Increased expression and levels of human β defensins ( hBD 2 and hBD 4 ) in adults with dental caries

© 2013 Girolamo Jose Barrera et al.; licensee University of Sarajevo Faculty of Health Studies. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. UNIVERSITY OF SARAJEVO FACULTY OF HEALTH STUDIES ABSTRACT


INTRODUCTION
Saliva plays a central role in oral health (1,2).Saliva is produced and secreted from salivary glands.Th e basic secretory units of salivary glands are clusters of cells called acini.Th ese cells secrete a fl uid that contains water, electrolytes, mucus, enzymes and antimicrobial peptides, all of which fl ow out of the acinus into collecting ducts.Antimicrobial peptides are important members of the host defense system.Th ey have a broad ability to kill microbes (3).In human the two main antimicrobial peptide families are defensins and cathelicidins.Defensins are small anti-microbial peptides act by disrupting the structure or function of microbial cell membranes, and are found in saliva and others compartments of the body (3).Evidence is accumulating that defensins play an important role in defense against pathogens and they are considered as part of innate immune response (3).Th ey have generally been considered to contribute to mucosal health; however, it is possible that these peptides can be considered biological factors that infl uence the appearance of caries.Defensins are cysteine-rich, cationic peptides with β-pleated sheet structures.Mammalian defensins are classifi ed into three subfamilies, the α-, β-and θ-defensins, which diff er in their distribution of and disulphide bonds between the six conserved cysteine residues (4).Defensins have activity against a wide variety of bacteria, fungal, and viral targets.Under optimal conditions, antimicrobial activity of defensins is observed at concentrations as low as 1-10 μg/ mL.Th e major mechanism of antimicrobial activity of all defensins is thought to occur through interaction with the membrane of the invading microbe resulting in a release of the cell contents (5).Model bacteria (Escherichia coli ML-35) that were treated by defensins became permeable to small molecules.In bacteria, permeabilization coincided with the inhibition of RNA, DNA and protein synthesis and decreased bacterial viability as assessed by the colony forming assay.Conditions that interfered with permeabilization also prevented the loss of bacterial viability, indicating that permeabilization is essential for bacterial killing (6).Th e human β defensins are widely expressed in oral tissues including gingival epithelium, salivary glands, ducts and saliva (3,4).It is known that these peptides are involved in defense against bacteria that can colonize the oral mucosa.Th e presence of defensins in saliva implies their potential role in protecting tooth structure from bacterially-induced caries.Th e hBDs have broad antimicrobial activity against oral microorganisms such as Streptococcus mutans, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans (3,5).Th e amount of hBDs expressed in saliva varies between individuals.Th is has been previously demonstrated for α-and β -defensins, histatin, and proline-rich proteins (7,8).Th is study shows that the levels of these peptides in unstimulated saliva vary greatly between individuals, even when diff erences in total salivary protein are considered.To date there are no reports of normal values of defensins in human saliva.It has been proposed that the variation in concentration of defensins in saliva could be attributed to the genetic factors.Th e genes for hBDs lie in a cluster on human chromosome 8.Several genes in this region can occur as multiple repeated copies (9).It is not well known if human with several copies of hBD2, for example, produce more defensins than other peoples.In the same way, individual diff erences in the quantity of α-and β -defensins may be genetically determined.Th ere is also data for genetically determined factors in susceptibility to caries.Some evidence suggests individual diff erences in caries experience in patients within the same family (9).Th ese individual diff erences suggest that genetic factors may play an important role in caries resistance or susceptibility.Th e purpose of this study was to determine a possible correlation between hBDs levels in saliva and caries experience in adults.We show high levels and high expression of hBDs in adults with caries experience.Our fi ndings suggest that high salivary levels of hBD2 and hBD4 may contribute to caries response.

Patients
Th is study was approved by the Institutional Review Board of the Applied Biotechnology Laboratory.Written informed consent was obtained from each participants.One hundred subjects participated in the study.Oral examinations were performed by trained calibrated clinicians using standardized procedures.All samples were obtained with informed consent.Examiners were instructed to rank subjects separately for active caries as follows: 0, caries-free group; 1, mild (one to three caries); 2, moderate (four to six caries); 3, severe (more than six caries).All patients were apparently healthy and were excluded from the study patients with a history of systemic disease or taking medications likely to infl uence periodontal health.

