Fusobacterium nucleatum and human beta defe nsins...

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Fusobacterium nucleatum and human beta defensins modulate the 1

release of antimicrobial chemokine CCL20/Mip-3α 2

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Santosh K. Ghosh, Sanhita Gupta, Bin Jiang and Aaron Weinberg* 5

Department of Biological Sciences, School of Dental Medicine, Case Western Reserve 6

University, Cleveland, OH 44106 7

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Running title: Fusobacterium nucleatum modulate the release of CCL20 10

Key words: Fusobacterium nucleatum, CCL20, Mip-3α, Human beta defensins, Oral 11

epithelial cells. 12

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*Correspondence: 16

Case Western Reserve School of Dental Medicine 17

2124 Cornell Road 18

Cleveland, OH 44106-4905 19

phone: 216-368-6729 20

fax: 216-368-0145 21

E-mail: [email protected] 22

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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.05586-11 IAI Accepts, published online ahead of print on 12 September 2011

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ABSTRACT: Cells of the innate immune system regulate immune responses through the 24

production of antimicrobial peptides, chemokines and cytokines, including human beta 25

defensins (hBDs) and CCL20. In this study, we examined the kinetics of primary human 26

oral epithelial cells’ (HOECs) production of CCL20 and hBDs, in response to 27

Fusobacterium nucleatum, a commensal bacterium of the oral cavity, which we previously 28

showed promotes HOEC induction of hBDs. HOECs secrete maximal levels of CCL20 at 29

6h, following stimulation by F. nucleatum cell wall (FnCW). Kinetics of CCL20 release is 30

distinct from that of hBD-2 and -3, which peaks after 24h and 48h FnCW stimulation, 31

respectively. FnCW induced release of CCL20 by HOECs requires both transcriptional 32

and translational activation. Release of CCL20 by HOECs is inhibited by brefeldin A, 33

suggesting that it is secreted through a vesicle transport pathway. Other epithelial 34

derived agents that FnCW induces, such as hBD-2, hBD-3, TNF-α and IL-1β, are also 35

able to release CCL20. By focusing on MAPKs, we show that both ERK1/2 and p38, but 36

not JNK, are required for hBDs, TNF-α and IL-1β induced secretion of CCL20 by 37

HOECs. The ability of FnCW and its induced hBDs, to produce proinflammatory 38

cytokines and CCL20, suggests the broad role of F. nucleatum and human antimicrobial 39

peptides in primary immune responses elicited by oral epithelium. 40

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INTRODUCTION 48

Epithelial cells are the host's first line of defense against microbial invasion and pathogenesis 49

[9, 12, 13,18]. At mucosal surfaces, including those of oral epithelia, cells have evolved as part 50

of the innate immune system, to possess antimicrobial activity and to “cross-talk” with the 51

adaptive immune system. Cells of the innate immune system regulate immune responses 52

through the production of chemokines and cytokines, including macrophage inflammatory 53

protein 3α (Mip-3α), also known as CCL20. CCL20 is a 70 amino-acid chemokine that attracts 54

immature dendritic cells and T cells via the chemokine receptor CCR6 [55], as has been 55

reported for the epithelial cell derived antimicrobial peptides (AMPs) human beta defensins 56

(hBD) 1 and -2 [60]. We and others have reported on the ability of hBDs to “cross-talk” with 57

the adaptive immune system [21,25,52] and, most recently we showed that overexpression of 58

hBD-3 is linked to oral cancer [34,35]. CCL20 has also been linked to a variety of diseases, 59

including cancer [39], rheumatoid arthritis [54] and may have an important regulatory role in 60

specific lymphocyte migration into inflamed periodontal tissues [30]. By in situ hybridization, 61

Abiko et al [2] showed that CCL20 expression in oral squamous cell carcinoma (OSCC) is 62

localized primarily at the epithelial pearls corresponding to the spinous layer. A recent study 63

[6] also suggests that CCL20 may represent a potential immunohistochemical marker for 64

prognostic prediction of OSCC. In periodontal diseased tissue, CCL20 has been shown to be 65

distributed in the basal layer of gingival epithelial cells [30]. The protein has direct 66

antibacterial, antifungal, and antiviral activities; again comparable to hBDs [11, 37, 46, 61]. 67

Therefore, CCL20, along with hBDs, may play an important role in bridging between innate 68

and adaptive immune responses. 69

Oral mucosa is in continuous contact with heterogeneous populations of diverse 70

commensal and opportunistic microorganisms and yet, for the most part, it remains healthy and 71

uninflamed. Recent research has highlighted the possibility that oral epithelium not only serves 72


