Wild Blueberry (Vaccinium angustifolium Ait.) Polyphenols Target

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Journal of Agricultural and Food Chemistry

Wild blueberry (Vaccinium angustifolium Ait.) polyphenols target Fusobacterium nucleatum and the host inflammatory response: Potential innovative molecules for treating periodontal diseases

Amel Ben Lagha†, Stéphanie Dudonné‡, Yves Desjardins‡, and Daniel Grenier†*

†

Oral Ecology Research Group, Faculty of Dentistry, Université Laval, Quebec City, QC, Canada

‡

Institute of Nutrition and Functional Foods, Université Laval, Quebec City, QC, Canada

*Corresponding author: Dr. Daniel Grenier, Oral Ecology Research Group, Faculty of Dentistry, Université Laval, 2420 rue de la Terrasse, Quebec City, QC, Canada, G1V 0A6 Telephone: 418-656-7341. Fax: 418-656-2861. Email: [email protected]

Short title: Blueberry polyphenols and periodontal diseases

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ABSTRACT

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Blueberries contain significant amounts of flavonoids to which a number of beneficial

3

health effects on humans have been associated. In the present study, we investigated the

4

effect of a polyphenol-rich lowbush blueberry (Vaccinium angustifolium Ait.) extract on

5

the two main etiologic components of periodontitis, a multifactorial disorder affecting the

6

supporting structures of the teeth. Phenolic acids, flavonoids (flavonols, anthocyanins,

7

flavan-3-ols), and procyanidins made up 16.6%, 12.9%, and 2.7% of the blueberry

8

extract, respectively. The blueberry extract showed antibacterial activity (MIC = 1

9

mg/mL) against the periodontopathogenic bacterium Fusobacterium nucleatum. This

10

property may result from the ability of blueberry polyphenols to chelate iron. Moreover,

11

the blueberry extract at 62.5 µg/mL inhibited F. nucleatum biofilm formation by 87.5 ±

12

2.3%. We then investigated the ability of the blueberry extract to inhibit the NF-κB

13

signaling pathway in U937-3xκB cells. The blueberry extract dose-dependently inhibited

14

the activation of NF-κB induced by F. nucleatum. In addition, a pre-treatment of

15

macrophages with the blueberry extract (62.5 µg/mL) inhibited the secretion of IL-1β,

16

TNF-α and IL-6 by 87.3 ± 1.3%, 80.7 ± 5.6% and 28.2 ± 9.3% respectively, following a

17

stimulation with F. nucleatum. Similarly, the secretion of MMP-8 and MMP-9 was also

18

dose-dependently inhibited. This dual anti-bacterial and anti-inflammatory action of

19

lowbush blueberry polyphenols suggests that they may be promising candidates for novel

20

therapeutic agents.

21

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KEYWORDS: Biofilm, blueberry, cytokine, matrix metalloproteinase, periodontal

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disease 2 ACS Paragon Plus Environment

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INTRODUCTION

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Periodontitis is a multifactorial disorder affecting the supporting structures of the teeth,

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including the periodontal ligament and the alveolar bone. Depending on the age group, up

27

to 15% of the population is affected by severe forms of the disease which, if left

28

untreated, may result in tooth loss 1. Periodontitis is initiated following the accumulation

29

of predominantly anaerobic Gram-negative bacteria in the subgingival plaque 2. More

30

specifically, Fusobacterium nucleatum has been associated with various forms of

31

periodontitis

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between commensal Gram-positive “early colonizers” and periodontopathogenic Gram-

33

negative “late colonizers” 4. In addition to be considered as an etiologic agent of

34

periodontitis, F. nucleatum can also cause a variety of extra-oral infections, including

35

endocarditis, inflammatory bowel disease, and brain abscesses 5.

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Host-bacteria interactions that result in a complex inflammatory response play a crucial

37

role in the progression and severity of periodontitis 6. Macrophages and monocytes are

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the first line of host defence against bacterial infections and play a key role in the

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initiation of an adaptive immune response 7. These cells secrete many cytokines,

40

including tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-

41

6), and interleukin-8 (CXCL8), in response to stimulation by periodontopathogens such

42

as F. nucleatum

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homeostasis, uncontrolled and excessive stimulation may result in the maintenance of a

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chronic inflammatory condition due to the secretion of large amounts of inflammatory

45

mediators and matrix metalloproteinases (MMPs) by mucosal and immune cells,

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contributing to periodontal tissue destruction 10- 11.

3

and is one of the main bacterial species involved in physical interactions

8- 9

. While this host response is often involved in gingival tissue



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The nuclear factor kappa B (NF-κB) signaling pathway plays a key role in a wide range

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of pathological processes such as inflammatory diseases, cancers, and atherosclerosis

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NF-κB is activated by a wide variety of stimuli, including bacterial pathogens

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has many target genes, including those encoding cytokines, adhesion molecules, and

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MMPs 15. NF-κB is thus a central player in inflammatory diseases such as periodontitis 16

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and, as such, the inhibition of its activation could be a promising therapeutic strategy 17-

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18

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Proper nutrition may be very important in the management of periodontal diseases 19. In

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fact, based on our current understanding of the etiology and pathogenesis of periodontal

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diseases, polyphenols found in fruits and vegetables may be of high interest for use in

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adjunctive periodontal therapies. The blueberry is cultivated in many regions of the world

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and is one of the most commonly consumed berries in the US, ranking second after

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strawberry in popularity

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including flavanols, flavonols, and anthocyanins

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reported beneficial health effects on humans. Studies have shown that blueberry fruits

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have a positive effect on brain aging, diabetes, cancers, and vascular diseases 23-25. To the

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best of our knowledge, no one has investigated the potential beneficial effects of

64

blueberry polyphenols on periodontal disease. In the present study, we investigated the

65

effect of a polyphenol-rich lowbush blueberry (Vaccinium angustifolium Ait.) extract on

66

the growth and biofilm formation of F. nucleatum. We also assessed the inhibitory effect

67

of the blueberry extract on the NF-κB signaling pathway and cytokine/MMP secretion in

68

monocytes and macrophages.

