Smoking and periodontal disease: discrimination of antibody responses to pathogenic and commensal oral bacteria - Hayman - 2011 - Clinical amp; Experimental Immunology - Wiley Online Library Free Access Smoking and periodontal disease: discrimination of antibody responses to pathogenic and commensal oral bacteria L. Hayman, Center for Oral Health Research, andSearch for more papers by this authorM. J. Steffen, Center for Oral Health Research, andSearch for more papers by this authorJ. Stevens, Center for Oral Health Research, andSearch for more papers by this authorE. Badger, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorP. Tempro, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorB. Fuller, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorA. McGuire, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorMohanad Al-Sabbagh, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorM. V. Thomas, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorJ. L. Ebersole, Corresponding Author Center for Oral Health Research, and Department of Oral Health Practice, University of Kentucky, Lexington, KY, USAJ. L. Ebersole, Center for Oral Health Research, HSRB422, College of Dentistry, University of Kentucky, Lexington, KY 40536, USA.E-mail: jleber2@uky.eduSearch for more papers by this author L. Hayman, Center for Oral Health Research, andSearch for more papers by this authorM. J. Steffen, Center for Oral Health Research, andSearch for more papers by this authorJ. Stevens, Center for Oral Health Research, andSearch for more papers by this authorE. Badger, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorP. Tempro, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorB. Fuller, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorA. McGuire, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorMohanad Al-Sabbagh, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorM. V. Thomas, Department of Oral Health Practice, University of Kentucky, Lexington, KY, USASearch for more papers by this authorJ. L. Ebersole, Corresponding Author Center for Oral Health Research, and Department of Oral Health Practice, University of Kentucky, Lexington, KY, USAJ. L. Ebersole, Center for Oral Health Research, HSRB422, College of Dentistry, University of Kentucky, Lexington, KY 40536, USA.E-mail: jleber2@uky.eduSearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary Smoking is an independent risk factor for the initiation, extent and severity of periodontal disease. This study examined the ability of the host immune system to discriminate commensal oral bacteria from pathogens at mucosal surfaces, i.e. oral cavity. Serum immunoglobulin (Ig)G antibody reactive with three pathogenic and five commensal oral bacteria in 301 current smokers (age range 21–66 years) were examined by enzyme-linked immunosorbent assay. Clinical features of periodontal health were used as measures of periodontitis. Antibody to the pathogens and salivary cotinine levels were related positively to disease severity; however, the antibody levels were best described by the clinical disease unrelated to the amount of smoking. The data showed a greater immune response to pathogens than commensals that was related specifically to disease extent, and most noted in black males. Significant correlations in individual patient responses to the pathogens and commensals were lost with an increasing extent of periodontitis and serum antibody to the pathogens. Antibody to Porphyromonas gingivalis was particularly distinct with respect to the discriminatory nature of the immune responses in recognizing the pathogens. Antibody responses to selected pathogenic and commensal oral microorganisms differed among racial groups and genders. The antibody response to the pathogens was related to disease severity. The level of antibody to the pathogens, and in particular P. gingivalis, was correlated with disease severity in black and male subsets of patients. The amount of smoking did not appear to impact directly serum antibody levels to these oral bacteria. Introduction Successful colonization of the oral cavity depends upon the presence of bacterial attachment sites on the conditioning layer derived from saliva and gingival crevicular fluid coating the oral hard and soft tissues surfaces 1 and microbial accumulation by autogenic and allogenic succession. Initial bacterial colonization by pioneering microorganisms alters the environment and enhances subsequent colonization by species more suited for the new environment (autogenic succession). Allogenic succession also occurs with environmental changes driven by a factor(s) other than those derived from the pioneer microorganisms, including those host-controlled factors 2, 3. The resulting microbial communities or biofilms are complex ecosystems of bacteria that develop over time and are somewhat unique to various ecological niches 2, 4, 5. The ecology in an individual evolves over time at the level