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a barrier function as the first physical line of defense against colonizing pathogens, but also 73

orchestrates local immune responses [14,26]. Of the 700 species of oral bacteria estimated to 74

colonize the oral cavity [1, 20, 48], a few have demonstrated the capacity to induce epithelial 75

cell innate responses, while others have been more stealth-like [23, 40, 56]. The ubiquitous oral 76

bacterium Fusobacterium nucleatum (Fn) and its cell wall extract (FnCW) induces expression 77

of hBD-2 and -3 transcript in gingival epithelial cells (GECs) in vitro [15,32,33,41]. Recently, 78

we have identified an FnCW associated peptide that induces hBD-2 in human oral epithelial 79

cells (HOECs) [29]. 80

FnCW has been shown to induce CCL20 transcript, and indeed induction of CCL20 by 81

FnCW is highest (72.7 fold in 6h) amongst all the other cytokines/chemokines studied [62]. In 82

the present communication, we further characterized F. nucleatum induced release of CCL20 83

by human oral epithelial cells (HOECs), the kinetics of beta defensin release by HOECs upon 84

stimulation with F. nucleatum, and their effect on CCL20 release by the host cells. The effects 85

of pro-inflammatory cytokines on HOEC regulation of CCL20 are also looked into. Finally, we 86

investigated the MAP kinase pathways regulating hBD and cytokine induced secretions of 87

CCL20 by HOECs. 88

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METHODS AND MATERIALS 98

Chemicals and reagents 99

Synthetic hBD2 and hBD-3 were purchased from Peptides International [Kentucky, 100

USA]. Recombinant TNF-α and recombinant IL-1β and were purchased from Peprotech Inc 101

(NJ, USA). All the MAPK inhibitors used in this study were purchased from Calbiochem (NJ, 102

USA). Actinomycin D, brefeldin A, cyclohexamide and DMSO were purchased from Sigma 103

Aldrich (St. Louis, USA). 104

Human primary oral epithelial cells culture and stimulation 105

Healthy human gingival tissue overlying impacted third molars was obtained from oral surgery 106

clinics, in the greater Cleveland area, in accordance with the Case Western Reserve University 107

Institutional Review Board approved protocol. Primary HOECs were isolated and grown in 108

keratinocyte basal medium with supplements as previously described [10, 40, 41]. To 109

overcome interpersonal variability, epithelial cells from three donors were mixed at a ratio of 110

1:1:1 and grown to 80% confluence prior to challenge. Results were compared to HOECs 111

grown to similar confluence from single donors. To avoid the effects of changes in epithelial 112

cell physiology during growth in culture, each time point had a corresponding untreated, 113

control group. Each experimental condition was conducted in triplicate. 114

Preparation of F. nucleatum cell wall fractions. 115

Cell wall from F. nucleatum was prepared as described previously [36]. Briefly, F. nucleatum 116

(ATCC 25586) was grown anaerobically in Columbia broth (BD Biosciences) overnight. 117

Crude cell wall preparations were prepared by French pressure cell disruption of freshly 118

harvested whole cells in phosphate-buffered saline (PBS; pH 7.2) at 15,000 pounds/inch2. The 119

cell walls were recovered after low speed centrifugation (1,000 x g, 15 min), followed by high 120

speed centrifugation (138,000 x g, 30 min) of the supernatant. 121

Quantification of CCL20 in cell free supernatant and in cell lysates. 122


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CCL20 was quantified in culture supernatants and in cell lysates by sandwich ELISA using 123

antibody pairs from R&D Systems (MN, USA) following the vendor’s instructions. CCL20 124

concentration was calculated from standard curves using recombinant human CCL20 (R&D 125

Systems, MN). The detection threshold was 250 pg/ml of CCL20. 126

Detection of hBD-2 and hBD-3 peptides in supernatant. 127

Human beta-defensins were measured in supernatants from FnCW stimulated HOECs 128

following our previously published protocol [27, 29]. 129

RNA preparation and PCR. 130

Cells were lysed using Trizol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated in 131

accordance with the manufacturer’s instructions. RNA concentration was measured by UV 132

absorbance using the Nanodrop 1000 spectrophotometer (Thermo Fisher Scientic inc., USA). 133

RT PCR was done with the Superscript III One Step RT PCR system (Invitrogen,USA) 134

according to the manufacturer’s protocol and the amplified product was analyzed on a 1.5% 135

agarose gel. Real-time PCR was done using the BIO-RAD (Hercules, CA) MyIQ system with 136

iScript cDNA Synthesis Kit and iQ SYBR Green Supermix according to the manufacturer’s 137

protocol. The results were analyzed to calculate fold induction of CCL20 with human keratin 5 138

(HK5) as the internal control. The following primers were used to amplify CCL20 and HK-5: 139

For CCL20 5’-AATTTATTGTGGGCTTCACACG-3’, 5’-140

ACCCAAGTCTGTTTTGGATTTG-3’ and for HK5 5’-GTC CTC TCC ATG GAC AAC 141

AAC- 3’, 5’-TGT CAA TCT CGG CTC TCA GCC-3’. 142

PathScan® MAP Kinase Multi-Target Sandwich ELISA. 143

Mixed donor (N=3) HOECs were treated by adding fresh media containing the regulator for 144

the desired time, rinsed with ice-cold PBS and lysed with ice-cold 1X lysis buffer (Cell 145

