© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

J Periodont Res 2015 All rights reserved

JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12271

Expression of antimicrobial peptides and interleukin-8 during early stages of inflammation: An experimental gingivitis study Dommisch H, Staufenbiel I, Schulze K, Stiesch M, Winkel A, Fimmers R, Dommisch J, Jepsen S, Miosge N, Adam K, Eberhard J. Expression of antimicrobial peptides and interleukin-8 during early stages of inflammation: An experimental gingivitis study. J Periodont Res 2015; doi: 10.1111/jre.12271. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

H. Dommisch1,2,3,a, I. Staufenbiel4,a, K. Schulze5, M. Stiesch5, A. Winkel5, R. Fimmers6, J. Dommisch3, S. Jepsen3, N. Miosge7, K. Adam5, J. Eberhard5 1 Department of Periodontology and Synoptic  – University Medicine Berlin, Dentistry, Charite Berlin, Germany, 2Department of Oral Health Sciences, Health Science Center, University of Washington, Seattle, WA, USA, 3Department of Periodontology, Operative and Preventive Dentistry, University Hospital Bonn, Bonn, Germany, 4Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, Hannover, Germany, 5Clinic for Dental Prosthetics and Biomedical Materials Science, Hannover Medical School, Hannover, Germany, 6Institute of Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany, and 7Research Group for Oral Biology and Tissue Regeneration, Department of Prosthetic Dentistry, University Hospital €ttingen, Go €ttingen, Germany Go

Background and Objectives: In the oral cavity, the epithelial surface is constantly exposed to a number of different microorganisms that are organized in a well-structured biofilm. The aim of this study was to monitor gingival expression of antimicrobial peptides (AMPs) and interleukin-8 (IL-8) in an early gingivitis model. Material and Methods: Experimental gingivitis was allowed to develop in healthy volunteers (n = 17). Bleeding on probing (BOP%) and gingival crevicular fluid volume (GCF) were assessed at baseline and day 1, 3, 5, 7 and 14. Expression of AMPs (human beta-defensin-2, hBD-2; CC-chemokine ligand 20, CCL20; psoriasin, pso/S100A7) and IL-8 was analyzed by immunohistochemistry in gingival biopsies. In addition, hBD-2 and IL-8 protein expression was monitored in GCF using the ELISA technology. Results: Experimental gingivitis gradually developed with an increase in BOP scores and GCF volume over time. In GCF, elevated concentrations of hBD-2 and IL-8 were monitored at day 1, 5 and 7 (p ≤ 0.0002). Immunohistochemical analysis of gingival sections demonstrated increased staining for hBD-2 at day 3, whereas the CCL20, pso/S100A7, and IL-8 expression was increased at later time points (p < 0.05). Conclusion: For the first time, this study showed the time-dependent regulation of AMPs, following clinical signs of experimentally induced gingival inflammation. Differential temporal expression for AMPs may ensure a constant antimicrobial activity against changes in the bacterial composition of the growing dental biofilm.

The surfaces of the oral cavity are constantly exposed to a high variety of microorganisms, which are capable

of forming biofilms on teeth, the oral mucosa, but also prostheses (1,2). A basic function of the mucosal barrier

Henrik Dommisch, DDS, PhD, Department of Periodontology and Synoptic Dentistry, University Medicine Berlin, Aßmannshauser Straße 4 – 6, 14195 Berlin, Germany Tel: +49 30 450 562322 Fax: +49 30 450 7562322 e-mail: [email protected] a These authors contributed equally to this work. Key words: antimicrobial peptides; CCL20;

experimental gingivitis; hBD-2; innate immunity; pso/S100A7 Accepted for publication February 2, 2015

is to provide protection against pathogenic microorganisms by maintaining a mechanical and a chemical

2

Dommisch et al.

barrier. The chemical barrier is represented by the gingival synthesis of antimicrobial peptides (AMPs), which are part of the innate immune system. Another important function of the epithelial cell layer is the induction of tolerance to antigens commonly present on mucosal surfaces for the maintenance of a healthy oral homeostasis. This aspect is of importance, because of the highly diverse and dynamic microbial flora in the oral cavity (1). This unresponsiveness or “tolerance” necessitates negative regulatory mechanisms to avoid unintended stimulation by bacteria colonized on anatomical and artificial surfaces (3). While the physical barrier is represented by rigid intercellular connections, the chemical barrier is composed of a number of AMPs, such as human beta-defensins 1–4 (hBDs), psoriasin (pso/S100A7), ribonuclease 7 (Rnase-7) or the cathelicidin LL-37 (4–7). These AMPs are part of the innate immune system and, thereby, mainly responsible for maintaining oral health by fighting pathogenic bacteria (8). In addition to the mucosal epithelium, AMPs are also synthesized by oral salivary glands and secreted into the saliva (9– 11), which covers the oral epithelium by forming a thin pellicle-like layer. The hBD-2 belongs to a group of small, cationic AMPs expressed by various oral epithelial tissues, and it is highly effective against gram-negative bacteria (8,12–14). The C-C-chemokine ligand 20 (CCL20) is a chemokine that also exhibits antimicrobial activity against gram-negative bacteria (15,16), and similar to hBD-2, it links innate and adaptive immune responses by interacting with the chemokine receptor 6 (CCR6) and, thereby, activates immature dendritic cells (17,18). In addition, both hBD-2 and CCL20 act as chemoattractants for immature dendritic cells (18,19). In gingival epithelial cells, increased hBD-2 and CCL20 mRNA expression was demonstrated when cells were exposed to Porphyromonas gingivalis whole cells, supernatant as well as the arginine gingipain B (RpgB) (20–22). In addition, naturally formed oral