13- 14

12

.

, and

.

20

. Blueberries contain significant amounts of flavonoids, 21- 22

, which likely contribute to the

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MATERIALS AND METHODS

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Plant material

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A blueberry extract (BlueCan NV Pure) prepared from the fruit of V. angustifolium Ait.

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was kindly provided by Nutra Canada (Champlain, QC, Canada). BlueCan NV Pure is a

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70% ethanolic extract of whole blueberry from which sugar was removed passing the

75

supernatant on a XAD-7 chromatographic column. According to the company data sheet,

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the total phenolic content of the extract is 40%, as determined by the Folin-Ciocalteu

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method.

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Chemicals

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The phenolic standards gallic acid, chlorogenic acid, protocatechuic acid, p-

80

hydroxybenzoic acid, p-coumaric acid, m-coumaric acid, caffeic acid, ferulic acid,

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quercetin, quercetin 3-glucoside, kaempferol, myricetin, isorhamnetin, catechin, and

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epicatechin were purchased from Sigma-Aldrich Canada Co. (Oakville, ON, Canada).

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Delphinidin 3-glucoside was from Extrasynthèse (Lyon, France). Liquid chromatography

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grade solvents acetone, methanol, and acetonitrile were from EMD Millipore Canada

85

(Mississauga, ON, Canada), and formic acid was from VWR International (Mississauga,

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ON, Canada). Ultrapure water was obtained using a Millipore Milli-Q water purification

87

system (EMD Millipore Canada).

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Phenolic characterization of the blueberry extract

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Characterization of anthocyanins and procyanidins by HPLC

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Anthocyanins and procyanidins were characterized as described previously

91

anthocyanins were separated and analyzed by reverse-phase analytical HPLC with an

26

. Briefly,



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Agilent 1100 series system (Agilent Technologies Canada Inc.; Mississauga, ON,

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Canada) using the following gradient of 5% formic acid in ultrapure water (solvent A)

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and methanol (solvent B): 0-2 min, 5% B; 2-10 min, 5-20% B; 10-15 min, 20% B; 15-30

95

min, 20-25% B; 30-35 min, 25% B; 35-50 min, 25-33% B; 50-55 min, 33% B; 55-65

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min, 33-36% B; 65-70 min, 36-45% B; 70-75 min, 45-53% B; 75-80 min, 53-55% B; 80-

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84 min, 55-70% B; 84-88 min, 70-5% B; 88-90 min, 5% B. Chromatographic data were

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acquired at 520 nm. Anthocyanins were quantified using an external calibration curve of

99

delphinidin 3-glucoside standard, which LOD and LOQ were respectively 0.03 and 0.08

100

ppm. Procyanidins were separated and analyzed by normal-phase analytical HPLC using

101

an Agilent 1260/1290 infinity system equipped with a fluorescence detector. The elution

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was performed using the following linear gradient of acetonitrile/acetic acid 98/2 (v/v)

103

(solvent A) and methanol/ultrapure water/acetic acid 95/3/2 (v/v/v) (solvent B): 0% to

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40% B for 35 min, 40% to 100% B for 5 min, 100% isocratic B for 5 min, and 100% to

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0% B for 5 min. Fluorescence was monitored at the excitation and emission wavelengths

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of 230 and 321 nm. Procyanidins, which eluted based on their degree of polymerization,

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were quantified using epicatechin monomer as a standard.

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Characterization of phenolic acids and flavonoids by UHPLC-MS/MS

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Phenolic acids and flavonoids were characterized as previously described

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Acquity UHPLC-MS/MS coupled to a TQD mass spectrometer equipped with a Z-spray

111

electrospray interface (Waters Ltd.; Mississauga, ON, Canada). The phenolic acids and

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flavonoids were eluted using the following gradient of 0.1% formic acid in ultrapure

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water (solvent A) and acetonitrile (solvent B): 0-4.5 min, 5-20% B; 4.5-6.45 min, 20% B;

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6.45-13.5 min, 20-45% B; 13.5-16.5 min, 45-100% B; 16.5-19.5 min, 100% B; 19.5-

26

using an


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19.52 min, 100-5% B; 19.52-22.5 min, 5% B. The MS/MS analyses were performed in

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negative mode using the following electrospray source parameters: electrospray capillary

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voltage, 2.5 kV; source temperature, 140°C; desolvation temperature, 350°C; and cone

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and desolvation gas flows, 80 l/h and 900 l/h, respectively. Data were acquired through

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multiple reaction monitoring using Waters Masslynx V4.1 software. Phenolic standards

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were analyzed using the same parameters and were used for the quantification.

