|Year : 2019 | Volume
| Issue : 2 | Page : 49-55
Interrelationship between Porphyromonas gingivalis and complement C3 and factor B Levels in chronic periodontitis individuals
Srirangarajan Sridharan, Paruchuri M Sravani
Department of Periodontics, Bangalore Institute of Dental Sciences and Post Graduate Research Centre, Bengaluru, Karnataka, India
|Date of Submission||21-Jun-2019|
|Date of Decision||04-Sep-2019|
|Date of Acceptance||04-Sep-2019|
|Date of Web Publication||22-Oct-2019|
Department of Periodontics, Bangalore Institute of Dental Sciences and Post Graduate Research Center, 5/3, Hosur Road, Bengaluru - 560 029, Karnataka
Source of Support: None, Conflict of Interest: None
Background: To evaluate the effect of nonsurgical periodontal therapy on gingival crevicular fluid (GCF) and serum levels of complement C3 and factor B and their correlation with Porphyromonas gingivalis levels in chronic periodontitis (CP) individuals. Materials and Methods: Thirty individuals were divided into three groups. Clinical parameters such as Plaque Index (PI), Gingival Index (GI), Probing depth (PD) and Clinical attachment level (CAL) were recorded and P. gingivalis levels were measured at baseline in Group I (periodontally healthy) and II( chronic periodontits) and 3 months post scaling and root planning (SRP) in Group III subjects. Serum and GCF samples were collected from all the individuals at baseline 3-month post-SRP to evaluate complement C3 and complement factor B levels by spectrophotometer and enzyme-linked immunosorbent assay, respectively. Levels of P. gingivalis were determined by anaerobic culture. Results: Complement C3 and factor B levels in serum and GCF along with P. gingivalis levels in plaque samples showed statistical significance between the groups (P < 0.001). SRP resulted in decrease in C3, factor B, and P. gingivalis levels after 3 months in CP group at P < 0001. Among the periodontal parameters, PD showed significant correlation with biochemical and microbiological parameters in both the groups before and after periodontal therapy. Conclusion: P. gingivalis correlated strongly with factor B values in CP and SRP resulted in reduction of these values suggesting a possible modulation of alternate complement pathway by P. gingivalis.
Keywords: Complement, periodontitis, Porphyromonas gingivalis
|How to cite this article:|
Sridharan S, Sravani PM. Interrelationship between Porphyromonas gingivalis and complement C3 and factor B Levels in chronic periodontitis individuals. Dent Med Res 2019;7:49-55
|How to cite this URL:|
Sridharan S, Sravani PM. Interrelationship between Porphyromonas gingivalis and complement C3 and factor B Levels in chronic periodontitis individuals. Dent Med Res [serial online] 2019 [cited 2019 Dec 16];7:49-55. Available from: http://www.dmrjournal.org/text.asp?2019/7/2/49/269667
| Introduction|| |
The complement system comprises of more than forty different soluble and membrane-bound proteins. It is a part of innate immune system which proceeds via controlled limited proteolysis. Two distinct pathways for activation of the complement system have been defined: the classical pathway and the alternative pathway. Activation of the classical pathway is generally associated with antigen-antibody complex formation, and the activation of the alternative pathway can be initiated by complex polysaccharides associated with the surfaces of bacteria, virus-infected cells, and fungi. On the contrary, the alternative pathway is autoactivated by binding of hydrolyzed C3 with factor B. In addition, the alternative pathway amplifies complement activation initiated through the classical or lecithin pathways. Thus, increased production of the Bb fragment can be suggestive of activation of the alternate pathway. Chronic periodontitis (CP) is a result of host–bacterial interaction leading to destruction of connective tissue and bone. These destructive processes occurring in CP emphasize the potential importance of complement activation in this disease process.
Complement split products are, in general, either absent or present at low concentrations in the gingival crevicular fluid (GCF) of healthy individuals, but abundantly present in both the GCF and serum of patients with periodontitis. Thus, C1q, factor B, factor Bb, C3, C3a, C3b, C3c, C3d, C4, C5, C5a, C5b, and C9 have all been detected in diseased periodontal tissue, on the surface of subgingival bacteria and in the GCF from patients with established periodontitis. Recently, animal model studies have given us insights as to how CP could be mediated by complement proteins, and according to these studies complement targeted immune subversion occur which could influence periodontal pathogens specifically.
Porphyromonas gingivalis is thought to cause complement- dependent damage to the periodontium., Increased local activation of complement in the periodontal tissues increases the intensity of the local inflammatory response, resulting in excessive release of reactive oxygen species, proteolytic enzymes, and interleukins (ILs)., P. gingivalis requires peptides and hemin for its growth and thus is dependent on host inflammatory serum exudates for its continued existence in the periodontal niche. To achieve this, P. gingivalis inhibits the complement cascade by enzymatic breakdown of C3, mediated by a group of cysteine proteases called gingipains.,
Complement C3 is the most significant component of the complement system accounting for about one-third of the total complement. The degree of C3 complement conversion in both gingival fluid and serum may reflect the state of disease severity. Studies have shown the evidence of C3 proactivator (factor B) conversion to C3 activator Bb as a part of alternate pathway of complement activation in periodontal pocket. The need to more fully understand the host–parasite interactions of CP has led investigators to evaluate relationships between the microbial insult of dental plaque and the inflammatory response of the host.,
However, till date, no study has been done to evaluate and correlate the levels of P. gingivalis and complement factors in GCF and serum before and after periodontal therapy in CP individuals. Thus, the current study was designed to evaluate the effect of scaling and root planing (SRP) on GCF and serum levels of complement C3 and factor B and their correlation with P. gingivalis levels in CP individuals.
| Materials and Methods|| |
Selection of individuals
The study included thirty patients visiting the outpatient department of Bangalore Institute of Dental Sciences and Hospital and Research Centre, Bengaluru, from July 2015 to 2016. The study protocol was prepared in accordance with the Declaration of Helsinki of 1973 (as revised in 2002) and was approved by the ethical committee of the institution.
