ABSTRACTObjective: The role of human beta-defensin-1 (HBD-1) as an antimicrobial peptide in a smoker's periodontitis is still unclear, this study aimed toanalyze the association of the levels of HBD-1 in smokers and non-smokers with chronic periodontitis.Methods: In total, 104 male subjects, 33-78 years old, were diagnosed to have chronic periodontitis in the Department of Periodontology, Oral DiseaseSpecial Clinic of the University of Indonesia. This cross-sectional study included clinical and laboratory examination. The data collected included thosefrom anamnesis, clinical examination (oral hygiene index Siller, pocket depth, clinical attachment level), and smoking status. The samples were storedat âˆ’20Â°C until testing for HBD-1 level by enzyme-linked immunosorbent assay.Results: The median (min-max) HBD-1 level in the group of mild-moderate chronic periodontitis was 57.61 (0.87-343.58) pg/ml and in the groupof severe periodontitis 15.27 (0.94-198.03) pg/ml (p=0.087). The median (min-max) HBD-1 level in non-smokers with chronic periodontitis was27.82 (0.92-200.58) pg/ml, and in smokers 25.04 (0.87-343.58) pg/ml (p=0.457).Conclusion: There were no significant differences in the HBD-1 levels in subjects with chronic periodontitis regardless of the smoking status orseverity of the disease.Keywords: Periodontitis, Î²-defensin, Smokers