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Bacterial strain and growth conditions

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F. nucleatum ATCC 25586 was grown anaerobically (80% N2, 10% CO2, 10% H2) for 24

123

h at 37°C in Todd-Hewitt broth (THB; BD-Canada, Mississauga, ON, Canada)

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supplemented with 0.001% hemin and 0.0001% vitamin K.

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Determination of minimal inhibitory and minimal bactericidal concentrations

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A microplate dilution assay was used to determine the minimal inhibitory concentration

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(MIC) and minimal bactericidal concentration (MBC) values of the blueberry extract

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against F. nucleatum. To determine the MIC, a 24-h culture of F. nucleatum was diluted

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in fresh culture medium to obtain an optical density of 0.2 at 660 nm (OD660). Equal

130

volumes (100 µL) of bacterial suspension and serial dilutions of the blueberry extract

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(1000 to 15.63 µg/mL) in culture medium were added to the wells of 96-well plates.

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Wells with no F. nucleatum or no blueberry extract were used as controls while

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tetracycline was used as a reference antibiotic. After a 48-h incubation at 37°C under

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anaerobic conditions, bacterial growth was monitored by recording the OD660 using a

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Synergy 2 microplate reader (BioTek Instruments, Winooski, VT, USA). The MIC value

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was the lowest concentration of the blueberry extract that completely inhibited the growth

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of F. nucleatum. To determine the MBC, 5-µL aliquots from the wells with no visible 7 ACS Paragon Plus Environment


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growth were spread on sheep blood-supplemented THB agar plates, which were

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incubated for 3 days at 37°C. The MBC value was the lowest concentration at which no

140

colony formation occurred. All assays were performed in triplicate to ensure

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reproducibility.

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Determination of siderophore activity

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The universal siderophore assay of Schwyn and Neils

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chelating activity of the blueberry extract. Ferrichrome (Sigma-Aldrich Canada Ltd.), a

145

siderophore produced by Ustilago sphaerogena, was used as the positive control 28.

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Effect of the blueberry extract on biofilm formation

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Microplate assay

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F. nucleatum was grown for 48 h under anaerobic conditions in the absence or presence

149

of the blueberry extract (1000 to 15.63 µg/mL) as described above. The medium

150

containing free-floating bacteria was removed by aspiration using a 26G needle. Then,

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the wells were washed three times with distilled water, and the biofilms were stained with

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100 µL of 0.05% crystal violet. After a 15-min incubation, the wells were washed three

153

times with distilled water and were dried for 2 h at 37°C. Ethanol (100 µL, 95% [v/v])

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was added to each well, and the plate was shaken for 10 min to release the dye from the

155

biofilms. The absorbance at 550 nm (A550) was recorded.

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Scanning electron microscopy

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The effect of the blueberry extract on F. nucleatum biofilm formation was also examined

158

by scanning electron microscopy. One mL of F. nucleatum suspended to an OD660 of 0.2

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in culture medium, with (62.5, 125, 250, or 500 µg/mL) or without the blueberry extract,

27

was used to measure the iron


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was added to the wells of a 12-well plate containing a 13-mm-diameter plastic coverslip.

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After a 48-h incubation under anaerobiosis, the medium and free-floating bacteria were

162

removed by aspiration, and the plastic coverslips were washed twice with 0.1 M

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cacodylate buffer (pH 7.2). The biofilm-coated coverslips were incubated for 3 h in

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fixation buffer (2.5% [w/v] glutaraldehyde [grade I], and 1 mM CaCl2 in 0.1 M

165

cacodylate buffer, pH 7.2), washed three times (20 min each time) with 0.1 M cacodylate

166

buffer (pH 7.2), and post-fixed for 90 min at room temperature in 1% [w/v] osmic acid

167

containing 2 mM potassium ferrocyanide and 6% [w/v] sucrose in cacodylate buffer.

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Samples were dehydrated using a graded series of ethanol (50, 70, 95, 100%), critical

169

point-dried, gold-sputtered, and examined using a JEOL JSM6360LV scanning electron

170

microscope operating at 30 kV.

171

Determination of quorum sensing inhibitory activity

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The bioluminescence assay using the reporter strain V. harveyi BB170 (ATCC BAA-

173

1117) was performed as previously described

174

was grown overnight in autoinducer bioassay (AB) medium at 30°C with agitation and

175

was then diluted 1:100 in fresh culture medium containing the blueberry extract at

176

concentrations (31.25, 62.5, 125, 250 µg/mL) that did not interfere with the bacterial

177

growth (data not shown). After a 16-h incubation at 30°C, the supernatant was recovered

178

by centrifugation at 10 000 x g for 10 min, filter-sterilized (0.22 µm), and stored at -80°C.

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The presence of autoinducer-2 (AI-2) was then assayed by adding 20 µL of cell-free

180

supernatant from each test sample to the wells of a 96-well, clear bottom, black wall

181

microplate. An overnight culture of V. harveyi BB170 diluted 1:5000 (180 µL) was added

182

to each well containing a supernatant sample. The positive control consisted of 20 µL of

29

. V. harveyi BB120 (ATCC BAA-1116)



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cell-free supernatant from V. harveyi BB120 grown in the absence of blueberry extract

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while the negative control consisted of AB medium. The plate was incubated for 15 h in a

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Synergy 2 microplate reader at 30°C with agitation (200 rpm). The bioluminescence and

186

OD660 were measured every 15 min during the incubation.