Collection of gingival samples
Tissue samples from caries group (n=4) were taken from interproximal sites showing redness and/or bleeding on probing, but no clinical attachment loss.Th e control specimens (caries-free group) (n=4) were collected during impacted third molar extraction surgery.All tissue samples were placed in Trizol solution and stored at -80ºC until the analysis.

Collection of saliva
Saliva samples were collected (4 to 6 ml) in a tube containing Nonidet P-40 to a fi nal concentration of 0.2% v/v.Saliva was cleared by centrifugation twice at 3000 x g for 20 min at 4°C.Total protein concentration was evaluated in the supernatant by bradford assay (10).Cleared unfractionated saliva was used for pH and buff er capacity determinations.Aliquots (200 μl) of supernatant were acid extracted by the addition of an equal volume of 1 M HCl/1% trifl uoroacetic acid overnight at 4ºC (11).Th e sample was centrifuged, and the supernatant was concentrated by vacuum evaporation and resuspended in distilled water equal to the starting sample volume (11).Acid-extracted saliva was used for immunoassay (ELISA).

pH and buffer capacity determinations
Cleared unfractionated saliva was used for pH determination with a portable pH-meter (Cole Parmer ACCUMET AB15).Th e buff er capacity was determined by titration using 1 mL of saliva, with 0.01 M HCl and after each addition of acid the change in pH was monitored up to pH 5.0.Th e buff er capacity was analyzed by ranges of pH.Th e volume of acid added to the saliva was calculated for each interval considered: initial pH-7.0,pH 7.0-6.0,and pH 6.0-5.0.Th e buff er capacity was expressed in volume (mL) of the acid added to 1 mL of saliva in the pH range considered, instead of equivalents of H.

Activity of peroxidase
Peroxidase activity was measured in the patients' saliva according to the 2-nitrobenzoic acid-thiocyanate (NBS-SCN) assay as previously described (12).Briefl y, the calorimetric change induced by the reaction between the enzyme and the substrate, Dithiobis 2-Nitrobensoic Acid (DTNB) in the presence of mercapto-ethanol, was read at a wavelength of 412 nm for 20 s.One unit of enzyme activity was defi ned as the level of enzyme activity needed to cleave 1 μmol of NBS/min at 22°C, using a molar extinction coeffi cient of 12,800.

ELISA
We coated 96-well immunoplates (MaxiSorp™; Nunc) with 100 μL of Acid-extracted saliva diluted in 0.05 mol/L carbonate buff er, pH 9.6, 4 °C, for 12 h.Subsequently, we blocked the wells with 200 μL of 1% bovine serum albumin (BSA) in PBS at room temperature for 2 h.After washing 5 times with 200 μL PBS containing 1 mL/L Tween 20, we incubated 100 μL/well with PBS containing 1% BSA and a 1:1000 dilution of anti-human BD2, BD4 or HNP4 (Santa Cruz Biotechnology) at room temperature for 2 h.Th e plates were washed 5 times with PBS containing 1 mL/L Tween 20, and wells were incubated at room temperature with 100 μL of peroxidase-coupled secondary antibody (Santa Cruz Biotechnology, cat.No. sc-2350 (for BD2 and HNP4) or cat.No. sc-2370 (for BD4) diluted to 1:5000 in PBS plus 1 mL/L Tween 20 for 30 min.Plates were washed 5 times as described above, and incubated with 100 μL of substrate (0.2M Na2H-PO4, 0.1M citric acid, 0.1% H 2 O 2 , 15mg O-phenylenediamine dihydrochloride) to each well in the dark at room temperature for 10 min.Stop solution (100 μl, 0.5M H 2 SO 4 ) was added to each well.Absorbance was measured at 405nm using a microtiter plate spectrophotometer Synergy HT (BioTek Instruments, Winooski, VT, USA).We quantifi ed hBDs by simultaneous ELISA runs using recombinant hBDs as calibrators.

Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from all tissue samples by Trizol TM (Invitrogen) as previously descried (12,13).RNA concentration and purity were measured using a spectrophotometer Synergy HT (BioTek Instruments, Winooski, VT, USA).Total RNA (1 μg) was reverse transcribed into cDNA using a commercial kit (Invitrogen Th ermoScript TM RT-PCR System), according to the manufacturer's instructions.Control reactions to check for DNA contamination were run in parallel with samples processed without reverse transcriptase.PCR was performed in a fi nal vol-ume of 25 μl containing 1 μl of the reverse transcription reaction, 50 μM deoxynucleotide triphosphates, 1.5mM MgCl2, 50mM Tris-HCl (pH 8.0), 1 IU Taq DNA polymerase and 0.2 μM each of sense and antisense hBD2 and hBD4 primers (see sequences below).PCR was performed in an Eppendorf Mastercycler STM thermocycler for 35 cycles consisting of denaturation at 94 o C (1min), annealing at 60 o C (1min) and extension at 72 o C (1min).Amplifi cation was terminated by a fi nal extension step at 72 o C for 5min.A negative control without the cDNA template was run with every assay to evaluate the overall specifi city.Th e integrity of the template RNA was checked by confi rming expression of β-actin mRNA.Th e primer sequences were: β-actin sense, CAC-GCCATCCTGCGTCGGAC; β-actin antisense, CATGCCATCCTGCGTCTGGAC; hBD2 sense, TTCCTGATGCCTCTTCCA; and hBD2 antisense, ATGTCGCACGTCTCTGA; hBD4 sense, GGCAGTCCCATAACCACATATTC; and hBD4 antisense, TGCTGCTATTAGCCGTTTCTCTT, hNP4 sense, TGCCGGCGAACAGAACTTC-GT; and hNP4 antisense, ACCGATGATGGC-GTTCCCAGC, Aliquots (10 μl) of the polymerase chain reaction products were electrophoresed on 1.5% agarose gels and stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Invitrogen TM ).Densitometric analyses were performed using the image analysis software Quantity One (Bio-Rad laboratories, Hercules, CA, USA).Briefl y, the digital image was analyzed to determine the pixel intensity of each band.Relative quantities of hBD2 and hBD4 mRNA among diff erent preparations were calculated as the ratio of the hBD2: β-actin and hBD4: β-actin pixel intensities from three independent RT-PCR experiments.Positive results were based on the presence of DNA bands of the expected size.

Caries experience
Fifty-four females and 46 males participated in the study.All subjects were between 18 and 32 years of age.Overall, the adults were healthy, with 90% having no history of disease.Oral examination showed that 15% had loose teeth.Sixty-one percent of the population reported having regular dental care.Gin-FIGURE 1. pH, Buffer capacity and oral peroxidase activity in saliva.Cleared unfractionated saliva was used for pH determination.The buffer capacity was determined by titration using 1 mL of saliva, with 0.01 M HCl and after each addition of acid the change in pH was monitored up to pH 5.0.The buffer capacity was analyzed by ranges of pH.The volume of acid added to the saliva was calculated for each interval considered: initial pH-7.0,pH 7.0-6.0,and pH 6.0-5.0.No difference in the saliva pH between the groups was noted (A).Considering the pH intervals analyzed, the buffer capacity showed no difference between the groups either in the initial interval pH -7.0 or pH 7.0-6.0.In the interval pH 6.0-5.0 the caries-free group showed a higher value than the control group (P<0.05)(B).Peroxidase activity was measured as previously described (12).The caries-free group showed significantly higher oral peroxidase activity than each of the groups with caries (*, P<0.05).