Auerkari, Elza Ibrahim

Lessang, Robert

Masulili, Sri Lelyati C

Soeroso, Yuniarti

Sulijaya, Benso

English

Innovare Academic Sciences: E-Journals

Online - 2455-3891 Print - 0974-2441Vol 9, Suppl. 1, 2016THE HUMAN BETA-DEFENSIN-1 LEVEL FROM SMOKERS WITH CHRONIC PERIODONTITISBENSO SULIJAYA1, SRI LELYATI C MASULILI1, ROBERT LESSANG1, YUNIARTI SOEROSO1,  ELZA IBRAHIM AUERKARI2*1Department of Periodontology, Faculty of Dentistry, University of Indonesia, Jakarta 10430, Indonesia, 2Department of Oral Biology, Faculty of Dentistry, University of Indonesia, Jakarta, Indonesia. Email: eauerkari@yahoo.comReceived: 13 May 2016, Revised and Accepted: 24 May 2016ABSTRACTObjective: The role of human beta-defensin-1 (HBD-1) as an antimicrobial peptide in a smoker’s periodontitis is still unclear, this study aimed to analyze the association of the levels of HBD-1 in smokers and non-smokers with chronic periodontitis.Methods: In total, 104 male subjects, 33-78 years old, were diagnosed to have chronic periodontitis in the Department of Periodontology, Oral Disease Special Clinic of the University of Indonesia. This cross-sectional study included clinical and laboratory examination. The data collected included those from anamnesis, clinical examination (oral hygiene index Siller, pocket depth, clinical attachment level), and smoking status. The samples were stored at −20°C until testing for HBD-1 level by enzyme-linked immunosorbent assay.Results: The median (min-max) HBD-1 level in the group of mild-moderate chronic periodontitis was 57.61 (0.87-343.58) pg/ml and in the group of severe periodontitis 15.27 (0.94-198.03) pg/ml (p=0.087). The median (min-max) HBD-1 level in non-smokers with chronic periodontitis was 27.82 (0.92-200.58) pg/ml, and in smokers 25.04 (0.87-343.58) pg/ml (p=0.457).Conclusion: There were no significant differences in the HBD-1 levels in subjects with chronic periodontitis regardless of the smoking status or severity of the disease.Keywords: Periodontitis, β-defensin, Smokers.INTRODUCTIONBased on the National Health Survey in Indonesia 2010, periodontal disease took the second place of the dental health problems, with 42.8% of the population in Indonesia [1]. Chronic periodontitis occurs as a result of the host response to the aggregation of bacteria on the tooth surface. The disease is characterized with absolute periodontal pockets and alveolar bone damage [2]. Any involvement of systemic factors such as diabetes, smoking, and stress will accelerate the progress of periodontal disease because of changes in the host response to the accumulation of plaque [2].Smoking is a known risk factor of periodontal disease [3]. Smoking periodontal patients show a more extensive alveolar bone and tooth loss compared with non-smoking patients [4]. The severity of the disease depends on the intensity and duration of smoking [4]. Cigarette smoke will modulate the immune response of the host to the bacterial plaque, indicated by aggressive periodontal tissue destruction [4].In response to the invasion of bacteria, the body will synthesize pro-inflammatory cytokines such as interleukin (IL)-1, IL-8, tumor necrosis factor-α, and also antimicrobial peptides such as human beta-defensin-1 (HBD-1) [4]. HBD-1 is an antimicrobial molecule that acts in the innate immune system and provides signaling for the adaptive immune system [5,6]. The role of HBD-1 is to pull the immature dendritic cells, T cells (memory) CD4/CD45RO, and CD8 T cells by binding to chemokine receptors, namely, CCR6 that will facilitate the destruction of Antigen (Ag) by making a “defensin-Ag” complex [7]. The expression of HBD-1 has been seen in the gingival epithelium, buccal mucosal epithelium, pulp and also in glandular salivary tissue [8]. Many studies suggest that HBD-1 is keeping the homeostasis of microbial pathogens by preventing bacterial colonization and viral infection [8]. HBD-1 is expressed in infected epithelial tissue, but also plays a role in wound healing and tumor suppression [9]. HBD-1 is expected to be a potential tool for therapeutic approaches and to stimulate wound healing [9]. In particular, HBD-1 has a large effect on the proliferation and differentiation of osteoblasts. HBD-1 has also been suggested as a vaccine against HIV-1 as well as a diagnostic biomarker for cancer [9]. There is a controversy in the expression of HBD-1 in inflamed gingival tissue and healthy tissue [6]. Immunohistochemical testing shows the presence of HBD-1 on inflamed marginal and epithelial sulcus exposed to dental microbial plaque [6]. There was an improvement regulation of HBD-1 in inflamed epithelium [10].The present research aimed to analyze the association of the levels of HBD-1 in smokers and non-smokers with chronic periodontitis. Subjects with chronic periodontitis and smoking will show symptoms of inflammation in periodontal tissues, as well as increased levels of HBD-1 in the oral epithelial lining of the periodontal tissues. The selection of sampling to detect HBD-1 from the gingival epithelium was done from periodontal lesions on mucosal areas exposed to tobacco smoke. As the treatment of periodontal disease in smokers can be challenging, it is worthwhile to explore the potential benefits of HBD-1 as inflammation biomarker and antimicrobial agent.METHODSSubjectsIn total, 104 male subjects, 33-78 years old, were diagnosed with chronic periodontitis in the Department of Periodontology, Oral and Dental Special Clinic of the University of Indonesia. The subjects had signed the written declaration of informed consent and had not received any periodontal treatment before. An anamnesis, clinical examination (oral hygiene index Siller, pocket depth, clinical attachment level (CAL)), and survey of smoking status were conducted on each subject. Tissue sampling is taken from the gingival epithelium. It was following the procedures with ethical approval from the Ethics Committee of the Faculty of Dentistry, University of Indonesia. The sampling was conducted before the subjects received any kind of periodontal therapy, from the pockets of chronic periodontitis with pocket depth <5 mm Research ArticleAsian J Pharm Clin Res, Vol 9, Suppl. 