187

Effect of the blueberry extract on the activation of the NF-κB transcription factor

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The human monoblastic leukemia cell line U937 3xκB-LUC, a subclone of the U937 cell

189

line stably transfected with a luciferase gene coupled to a promoter of three NF-κB-

190

binding sites, was kindly provided by Dr. Rune Blomhoff (University of Oslo, Norway)

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30

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(RPMI-1640; Life Technologies Inc., Burlington, ON, Canada) supplemented with 10%

193

heat-inactivated fetal bovine serum (FBS), 100 µg/mL of penicillin G/streptomycin, and

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75 µg/mL of hygromycin B at 37°C in a 5% CO2 atmosphere. First, the effect of the

195

blueberry extract at concentrations ≤ 500 µg/mL on U937 3xκB-LUC viability was

196

determined using an MTT (3-[4,5-diethylthiazol-2-yl]-2,5diphenyltetra-zolim bromide)

197

assay, according to the manufacturer’s instructions (Roche Diagnostics, Laval, QC,

198

Canada). Briefly, U937 3xκB-LUC cells were treated with the blueberry extract for 6.5 h

199

prior to monitor viability. To induce activation of the NF-κB transcription factor, the

200

U937 3xκB-LUC cells (106 cells/well) were placed in the wells of a 96-well black bottom

201

microplate (Greiner Bio-One North America Inc.; Monroe, NC, USA) and were

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stimulated for 6 h (optimal activation based on preliminary assays) with F. nucleatum

203

cells at a multiplicity of infection (MOI) of 2, 10, 50, or 100. To assess the effect of the

204

blueberry extract on the pro-inflammatory potential of F. nucleatum, an overnight culture

205

was pre-incubated with various concentrations of the blueberry extract (31.25 to 250

. Cells were routinely cultivated in Roswell Park Memorial Institute 1640 medium


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µg/mL) for 30 min with shaking. The F. nucleatum cells were then harvested by

207

centrifugation (10 000 g for 10 min), washed three times, and suspended in RPMI

208

supplemented with 1% FBS. They were then added to wells containing U937 3xκB-LUC

209

cells at an MOI of 100. Lastly, to investigate the effect of the blueberry extract on F.

210

nucleatum-induced NF-κB activation, U937 3xκB-LUC cells were pre-incubated with the

211

extract (15.6 to 500 µg/mL; in RPMI containing 1% FBS) for 30 min and were then

212

stimulated for 6 h with bacteria at an MOI of 100. Wells with no F. nucleatum or no

213

blueberry extract were used as controls to measure basal NF-κB activity. Assays using

214

Escherichia coli lipopolysaccharide (LPS) or a commercial inhibitor (BAY-11-7082; 25

215

µM; EMD Millipore Canada) were used as positive and negative controls of the NF-κB

216

signaling pathway, respectively. NF-κB activation was determined by measuring

217

luciferase activity following the addition Bright-Glo reagent (Promega Corporation,

218

Madison, WI, USA) in accordance with manufacturer’s protocol. Luminescence was

219

monitored using a Synergy 2 microplate reader.

220

Effect of the blueberry extract on cytokine and MMP secretion by macrophages

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U937 human monocytes (ATCC CRL-1593.2) from the American Type Culture

222

Collection (Manassas, VA, USA) were cultivated in RPMI-1640 supplemented with 10%

223

FBS and 100 µg/mL of penicillin G/streptomycin at 37°C in a 5% CO2 atmosphere. The

224

monocytes (2.5 x 105 cells/mL) were then incubated in RPMI-10% FBS containing 100

225

ng/mL of phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich Canada Ltd.) for 48 h

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to induce differentiation into adherent macrophage-like cells

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like cells were harvested by scraping and were centrifuged at 1200 g for 5 min. The cells

228

were washed, suspended in RPMI-1% FBS at a concentration of 1 x 106 cells/mL, seeded

31

. Adherent macrophage-



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into the wells of a 12-well microplate (1 x 106 cells/well), and incubated overnight at

230

37°C in a 5% CO2 atmosphere. The macrophage-like cells were treated with the blueberry

231

extract (62.5 to 500 µg/mL) for 2 h. They were then stimulated with F. nucleatum at an

232

MOI of 100. After a 24-h incubation at 37°C in a 5% CO2 atmosphere, the culture

233

medium supernatants were collected and stored at -20°C until used. Cells incubated in

234

culture medium with or without the blueberry extract and stimulated or not with bacteria

235

were used as controls. Enzyme-linked immunosorbent assay (ELISA) kits (eBioscience

236

Inc., San Diego, CA, USA) were used to quantify IL-1β, IL-6, CXCL8, TNF-α, MMP-8,

237

and MMP-9 concentrations according to the manufacturer’s protocols. To exclude the

238

possibility that cell toxicity due to the blueberry extract might have been responsible for a

239

decrease in cytokine and MMP levels, the viability of the blueberry extract-treated

240

macrophages was assessed using an MTT test and by Trypan blue exclusion.