FIGURE 2.
Defensins levels in saliva as a function of caries score.Saliva was thawed and cleared by centrifugation twice at 3000 x g for 20 min at 4°C, proteins were precipitated with HCl-TCA.Defensins concentrations were determined by ELISA using anti-hBD2, anti-hBD4, or anti-hNP4 as primary antibody, as indicated in Materials and methods (12).The fi gure shows the measured concentrations of defensins expressed as μg/ml (A), and relative to salivary protein in μg/mg protein (B).hBD2, hBD4 and hNP4 concentrations in saliva, expressed as μg/ml; (C, E and G) and relative to salivary protein in μg/mg protein (D, F and H).The caries group showed signifi cantly higher hBD2 and hBD4 concentration (A and B) than each of the groups with no caries (**, P<0.01).Each assay was carried out in three independent experiments, and results are reported as mean±S.D.

Salivary analysis
Th e median protein concentration of unstimulated saliva samples (n=44) were 1.35 mg/ml (range from 0.63 to 2.67 mg/ml) for caries-free group and 1.29 mg/mL (range from 0.79 to 2.89 mg/mL) for all subjects with evidence of caries (n=56).Th e salivary protein concentration showed no correlation with age, gender, or caries score (data not shown).No diff erence in the saliva pH between the groups was noted (Figure 1A).Considering the pH intervals analyzed, the buff er capacity showed no diff erence between the groups either in the initial interval pH -7.0 or pH 7.0-6.0.In the interval pH 6.0-5.0 the caries-free group showed a higher value than the control group (P<0.05)(Figure 1B).Th e peroxidase activity was reduced for all subjects with evidence of caries when compared with the control group (P< 0.05) (Figure 1C).Finally, the levels of salivary defensins hBD2, hBD4 and hNP4 were in the μg/ml range (Figure 2A).hBD levels were also normalized to the protein concentration in whole saliva for each sample (Figure 2B).

Association between defensins and caries experience
In order to evaluate the relationship of defensins levels and caries experience in the population, we used the Kruskal-Wallis nonparametric test based on rank as previously reported (11,14).We found a signifi cant diff erence in the level of hBD2 and hBD4 among diff erent caries groups (P < 0.01).Diff erences were observed for both the median level of salivary defensins concentration (μg/ml) and salivary defensins relative to salivary protein (μg/ mg) (Figs.2C-2F).On the other hand, there was not diff erence in the level of hNP4 among diff erent group (Figs.2G and 2H).Th e median salivary levels of hBD2, hBD4 and hNP4 were 7.26 μg/ml, 4.25 μg/ml and 4.52 μg/ml respectively for the caries group (n=44) and 1.88 μ/ml (hBD2), 0.86 μg/ ml (hBD4) and 3.91 μg/ml (hNP4) for all subjects with no evidence of caries (n=56).Th e defensins value relative to total salivary protein was 3.53 μg/ mg (hBD2), 2.07 μg/mg (hBD4) and 2.22 μg/mg (hNP4) protein in the caries group and 0.96 μg/ mg (hBD2), 0.44 μg/mg (hBD4) and 1.98 μg/mg (hNP4) protein in the caries-free group (P < 0.01).hBD2 and hBD4 concentration was positively correlated with caries score (r = 0.7525 and r = 0,7201 respectively), and the correlation is signifi cant at the 0.0001 level (P < 0.0001).No correlation was found between caries level and hNP4 concentration.Additionally, semi-quantitative RT-PCR was used to de-termine whether the caries group present an increase in oral epithelial cell expression of hBD2 and hBD4 mRNA.As shown in Figure 3, in the caries group there was a signifi cantly higher expression of hBD2 and hBD4 compared with defensin levels in cariesfree group.On the other hand, there was no diff erence in the expression of alpha defensin 4 (hNP4).Together, the results demonstrate that hBD2 and Note that an increasing proportion of subjects had caries as the defensins (hBD2 and hBD4) concentration increased.hNP4 analysis showed no signifi cant differences among the population, with the same levels of hNP4 in caries and caries free group.
hBD4 were upregulated in caries group.To further examine the relationship of defensins with caries, the hBD2, hBD4 and hNP4 concentration range was evaluated in subjects with no caries compared to those with caries (Figure 4A-C).hBD2 analysis showed an increasing proportion of subjects had caries as the defensins concentration increased; 86% of the subjects with defensins levels lower than 3.0 μg/ml (n = 48) had no caries, but only 15% of the subjects with hBD2 levels greater than 3.0 μg/ml (n = 8) had no caries (Figure 4A).Similar analysis for hBD4 is shown in Figure 4B.Th e results showed the same trend with higher levels of hBD4 in the caries group than in those with no caries.On the other hand, hNP4 analysis showed no signifi cant diff erences among the population with the same levels of hNP4 in caries and caries free group (Figure 4C).