Signaling, Danvers, MA) plus 1mM phenylmethylsulfonyl fluoride (PMSF) for 10 min on ice. 146

Cells were scraped off the plate, transferred to a microfuse tube, sonicated on ice and clarified 147


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by centrifugation (13,000 rpm, 4°C, 15 min). The PathScan® Multi-Target (phospho-p38, 148

phospho-ERK1/2, phospho-MEK1/2, phospho-SAPK/JNK and MEK1) ELISA was then 149

performed following the vendor’s (Cell signaling, MA) instruction on the clarified supernatant. 150

Statistical analysis 151

Statistical analyses were performed using either unpaired Student’s t-test or ANOVA test. The 152

differences were considered significant when the probability value was less than 5% (P < 0.05). 153

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RESULTS 173

F. nucleatum induces release of CCL20 by HOECs. 174

To determine baseline release of CCL20 by unstimulated HOECs, cell free supernatants from 175

epithelial cell cultures were collected at different time intervals and CCL20 concentration was 176

quantified by ELISA. Figure 1A demonstrates release of CCL20 protein by HOECs as early as 177

1 hr after the addition of fresh media and increases gradually with time. To examine the release 178

of CCL20 protein upon stimulation with F. nucleatum, HOECs were treated with F. nucleatum 179

cell wall (FnCW; 10 µg/ml) for 1–48h. Time course studies demonstrated a significant increase 180

in CCL20 release at 4h following F. nucleatum stimulation, with a plateauing of CCL20 181

release after 6 hr of stimulation [Fig. 1A]. We observed that the fold increase in secreted 182

CCL20 from FnCW treated cells, when compared to untreated quiescent cells, reached its 183

maximum by 6 hrs post treatment [Fig. 1B, derived from Fig 1A], and that interpersonal 184

variability was demonstrated in HOECs from different donors towards release of CCL20 upon 185

F. nucleatum challenge; i.e., from 2.8 to 8.5 fold above baseline (N=5; variation 186

coefficient, %CV= 37) [Fig. 1C] 187

CCL20 release requires rapid de novo generation and is inhibited by brefeldin A. 188

To determine if CCL20 is synthesized by HOECs in response to FnCW, near confluent HOEC 189

cultures were pretreated with actinomycin D (10μg/ml) or cyclohexamide (10μg/ml) for 5 min. 190

HOECs were then cultured for an additional 6 h with FnCW, as that was shown to be the 191

optimal time for CCL20 release (Fig. 1A, B). Controls included HOECs with media alone (or 192

DMSO in media), with FnCW alone, or with either actinomycin D or cyclohexamide alone. 193

After 6 h of exposure, media were collected and analyzed for the presence of CCL20. As 194

shown in Figure 2A, FnCW is unable to induce the release of CCL20 by HOECs in the 195

presence of actinomycin D or cyclohexamide, indicating that the release of CCL20 by FnCW 196

is transcriptionally and translationally regulated. To examine the effect of brefeldin A (BFA), a 197


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protein release inhibitor, on FnCW induced release of CCL20 by HOECs, near confluent 198

HOECs were treated with BFA (10 μg/ml) for 5 min followed by FnCW (10 μg/ml) for 6h, or 199

with either agent alone for up to 6h; supernatant and pellet samples were collected and CCL20 200

levels were measured by ELISA. BFA inhibited CCL20 release from HOECs in the presence 201

of FnCW stimulation [Fig. 2B]. In contrast, BFA alone or in combination with FnCW caused 202

an increase in intracellular CCL20 [Fig. 2C]. Comparison of CCL20 levels in supernatants and 203

in cell pellets indicates that the CCL20 is rapidly released upon synthesis. 204

Release of beta defensins in HOECs by FnCW. 205

Cell free supernatants from FnCW (10μg/ml) treated HOECs were collected at different time 206

points (1 – 72 hr) and hBD-2 and -3 peptide concentrations were quantified by ELISA. Figures 207

3A and 3B show the kinetics of hBD-2 and -3 released from untreated vs. FnCW treated 208

HOECs over the designated time periods, respectively. When compared to the untreated cells, 209

hBD-2 fold increases of 1.1, 7.5, 13.5, 3.9 and 2.7 were observed at 4, 18, 24, 48 and 72 h, 210

respectively [Fig. 3C]. Fold increases for hBD-3 [Fig. 3E] were 1.1, 1.4, 2.3, 2.9 and 2.7 at the 211

same respective time points as for hBD-2. Interpersonal variability was evident for hBD-2 and 212