biofilms induce the gene expression of hBD-2 in gingival epithelia cells (5). pso/S100A7 has been identified as members of the S100 protein super family, which is expressed by epithelial cells and exhibits antimicrobial activity (5,6,23). In gingival epithelial cells, the gene expression of pso/ S100A7 is inducible by naturally formed oral biofilms (5). Interleukin-8 (IL-8) is synthesized in a large number of human cells, such as macrophages, monocytes, fibroblasts and epithelial cells, and it represents a chemokine that specifically chemoattracts and activates neutrophils (24). In recent studies, expression profiles for IL-8 were demonstrated in gingival crevicular fluid and gingival biopsies in the course of experimental gingivitis, but at later time points (> day 7) (25,26). The exact role of gingival epithelial cells regarding the physiological expression profiles of AMPs and/or proinflammatory mediators in response to natural biofilms is not yet apparent. A number of studies showed the expression profiles of both AMPs and proinflammatory mediators in gingival epithelial cells exposed to bacteria that have been cultured separately (8,27–29). However, these stimulation experiments using cytokines, bacterial cell wall products and single species or multispecies biofilms may not reflect the environmental complexity of sequential interactions between bacterial biofilms and mucosal surfaces. To the best of our knowledge, there is no information regarding the time-dependent expression profile of AMPs during the early stages of inflammation in vivo. The aim of the present study was to determine how the expression of AMPs, such as hBD-2, CCL20, and pso/S100A7, is regulated during early gingival inflammation in vivo. Therefore, the human model of experimental gingivitis was employed to study different phases of bacterial induced gingival inflammation. It was hypothesized that the expression of hBD-2, CCL20 and pso/S100A7 is regulated parallel to the clinically measurable (plaque and bleeding indices) development of early gingivitis. Furthermore, this study

aimed to compare tissue expression with gingival crevicular fluid measurements of hBD-2 and IL-8.

Material and methods Participants

The study was designed as a prospective clinical trial. Participants were selected according to the following inclusion criteria: (i) 20–35 years old; (ii) non-smokers; (iii) no clinical signs of gingival inflammation (redness, swelling, bleeding); (iv) no probing pocket depth ≥ 2 mm at any site; (v) no clinical attachment loss; and (vi) gingival index (GI) = 0 at baseline. Exclusion criteria were: (i) systemic diseases; (ii) pregnancy (to determine pregnancy before the study, a pregnancy test was performed by female participants) or breastfeeding; (iii) physical or mental handicaps that can interfere with adequate oral hygiene procedures; (iv) history of drug abuse; (v) allergic diathesis; (vi) medications (in particular current medication with non-steroidal or steroidal anti-inflammatory drugs, analgesic or antibiotics) within 6 wk before start of the study; (vii) untreated carious lesions and/or insufficient restorations, implants, crowns on teeth in the maxillary right quadrant; (viii) ongoing dental treatment; (ix) maxillary orthodontic appliances, fixed or removable (permanent retainers on oral sites of maxillary incisors were accepted); and (x) mouth breathing. Subjects eligible to participate were asked to complete a questionnaire on their medical and dental history. Precise instructions about the experimental procedures were given and an explanatory pamphlet with details on the study design was handed out. All subjects signed an informed consent. Participants who preliminarily met the inclusion criteria were scheduled for a dental screening appointment. All participants received scaling and polishing of all tooth surfaces to remove all supra- and subgingival plaque, stain and calculus 14 d before the start of the study. Individualized brushing and oral hygiene instructions were given to each subject. The study

Gingivitis and antimicrobial peptides protocol was approved by the Ethical Committee of Hannover Medical School (no. 5253). Experimental protocol

At baseline the oral hygiene status of the participants was evaluated and the participants received a toothpaste without fluoride to clean the teeth, which were not included in the study (Kinderzahngel, Weleda, Schw€abisch Gm€ und, Germany), and were instructed to refrain from using any other commercial toothpastes and mouth rinses. The clinical trial included a 14 d experimental nonbrushing period, during which the subjects were not allowed to perform any oral hygiene on six teeth of the right side of the upper jaw. The subjects were clinically examined at baseline as well as on days 1, 3, 5, 7 and 14. At the end of the study, professional tooth cleaning and topical fluoride application was performed. Clinical measurements

The following clinical parameters were evaluated at each time point in the listed order to characterize the inflammatory status of the gingival tissues:





Bacterial plaque accumulation on the tooth surfaces was assessed using a modification of the Silness–L€ oe plaque index (PI) (30) at the buccal and oral sites of selected teeth. The dental plaque was categorized adjacent to the gingival tissues by four degrees without staining as follows: 0 = no plaque; 1 = sparse accumulation of plaque deposits visible with magnifying glasses; 2 = moderate accumulation of plaque deposits visible to the naked eye; 3 = heavy accumulation of soft material filling the niche between the gingival margin and tooth surface. The PI was calculated by dividing the total score by the total number of scored surfaces. GI (31) was assessed as follows: 0 = normal gingiva, no inflammation, discoloration or bleeding;





1 = mild inflammation, slight color change, mild alteration of gingival surface, no bleeding on pressure; 2 = moderate inflammation, erythema and swelling, bleeding on pressure; 3 = severe inflammation, erythema and swelling, tendency to spontaneous bleeding, perhaps ulceration. Gingival crevicular fluid was collected after gentle drying of the tooth for 10 s followed by using a filter paper strip (Periopaper; Pro Flow Incorporated, Amityville, NY, USA) that was inserted for 30 s into the gingival sulcus at four sites of the upper right first premolar at the mesio-buccal, disto-buccal, mesio-oral and distooral sites. The gingival crevicular fluid was measured with a calibrated Periotron 6000 gingival fluid meter (Pro Flow Incorporated) and expressed in Periotron Units (PU). Bleeding frequency of the gingival tissues after gentle probing (BOP) was recorded at four sites of all selected tooth sites: mesio-buccal, disto-buccal, mesio-oral and distooral. The presence or absence of bleeding was recorded after gingival crevicular fluid collection. The sulcus was gently probed with a pressure-calibrated probe (TPS probe; Vivacare, Schaan, Liechtenstein). The tip of the probe had a diameter of 0.5 mm and the probing force was set to 20 g.