1, 2016, 174-176 Sulijaya et al. 175for the mild-moderate periodontitis group, and pocket depth ≥5 mm for the severe periodontitis group. Then, the samples were stored into Eppendorf tubes containing sterile phosphor buffered saline. The samples were stored at −20°C until enzyme-linked immunosorbent assay (ELISA) testing.ELISA protocolAbout 76 samples were selected by consecutive sampling. HBD-1 ELISA kit, pink-ONE, KOMA BIOTECH INC (Korea) was used in this study. For the ELISA procedure, the samples were thawed at room temperature. Each 100 mL of samples and standards were inserted into the well plate and incubated at room temperature for 2 hrs, and then aspirated well to dispense fluid and to wash the well. A 100 mL batch of diluted detection antibody (0.5 mg/ml) was added per well and incubated at room temperature for 2 hrs, aspirated back and rinsed. Add 100 mL diluted color development enzyme (1:20 dilution) per well, and incubate for 30 minutes at room temperature (37°C for 30 minutes). Aspirate back and the rinse. For the last, added 100 mL pink-ONE TMB color development reagent to each well, incubated for 1-8 minutes to form a color changing, add 100 mL stop solution to stop the color changing happens. Then, well plate is read by the plate reader at a wavelength of 450 nm. Once the well plate is inserted into the plate reader (Accu Reader) and it will show the optical density and the concentration of HBD-1 from each well.Statistical analysisData were analyzed with SPSS version 20. The Kolmogorov-Smirnov was used to test normality. For normally distributed data, the independent t-test can be applied; otherwise, the Mann-Whitney test is selected.RESULTSAll research subjects were diagnosed to have chronic periodontitis. The age is ranging from 33 until 78 years old, with a mean of 51.17 years and standard deviation of 9.8 years.Table 1 shows a balanced number of subjects between mild-moderate and severe levels of periodontitis. Considering the smoking status, the number of smokers is slightly higher than non-smokers. The distribution of mean CAL on clinical examination was 5.54±2.32 mm with a minimum of two and a maximum of 12 mm.Table 2 shows the results of normality testing. The distribution of HBD-1 in the mild-moderate group appears normal (p=0.09), but the levels of HBD-1 in severe periodontitis or the levels of HBD-1 in smokers or non-smokers are not normally distributed. Nonparametric Mann-Whitney test was applied to determine the association of the levels of HBD-1, severity of periodontitis, and smoking status (Tables 3 and 4).The mean levels of HBD-1 in mild-moderate periodontitis group were higher (69.97±71.76 pg/ml) than the severe periodontitis group (40.50±53.37 pg/ml), although the difference was not indicated as statistically significant.The mean and median levels being fairly similar, it appears that there is no significant difference in the levels of HBD-1 between smokers and non-smokers (p=0.457).DISCUSSIONIn this study, no significant difference was found in the levels of HBD-1 between mild-moderate periodontitis and severe periodontitis groups (p=0.087). This is consistent with the findings of Bissell et al. (2004) suggesting that the differences in the levels of HBD-1 between healthy gingival and pathologic conditions were not significant [11]. A possible reason is that HBD-1 can activate the expression of pro-inflammatory mediators in infected areas, whereas in healthy areas there is also a pro-inflammatory mediator expressed as a growth factor. Another reason for the HBD-1 levels appearing not significantly different could be the additional genetic variation of the HBD-1 levels. Yet another possibility for the pathological condition is that HBD-1 is degraded by cathepsin cysteine proteases to become inactivated. When an infection occurs, then an increase of cathepsin will cause the degradation of HBD-1 directly, leading to bacterial colonization and infection. Bissell et al. (2004) also noted that there is no association of the expression of HBD-1 with age and sex [11]. On the other hand, the results of Lu et al. (2004) showed that increased HBD-1 expression in the walls of the pocket compared to healthy tissue (p<0.05) [6]. Inflammatory mediators would affect the expression of HBD-1. Increased IL-4 and IL-13 levels will inhibit HBD-2, whereas IL-1 will stimulate both HBD-1 and HBD-2 [6]. Sengul et al. (2007) found low regulation of HBD-1 in gingivitis and high regulation of HBD-1 in chronic periodontitis [12]. In this study, the level of HBD-1 was positively observed in all samples, but not comparing the condition of gingivitis to periodontitis.The potential of HBD-1 as a biomarker of periodontal inflammation is still not fully supported by the evidence. Winter et al. (2012) argue that the HBD-1 will increase the proliferation and migration of keratinocyte cells and has a positive effect on wound closure [9]. Studies by Bissell et al. (2004) found elevated levels of HBD-1 in healthy tissue (p>0.50) [11], while Sengul et al. (2007) found no increase of Table 1: Subject distribution based on severity and smoking statusSubject n (76) Percentage (100)PeriodontitisMild-moderate 