-3 release from HOECs induced by FnCW for 24h [Fig. 3D and 3F; N=5 donors; variation 213

coefficient, % CV=38.5 and 27.5, for hBD-2 and hBD-3, respectively]. 214

In contrast to hBD-2 and -3, we observed no differences in the release of hBD-1 215

between FnCW stimulated and unstimulated control cells (data not shown), supporting the 216

constitutive nature of hBD-1 expression in HOECs. However, linear accumulation of hBD-1 in 217

both stimulated and unstimulated culture media over time was observed with R2 values 0.94 218

and 0.97, respectively. 219

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Induction of CCL20 by beta defensins (-2 and-3). 222

Since hBD-2 (2μg/ml) has been shown to induce CCL20 transcript in primary oral epithelial 223

cells [62], we looked for hBD-2 induced CCL20 release by HOECs. HOECs were incubated 224

with synthetic hBD-2 (0.1 to 10 μg/ml) for 6 hr. An hBD-2 dose dependent release of CCL20 225

was evidenced, with the maximum release noted with 5 μg/ml hBD-2 [Fig. 4A]. Moreover, 226

CCL20 was seen to increase in the spent supernatant over time (1-24 hrs) when hBD-2 was 227

added to HOECs at the optimal concentration of 5 μg/ml [Fig. 4B]; a kinetic profile different 228

from that seen with FnCW (Fig. 1A). To investigate the inductive capacity of hBD-3 to 229

promote CCL20 expression and subsequent release from HOECs, we incubated near confluent 230

HOECs with increasing concentrations of hBD-3 (0.1 to 10 µg) for 6 hrs, followed by isolation 231

of total RNA and amplification of CCL20 mRNA by PCR and quantification of released 232

CCL20 by ELISA, respectively. The increase in CCL20 mRNA expression was also 233

determined by real time PCR. There was a direct correlation between the increase in CCL20 234

transcript expression [Fig. 5B] and protein release from HOECs with hBD-3 peptide 235

concentrations used over the 6 hr period [Fig. 5C]. CCL20 was seen to also increase in the 236

spent supernatant over time (1-24 hrs) when hBD-3 was added to HOECs at the optimal 237

concentration of 5 μg/ml, as seen with hBD-2, but different from that seen with FnCW (Fig. 238

1A). Interestingly, hBD-3 induced higher levels of released CCL20 than did hBD-2 (compare 239

Fig. 4B and 5D) and there appeared to be an approximately 6 fold increase in CCL20 in the 240

spent supernatant induced by hBD-3 at around 18 hrs of incubation (Fig. 5D). 241

No lactate dehydrogenase activity was detected in the supernatants after stimulation 242

with hBD-2 or hBD-3 (0.1 to 10 μg/ml) for 24 hrs (data not shown), confirming that cellular 243

viability was not impaired by hBD-2 or -3 at the concentration range used and time periods 244

studied. 245


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Cytokines (IL-1β, TNF-α) induce secretion of CCL20 by HOECs. 246

We and others have previously shown that FnCW induces TNF-α and IL-1β transcript 247

expression in HOECs [41,62], and that these two cytokines have been shown to induce 248

CCL20 release from several epithelial cell lines [8,49,51]. Here, we looked to see if these 249

cytokines could induce the release of CCL20 from HOECs. We incubated HOEC monolayers 250

with 10 ng/ml of either IL-1β or TNF-α for three different time periods (6, 18 and 24 hrs), 251

followed by ELISA analysis. We observed that both cytokines are capable of releasing 252

increased amounts of CCL20 compared to the untreated control [Fig. 6]. When cells were 253

stimulated with a combination of TNF-α and IL1β, an additive effect was observed in the 254

release of CCL20. Since F. nucleatum and hBD induced CCL20 production is a relatively early 255

event, it is unlikely that the production of intermediary cytokines are involved in F. nucleatum 256

or hBD induced release of CCL20. We corroborated this by using antibodies against TNF-α 257

and IL1β in our assay system, and saw that FnCW and hBDs, respectively, were not prevented 258

from inducing CCL20 in HOECs (data not shown). 259

Effect of MAP kinase inhibitors on defensin and cytokine induced release of CCL20 by 260

HOECs. 261

Different MAP kinases exist and their impact on CCL20 production in each cell line is 262

different [31, 43,51,57]. Hence, the signaling pathway involved in CCL20 production in 263

HOECs needs further investigation. Since it was reported that beta defensins may activate 264

MAPK pathways [47], we used inhibitors of these pathways to see whether any are involved in 265

hBD induced release of CCL20. HOEC monolayers were pretreated for 1 hr with different 266

inhibitors before 18 hr incubation with stimulants. As shown in Fig. 7A and 7B, ERK1/2 267

activation inhibitors [UO126 (20μM) or PD98059 (20μM)] were effective in suppressing beta-268

defensin (hBD-2 or hBD-3) induced release of CCL20 by HOECs. The specific p38 mitogen 269


