 1990; 160:

259–269.

17. Dierickx K, Pauwels M, Van Eldere J, et al. Viability of cultured

periodontal pocket epithelium cells and Porphyromonas gingivalis

association. J Clin Periodontol 2002; 29: 987–996.

18. Suzuki YJ, Mizuno M, Tritschler HJ and Packer L. Redox regu-

lation of NF-kappa B DNA binding activity by dihydrolipoate.

Biochem Mol Biol Int 1995; 36: 241–246.

19. Zang W and Frei B. A-Lipoic acid inhibits TNF-a-inducedNF-kB activation and adhesion molecule expression in human

aortic endothelial cells. FASEB J 2001; 15: 2423–2432.

20. Rodrigues A, Queiroz DB, Honda L, et al. Activation of toll-like

receptor 4 (TLR4) by in vivo and in vitro exposure of rat epi-

didymis to lipopolysaccharide from Escherichia coli. Biol Reprod

2008; 79: 1135–1147.

Milward et al. 11




21. Guo Q, Tirosh O and Packer L. Inhibitory effect of alpha-lipoic

acid and its positively charged amide analogue on nitric oxide

production in RAW 264.7 macrophages. Biochem Pharmacol

2001; 61: 547–554.

22. Van Dyke TE. The management of inflammation in periodontal

disease. J Periodontol 2008; 79: 1601–1608.

23. Chapple IL and Matthews JB. The role of reactive oxygen and

antioxidant species in periodontal tissue destruction. Periodontol

2000 2007; 43: 160–232.

24. Ryan KA, Smith Jr MF, Sanders MK and Ernst PB. Reactive

oxygen and nitrogen species differentially regulate Toll-like

receptor 4-mediated activation of NF-kappa B and interleukin-

8 expression. Infect Immun 2004; 72: 2123–2130.

25. Fuchs J and Milbradt R. Antioxidant inhibition of skin

inflammation induced by reactive oxidants: evaluation of the

redox couple dihydrolipoate/lipoate. Skin Pharmacol 1994; 7:

278–284.

26. Larghero P, Vene R, Minghelli S, et al. Biological assays and

genomic analysis reveal lipoic acid modulation of endothelial

cell behavior and gene expression. Carcinogenesis 2007; 28:

1008–1020.

27. Cao Z, Tsang M, Zhao H and Li Y. Induction of endogenous

antioxidants and phase 2 enzymes by alpha-lipoic acid in rat

cardiac H9C2 cells: protection against oxidative injury.

Biochem Biophys Res Commun 2003; 310: 979–985.

28. Kiemer AK, Muller C and Vollmar AM. Inhibition of LPS-

induced nitric oxide and TNF-alpha production by alpha-lipoic

acid in rat Kupffer cells and in RAW 264.7 murine macrophages.

Immunol Cell Biol 2002; 80: 550–557.

29. Handleman GJ, Han D, Tritschler H and Packer L. Alpha-lipoic

acid reduction by mammalian cells to the dithiol form, and

release into the culture medium. Biochem Pharmacol 1994; 47:

1725–1730.

30. Chapple ILC, Mason GI, Garner I, et al. Enhanced chemilumin-

escence assay for measuring total antioxidant capacity of serum,

saliva and crevicular fluid. Ann Clin Biochem 1997; 34: 412–421.

31. Maki JM, Sormunen R, Lippo S, et al. Lysyl oxidase is essential

for normal development and function of the respiratory system

and for the integrity of elastic and collagen fibers in various tis-

sues. Am J Pathol 2005; 167: 927–936.

32. Shay KP, Moreau RF, Smith EJ, et al. Alpha-lipoic

acid as a dietary supplement: molecular mechanisms and

therapeutic potential. Biochim Biophys Acta 2009; 1790:

1149–1160.

33. Karakoyun B, Yuksel M, Ercan F, et al. Alpha-lipoic acid

improves acetic acid-induced gastric ulcer healing in rats.

Inflammation 2009; 32: 37–46.

34. Nemeth J, Stein I, Haag D, et al. S100A8 and S100A9 are novel

nuclear factor kappa B target genes during malignant progression

of murine and human liver carcinogenesis. Hepatology 2009; 50:

1251–1262.

35. Vega-Lopez S, Devaraj S and Jialal I. Oxidative stress and anti-

oxidant supplementation in the management of diabetic cardio-

vascular disease. J Investig Med 2004; 52: 24–32.




Micronutrient modulation of NF-κB in oral keratinocytes exposed to periodontal bacteria

Documents

Transcript of Micronutrient modulation of NF-κB in oral keratinocytes exposed to periodontal bacteria