3

papers were centrifuged at 4°C at 905 g for 15 min. This procedure was repeated three times. Protein concentrations of hBD-2 and IL-8 were determined by commercially available enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions (PeproTech, Hamburg, Germany). Protein concentrations were analyzed from baseline to day 7 during the experimentally induced gingivitis. Biopsy sampling

Biopsies (1 9 1.5 mm) were collected from the marginal gingiva located adjacent to the tooth surface in the interdental region. This location was selected because it represents the contact area between the growing biofilms and the epithelial tissues. For every biopsy, a different tooth was selected. Before surgically removing the tissues, local anesthesia (0.2 mL, UBS forte; Boehringer Ingelheim, Ingelheim, Germany) was administered to an area approximately 15 mm away from the sampling area to avoid any effect of the anesthetic. Two incisions were made into the free gingiva before the tissue was gently mobilized and immediately fixed in 10% formalin. Immunohistochemical preparation of the paraffin-embedded tissues

All measurements were carried out under the same conditions by two calibrated investigators at baseline as well as on days 1, 3, 5, 7 and 14 at four sites per tooth, with the exception of gingival crevicular fluid that was only measured on the upper right first premolar.

All specimens were immediately fixed in 4% buffered formalin and embedded in paraffin. Serial histological sections of 2–4 lm were deparaffinized and rehydrated using graded ethanol and deionized water, respectively. Afterwards, the endogenous peroxidase activity was blocked using a mixture of 0.3% hydrogen peroxide (700 lL) and methanol (70 mL).

Analysis of human beta-defensin-2 and interleukin-8 in gingival crevicular fluid

CC-chemokine ligand 20, human beta-defensin-2, psoriasin and interleukin-8 staining

After the clinical measurements, the filter paper strips were stored at 80°C. Peptides were extracted by adding 50 mL phosphate-buffered saline to the filter paper strips. The filter

The following antibodies were used in this study: hBD-2 (rabbit, polyclonal; sc-20798; dilution 1 : 25; Santa Cruz Biotechnology Inc., Dallas, TX, USA), CCL20 (rabbit, polyclonal,

4

Dommisch et al.

antimacrophage inflammatory protein 3 alpha antibody, ab9829; dilution 1 : 50; Abcam, Cambridge, MA, USA), pso/S100A7 (rabbit, polyclonal; sc-67047; dilution 1 : 100; Santa Cruz Biotechnology Inc.) and IL-8 (rabbit, polyclonal, sc-7922; dilution 1 : 50; Santa Cruz Biotechnology Inc.). Each histologic slide was incubated with the primary antibody overnight at 4°C. Antibodies were diluted using 1% tris-buffered saline/bovine serum albumin. Subsequently, sections were either incubated with the secondary biotinylated antirabbit IgG antibody (DAKO EnVision+ System-horseradish peroxidase; Dako Denmark A/S, Glostrup, Denmark; for hBD-2, CCL20 and IL8) or with the antirabbit (Dianova GmbH, Hamburg, Germany; for pso/ S100A7) and the avidin–biotin–peroxidase reaction was carried out following standard protocols (32). After the color reaction with aminoethylcarbazide solution (Merck, Darmstadt, Germany), tissues were counterstained with hematoxylin (1 : 2 dilution with H2O; Merck). For each staining procedure, negative controls were performed omitting the primary antibody (Fig. S1). Staining was performed using master mixes for each methodological step, so that all sections from each volunteer were treated simultaneously. Sections from a total of three donors were therefore stained at the same time using the same antibody mix. Thus, methodological errors, such as inhomogeneous pipetting and/or inconsistency in incubation time, could be avoided. Analysis and scoring of immunohistochemical staining

Immunohistological sections were analyzed in a standardized manner. First, each section was inspected under a microscope (AxioImager A1; Carl Zeiss, Jena, Germany) to verify specific immune reactions in comparison to corresponding methodological negative controls. In a second step, standardized digital images of each slide were taken at the same magnification (209). It was essential to capture the epithelial cell layer as well as

the underlying connective tissue when present. Each image was captured using constant exposure time and brightness. Subsequently, images were cataloged by lab numbers and stored on an external hard drive. For analysis, all high-resolution (1388 9 1040 dpi) non-edited images were copied into a presentation program (KeynoteÒ; Apple Software, Cupertino, CA, USA) using a neutral light gray background. Evaluation was performed using the method of intermodal intensity comparison. Each image was scored by four blinded investigators (I.S., A.W., K.W. and J.E.). Investigators were not aware of the order of appearance (meaning different experimental time points) as well as the stained molecule (meaning the antibody used for staining). In addition, each investigator scored all images using the same high-resolution monitor. For scoring, investigators were asked to use a color scale, where each color represents a score between “0” and “9” (NumbersÒ; Apple Software). This color scale allowed scoring without knowing the exact score number and an individual bias could therefore be avoided. A score of “0” was equal to no visible staining, whereas a score of “9” represented maximum immune reaction within the section. For analysis, total staining of the epithelial cell layers was scored and summarized for all volunteers (n = 17). Statistical methods

The statistical unit was the individual subject and for all clinical and laboratory recordings, means and standard deviations were calculated. For analysis of immunohistological scores, the data set was tested for dependencies regarding repeated measurements as well as inter-rater dependency using a general linear model. Before analysis, rates for BOP measurements were logit-transformed. ANOVA including correction for multiple testing (Friedman test) was performed. Subsequently, the Wilcoxon signed-rank test was applied to identify individual p-values. Statistical evaluation was computed using SPSS 20.0 (SPSS

Inc., Chicago, IL, USA) and GraphPad PrismÒ (GraphPad Software, La Jolla, CA, USA). The significance level was set at p ≤ 0.05.