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kinase inhibitor, SB203580 (20μM), also had a modest inhibitory effect on defensin induced 270

CCL20 release. In contrast, the JNK specific inhibitor, SP600125 (20μM), had no inhibitory 271

effect on defensin induced CCL20 release. Similar experiments were conducted to assess the 272

importance of MAPK in TNF-α or IL-1β induced release of CCL20 by HOECs. We found that, 273

like beta defensins, these cytokines induce CCL20 via ERK1/2 and p38 but not via JNK [Fig. 274

7C and 7D]. Interestingly, all four MAPK inhibitors showed little or no effect on FnCW 275

induced CCL20 release by HOECs (data not shown), suggesting that the signaling pathway(s) 276

involved for FnCW induced secretion of CCL20 is/are different from defensins and cytokine 277

induced release of CCL20 by HOECs. 278

Since the inhibitor studies showed that beta defensins ( hBD-2 or hBD-3) and cytokines 279

(TNFα or IL-1β) induced release of CCL20 via ERK1/2 and p38 but not via JNK, we used 280

PathScan® MAP Kinase Multi-Target Sandwich ELISA to compare the levels of phospho-281

ERK1/2 (Thr202/Tyr204), phospho-MEK1/2 (Ser217/221), phospho-p38 MAPK 282

(Thr180/Tyr182) and phospho-SAPK/JNK (Thr183/Tyr185) in the cell lysates of HOECs after 283

stimulating the cells with TNFα, IL-1β, hBD-2 and hBD-3, respectively. As shown in Fig. 8, 284

phosphorylation levels of ERK1/2, MEK1/2 and p38, but not of SAPK/JNK, were enhanced in 285

HOECs following 30 or 60 min of challenge with the above mentioned stimuli. 286

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DISCUSSION 294

Commensal bacteria are usually regarded as beneficial to the host by displacing pathogens 295

from a microbial niche or providing protection by continually stimulating epithelial surfaces to 296

express and secret antimicrobial peptides (AMPs) at levels that kill opportunistic/pathogenic 297

organisms [4]. The release of antimicrobial chemokine CCL20 [51] requires stimulation for its 298

release and the discrete stimuli that lead to its up regulation may be cell specific [17, 31, 31, 299

57,58]. In these studies, we demonstrated the ability of primary oral epithelial cells to release 300

CCL20 in response to the oral commensal bacterium F. nucleatum and other epithelial derived 301

agents that it also induces; i.e., hBD-2, hBD-3, TNF-α and IL-1β. The kinetic studies of 302

CCL20 release by HOECs suggest that this is an early event subsequent to challenge by any of 303

the agents used. FnCW caused rapid release of CCL20 from HOECs; i.e., 6 hr post challenge 304

resulted in a 6 fold greater presence of CCL20 in the spent supernatant than control supernatant 305

and twice the increase of a 24 hr challenge (6 fold vs ~3 fold). This is in agreement with a 306

recent study that showed that F. nucleatum elicits rapid induction of the CCL20 gene (72.7 307

fold in 6 hr compared to 21.7 fold in 24hr [62]. Lin et al [43] also showed early release of 308

CCL20 by human mast cells in response to Pseudomonas aeruginosa, which also peaked at 6 309

hr. Adenylate and uridylate (A+U)-rich elements (AREs)-mediated mRNA turnover is an 310

important regulatory component of gene expression for innate and specific immunity [24]. The 311

transient and rapid up-regulation of CCL20 gene expression by FnCW could be due to 312

posttranscriptional regulation of mRNA stability via AREs [53,19]. Indeed, when we looked 313

for AREs within the 3’untranslated region of CCL20 mRNA, using “ARE site” 314

[http://rna.tbi.univie.ac.at/AREsite., 28], we found three ATTTA domains [Supplementary 315

Figure S1]. Another rapidly induced gene by FnCW in HOECs is CXCL5 (40.5 fold in 6h) 316

[62] and using the ARE site we could find eleven ATTTA domains within its 3’UTR 317


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[Supplementary Figure S1]. We could not find any AREs within the 3’UTR of DEFB4 318

(hBD-2) or in DEFB103 (hBD-3) and, not surprisingly, these defensins are not released as 319

rapidly as CCL20 by FnCW in HOECs. 320

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In the present study, we found that actinomycin D and cyclohexamide inhibit FnCW 322

induced release of CCL20 by HOECs, indicating that CCL20 release requires transcriptional 323

and translational activation. Brefeldin A (BFA) is a fungal metabolite able to rapidly and 324

reversibly block intracellular vesicle transport from the endoplasmic reticulum to the Golgi 325

apparatus, and therefore, inhibit protein secretion with minimal effect on protein synthesis [38]. 326