of the quantity and quality of phyla, genera and species 6-8, as well as the genomic profile of the individual species 9-12. However, this evolution generally leads to equilibrium between the microbiota and the environment as a climax community. Climax biofilm communities are thought to be unique to each individual and ecological niche in the oral cavity 2, 3. Microorganisms that colonize the oral soft and hard tissues and maintain a symbiotic relationship with the host are referred to as commensal bacteria 13. Pathogenic bacteria are those that are harmful to the host. Microbial biofilm communities on the subgingival tooth surfaces subjacent to the gingival tissues are composed of approximately 700 species 8, 14. The microbial ecology of the subgingival environments of periodontally healthy and periodontally diseased sites are distinct 6, 8, 14. Accumulation of tooth-associated bacterial biofilm (plaque) elicits gingival inflammation as a result of bacterial virulence factors and vascular dilation. In sites colonized by pathogen-dominated biofilms the inflammatory response results in destruction of connective tissue and alveolar bone, the classic features of periodontitis. The tissue destruction is actually a result of the host response elicited by the pathogens, rather than direct toxic/noxious actions of the bacterial virulence factors 15, 16. The immune system is comprised of both innate and adaptive immune responses that are used to manage bacterial infections. The adaptive immune response results from a cognate interaction of receptors on immune cells engaging antigens as ligands, resulting in the initiation of cell-mediated and/or humoral immune responses. Antigenic triggering of immunoglobulin receptors on B cells leads to maturation and differentiation into plasmacytes that produce antibody are the effector molecules of humoral immunity 16, 17. Important to the objectives of this project, the host oral cavity is colonized routinely by a range of commensal bacteria, as well as a varied number of potentially pathogenic species. While these bacteria all represent ‘non-self’, it remains unclear how the immune system differentiates commensals that are important to maintain for health from those bacteria with greater virulence capabilities 8. It has been suggested that the immune system has the ability to recognize commensal bacteria differently from pathogens, thus leading to a different type of immune response 13, 17, 18. However, the details of these characteristics, specifically with regard to the oral cavity, remain to be determined. Various environmental factors affect the microbial composition in the oral cavity, as well as the host response. While smoking is a well-recognized risk factor for periodontal attachment loss, smokers often exhibit less gingival bleeding than would be predicted 19. This is due probably to effects of the toxic cigarette chemicals on the local vascular functions 19, 20. Minimal data are available to compare the potential effect of smoking on the immune system discrimination of commensals from pathogenic oral bacteria. Data analysis was performed to address two central objectives for the study: (i) to determine the level of immunoglobulin (Ig)G antibody to periodontal pathogens and oral commensal bacteria in smokers; and (ii) to determine how antibody responses are affected by the extent of smoking and degree of periodontal disease. It was hypothesized that the level of IgG antibody to periodontal pathogens would be greater than that observed to oral commensal bacteria. The extent of smoking and/or periodontal disease was expected to modify this relationship (i.e. greater antibody to pathogens, lower antibody to commensals) and contribute to a greater risk of progressing periodontitis. An array of oral microorganisms were used in the assays, cultivated under standard conditions, and prepared for antigens as described previously 21. The bacteria included the proposed periodontopathogens: Aggregatibacter actinomycetemcomitans (Aa) strain JP2, Porphyromonas gingivalis (Pg) American Type Culture Collection (ATCC) 33277, Treponema denticola (Td) ATCC 35405 and a group of oral commensal bacteria that included Streptococcus sanguis (Ss) ATCC 10556, Actinomyces naeslundii (An) ATCC 49340, Prevotella loescheii (Pl) ATCC 15930, Veillonella parvula (Vp) ATCC 10790 and Capnocytophaga ochracea (Co) ATCC 33596. Full-mouth mean pocket depth (PD), measured in millimetres (mm), and bleeding on probing (BOP), measured by percentage of sites in the mouth that bleed, were determined at six sites/tooth excluding third molars 22. The measurements were taken and recorded by a single examiner. Serum from a venipuncture blood sample was obtained from a group of 301 smokers (age 21–65 years, 34 black males, 48 black females, 72 white males, 147 white females). The protocol for these studies was approved by the University of Kentucky Institutional Review Board and all participants