Results Experimentally induced gingival inflammation

Seventeen participants (10 female and seven male) aged between 20 and 33 years (mean 24.53  3.41 years) were examined and all of them completed the study. No adverse effects were reported during the 14 d period of the study. The clinical outcome variables of all measurements regarding PI, GI, gingival crevicular fluid and BOP at baseline and days 1, 3, 5, 7 and 14 are presented in Fig. 1. Contemplating the differences between baseline and day 14, the PI scores (Fig. 1) significantly increased during the experimental gingivitis period from 0.0 (median; minimum: 0.0; maximum: 1.17) to 2.23 (median; minimum: 0.71; maximum: 3.0) (p < 0.0001). The GI values significantly increased from 0.0 (median; minimum: 0.0; maximum: 0.14) at baseline to 1.14 (median; minimum: 0.0; maximum: 2.43) at day 14 (p < 0.0001). The differences between the gingival crevicular fluid volumes at baseline (median: 3.0 PU; minimum: 0.0 PU; maximum: 48 PU) and day 14 28.0 PU (median; minimum: 2.0 PU; maximum: 108.0 PU) were statistically significant (p < 0.0001). At baseline, the BOP values were 11.0% (median; minimum: 0.0%; maximum: 32.0%) and significantly increased to 36.0% (median; minimum: 14.0%; maximum: 57.0%) at day 14 (p < 0.0001). The clinical values for PI, GI, gingival crevicular fluid and BOP increased in a timedependent manner (Fig. 1A–D). Human beta-defensin-2 and interleukin-8 concentration in gingival crevicular fluid

The hBD-2 and IL-8 concentrations were determined in gingival crevicular fluid at baseline and days 1, 3, 5 and 7 by enzyme-linked immunosorbent

4

Dommisch et al.

antimacrophage inflammatory protein 3 alpha antibody, ab9829; dilution 1 : 50; Abcam, Cambridge, MA, USA), pso/S100A7 (rabbit, polyclonal; sc-67047; dilution 1 : 100; Santa Cruz Biotechnology Inc.) and IL-8 (rabbit, polyclonal, sc-7922; dilution 1 : 50; Santa Cruz Biotechnology Inc.). Each histologic slide was incubated with the primary antibody overnight at 4°C. Antibodies were diluted using 1% tris-buffered saline/bovine serum albumin. Subsequently, sections were either incubated with the secondary biotinylated antirabbit IgG antibody (DAKO EnVision+ System-horseradish peroxidase; Dako Denmark A/S, Glostrup, Denmark; for hBD-2, CCL20 and IL8) or with the antirabbit (Dianova GmbH, Hamburg, Germany; for pso/ S100A7) and the avidin–biotin–peroxidase reaction was carried out following standard protocols (32). After the color reaction with aminoethylcarbazide solution (Merck, Darmstadt, Germany), tissues were counterstained with hematoxylin (1 : 2 dilution with H2O; Merck). For each staining procedure, negative controls were performed omitting the primary antibody (Fig. S1). Staining was performed using master mixes for each methodological step, so that all sections from each volunteer were treated simultaneously. Sections from a total of three donors were therefore stained at the same time using the same antibody mix. Thus, methodological errors, such as inhomogeneous pipetting and/or inconsistency in incubation time, could be avoided. Analysis and scoring of immunohistochemical staining

Immunohistological sections were analyzed in a standardized manner. First, each section was inspected under a microscope (AxioImager A1; Carl Zeiss, Jena, Germany) to verify specific immune reactions in comparison to corresponding methodological negative controls. In a second step, standardized digital images of each slide were taken at the same magnification (209). It was essential to capture the epithelial cell layer as well as

the underlying connective tissue when present. Each image was captured using constant exposure time and brightness. Subsequently, images were cataloged by lab numbers and stored on an external hard drive. For analysis, all high-resolution (1388 9 1040 dpi) non-edited images were copied into a presentation program (KeynoteÒ; Apple Software, Cupertino, CA, USA) using a neutral light gray background. Evaluation was performed using the method of intermodal intensity comparison. Each image was scored by four blinded investigators (I.S., A.W., K.W. and J.E.). Investigators were not aware of the order of appearance (meaning different experimental time points) as well as the stained molecule (meaning the antibody used for staining). In addition, each investigator scored all images using the same high-resolution monitor. For scoring, investigators were asked to use a color scale, where each color represents a score between “0” and “9” (NumbersÒ; Apple Software). This color scale allowed scoring without knowing the exact score number and an individual bias could therefore be avoided. A score of “0” was equal to no visible staining, whereas a score of “9” represented maximum immune reaction within the section. For analysis, total staining of the epithelial cell layers was scored and summarized for all volunteers (n = 17). Statistical methods

The statistical unit was the individual subject and for all clinical and laboratory recordings, means and standard deviations were calculated. For analysis of immunohistological scores, the data set was tested for dependencies regarding repeated measurements as well as inter-rater dependency using a general linear model. Before analysis, rates for BOP measurements were logit-transformed. ANOVA including correction for multiple testing (Friedman test) was performed. Subsequently, the Wilcoxon signed-rank test was applied to identify individual p-values. Statistical evaluation was computed using SPSS 20.0 (SPSS

Inc., Chicago, IL, USA) and GraphPad PrismÒ (GraphPad Software, La Jolla, CA, USA). The significance level was set at p ≤ 0.05.