241

Effect of the blueberry extract on MMP-9 activity

242

Human recombinant MMP-9 (active form) purchased from AnaSpec (Fremont, CA,

243

USA) was diluted in reaction buffer (300 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl2, 20

244

µM ZnCl2, pH = 7.5) to a final concentration of 50 ng/mL and was incubated for 4 h in

245

the absence or presence of the blueberry extract (7.9 - 500 µg/mL) and the fluorogenic

246

substrate Gelatin DQTM (Molecular Probes, Eugen, OR, USA) at a concentration of 100

247

µg/mL. The fluorogenic substrate alone or with blueberry extract was used as controls.

248

An assay using a specific inhibitor of MMP (1 µM; GM6001; Calbiochem, San Diego,

249

CA, USA) was also performed. The assay mixtures were incubated in the dark for 2 h at

250

37°C. The fluorescence was measured using a fluorometer with excitation and emission


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wavelengths set at 495 nm and 525 nm, respectively. Two independent assays performed

252

in triplicate were performed.

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Statistical analysis

254

Unless indicated otherwise, all experiments were performed in triplicate. The data are

255

expressed as means ± standard deviations (SD). Statistical analyses were performed using

256

a one-way analysis of variance with a post hoc Bonferroni multiple comparison test

257

(GraphPad Software Inc.; La Jolla, CA, USA). All results were considered statistically

258

significant at p ˂ 0.001 or p < 0.05.

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RESULTS

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Phenolic composition of the blueberry extract

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The phenolic composition of the blueberry extract is presented in details in Table S1.

265

Phenolic acids, flavonoids (flavonols, anthocyanins, flavan-3-ols), and procyanidins

266

made up 16.6%, 12.9%, and 2.7% of the blueberry extract, respectively. Chlorogenic acid

267

(67%) was the most abundant phenolic acid, while flavonols and anthocyanins accounted

268

for 99% of the total flavonoids, and quercetin and its sugar-conjugated derivatives for

269

98% of the flavonols. The procyanidin content was mostly monomers, dimers, and

270

polymers with a degree of polymerization >10.

271

Antibacterial and anti-biofilm properties of the blueberry extract

272

The ability of the blueberry extract to interfere with the growth of F. nucleatum was

273

assessed first. The MIC of the blueberry extract against F. nucleatum was 1 mg/mL

274

(Figure 1). This concentration also corresponded to the MBC. The blueberry extract at a

275

concentration of 500 µg/mL reduced the growth of F. nucleatum by 29.9 ± 12.3% while it

276

had no growth inhibitory effect at concentrations ≤ 250 µg/mL. Tetracycline used as a

277

reference antibiotic showed a MIC of 0.39 µg/mL and a MBC of 1.56 µg/mL (data not

278

shown). In order to identify the mechanism by which the blueberry extract exerted its

279

antimicrobial activity against F. nucleatum, we investigated its ability to chelate iron. The

280

blueberry extract dose-dependently chelated iron in a universal siderophore assay (Figure

281

2). This activity was comparable to that of ferrichrome, the positive control, a

282

siderophore produced by U. sphaerogena.

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We then tested the effect of the blueberry extract on biofilm formation by F. nucleatum

285

(Figure 1). Blueberry extract concentrations ranging from 500 to 62.5 µg/mL had a

286

significant effect on biofilm formation. More specifically, 62.5 µg/mL of the blueberry

287

extract, which did not reduce the growth of F. nucleatum, inhibited biofilm formation by

288

87.5 ± 2.3%. The effect of the blueberry extract on biofilm formation by F. nucleatum

289

was visualized by scanning electron microscopy. Electron micrographs clearly showed

290

that there was a marked reduction in mature biofilm and that the architecture was

291

disrupted when F. nucleatum was grown in the presence of the blueberry extract

292

(Figure 3).

293

Given that the blueberry extract reduced biofilm formation by F. nucleatum, we then

294

investigated the quorum sensing inhibitory activity of the blueberry extract. V. harveyi

295

BB120 was grown in the presence of various concentrations of blueberry extract, and the

296

supernatants were then collected. The levels of secreted AI-2 were determined by

297

monitoring bioluminescence using the reporter strain V. harveyi BB170. The blueberry

298

extract dose-dependently inhibited AI-2-mediated bioluminescence (Figure 4). Quorum

299

sensing activity was inhibited at concentrations that had no effect on the growth of

300

V. harveyi BB120 (data not shown).

301

Effect of the blueberry extract on NF-κB activation in monocytes

302

We first showed that F. nucleatum dose-dependently activates the NF-κB transcription

303

factor, as indicated by the increase in luciferase activity (Figure S1). The strongest

304

activity was obtained with an MOI of 100. A 30-min pre-incubation of bacterial cells

305

(MOI of 100) with the blueberry extract reduced the ability of F. nucleatum to activate



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the NF-κB pathway (Figure 5). More specifically, 250 and 125 µg/mL of the blueberry

307

extract decreased NF-κB activation by 33.19 ± 7.48% and 17.46 ± 0.32%, respectively.

308

We then determined whether the presence of blueberry extract prevented F. nucleatum-

309

induced NF-κB activation in U937-3xκB cells. The blueberry extract dose-dependently

310

inhibited the activation of NF-κB induced by F. nucleatum at an MOI of 100 (Figure 6).