signed an appropriate consent form. A comprehensive oral examination was completed to evaluate the presence and severity of periodontitis. The serum samples were stored at −80°C until the assays were performed. An enzyme-linked immunosorbent assay (ELISA) was used to determine the level of IgG antibody to the bacteria 22. Purified human IgG was bound to the plate to produce a standard curve. Sample data were extrapolated from this curve, using a four-parameter logistic curve fit 23. Certain comparisons were based upon disease extent/severity of the patients. Thus, the population was also stratified based upon full-mouth mean pocket depths into 3·0-mm, 3·0–4·0-mm and 4-mm groups. Additionally, to assess the relationship of antibody levels to gingival inflammation, the population was stratified into groups based upon the frequency of sites with BOP (as a dichotomous index) into groups of 20%, 20–50% and greater than 50% bleeding sites. Unstimulated saliva was collected from each individual in the sample population. Each sample was centrifuged at 1500 g and frozen at −80°C until needed for data collection. Cotinine levels were measured for each sample using a standard procedure with the Salimetrics\' High Sensitivity Salivary Cotinine Quantitative enzyme immunoassay (EIA) kit. Analyses of any differences among clinical parameters, IgG antibody levels and extent of smoking was conducted via a Kruskal–Wallis analysis of variance (anova) with post-hoc testing of paired groups using Dunn\'s method (SigmaStat; Systat Software, Inc., Richmond, CA, USA). Evaluation of the significance of correlation data was performed using Spearman\'s correlation test. Data with an alpha of 0·05 (after being adjusted for the multiple comparisons) were accepted as statistically significant. The comparisons for each parameter by race and gender are shown in Fig. 1. The black male group showed significantly greater extent and severity of destructive disease (e.g. pocket depth) and significantly greater gingival inflammation (e.g. bleeding) than any of the other patient subsets. Measures of the extent of inflammation [% of sites with bleeding on probing (BOP)] and periodontitis [full-mouth mean pocket depth (PD) and % of sites with pocket depth (PD)   4 mm or 5 mm)] in subsets of patients stratified on race and gender. Bars show the group mean values and brackets identify 1 standard deviation.Figure 2 shows that the level of salivary cotinine was increased significantly with increasing disease, although no correlation between the cotinine levels or pack-years of smoking and antibody to the pathogens, commensals or any individual microorganism was observed (data not shown). Salivary cotinine levels in the population stratified into categories based upon periodontitis severity. Bars show the mean group values and with brackets identify 1 standard deviation. The mean IgG responses to each of the oral pathogens is depicted Fig. 3. The results demonstrate higher antibody in black patients to all three pathogens when compared to levels in white patients; however, antibody to Aa and Pg were elevated significantly in black male patients compared to all other groups. Figure 3 also summarizes the serum IgG antibody response to each commensal species across the four subsets of patients based upon race and gender. Antibody levels to Pl were increased significantly in both genders of black subjects compared to the white subjects, with no difference in levels of this antibody within the black population. No significant differences were observed in response to Co, Vp, Ss or An. Interestingly, the magnitude of differences to these commensals among the groups was substantially less that the disparate responses to the pathogenic bacteria. Serum antibody levels to the pathogens: Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Treponema denticola (Td) and to the commensals: Veillonella parvula (Vp), Streptococcus sanguis (Ss), Prevotella loescheii (Pl), Actinomyces naeslundii (An) and Capnocytophaga ochracea (Co) in each subset of the population, separated by race and gender. Bars show the mean group values and brackets depict 1 standard error of the mean. The characteristics of the serum antibody responses to the oral pathogens and commensal bacteria related to the extent of periodontal disease in this population of smokers (measured by pocket depth) were also evaluated. The patients were stratified based upon their whole-mouth mean pocket depths into 3·0, 3·0–4·0 and 4·0 mm. The results in Fig. 4 present the relationship of antibody to the oral bacteria and periodontal disease using two formats. First, a significant increase in the summation (Σ) of antibody levels to the three oral pathogens (P. gingivalis, T. denticola, T. forsythia) is shown with increasing disease, with no similar increase in the sum of antibody to the five commensal bacteria. The additional graph compares the average antibody to the three pathogens and the