BFA blocks the release of newly synthesized proteins, but has no effect on preformed granule-327

bound proteins [45]. Although BFA has been widely used as an inhibitor of protein secretion, 328

the information concerning its effect on CCL20 release has not been reported. Our study 329

showed that BFA not only inhibited the FnCW induced release of CCL20 by HOECs, but also 330

constitutive (unstimulated) release of CCL20. The increased levels of intracellular CCL20 in 331

unstimulated and FnCW stimulated cell lysates from BFA pretreated HOECs further 332

corroborates the inhibition of CCL20 release by BFA. Given the well-characterized properties 333

of BFA, it is reasonable to conclude that CCL20 is secreted through the vesicle transport 334

pathway. 335

Certain commensal bacteria are excellent inducers of beta defensins [41,59, 22], 336

suggesting that the commensal bacterial community may act by priming innate immune 337

readiness of oral epithelia. We and others have shown that F. nucleatum can induce innate 338

response elements, such as hBDs, in HOECs [29, 32, 41, 50,]. More recently, several 339

immunoregulatory functions with the adaptive immune system have been attributed to hBDs in 340

addition to their antimicrobial capacity; i.e., their ability to cross-talk with the adaptive 341

immune system by acting as chemokines [52, 34], and by changing a cell’s phenotype, either in 342


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activation as seen with antigen presenting cells [25] or antagonism, as seen with T cells [21]. 343

These findings conform to reports in the literature that innate responses usually precede, and 344

are necessary for, the establishment of adaptive immunity [44]. Upon stimulation by 345

antimicrobial peptides, different immune and inflammatory cells have been shown to produce 346

cytokines and/or chemokines. For example, the α-defensins, human neutrophil peptides-1 and -347

3 stimulate the production of IL-1, -4, -6, TNF-α, and IFN-γ in monocytes [5]. Moreover, 348

hBDs and LL-37 have been reported to enhance the generation of IL-18 in keratinocytes [47]. 349

In this study, we demonstrated that the inducible defensins, hBD-2 and hBD-3, can induce the 350

release of other AMPs/ chemokines, i.e., CCL20, by HOECs. Therefore, the ability of hBDs to 351

promote the release of the antimicrobial chemokine CCL20 suggests the broad role of human 352

antimicrobial peptides in primary immune responses by oral epithelial cells. This regulatory 353

loop could act in a complimentary manner to provide added protection against microbial 354

assault. 355

To gain initial insight into the cellular pathways through which defensins induce 356

CCL20 release by HOECs, we focused on three major downstream MAPK cascades: ERK, 357

p38 and JNK. In cells, these kinase cascades act as signal sorters for a variety of upstream 358

signals before entering the nucleus. The p38, ERK, and JNK kinase cascades have been shown 359

to be involved in a large variety of cellular activities [3, 7]. Defensins have been shown to 360

activate primary human keratinocytes through p38 and ERK1/2 [47]. By focusing on these 361

three MAPKs, we demonstrated that both ERK1/2 and p38, but not JNK are required for hBD 362

induced CCL20 secretion by HOECs. The involvement of similar MAPKs was also observed 363

in the release of CCL20 by TNF-α or IL-1β stimulated oral epithelial cells. The p38 mitogen-364

activated protein kinase (MAPK) pathway plays an important role in the post-transcriptional 365

regulation of inflammatory genes and has been found to regulate both the translation and the 366


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stability of inflammatory mRNAs. The mRNAs regulated by p38 are known to have AU-rich 367

elements (ARE) present in their 3’-untranslated regions [ 16,24] and thus, the presence of ARE 368

within the 3’UTR of CCL20 supports the involvement of p38 in it’s induction by defensins and 369

cytokines. Surprisingly, ERK1/2 and p38 inhibitors were found to have a negligible effect on 370

FnCW induced release of CCL20 by HOECs. Most recently, Yin and Chung [63] found the 371

lack of involvement of p38 in F. nucleatum induced CCL20 mRNA induction. At this stage, 372

however, it is not clear why, in contrast to induction of CCL20 by defensins and cytokines, the 373

induction of CCL20 by FnCW does not occur via p38, despite the fact that FnCW is capable of 374

causing rapid and transient activation of p38 MAPK [42] in HOECs. Nevertheless, these 375

observations further corroborate the fact that the induction of CCL20 by FnCW is not 376

dependent on defensins (hBD-2 and-3), TNF-α or IL-1β. 377

Kinetics of FnCW induced CCL20 and β-defensin release are distinct; i.e., CCL20 378

release peaks at 4 to 6 h [ Fig. 1B] by FnCW, while peak time is 24 h for hBD-2 [Fig 3C] and 379

48 h for hBD-3 [ Fig 3D]. Thus, coordinated stimulation of CCL20 and β-defensins by FnCW 380

on oral epithelial cells, might suggest a complementary activity in exerting the innate nature of 381