311

More specifically, 250, 125, 62.5, and 31.25 µg/mL of the blueberry extract reduced NF-

312

κB activity by 97 ± 0.56%, 88.6 ± 0.9%, 68.8 ± 5.7%, and 48.5 ± 2%, respectively. As

313

expected, the commercial inhibitor BAY-11-7082 (25 µM) completely prevented NF-κB

314

activation. The blueberry extract alone (in the absence of F. nucleatum) was not cytotoxic

315

and had no effect on NF-κB activity (data not shown).

316

Effect of the blueberry extract on cytokine and MMP secretion by macrophages

317

We used a macrophage-like model (PMA-treated U937 cells) stimulated with F.

318

nucleatum to determine whether the ability of the blueberry extract to attenuate NF-κB

319

activation in U937-3xκB cells also reduced inflammatory mediator secretion. Adherent

320

macrophage-like cells were pre-treated for 2 h with the blueberry extract and were then

321

stimulated for 48 h with F. nucleatum (MOI of 100). We then measured the secretion of

322

cytokines (IL-6, IL-1β, TNF-α, CXCL8) and MMPs (MMP-8, MMP-9) by the adherent

323

macrophage-like cells. Using an MTT test and by Trypan blue exclusion, the blueberry

324

extract displayed no cytotoxicity and had no effect on cell viability, which was ≥ 98 ±

325

6.8% for all concentrations tested compared to the untreated controls (data not shown).

326

Therefore, a decrease in cytokine and MMP levels cannot result from loss of cell

327

viability. F. nucleaum significantly increased the secretion of IL-6 (234.6 fold), IL-1β


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328

(5.2 fold), TNF-α (215 fold), CXCL8 (6.2 fold), MMP-8 (1.8 fold), and MMP-9 (1.3

329

fold) by macrophages (Figures 7 and 8). The secretion of pro-inflammatory cytokines

330

(IL-1β, IL-6, and TNF-α) by macrophages stimulated with F. nucleatum was significantly

331

and dose-dependently attenuated by the blueberry extract compared to control cells. At a

332

concentration of 62.5 µg/mL, the blueberry extract reduced the secretion of IL-1β by 87.3

333

± 1.3% (Figure 7A), TNF-α by 80.7 ± 5.6% (Figure 7B), and IL-6 by 28.2 ± 9.3%

334

(Figure 7C). While the secretion of the chemokine CXCL8 was not affected by 62.5

335

µg/mL of the blueberry extract, 500, 250, 125 µg/mL of the blueberry extract decreased

336

CXCL8 secretion by 79 ± 6.3%, 57.9 ± 0.2%, and 11.2 ± 0.3%, respectively (Figure 7D).

337

Treating the cells with the highest concentration of the blueberry extract (500 µg/mL)

338

decreased IL-1β, TNF-α IL-6, and CXCL8 secretion to the same degree as the

339

commercial inhibitor (Figure 7). The blueberry extract in the absence of F. nucleatum

340

had no effect on the basal levels of secreted cytokine (data not shown). Lastly, MMP-8

341

and MMP-9 secretion by F. nucleatum-stimulated macrophages was also attenuated by

342

the blueberry extract, in some cases below basal levels. More specifically, 500, 250, and

343

125 µg/mL of the blueberry extract reduced the secretion of MMP-8 by 67.7 ± 0.4%, 66.9

344

± 0.4%, and 60.2 ± 0.2%, respectively, and the secretion of MMP-9 by 93.0 ± 2.24%,

345

62.7 ± 1.7%, and 26.7 ± 7.8%, respectively (Figure 8).

346

Effect of the blueberry extract on MMP-9 activity

347

After demonstrating that the blueberry extract can decrease MMP-9 secretion in a

348

macrophage model, we evaluated its effect on the gelatinase activity of MMP-9. As

349

reported in Table 1, the blueberry extract at 500, 250 and 125 µg/mL reduced MMP-9

350

activity by 99.7 ± 1.8%, 82.6 ± 2.4% and 56.7 ± 5.5% respectively. 17 ACS Paragon Plus Environment


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352

DISCUSSION

353

Conventional periodontal treatments rely primarily on removing the subgingival dental

354

biofilm that modulates the host inflammatory response associated with periodontal tissue

355

destruction. While physical removal of the biofilm by scaling and root planing is usually

356

sufficient to reverse the progression of the disease, adjunctive treatments, such as

357

antibiotics, are occasionally required. Over the past two decades, natural compounds

358

exhibiting both antibacterial and anti-inflammatory properties have received considerable

359

attention as potential new therapeutic agents for the prevention and treatment of

360

periodontal infections

361

recognized for their health benefits

362

blueberries: highbush (Vaccinium corymbosum L.) and lowbush (V. angustifolium Ait.).

363

Lowbush blueberries, also known as wild blueberries, are often harvested from wild

364

patches and are endemic to the boreal forests. They are smaller in size than V.

365

corymbosum L. and have a distinct intense flavor and aroma. Lowbush blueberries are

366

also grown commercially, especially in Maine, Atlantic Canada, and Quebec. Many

367

studies have reported that blueberries contain health-promoting compounds, including

368

phenolic acids and flavonoids such as chlorogenic acid, ellagic acid, quercetin,

369

anthocyanins, and procyanidins, which account for their pronounced antioxidant, anti-

370

inflammatory, and immunomodulatory properties 33- 34. The blueberry extract used in our

371

study was particularly rich in a variety of polyphenols and more specifically chlorogenic

372

acid, flavonols, as well as monomers, dimers and polymers of procyanidins. Given that

373

blueberry polyphenols are potential candidates for the prevention and treatment of

374

periodontal diseases, the present study was aimed at investigating the effects of a

32

. Blueberries are mainly native to North America and are 23- 24

. There are two main commercial species of



Page 20 of 45

375

lowbush blueberry extract (V. augustifolium Ait.) on various aspects of the

376

etiopathogenesis of periodontitis.