DISCUSSION
Salivary constituents are potential candidates as biological factors infl uencing caries risk.Many salivary protein components, such as glycoprotein, immunoglobulins, agglutinin, lactoferrin, and defensins are thought to have a role in defense in the oral cavity (15).Th e salivary protein concentration showed no correlation with age, gender, or caries score.According to Rudney et al. (16) a high protein concentration in the saliva contributes to greater adherence of S. mutans, the fi rst resident of dental plaque, however, in this work there was no diff erence in protein levels between caries and no caries groups.Th e mean saliva pH values of the 2 groups were similar.In literature results are confl icting with respect to saliva pH.Factors such as collection methods (sites in the oral cavity), the ages, and diet can infl uence results (17).In the range of pH 7.0-6.0 the buff er capacity of saliva of the two groups was no diff erent.In fact, the range pH 7.0-6.0constitutes the most important pH interval related to dental cavity formation, since in this range two pKs of two buff er systems are found, namely, the bicarbonate/carbonate system with a pK around 6.1 and the phosphate buff er system with a pK around 6.8.Th e presence of these two buff er systems in this range is the cause of the higher acid consumption in this pH interval.However, in the interval pH 6.0-5.0 the caries-free group showed a higher value than the control group (P<0.05).It is recommended to continue doing investigations in order to assess the levels of sodium bi-carbonate in plasma because there are evidence that the bicarbonate is the most important buff er system of the saliva (18).In the same way, as Smith suggests (19), when the concentrations of bicarbonate of sodium in plasma are high, this excess can be excreted by the salivary glands, probably by the acinar cells.On the other hand, Lamanda (20) demonstrated that salivary buff ering between pH 3.4 and 5 was not based on hydrogencarbonate and dihydrogenphosphate but rather on proteins.However, further studies have to be undertaken to identify the protein buff er components in the human salivary proteome.Th e presence of the low capacity buff er in caries group evaluated could be related to high dental caries risk, which might cause modifi cations in the acid-base physiologic homeostasis, causing a decrease of the systemic buff er system and so, of the capacity salivary buff er.Th e peroxidase activity is significantly greater in caries-free adults than in those with caries.Oral Peroxidase (OPO) is composed of two peroxidase enzymes, salivary peroxidase (SPO) and myeloperoxidase (MPO).Th e SPO secreted from the major salivary glands, mainly the parotid gland (21), contributes 80% of OPO activity, while MPO, produced by leukocytes in infl ammatory regions of the oral cavity (22).Oral peroxidase is an enzyme with antimicrobial properties, and in the mouth, it is secreted by salivary glands and catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce on oxidized form of thiocyanate.Th e product of the reaction catalyzed by peroxidase inhibits bacterial growth (23).In this investigation, the decrease of peroxidase activity observed in caries group may be linked to the increase of dental caries risk.Numerous studies have investigated the correlation among these salivary proteins and caries experience, but no studies have shown reliable association between a single salivary component and caries experience.Th e expression of defensins in saliva and throughout the oral cavity suggests that they may have a central role in protecting tooth structure from dental caries as well as protecting oral mucosa.Several reasons for this proposal are 1) Defensins have broad antimicrobial activity; 2) they stimulate the acquired immune system and could function to enhance IgA production as well as IgG production (24); 3) these defensins may function to keep overall bacteria in check and to help prevent biofi lm formation.Th us, oral defensins may provide a natural antibi-otic barrier.Th ere are several new fi ndings of this study.First, hBD2, hBD4 and hNP4 are detectable in saliva but show extensive variation in concentration between subjects (Figure 2A-B).Th e concentration of defensins (BD2 and BD4) in unstimulated saliva of adults has not been previously reported, although healthy adults had a mean value of 0.8 μg/ ml for other antimicrobial peptide such as human defensins-1 (25).Second, salivary defensins (hBD2 and hBD4) are signifi cantly greater in caries adults than in those with caries-free, however there are no diff erence in hNP4 levels between groups (Figure 2G-H).Th ird, the defensins levels found in saliva in this study are in the range of eff ective antimicrobial function, especially considering the low salt concentration in saliva and the synergistic action of the peptides.Fourth, the correlation of a salivary cationic defensins with caries experience suggests the possible protective eff ect of hBD2 and hBD4.Conversely, low levels of defensins may result in increased susceptibility to caries.In this work, salivary defensins concentrations showed large variation between individuals, with a signifi cantly higher level of salivary defensins in adults with caries.Finally, this study shows the simultaneous expression of human hBD2 and hBD4 in caries and caries free gingival tissue samples detected by semi-quantitative RT-PCR.Previous studies demonstrated the constitutive expression of hBD2 in oral tissues (26).Our analysis of gene expression in caries and caries free group showed diff erential transcriptional levels for the defensins.In samples isolated from caries group, hBD2 and hBD4 expression was at a higher level than caries-free group.Th e lower expression of hBD2 and hBD4 in caries-free group could explain the lower concentration of these antimicrobial peptides in saliva.Th e salivary levels of hBD2 and hBD4 may represent a genetically determined factor that contributes to caries susceptibility.Th e large variation in the concentration of defensins in saliva could be due to previously demonstrated polymorphisms in sequence and copy number in the genes encoding these peptides (27).Saliva is an easily available sample which can be collected noninvasively and used to measure and monitor the risk for caries (28).Th e oral cavity, which is colonized by numerous microorganisms, contains a wide selection of antibacterial peptides that play an important role in maintaining its complex eco-logical homeostasis.We have shown that adults with caries have a signifi cantly higher expression and levels of defensins (hBD2 and hBD4) based on both the RT-PCR and ELISA (Figure 2-3).Future studies could lead to development of means to enhance endogenous oral peptide expression, utilization of these peptides as therapeutics, and to a simple test for clinical evaluation of caries risk.