Results Experimentally induced gingival inflammation

Seventeen participants (10 female and seven male) aged between 20 and 33 years (mean 24.53  3.41 years) were examined and all of them completed the study. No adverse effects were reported during the 14 d period of the study. The clinical outcome variables of all measurements regarding PI, GI, gingival crevicular fluid and BOP at baseline and days 1, 3, 5, 7 and 14 are presented in Fig. 1. Contemplating the differences between baseline and day 14, the PI scores (Fig. 1) significantly increased during the experimental gingivitis period from 0.0 (median; minimum: 0.0; maximum: 1.17) to 2.23 (median; minimum: 0.71; maximum: 3.0) (p < 0.0001). The GI values significantly increased from 0.0 (median; minimum: 0.0; maximum: 0.14) at baseline to 1.14 (median; minimum: 0.0; maximum: 2.43) at day 14 (p < 0.0001). The differences between the gingival crevicular fluid volumes at baseline (median: 3.0 PU; minimum: 0.0 PU; maximum: 48 PU) and day 14 28.0 PU (median; minimum: 2.0 PU; maximum: 108.0 PU) were statistically significant (p < 0.0001). At baseline, the BOP values were 11.0% (median; minimum: 0.0%; maximum: 32.0%) and significantly increased to 36.0% (median; minimum: 14.0%; maximum: 57.0%) at day 14 (p < 0.0001). The clinical values for PI, GI, gingival crevicular fluid and BOP increased in a timedependent manner (Fig. 1A–D). Human beta-defensin-2 and interleukin-8 concentration in gingival crevicular fluid

The hBD-2 and IL-8 concentrations were determined in gingival crevicular fluid at baseline and days 1, 3, 5 and 7 by enzyme-linked immunosorbent

6

Dommisch et al.

A

B

C

D

Fig. 3. In vivo protein expression of hBD-2, CCL20, pso/S100A7 and IL-8 in gingival epithelial tissue (n = 17). (A) hBD-2 expression scores; (B) CCL20 expression scores; (C) pso/ S100A7 expression scores; (D) IL-8 expression scores. Box plots represent median, 25 and 75 percentiles, minimum, and maximum values. *p ≤ 0.044; **p ≤ 0.0089; ***p ≤ 0.001; ****p < 0.0001. BL, baseline; CCL20, CC-chemokine ligand 20; hBD-2, human betadefensin-2; IL, interleukin; pso/S100A7, psoriasin.

gingivitis was applied (33). The termination of oral hygiene procedures led to an accumulation of bacterial deposits on the tooth surfaces and soft tissues with a concomitant increase of the bleeding frequency and gingival sulcus fluid volume, which are indicative of a local inflammatory host response (34,35). Contrary to cross-sectional clinical studies, the experimental gingivitis model comprises several advantages (33). Among others, it is possible to control tightly risk factors that may affect plaque formation or inflammatory host responses. Further, it is possible to document accurately the association between bacterial biofilms and clinical inflammatory reactions by sensitive clinical parameters (36–38).

In this study, only young, healthy, non-smoking individuals were included and the inflammatory reaction was a consequence of the lack of oral hygiene procedures, which was indicated by several clinical parameters. For the present study, biopsies were taken and analyzed by immunohistochemistry, which is a suitable method to reveal the cellular source of protein synthesis in tissues. For hBD-2, it was found that its expression was detectable at baseline and with an increasing immunohistological score at 3, 7 and 14 d. Despite the fact that all volunteers presented clinically with healthy gingival tissue and meticulous oral hygiene, as they were all dental students, the expression of hBD-2 was

detectable even at baseline. For human skin, it was shown that hBD2 is only expressed in the cause of inflammatory reactions, but not in healthy skin (12). The detection of hBD-2 protein in gingival epithelial cells supports earlier reports on hBD-2 mRNA expression in healthy gingival tissues, however, without indicating the cellular source of the AMP (39,40). In addition, the expression of CCL20 and pso/ S100A7 was found at baseline, and it slightly increased over time. All three AMPs showed high immunohistological scores at day 14 when the biofilm became mature, and volunteers showed signs of gingival inflammation. These data indicate that hBD-2 is highly expressed at earlier time points during the development of gingivitis when compared to the expression of CCL20 and pso/ S100A7. In cell culture experiments, it was shown that both AMPs, hBD2 and CCL20, were upregulated in response to the combined stimulation with the commensal Streptococcus gordonii and pathogen P. gingivalis (22). It may be suggested that interactions between the commensal bacterial flora and the epithelial cells are accompanied by the transient synthesis of AMPs during phases of no obvious clinical signs of inflammation in the oral cavity, supporting the concept of immune tolerance to commensals. In this context, it may be speculated that the increased expression of AMPs, such as CCL20, which bind to receptors on immune cells, not only fight the bacterial infection, but also initiate adaptive immune responses via, e.g. the activation of immature dendritic cells (17,41,42). In a long-term perspective, this mechanism may facilitate the dysbiotic biofilm development, and subsequently progression of periodontal inflammation when the biofilm remains untreated (42,43). In vitro it has been shown that hBD-2 chemoattracts macrophages (44), and additionally, both hBD-2 and CCL20 activate immature dendritic cells via the cell surface receptor CCR6 (17,41). In general, besides

Gingivitis and antimicrobial peptides A

B

C

D

E

F

7

Fig. 4. Immunohistological staining for the human beta-defensin-2 antibody. (A) Expression at baseline; (B) day 1; (C) day 3; (D) day 5; (E) day 7; and (F) day 14. Immune reaction for the human beta-defensin-2 antibody was pronounced after 3 d and remained at this increased level over time, and its expression was found mainly in epithelial cells, but also in fibroblasts from connective tissue. Representative slides from volunteer 6. Scale bar represents 100 lm.

their antimicrobial activity, AMPs are involved in a number of different cell, tissue and/or immune processes (45). It is conceivable that increasing synthesis of AMPs, such as hBD-2 and CCL20, may elevate the tissue level to a certain point where AMPs not only show antimicrobial but also mediatorlike activity. For gingival epithelial tissues, AMPs and IL-8 are synthetized by different cellular sources. In-vitro studies showed that gingival epithelial cells and gingival fibroblasts express hBD-2, CCL20 and IL-8, whereas expression of pso/S100A7 has been found in gingival epithelial cells only (5,21–23). In this study, immune reactions were detected for antibodies against hBD-2, CCL20 and IL-8 in epithelial cells as well as gingival fibroblasts embedded in the subepithelial connective tissue. In contrast, immune reactions using anti-pso/