five commensals across patient groups stratified with respect to mean pocket depth measures. In this case, the average antibody level to the pathogens was significantly greater than the antibody levels to the commensals only in the patient subgroup with full-mouth mean pocket depths consistent with periodontal disease. Figure 5 provides a summary of the antibody comparisons for the individual pathogenic and commensal bacteria as disease-related severity. It was clear that antibody to P. gingivalis differed significantly with increasing disease, manifest in the response differences to the pathogens. No significant differences were noted with any of the commensal bacteria. Serum antibody levels in patients grouped according to mean pocket depth. The comparisons are made using summed (Σ) antibody across all tested pathogenic bacteria (Path) and all oral commensal bacteria (Comm) or the average antibody to the pathogens or commensals. Bars show the mean group values and brackets depict 1 standard deviation. Summary of antibody comparisons across each individual pathogenic or commensal bacterial species (see Fig. 3 legend) with patients grouped based upon whole-mouth mean pocket depth. Bars show the mean group values and brackets depict 1 standard error of the mean. A fundamental question that was to be addressed was whether this smoking population with varying levels of oral disease responded differently to putative periodontal pathogens compared to members of the commensal oral microbiota. As such, we compared the average antibody response of each patient subset to the pathogens and commensals (Fig. 6). The results show a trend of greater responses to the pathogenic bacteria in each patient subset based on race and gender, with statistically significant elevations to the pathogens in black males reflective of the more severe disease in this group. Average serum antibody levels to pathogens and commensals in patients stratified by race and gender. Bars show the mean group values and brackets depict 1 standard deviation.Figure 7 displays the correlation characteristics between the sum of antibody to the pathogens and the sum of antibody to the commensals in each patient and demonstrates a significant positive correlation across the population. Thus, the data were analysed to identify relationships among these IgG responses and clinical parameters, focusing upon pocket depth as a measure of tissue destructive processes and BOP as an indicator of the magnitude of gingival inflammation in the individual patient. Figure 8 describes the relationship of antibody to the pathogenic and commensal bacteria stratified into subsets based upon the extent of inflammation, i.e. frequency of bleeding sites. The results show no significant differences in antibody levels to the pathogens or commensals based upon the gingival inflammation measure. Figure 9 summarizes the correlations of antibody to the pathogens and commensals in patient groups according to the mean mouth pocket depth. Correlation between the summation (Σ) of immunoglobulin G antibody levels to pathogens and commensals in the entire population. The linear regression and significance value is depicted. Relationship of serum antibody levels to pathogens and commensals with patients stratified into categories based upon frequency of sites with bleeding on probing (BOP). Bars show the mean group values and brackets depict 1 standard deviation. No statistical differences were observed in responses across the various bleeding subsets of patients. Correlations of the summation (Σ) of immunoglobulin G antibody levels to pathogens and commensals in subsets of patients stratified based upon mouth mean pocket depths. The linear regression is depicted for each subgroup. The results demonstrated positive correlations within the different disease groups although, as shown in Table 1, in the most diseased individuals the relationship of antibody to these groups of bacteria was less related than those observed in more periodontally normal patients. Additionally, the table demonstrates that stratifying the patients based upon the level of antibody to the pathogens showed a significant positive correlation in patients with low levels of antibody to the pathogens. As the patients respond with higher antibody levels to the pathogens, e.g. generally associated with more periodontal disease, the significance of the correlation of antibody between the pathogens and commensals is lost. Table 1. Correlation of antibody to pathogens and commensals related to pocket depth (PD) and level of antibody to the pathogens. Low antibody pathogens denotes sum of antibody to pathogens   50 µg/ml; medium antibody 50–125 µg/ml; and high antibody   125 µg/ml; n.s.: not significant. Finally, due to the antibody response to P. gingivalis providing a significant contribution to the anti-pathogen antibody profile in this population of adults, we evaluated the relationship between this