CONCLUSION
Salivary defensins are potential candidates as biological factors infl uencing caries response.Th e higher expression of defensins in saliva suggests that they may have a central role in protecting tooth structure from dental caries as well as protecting oral mucosa We conclude that high salivary levels and expression of beta defensins may represent a biological response of oral tissue to caries.However, these suggestions deserve further investigation.

FIGURE 3 .
FIGURE 3. Quantifi cation of differentially-expressed defensins mRNAs by RT-PCR.(A) Specifi c primers and annealing temperatures employed.(B) RT-PCRs for hBD2, hBD4 and hNP4 were carried out from gingival samples divided in two main groups: caries-free (1-4) and caries (5-8).The PCR-products were run onto 2% agarose gel electrophoresis.Control reactions without reverse transcriptase were carried out.PCR was performed in a fi nal volume of 25μl containing 1μl of the reverse transcription reaction, 50μM of dNTPs, 1.5mM MgCl2, 50mM Tris-HCl (pH 8.0), 1 IU Taq polymerase and 0.2μM each of sense and antisense primers.Specifi c PCR for a constitutively expressed gene (β-actin) was carried out as a positive control (data no shown).The relative amount of product was quantifi ed by densitometric analysis of DNA bands (C).Defensins-mRNA expression levels are shownnormalized to β-actin.Results are mean ± SEM of three independent experiments.

FIGURE 4 .
FIGURE 4. Defensins values and caries in the population.The number of subjects with no caries (open bars) compared to those with caries (fi lled bars) with hBD2 (A), hBD4 (B) and hNP4 (C) concentrations (μg/ml saliva) in the ranges indicated.Note that an increasing proportion of subjects had caries as the defensins (hBD2 and hBD4) concentration increased.hNP4 analysis showed no signifi cant differences among the population, with the same levels of hNP4 in caries and caries free group.