S100A7 revealed localized expression in gingival epithelial cells, but not gingival fibroblasts. In gingival crevicular fluid, the concentration of the hBD-2 peptide was higher at days 1, 3 and 5 when compared to the level at baseline. To the best of our knowledge, this study shows increased hBD-2 levels in the early stages of gingival inflammation (gingivitis) for the first time. For aggressive and chronic periodontitis as well as for smokers, low hBD-2 levels have been described (46–48). The data from the present study suggest that an increased hBD-2 concentration may only be detectable at time points as early as in the first 24–120 h of gingival inflammatory reaction when measured in gingival crevicular fluid. With increasing inflammation, the hBD-2 concentration decreased to the level at baseline after 7 d, which underlines the role of AMPs as a major factor in

the maintenance of a healthy oral homeostasis (8). In contrast, decreasing hBD-2 levels may be due to degradation by bacterial proteases. Parallel to the synthesis of AMPs, the epithelial expression of IL-8 increased over time. Compared to IL-8 levels measured in gingival crevicular fluid, the epithelial level appeared rather low. This may indicate that neutrophils are the main source of the produced IL-8 during early gingival inflammation, and gingival epithelial cells slightly contribute to the generally increased IL-8 level in gingival crevicular fluid. High IL-8 levels were reported in samples from patients with gingival and periodontal inflammation (49–51), and increasing IL-8 synthesis has been demonstrated during experimental gingivitis studies (25,26). As the expression of IL-8 has been shown in similar studies, the presence of IL-8 was determined as an internal control

6

Dommisch et al.

A

B

C

D

Fig. 3. In vivo protein expression of hBD-2, CCL20, pso/S100A7 and IL-8 in gingival epithelial tissue (n = 17). (A) hBD-2 expression scores; (B) CCL20 expression scores; (C) pso/ S100A7 expression scores; (D) IL-8 expression scores. Box plots represent median, 25 and 75 percentiles, minimum, and maximum values. *p ≤ 0.044; **p ≤ 0.0089; ***p ≤ 0.001; ****p < 0.0001. BL, baseline; CCL20, CC-chemokine ligand 20; hBD-2, human betadefensin-2; IL, interleukin; pso/S100A7, psoriasin.

gingivitis was applied (33). The termination of oral hygiene procedures led to an accumulation of bacterial deposits on the tooth surfaces and soft tissues with a concomitant increase of the bleeding frequency and gingival sulcus fluid volume, which are indicative of a local inflammatory host response (34,35). Contrary to cross-sectional clinical studies, the experimental gingivitis model comprises several advantages (33). Among others, it is possible to control tightly risk factors that may affect plaque formation or inflammatory host responses. Further, it is possible to document accurately the association between bacterial biofilms and clinical inflammatory reactions by sensitive clinical parameters (36–38).

In this study, only young, healthy, non-smoking individuals were included and the inflammatory reaction was a consequence of the lack of oral hygiene procedures, which was indicated by several clinical parameters. For the present study, biopsies were taken and analyzed by immunohistochemistry, which is a suitable method to reveal the cellular source of protein synthesis in tissues. For hBD-2, it was found that its expression was detectable at baseline and with an increasing immunohistological score at 3, 7 and 14 d. Despite the fact that all volunteers presented clinically with healthy gingival tissue and meticulous oral hygiene, as they were all dental students, the expression of hBD-2 was

detectable even at baseline. For human skin, it was shown that hBD2 is only expressed in the cause of inflammatory reactions, but not in healthy skin (12). The detection of hBD-2 protein in gingival epithelial cells supports earlier reports on hBD-2 mRNA expression in healthy gingival tissues, however, without indicating the cellular source of the AMP (39,40). In addition, the expression of CCL20 and pso/ S100A7 was found at baseline, and it slightly increased over time. All three AMPs showed high immunohistological scores at day 14 when the biofilm became mature, and volunteers showed signs of gingival inflammation. These data indicate that hBD-2 is highly expressed at earlier time points during the development of gingivitis when compared to the expression of CCL20 and pso/ S100A7. In cell culture experiments, it was shown that both AMPs, hBD2 and CCL20, were upregulated in response to the combined stimulation with the commensal Streptococcus gordonii and pathogen P. gingivalis (22). It may be suggested that interactions between the commensal bacterial flora and the epithelial cells are accompanied by the transient synthesis of AMPs during phases of no obvious clinical signs of inflammation in the oral cavity, supporting the concept of immune tolerance to commensals. In this context, it may be speculated that the increased expression of AMPs, such as CCL20, which bind to receptors on immune cells, not only fight the bacterial infection, but also initiate adaptive immune responses via, e.g. the activation of immature dendritic cells (17,41,42). In a long-term perspective, this mechanism may facilitate the dysbiotic biofilm development, and subsequently progression of periodontal inflammation when the biofilm remains untreated (42,43). In vitro it has been shown that hBD-2 chemoattracts macrophages (44), and additionally, both hBD-2 and CCL20 activate immature dendritic cells via the cell surface receptor CCR6 (17,41). In general, besides

Gingivitis and antimicrobial peptides developing biofilm in vivo. All AMPs investigated were found in clinically non-inflamed tissues supporting the hypothesis that “clinically healthy gingiva appears to deal with microbial challenges without progressing to a diseased state” (55). With maturing biofilms, the epithelial synthesis of AMPs increased potentially to maintain a healthy homeostasis. It was found that hBD-2 was upregulated during earlier stages of the inflammatory response when compared to CCL20 and pso/S100A7 highlighting different temporal antimicrobial and mediator actions. This study revealed the complexity of the epithelial response to initial phases of biofilm maturation that may open new diagnostic, preventive, as well as, therapeutic avenues.

Acknowledgements The authors kindly thank Mrs. I. Bay, Mrs. J. Eich and Ms. D. Lalaouni, for their excellent technical assistance. H.D. and S.J. were financially supported by the German Research Foundation (Clinical Research Unit 208, Deutsche Forschungsgemeinschaft, DFG). J.E. was funded by the intramural HILF funding of Hannover Medical School.