F. nucleatum in host defense. Since FnCW is a complex mixture of proteins, it is tempting to 382

speculate that different components within the FnCW fraction could be responsible for the 383

release of β-defensins and CCL20. We have recently identified a fusobacterium associated β-384

defensin inducer peptide (FAD-I), within FnCW, which is responsible for the induction of 385

hBD-2 in HOECs [29]. When we tested FAD-I’s ability to induce CCL20 release by HOECs, 386

we found it to be negligible when compared to FnCW [Fig. 9A], while its ability to induce 387

hBD-2 release was comparable to that of FnCW [Fig. 9B]. However, an isoelecric focusing 388

(IEF) fraction of crude FnCW, from which FAD-I was further isolated [29], is capable of 389

inducing CCL20 release by HOECs [Fig. 9A], indicating that the CCL20 inducing factor(s) 390


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is/are present in the IEF fraction. Results from Fig. 9 A, B appear to suggest this. Although the 391

IEF fraction is a semi purified fraction from crude FnCW, the phosphorylation pattern of 392

different MAPKs elicited by the IEF fraction is similar to that elicited by crude FnCW [Fig. 393

10], and not surprisingly, the effectiveness of the IEF fraction in inducing CCL20 or hBD-2 is 394

close to that of FnCW. Isolating the CCL20 inducing factor, in conjunction with FAD-I may 395

one day be used to bolster the innate defenses of vulnerable mucosae. 396

Acknowledgement: We thank Drs. J. R. Blakemore, E. K. Schneider, W. S. Blood and F. 397

Faddoul for providing normal human oral tissue. We appreciate helpful discussions with Drs. 398

Zhimin Feng, Ge Jin and Jennifer Greene, Department of Biological Sciences. This work was 399

supported by National Institutes of Health Grants R01DE016334 (AW) and RO1DE018276 400

(AW.). 401

402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430


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Figure Legends: 633

Fig. 1: Kinetics of FnCW induced release of CCL20 by HOECs: [A] HOECs (mixed 634

donors; N=3) were challenged with FnCW (10 μg/ml) for indicated time periods and the 635

amount of CCL20 in cell free supernatants were measured by ELISA. (Results are the mean 636

[+SD] of three independent experiments.) [B] Fold changes in CCL20 release by HOECs 637

(mixed donors; N=3) after challenge with FnCW (10μg/ml) for indicated time periods. (Results 638

are the mean [+SD] of three independent experiments) [C] Interpersonal variability in CCL20 639

release by HOECs from five different donors after challenge with FnCW (10 μg/ml) for 6hrs. 640

(Results are the mean [+SD] of three independent ELISA measurements) 641

642 Fig. 2: [A] Effect of actinomycin D (Act D) and cyclohexamide (CHX) on FnCW induced 643

release of CCL20 by HOECs: HOEC monolayers (mixed donor; N=3) were pretreated with 644

actinomycin D or cyclohexamide for 5 min and then incubated with or without FnCW (10 645

μg/ml) for an additional 6 h. As a negative control, cells were incubated with media or media 646

with vehicle control (DMSO) for actinomycin D. CCL20 levels in cell free supernatants were 647

measured by ELISA. [B] and [C] Effect of brefeldin A (BFA) on CCL20 release by HOECs. 648

HOEC monolayers (mixed donor; N=3) were pretreated with BFA (10 μg/ml) for 5 min, 649

followed by incubation with or without FnCW (10 μg/ml) for an additional 6 h. As a negative 650

control, cells were incubated with media alone or media with vehicle control (ethanol) for BFA. 651

CCL20 levels in cell free supernatants [B] and cell lysates [C] were measured by ELISA. 652

Results are the mean (+ SD) of three independent experiments (* p<0.05). For comparison of 653

CCL20 content in supernatants and in cell pellets the levels of CCL20 is expressed as ng/well 654

instead of ng/ml. One ml of media per well was used in a six well format. 655

656


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Fig. 3: Kinetics of FnCW induced release of hBD-2 and hBD-3 by HOECs: [A] and [B], 657

HOEC monolayers (mixed donor; N=3) were challenged with FnCW (10 μg/ml) for the 658

indicated time periods, and the levels of hBD-2 and hBD-3 in cell free supernatants were 659

determined by ELISA. . (Results are the mean [+SD] of three independent experiments) [C] 660

and [E] Fold changes (calculated from figures A and B) in hBD-2 and -3 release by HOECs 661

(mixed donor; N=3) after FnCW challenge for indicated time periods. (Results are the mean 662

[+SD] of three independent experiments) [D] and [F] Interpersonal variability in hBD-2 and-3 663

release by HOECs from five different donors after FnCW treatment for 24 hrs. (Results are the 664

mean [+SD] of three independent ELISA measurements) 665

Fig. 4: HBD-2 induced release of CCL20 by HOECs: [A] HOEC monolayers (mixed donor; 666