specific antibody and the race and gender subsets in the population. The results in Table 2 demonstrate significant correlations between this antibody and the extent of periodontal disease described as the frequency of sites with pocket depths 5 mm. As can be seen in the table, the significant correlations were noted only in the males and black subgroups in this population. Table 2. Correlation of clinical measures of periodontal disease and antibody to Porphyromonas gingivalis. This investigation examined the relationship of smoking, periodontitis and systemic antibody responses to oral bacteria described as pathogens or commensal members of the oral microbial ecology. Based upon existing data suggesting variations in antibody responses based upon race/ethnicity and gender, antibody levels were evaluated within subsets of the patients. Black males demonstrated more severe periodontitis than the other race/gender subsets for the clinical parameters of periodontitis. This was not unexpected, based upon other literature suggesting an increased severity of disease in minority populations and in males 24, 25. Cotinine levels in saliva samples provided a measure of an individual\'s exposure, either primary or second-hand, to nicotine in cigarette smoke, although with this population the levels of cotinine in saliva were related directly to the amount of current smoking. Stratifying the patients based upon pocket depth extent, i.e. mouth mean, showed a significant increase in disease severity with increased tobacco use. Interestingly, the black males did not demonstrate higher cotinine levels that would support that smoking was the single basis for this increased oral disease. There was no obvious association between smoking status and serum antibody levels to any of the oral bacteria. These observations appear generally similar to previous studies that have examined smoking and serum antibody to oral bacteria. In these reports, smoking was suggested to modulate B cell function, and thus antibody levels to specific bacteria have been noted to be altered in smokers, particularly related to race and generalized versus localized disease 26-29. However, these reports generally limited their data comparison to antibody levels and periodontal disease in smokers versus non-smokers, with minimal examination of data linking the antibody levels to an amount of ‘smoking challenge’. We then examined this population to test the hypothesis that IgG antibody levels to periodontal pathogens differed from the response to oral commensal bacteria at the individual level, and were not related simply to the overall microbial challenge to the immune system. This was observed particularly in the population of black males, which showed a significantly higher IgG response to the pathogens than to commensal oral bacteria. Examination of antibody response profiles to individual bacteria showed that blacks had significantly higher IgG responses to Aa, Pg, Pl and Co. More specifically, black males had significantly higher antibody levels to both Aa and Pg compared to all other subsets of the population of smokers. Similar results were noted in the patients with the most severe periodontitis, who demonstrated significantly higher antibody to the pathogens than to the commensals. These results support that the host immune system may discriminate actively between pathogens and commensals, preferentially eliciting more robust antibody response to defend against oral pathogens. This interpretation is further supported because, overall, these commensal bacterial species are detected in substantially larger quantities in both healthy and periodontitis patients compared to the oral burden with the pathogens 7, 30-33. Thus, it would be predicted that if the level of antibody responses were a function of the magnitude of antigenic challenge (i.e. the portion of the bioburden due to a particular species), the antibody response to the commensal bacteria should be substantially more robust than the response to the periodontal pathogens. Stratifying the patients into disease severity groups based upon mean pocket depth demonstrated that only the sum of antibody responses to the periodontal pathogens increased significantly with severity of periodontal disease, while the response to the commensals was similar across the disease groups. Additionally, comparing the antibody responses to the pathogens and commensals in the disease-stratified patients showed that in the most diseased patients the antibody levels to the pathogens were greater than antibody to the commensal bacteria. Comparison of the antibody levels to the individual bacterial species in disease-stratified groups demonstrated that among the pathogens, P. gingivalis was the only species that increased significantly with severity of disease. Therefore, in this adult population, antibody to P. gingivalis appears to provide a distinct marker of the current periodontal status, which is