3.

4.

5.

6.

7.

8.

9.

10.

Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. 1. Negative controls using only the secondary anti-rabbit antibody Dianova (A) and DAKO EnVision (B). Fig. 2. Immunohistological staining for the CCL-20 antibody. Fig. 3. Immunohistological staining for the IL-8 antibody.

References 1. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 2005;43:5721–5732. 2. Ramberg P, Sekino S, Uzel NG, Socransky S, Lindhe J. Bacterial colonization

11.

12.

13.

14.

15.

during de novo plaque formation. J Clin Periodontol 2003;30:990–995. Clavel T, Haller D. Molecular interactions between bacteria, the epithelium, and the mucosal immune system in the intestinal tract: implications for chronic inflammation. Curr Issues Intest Microbiol 2007;8:25–43. Doss M, White MR, Tecle T, Hartshorn KL. Human defensins and LL-37 in mucosal immunity. J Leukoc Biol 2010;87:79–92. Eberhard J, Menzel N, Dommisch H, Winter J, Jepsen S, Mutters R. The stage of native biofilm formation determines the gene expression of human b-defensin2, psoriasin, ribonuclease 7 and inflammatory mediators: a novel approach for stimulation of keratinocytes with in situ formed biofilms. Oral Microbiol Immunol 2008;23:21–28. Glaser R, Harder J, Lange H, Bartels J, Christophers E, Schroder JM. Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection. Nat Immunol 2005;6:57–64. Harder J, Schroder JM. RNase 7, a novel innate immune defense antimicrobial protein of healthy human skin. J Biol Chem 2002;277:46779–46784. Chung WO, Dommisch H, Yin L, Dale BA. Expression of defensins in gingiva and their role in periodontal health and disease. Curr Pharm Des 2007;13:3073–3083. Bonass WA, High AS, Owen PJ, Devine DA. Expression of beta-defensin genes by human salivary glands. Oral Microbiol Immunol 1999;14:371–374. Mathews M, Jia HP, Guthmiller JM et al. Production of b-defensin antimicrobial peptides by the oral mucosa and salivary glands. Infect Immun 1999;67: 2740–2745. Sahasrabudhe KS, Kimball JR, Morton TH, Weinberg A, Dale BA. Expression of the antimicrobial peptide, human b-defensin 1, in duct cells of minor salivary glands and detection in saliva. J Dent Res 2000;79:1669–1674. Harder J, Bartels J, Christophers E, Schroder JM. A peptide antibiotic from human skin. Nature 1997;387:861. Ouhara K, Komatsuzawa H, Yamada S et al. Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, b-defensins and LL37, produced by human epithelial cells. J Antimicrob Chemother 2005;55:888–896. Dunsche A, Acil Y, Dommisch H, Siebert R, Schroder JM, Jepsen S. The novel human beta-defensin-3 is widely expressed in oral tissues. Eur J Oral Sci 2002;110:121–124. Hoover DM, Boulegue C, Yang D et al. The structure of human macrophage inflammatory protein-3a/CCL20. Linking

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

9

antimicrobial and CC chemokine receptor-6-binding activities with human b-defensins. J Biol Chem 2002;277:37647– 37654. Yang D, Chen Q, Hoover DM et al. Many chemokines including CCL20/ MIP-3a display antimicrobial activity. J Leukoc Biol 2003;74:448–455. Yang D, Chertov O, Bykovskaia SN et al. b-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999;286: 525–528. Schutyser E, Struyf S, Van Damme J. The CC chemokine CCL20 and its receptor CCR6. Cytokine Growth Factor Rev 2003;14:409–426. Yang D, Chen Q, Chertov O, Oppenheim JJ. Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells. J Leukoc Biol 2000;68:9–14. Dommisch H, Chung WO, Rohani MG et al. Protease-activated receptor 2 mediates human beta-defensin 2 and CC chemokine ligand 20 mRNA expression in response to proteases secreted by Porphyromonas gingivalis. Infect Immun 2007;75:4326–4333. Dommisch H, Chung WO, Jepsen S, Hacker BM, Dale BA. Phospholipase C, p38/MAPK, and NF-jB-mediated induction of MIP-3a/CCL20 by Porphyromonas gingivalis. Innate Immun 2010;16:226– 234. Dommisch H, Reinartz M, Backhaus T, Deschner J, Chung W, Jepsen S. Antimicrobial responses of primary gingival cells to Porphyromonas gingivalis. J Clin Periodontol 2012;39:913–922. Eberhard J, Pietschmann R, Falk W, Jepsen S, Dommisch H. The immune response of oral epithelial cells induced by single-species and complex naturally formed biofilms. Oral Microbiol Immunol 2009;24:325–330. Schroder JM, Christophers E. The biology of NAP-1/IL-8, a neutrophil-activating cytokine. Immunol Ser 1992;57:387– 416. Offenbacher S, Barros SP, Paquette DW et al. Gingival transcriptome patterns during induction and resolution of experimental gingivitis in humans. J Periodontol 2009;80:1963–1982. Salvi GE, Franco LM, Braun TM et al. Pro-inflammatory biomarkers during experimental gingivitis in patients with type 1 diabetes mellitus: a proof-of-concept study. J Clin Periodontol 2010;37:9– 16. Chung WO, Dale BA. Innate immune response of oral and foreskin keratinocytes: utilization of different signaling pathways by various bacterial species. Infect Immun 2004;72:352–358.

10

Dommisch et al.