N=3) were challenged with hBD-2 (0.1 to 10 μg/ml) for 6 hrs; released CCL20 in cell-free 667

supernatants was determined by ELISA, followed by calculation of fold increase above 668

baseline. [B] HOEC monolayers (mixed donor; N=3) were challenged with hBD-2 (5 μg/ml) 669

for indicated time periods and the amount of released CCL20 in cell-free supernatants was 670

determined by ELISA. Results are the mean (+ SD) of three independent experiments [* 671

indicates p<0.05 with respect to control (no treatment)] 672

673 Fig. 5: HBD-3 induced up regulation of CCL20 transcript in HOECs ([A] and [B]). HOEC 674

monolayers (mixed donors; N=3) were treated with indicated amounts of synthetic hBD-3 for 6 675

hrs. Total RNAs from both treated and untreated cells were isolated, the hBD-3 transcripts 676

were amplified by RT-PCR [A] and the fold changes were quantified by real-time PCR [B]. 677

HK5, human keratin 5 has been used as housekeeping gene. HBD-3 induced release of 678

CCL20 by HOECs [C]: HOEC monolayers (mixed donors; N=3) were treated with indicated 679

amounts of synthetic hBD-3 for 6 hrs and the levels of CCL20 in the cell free supernatants 680

were determined by ELISA, followed by fold change determinations. Kinetics of hBD-3 681


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induced CCL20 release by HOECs [D]. HOEC monolayers (mixed donors; N=3) were 682

treated with hBD-3 (5μg/ml) for indicated time periods and release of CCL20 in cell free 683

supernatants for both treated and untreated cells was determined by ELISA. Results for B-D 684

are the mean (+ SD) of three independent experiments [* = p<0.05]. 685

686 Fig. 6: TNF-α and IL-1β induced release of CCL20 by HOECs. HOEC monolayers 687

(mixed donors; N=3) were separately treated with TNF-α (10 ng/ml), IL-1β (10 ng/ml), TNF-688

α+ IL-1β (5 ng/ml each) or media alone for indicated time periods, and the levels of released 689

CCL20 in the spent supernatants were determined by ELISA. Results are the mean (+ SD) of 690

three independent experiments. [* indicates p<0.05 with respect to control (no treatment)] 691

Fig. 7: Inhibition of ERK, p38 and JNK in hBD-2, hBD-3, TNF-α and IL-1β induced 692

release of CCL20 by HOECs. HOEC monolayers (mixed donor, N=3) were pretreated with 693

inhibitors of ERK [UO126 (20μM) or PD98059 (20μM)], p38 [SB203580 (20μM)] or JNK 694

specific inhibitors [SP600125 (20μM)], respectively, for 1 hr, followed by treatment with [A] 695

hBD-2 (5 μg/ml) or [B] hBD-3 (5 μg/ml) or [C] TNFα (10 μg/ml) or [D] IL1β (10 μg/ml) 696

for an additional 18 hrs. DMSO was used as the negative control. CCL20 levels in the spent 697

supernatants were measure by ELISA. Results are the mean (+ SD) of three independent 698

experiments. (* = p<0.05) 699

Fig. 8: PathScan® MAP Kinase Multi-Target Sandwich ELISA: HOEC monolayers 700

(mixed donor, N=3) were treated with either TNFα (10 ng/ml), IL-1β (10 ng/ml), hBD-2 (5 701

μg/ml) or hBD-3 (5 μg/ml) for 0, 30 and 60 minutes, respectively. The cell lysates were 702

analyzed for the indicated target, using PathScan® MAP Kinase Multi-Target Sandwich 703

ELISA kit (Cell Signaling, MA) according to the vendor’s instructions. 704


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Fig. 9: Comparison of FnCW, isoelectric focusing (IEF) fraction of FnCW and 705

recombinant FAD-I induced CCL20 and hBD-2 release. HOECs (mixed donor, n=3) were 706

treated with media alone, FnCW (10 μg/ml), IEF fraction (10 μg/ml) or recombinant FAD-I 707

(rFAD-I) (10 μg/ml) for indicated time periods. The levels of CCL20 [A] and hBD-2 [B] in 708

cell free supernatants were measured by ELISA. The IEF fraction and rFAD-I were prepared as 709

described by Gupta et al [29]. (* = p<0.05 and NS= none significant) 710

Fig. 10: PathScan® MAP Kinase Multi-Target Sandwich ELISA: HOEC monolayers 711

(mixed donor, N=3) were treated with either IEF (10 μg/ml ) or FnCW (10 μg/ml) for 0, 30 712

and 60 minutes, respectively. The cell lysates were analyzed for the indicated target, using 713

PathScan® MAP Kinase Multi-Target Sandwich ELISA kit (Cell Signaling, MA) according to 714

the vendor’s instructions. 715

716


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