377

Based on our previous studies showing that cranberry proanthocyanidins interfere with

378

the pathogenic properties of periodontopathogens

379

polyphenols may have a beneficial effect on periodontal diseases by attenuating the

380

growth and pathogenic properties of F. nucleatum, a bacterial species that has been

381

associated with various forms of the disease. The blueberry extract displayed potent

382

antibacterial activity against F. nucleatum, with MIC and MBC values of 1 mg/mL. This

383

finding was consistent with previous studies by Lacombe et al., who reported that North

384

American lowbush blueberry polyphenols exhibit marked antibacterial effects against

385

Gram-negative bacteria, including Escherichia coli 157:H7, and foodborne pathogens 36.

386

Blueberry polyphenols may alter the bacterial membrane by destabilizing the LPS and

387

increasing the efflux of ATP from the cytoplasm

388

acids (caffeic acid, chlorogenic acid, protocatechuic acid), and quercetin, which were

389

present in our blueberry extract, have been associated with antibacterial and membrane

390

permeabilizing activities

391

possess marked iron-chelating activity, which may be another antimicrobial mechanism.

392

Indeed, by chelating iron, which is an essential cofactor for bacterial growth, blueberry

393

polyphenols may compete with bacteria for iron, creating an iron-deficient, bacteriostatic

394

environment. Interestingly, Dastmalchi et al. investigated the antioxidative properties of a

395

variety of edible neotropical blueberries and identified iron-chelating activity in some

396

varieties of blueberry, although they did not test the lowbush blueberry

397

ferrous form of iron is responsible for the formation of ROS, this iron-scavenging

38

35

37

, we hypothesized that blueberry

. Moreover, anthocyanins, phenolic

. In the present study, we showed that blueberry polyphenols

39

. Since the


Page 21 of 45


40

398

property may contribute to the antioxidative activity of blueberry polyphenols

399

Moreover, given that ROS are known to activate MMPs, the iron-chelating property of

400

blueberry polyphenols may also contribute to decreasing proteolytic activity in diseased

401

periodontal sites.

402

Since F. nucleatum plays a critical role in subgingival biofilm formation by bridging

403

early and late colonizers, we investigated the anti-biofilm activity of the blueberry extract

404

using concentrations that did not inhibit bacterial growth. Based on the results of a

405

colorimetric microplate assay, the blueberry extract almost completely prevented F.

406

nucleatum biofilm formation at concentrations as low as 62.5 µg/mL. The anti-biofilm

407

activity of the blueberry extract was confirmed by scanning electron microscopy. The

408

above observations suggest that the blueberry extract may inhibit the attachment and

409

maturation of periodontal biofilms through dynamic and sequential processes. This may

410

help prevent or slow periodontal disease initiation and progression since biofilms, which

411

are structured microbial communities attached to oral surfaces, enable bacteria to evade

412

immune defenses, resist mechanical removal, and avoid chemotherapeutic agents. In

413

addition, the blueberry extract disrupted biofilm formation, a major virulence property of

414

F. nucleatum, at concentrations that were not antibacterial, indicating that bacteria may

415

not develop resistance to bioactive molecules in blueberry extracts. Zimmer et al. studied

416

hydroethanolic extracts of blueberries from various V. virgatum cultivars and reported

417

that the caffeic and chlorogenic acids in the extracts were likely responsible for the

418

inhibition of biofilm formation by Staphylococcus epidermidis and Pseudomonas

419

aeruginosa, two major human bacterial pathogens

420

present in our blueberry extract.

41

.

. These two compounds were also



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421

Given the fact that blueberry polyphenols interfere with biofilm formation by F.

422

nucleatum, we hypothesized that the blueberry extract might also possess anti-quorum

423

sensing activity. Quorum sensing refers to the coordinated regulation of virulence gene

424

expression modulated by cell density. Using the V. harveyi quorum sensing model, we

425

showed that the blueberry extract attenuated AI-2-mediated cell-cell signaling. Such an

426

attenuation of bacterial virulence by the inhibition of quorum sensing has been suggested

427

be a promising strategy for reducing antibiotic use 42.

428

While the colonization of subgingival sites and subsequent biofilm formation by

429

periodontopathogens such as F. nucleatum is the initial step in the pathogenesis of

430

periodontitis 2, the host immune response, which results in the overproduction of a large

431

variety of inflammatory mediators involved in tissue and bone destruction, is critical

432

11

433

health benefits of berries 43, we thus investigated the effect of the blueberry extract on the

434

inflammatory response of host cells. Macrophages are major immune cells involved in

435

the continuous and excessive host responses that result in the secretion of large amounts

436

of pro-inflammatory cytokines, chemokines, and MMPs, which in turn are involved in

437

tissue and bone destructive processes 7. In the present study, we showed that lowbush

438

blueberry polyphenols significantly reduce the secretion of IL-1β, TNF-α, IL-6, and

439

CXCL8 by F. nucleatum-stimulated macrophages. These results are in agreement with

440

those of Kang et al.