also a reflection of past disease experience in the patients. P. gingivalis has been implicated strongly as a periodontal pathogen, and it is biologically plausible that it might elicit a disproportionate antibody response. Examination of antibody levels, disease and smoking using correlation analysis provided some additional observations. Minimal correlation was noted between antibody levels BOP. While the extent of inflammation is generally related to the severity and extent of periodontitis, one explanation in this population could lie in the fact that all subjects in the study are current smokers. Smoking reduces BOP because the nicotine in cigarettes causes vasoconstriction in the gingiva, so this may alter the relationship between immune response capacity and the extent of BOP 34. Vasoconstriction also prevents white blood cells, and thus stimulation of IgG antibody production, from the microbial challenge in the gingiva. One might anticipate a different relationship in non-smoking subjects. This would be supported by existing literature describing differences in antibody levels in periodontitis versus control subjects that varied depending upon the smoking status of the subjects 35, 36. We identified a significant positive correlation between antibody levels to the pathogens and antibody levels to the commensal bacteria in the entire population of smokers. However, this relationship changed dependent upon the amount of periodontal disease and the amount of antibody to pathogens. Somewhat counterintuitively, in patients with more generalized periodontitis or having the highest level of antibody to the pathogens, the correlation in antibody levels to the pathogens and commensals were minimal. This finding supports the hypothesis that with chronic infection leading to oral tissue destruction, the host immune response is dysregulated and selectively recognizes and responds to the pathogens, while not responding as robustly to the multitude of commensal bacteria within the context of the large polymicrobial ecology 7, 30, 37. We did, however, observe a significant correlation between antibody levels to P. gingivalis and periodontal status. These relationships were noted in blacks and males within this population of smoking patients and correlated specifically with the frequency of disease sites, linking the antibody more directly to the infectious challenge. In summary, the data show an elevated immune response to pathogens compared to commensals within this smoking population and suggesting that the host immune system has the ability to discriminate between potential pathogenic versus commensal species in the complex biofilms. Response to the pathogens was also shown to be greatest in the subjects with the greatest extent of disease, comparable to previous findings in other populations and was most notable with antibody to P. gingivalis21, 38. The observation that black males demonstrated the most severe periodontal disease, which was not commensurate with their level of smoking, supports the need for additional studies to identify the factor(s) that could be contributing to disease susceptibility/expression. While we acknowledge that this was not an exhaustive study of antibody specificities to oral bacterial, the findings highlight processes by which the immune system recognizes pathogens such as P. gingivalis, and this response would be predicted to help to manage the periodontal disease immunopathology in adult populations. As importantly, it must be considered that antibodies are effector molecules in the host immune response and principal protective factors against extracellular bacterial pathogens. In that regard, previous studies have described antibody subclass distribution to oral pathogens 25, 39, 40 and suggested variations in the profiles related to the particular bacterial species. These findings were extended to potential success or failure of the antibodies to protect the host effectively. A range of studies have suggested that the immune response to oral pathogens does not mature effectively, as estimated via antibody avidity 41-46, and could contribute to lowered protective capacity. Furthermore, examination of the effector functions of antibodies to the oral pathogens has provided some challenge due to, for example, the gingipains from P. gingivalis effectively degrade antibodies and complement components in vitro47-49, and the leucotoxin from A. actinomycetemcomitans killing neutrophils 50-52 in test for opsonizing potential. Thus, studies of the antibody characteristics as they relate to subclass distribution and targeted functions, comparing the response to pathogens and commensals must be conducted to understand more fully the in vivo ramifications of the host discrimination of these bacteria coupled with the ability of the antibodies to modulate the oral microbial burden in health and disease. Jakubovics NS. 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