28. Krisanaprakornkit S, Kimball JR, Weinberg A, Darveau RP, Bainbridge BW, Dale BA. Inducible expression of human b-defensin 2 by Fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier. Infect Immun 2000;68: 2907–2915. 29. Krisanaprakornkit S, Weinberg A, Perez CN, Dale BA. Expression of the peptide antibiotic human b-defensin 1 in cultured gingival epithelial cells and gingival tissue. Infect Immun 1998;66:4222–4228. 30. Silness J, Loe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964;22:121–135. 31. Loe H, Silness J. Periodontal disease in pregnancy. I. prevalence and severity. Acta Odontol Scand 1963;21:533–551. 32. Lossdorfer S, Gotz W, Jager A. Immunohistochemical localization of receptor activator of nuclear factor kappaB (RANK) and its ligand (RANKL) in human deciduous teeth. Calcif Tissue Int 2002;71:45–52. 33. Loe H, Theilade E, Jensen SB. Experimental gingivitis in man. J Periodontol 1965;36:177–187. 34. Lamster IB, Oshrain RL, Celenti R, Levine K, Fine JB. Correlation analysis for clinical and gingival crevicular fluid parameters at anatomically related gingival sites. J Clin Periodontol 1991;18:272– 277. 35. Lamster IB, Oshrain RL, Celenti RS, Fine JB, Grbic JT. Indicators of the acute inflammatory and humoral immune responses in gingival crevicular fluid: relationship to active periodontal disease. J Periodontal Res 1991;26:261–263. 36. Heasman PA, Collins JG, Offenbacher S. Changes in crevicular fluid levels of interleukin-1 beta, leukotriene B4, prostaglandin E2, thromboxane B2 and tumour necrosis factor alpha in experimental gingivitis in humans. J Periodontal Res 1993;28:241–247.

37. Eberhard J, Heilmann F, Acil Y, Albers HK, Jepsen S. Local application of n-3 or n-6 polyunsaturated fatty acids in the treatment of human experimental gingivitis. J Clin Periodontol 2002;29:364–369. 38. Jepsen S, Eberhard J, Fricke D, Hedderich J, Siebert R, Acil Y. Interleukin-1 gene polymorphisms and experimental gingivitis. J Clin Periodontol 2003;30: 102–106. 39. Dale BA, Kimball JR, Krisanaprakornkit S et al. Localized antimicrobial peptide expression in human gingiva. J Periodontal Res 2001;36:285–294. 40. Dommisch H, Acil Y, Dunsche A, Winter J, Jepsen S. Differential gene expression of human beta-defensins (hBD-1, -2, -3) in inflammatory gingival diseases. Oral Microbiol Immunol 2005;20:186– 190. 41. Yang D, Biragyn A, Kwak LW, Oppenheim JJ. Mammalian defensins in immunity: more than just microbicidal. Trends Immunol 2002;23:291–296. 42. Hajishengallis G. Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response. Trends Immunol 2014;35:3–11. 43. Bartold PM, Van Dyke TE. Periodontitis: a host-mediated disruption of microbial homeostasis. Unlearning learned concepts. Periodontol 2000 2013;62:203– 217. 44. Soruri A, Grigat J, Forssmann U, Riggert J, Zwirner J. b-Defensins chemoattract macrophages and mast cells but not lymphocytes and dendritic cells: CCR6 is not involved. Eur J Immunol 2007;37:2474– 2486. 45. Lehrer RI. Primate defensins. Nat Rev Microbiol 2004;2:727–738. 46. Ertugrul AS, Sahin H, Dikilitas A, Alpaslan NZ, Bozoglan A, Tekin Y. Gingival crevicular fluid levels of human beta-defensin-2 and cathelicidin in smoker and non-smoker patients: a cross-sectional study. J Periodontal Res 2014;49:282–289. 47. Kuula H, Salo T, Pirila E et al. Human b-defensin-1 and -2 and matrix metallo-

48.

49.

50.

51.

52.

53.

54.

55.

proteinase-25 and -26 expression in chronic and aggressive periodontitis and in peri-implantitis. Arch Oral Biol 2008;53:175–186. Pereira AL, Holzhausen M, Franco GC, Cortelli SC, Cortelli JR. Human b-defensin 2 and protease activated receptor-2 expression in patients with chronic periodontitis. Arch Oral Biol 2012;57:1609–1614. Luo L, Xie P, Gong P, Tang XH, Ding Y, Deng LX. Expression of HMGB1 and HMGN2 in gingival tissues, GCF and PICF of periodontitis patients and periimplantitis. Arch Oral Biol 2011;56:1106– 1111. Kebschull M, Demmer R, Behle JH et al. Granulocyte chemotactic protein 2 (gcp-2/cxcl6) complements interleukin-8 in periodontal disease. J Periodontal Res 2009;44:465–471. Lee E, Yang YH, Ho YP, Ho KY, Tsai CC. Potential role of vascular endothelial growth factor, interleukin-8 and monocyte chemoattractant protein-1 in periodontal diseases. Kaohsiung J Med Sci 2003;19:406–415. Li J, Zhu HY, Beuerman RW. Stimulation of specific cytokines in human conjunctival epithelial cells by defensins HNP1, HBD2, and HBD3. Invest Ophthalmol Vis Sci 2009;50:644–653. Dommisch H, Winter J, Willebrand C, Eberhard J, Jepsen S. Immune regulatory functions of human beta-defensin-2 in odontoblast-like cells. Int Endod J 2007;40:300–307. Joly S, Organ CC, Johnson GK, McCray PB Jr, Guthmiller JM. Correlation between b-defensin expression and induction profiles in gingival keratinocytes. Mol Immunol 2005;42:1073–1084. Kinane DF, Berglundh T, Lindhe J. HostParasite Interactions in Periodontal Disease. In: Lindhe J, Karring T, Lang NP, eds. Clinical Periodontology And Implant Dentistry. UK: Blackwell Munksgaard, 2003:150–178.

Expression of antimicrobial peptides and interleukin-8 during early stages of inflammation: An experimental gingivitis study.

In the oral cavity, the epithelial surface is constantly exposed to a number of different microorganisms that are organized in a well-structured biofi...
884KB Sizes 0 Downloads 6 Views