441

lowbush blueberries and highbush blueberries reduced the production of IL-6 and TNF-α

442

by LPS-stimulated macrophages and that the anti-inflammatory activity of the lowbush

443

extract was greater than that of the highbush extract. They also provided evidence that the

8- 9-

. Given that polyphenols have anti-inflammatory properties that contribute to the human

20

, who showed that two phenolic acid extracts prepared from


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444

decreased production IL-6 and TNF-α was associated with the inhibition of the NF-κB

445

signaling pathway and that chlorogenic acid (67% of all phenolic acids in our blueberry

446

extract) was likely the main contributor to the anti-inflammatory property of blueberries

447

20

448

induced NF-κB signaling pathway, and reduces the secretion of the pro-inflammatory

449

cytokines TNF-α, IL-1β and IL-6 involved in mastitis. Cheng et al. provided proof that

450

that blueberry anthocyanins (petunidin, peonidin, malvidin, and cyanidin) inhibit the IL-

451

1β, IL-6, and IL-12 genes in LPS-stimulated RAW264 macrophages, a further indication

452

that blueberry extracts possess anti-inflammatory activity 45.

453

Inhibitors of the NF-κB transcription factor hold great promise for the prevention and

454

treatment of chronic inflammatory disorders

455

activating or inhibiting various cell signaling pathways by, for example, modulating

456

kinase activity and activating the NF-κB transcription factor 43. The mechanism by which

457

the blueberry extract used in the present study reduced inflammatory mediator secretion

458

may be related to its capacity to block NF-κB activation since this signaling pathway is

459

central to the inflammatory response

460

that the blueberry extract inhibited NF-κB activation induced by F. nucleatum, most

461

likely its cell surface LPS. While the exact bioactive molecule(s) in our blueberry extract

462

has not been identified, anthocyanins, which made up 23% of the total polyphenolic

463

content of the extract, may be involved. Indeed, Taverniti et al. 34 previously reported that

464

an anthocyanin-rich lowbush blueberry extract reduced IL-1β-induced NF-κB activation

465

in Caco-2 epithelial cells. In addition, cranberry proanthocyanidins reduce the LPS-

466

induced inflammatory response in oral epithelial cells via the NF-κB pathway 47.

. Ruifeng et al.

44

reported that chlorogenic acid inhibits the TLR4-mediated and LPS-

43

46

. Plant polyphenols act on host cells by

. Using the U937-3xκB cell model, we showed



Page 24 of 45

467

MMP-9 and MMP-8 have been strongly associated with the progression of periodontitis

468

48

469

secretion by F. nucleatum-stimulated macrophages. This result was in agreement with

470

those of a previous study by Matchett et al.

471

anthocyanins and proanthocyanidins down-regulate MMP-2 and MMP-9 activities in the

472

DU-145 prostate tumor model, and increase the expression of tissue inhibitors of

473

metalloproteinases 1 (TIMP-1). Matchett et al. suggested that this effect was mainly due

474

to proanthocyanidins and that it involved the protein kinase C (PKC) and mitogen-

475

activated protein

476

secretion by macrophages, we also showed that the blueberry extract inhibits the catalytic

477

activity of MMP-9 (gelatinase-2). Since MMPs are associated with periodontal tissue

478

destruction, our results suggested that blueberry polyphenols may contribute to reducing

479

host cell damage, including bone resorption . Interestingly, Zhang et al.

480

feeding rats with a blueberry diet inhibits bone resorption through suppression of receptor

481

activator of nuclear factor-κB ligand (RANKL).

482

In conclusion, we showed that polyphenols in a lowbush blueberry extract are active

483

against the two main etiologic components of periodontitis. On the one hand, the

484

blueberry extract inhibited the growth and biofilm formation of the periodontopathogenic

485

bacterium F. nucleatum. On the other, it reduced the secretion of cytokines and MMPs by

486

macrophages by blocking the activation of the NF-κB signaling pathway. This dual action

487

of lowbush blueberry polyphenols suggests that they may be promising candidates for

488

novel therapeutic agents.

. Interestingly, we showed that our blueberry extract reduced MMP-8 and MMP-9

kinase pathways

49

49

, who reported that lowbush blueberry

. In addition to attenuate MMP-8 and MMP-9

50

reported that

489 24 ACS Paragon Plus Environment

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490

Acknowledgments

491

We are grateful to Nutra Canada (Champlain, QC, Canada) for providing the blueberry

492

extract and to R. Blomhoff and H. Carlsen (University of Oslo, Norway) for providing

493

the U937-3xκB-LUC cell line. We thank G. LeBel for technical assistance. This study

494

was supported by the Laboratoire de Contrôle Microbiologique de l’Université Laval.

495

The authors report no conflicts of interest related to this study.

496 497 498

Supporting Information

499

Table S1 presents the detailed phenolic composition of the blueberry extract. Figure S1

500

presents the effect of F. nucleatum on NF-κB activation using the U937-3xκB cell model

501

(results are expressed as the means ± SD of triplicate assays from two independent

502

experiments; *, significant increase compared to non-stimulated control cells at p

Wild Blueberry (Vaccinium angustifolium Ait.) Polyphenols Target

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