38 50Severe 38 50Smoking statusSmokers 39 51.3Nonsmokers 37 48.7Table 2: Normality test on the level of human beta‑defensin‑1, severity of periodontitis, smoking statusVariable pHuman beta-defensin-1 levelMild-moderate 0.09*Severe 0.00Human beta-defensin-1 levelSmokers 0.00Nonsmokers 0.00Kolmogorov-Smirnov test; *p>0.05: Normal data distributionTable 3: Median, mean, and standard deviation of the level of human beta‑defensin‑1 with the severity of periodontitisHuman beta defensin‑1n Median (pg/ml)(minimum‑maximum)Mean±SD(pg/ml)pChronic periodontitisMild-moderate 38 57.61 (0.87-343.58) 69.97±71.76 0.087Severe 38 15.27 (0.94-198.03) 40.50±53.37Mann-Whitney U-test; p<0.05 significant difference, SD: Standard deviationTable 4: Median, mean and standard deviation of the level of human beta‑defensin‑1 with smoking statusHuman beta‑defensin‑1n Median (pg/ml)(minimum‑maximum)Mean±SD(pg/ml)pChronic periodontitisSmokers 39 25.04 (0.87-343.58) 52.12±69.30 0.457Nonsmokers 37 27.82 (0.92-200.58) 58.51±59.90Mann-Whitney U-test; p<0.05 significant difference, SD: Standard deviationAsian J Pharm Clin Res, Vol 9, Suppl. 1, 2016, 174-176 Sulijaya et al. 176HBD-1 in chronic periodontitis group compared with control group (p<0.001) [12]. Lu et al. (2004) argue that HBD-1 is increased in the epithelium pocket compared with healthy tissue [6]. The differences in outcome are likely to arise from the differences of the severity of periodontitis, research methods and possible genetic variations related to the expression of HBD-1. This study could not confirm the HBD-1 as a biomarker of inflammation because this study did not compare the pathological condition with a healthy one. The study is limited only to analyze the association of HBD-1 with the severity of periodontitis and smoking status.In this study, there was no significant difference in the levels of HBD-1 in smokers and non-smokers (p=0.457). This is not in accordance with the results of in-situ studies by Mahanonda et al. (2009) who found that cigarette extracts can reduce the expression of HBD [4]. Cigarette smoke is known contain carbon monoxide which will directly reduce the oxygen levels. The body will compensate the oxygen drop by vasoconstriction of blood vessels, which in turn will reduce the host response to inflammation. Smokers have been shown to have a risk of alveolar bone loss more severe than non-smokers [13]. Thaper et al. (2016) in his discussion also support that smoking could increase the risk of periodontitis about 10 times greater in systemic disease. [14]CONCLUSIONIn this study, no significant association was found between the levels of HBD-1 and the severity of chronic periodontitis or the smoking status. The author found a slightly higher level of HBD-1 in non-smokers than in smokers, but it was not statistically significant. The HBD-1 levels in mild-moderate periodontitis group were also higher than the severe periodontitis group, but this result was again not statistically significant.ACKNOWLEDGMENTSThe authors wish to gratefully acknowledge the financial support from the Ministry of Education and Culture of Republic of Indonesia through Directorate Research and Community Services, University Indonesia.REFERENCES1. Kasim E. Smoking as risk factor of periodontal disease. J Kedokter Trisakti 2001;19(1):1-7.2. Newman MG, Takei HH, Klokkevold PR, Carranza FA. Chronic periodontitis. In: Novak MJ, Novak KF, editors. Carranza’s Clinical Periodontology. 11th ed. St. Louis, Missouri: Elsevier; 2012. p. 160-76.3. Romao C, Wennström JL. Periodontal conditions in a Swedish city population of adolescents: A comparison between smokers and never-smokers. Oral Health Prev Dent 2007;5(2):105-12.4. Mahanonda R, Sa-Ard-Iam N, Eksomtramate M, Rerkyen P, Phairat B, Schaecher KE, et al. Cigarette smoke extract modulates human beta-defensin-2 and interleukin-8 expression in human gingival epithelial cells. J Periodontal Res 2009;44(4):557-64.5. Weinberg A, Jin G, Sieg S, McCormick TS. The yin and yang of human Beta-defensins in health and disease. Front Immunol 2012;3:294.6. Lu Q, Jin L, Darveau RP, Samaranayake LP. Expression of human beta-defensins-1 and -2 peptides in unresolved chronic periodontitis. J Periodontal Res 2004;39(4):221-7.7. van Dijk A, Veldhuizen EJ, Haagsman HP. Avian defensins. Vet Immunol Immunopathol 2008;124(1-2):1-18.8. Diamond G, Ryan L. Beta-defensins: What are they really doing in the oral cavity? Oral Dis 2011;17(7):628-35.9. Winter J, Wenghoefer M. Human defensins: Potential tools for clinical applications. Polymers 2012;4(1):691-709.10. Ganz T. Defensins: Antimicrobial peptides of innate immunity. Nat Rev Immunol 2003;3(9):710-20.11. Bissell J, Joly S, Johnson GK, Organ CC, Dawson D, McCray PB Jr, et al. Expression of beta-defensins in gingival health and in periodontal disease. J Oral Pathol Med 2004;33(5):278-85.12. Vardar-Sengul S, Demirci T, Sen BH, Erkizan V, Kurulgan E, Baylas H. Human beta defensin-1 and -2 expression in the gingiva of patients with specific periodontal diseases. J Periodontal Res 2007;42(5):429-37.13. Buduneli N, Biyikoglu B, Sherrabeh S, Lappin DF. Saliva concentrations of RANKL and osteoprotegerin in smoker versus non-smoker chronic periodontitis patients. J Clin Periodontol 2008;35(10):846-52.14. Thaper S, Thaper T, Priya VV, Thaper R, Thaper R. Prevalence of Periodontitis in Diabetic and Non-Diabetic Patients. Asian J Pharm Clin Res. 2016;9(1):329-31.

THE HUMAN BETA-DEFENSIN-1 LEVEL FROM SMOKERS WITH CHRONIC PERIODONTITIS

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THE HUMAN BETA-DEFENSIN-1 LEVEL FROM SMOKERS WITH CHRONIC PERIODONTITIS

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