- Age group 30–60 years
- Both male and female patients
- Periodontally healthy individuals with clinically healthy periodontium, gingival index (GI) = 0 (absence of clinical inflammation), probing depth (PD) <3 mm, and clinical attachment level (CAL) = 0, with no evidence of bone loss on radiographs
- CP individuals with pocket depth ≥5 mm, clinical attachment loss ≥3 mm and exhibiting radiographic signs of bone loss in more than one quadrant (AAP, 1999)
- Number of natural teeth present ≥20 teeth.
- Presence of any systemic conditions or debilitating diseases
- Pregnant or lactating women
- A recent history or presence of any acute or chronic infections
- Patients with the history of any drug intake including antibiotics, analgesics, or any other drugs in the last 3 months
- Patients who had undergone periodontal therapy in the last 6 months
- Patients who were smokers/paan/tobacco/betel nut users
- Patients who were physically or mentally challenged
- Patients who were not willing to take part in the study.
Based on the periodontal findings, the individuals were divided into the following two groups:
- Group I – 15 periodontally healthy individuals; with clinically healthy periodontium, GI = 0 (absence of clinical inflammation), PD < 3 mm, and CAL = 0, with no evidence of bone loss on radiographs
- Group II – 15 CP individuals with pocket depth ≥5 mm, clinical attachment loss ≥3 mm, and exhibiting radiographic signs of bone loss in more than one quadrant
- Group III – Group II CP individuals 3 months after SRP.
The purpose of the study was explained to the patient, and a written consent form was obtained agreeing to comply with maintenance and re-examination schedule. All thirty patients were subjected to clinical, microbiological, and biochemical analysis at baseline and at the end of 3 months.
The following clinical parameters were recorded for each patient.
- Plaque index (PI) by Silness and Loe (1964)
- GI (Loe and Silness) (1963)
- Probing pocket depth (PPD) was measured as the distance from the free gingival margin to the base of the pocket using University of North Carolina probe (UNC) (UNC 15 periodontal probe hufriedy IL, Chicago). Six sites were measured from each tooth, one each at mesiofacial, midfacial, distofacial, mesiolingual/mesiopalatal, mid-lingual/mid-palatal, and distolingual/distopalatal. The PD score of a person was obtained by adding all the individual scores and divided by the number of surfaces examined
- CAL was evaluated from the cementoenamel junction to base of pocket using UNC 15 probe.
Subgingival plaque sample collection
Plaque samples from subgingival area of healthy and CP patients were collected from the deepest periodontal pocket with the help of a sterile Gracey curette along the long axis of the tooth. Samples contaminated with blood were discarded. The collected plaque samples were stored in Eppendorf tubes containing sterile distilled water. Same procedure was performed for subgingival plaque sample collection after 3 months of SRP in CP group.
Serum sample collection
Blood collection was done at the same appointment for the purpose of serum sample collection. Two milliliters of blood was collected from the antecubital fossa by venipuncture using a 20G needle with 2-ml syringe and immediately transferred to the laboratory. The blood sample was allowed to clot at room temperature, and after 1 h, serum was separated from blood by centrifuging at 3000 g for 5 min. The serum was immediately transferred to a plastic vial and stored at −70°C until the time of assay. Same procedure was performed for serum collection after 3 months of SRP in CP group.
Gingival crevicular fluid sample collection
GCF sample collection was done from most inflammed sites. Test sites for GCF sample collection were selected based on the highest scored sites. Initially, the selected site was cleaned, isolated, and air-dried using sterile cotton rolls, and the supragingival plaque was removed gently using a Gracey curette so as to avoid contamination of the paper strips. The microcapillary pipettes were placed gently at the entrance of the gingival sulcus until the light resistance was felt, care being taken to avoid mechanical injury, and left in place for 60 s. Samples that were suspected to be contaminated with blood and saliva were excluded from the study to avoid any kind of bias in sample collection. After collection of the gingival fluid, the GCF from each individual were pooled and were immediately transferred in microcentrifuge tubes containing 200 μl of phosphate buffer saline and stored frozen at −70°C for subsequent analysis. SRP was performed for CP individuals at the same appointment after GCF collection by the operator. The same procedure was carried out after 3 months for the CP group.
Porphyromonas gingivalis assay
After incubation for 30 min, the plaque samples were inoculated onto blood agar plates. The plates were arranged in the rack; the rack was then placed in anaerobic jar. The anaerobic jar was kept in the incubator at 37°C for 5 days. Organisms were provisionally identified based on their colony characteristics, and the colony counting was done under light microscopy.
Assessment of complement C3
Estimation of complement C3 levels in serum and GCF was done by two beam spectrophotometry method done in biochemistry auto-analyzer (Meril diagnostics, Gujarat, India).
Assessment of factor B
The samples were assayed for complement factor B levels in GCF and serum using a highly sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the instructions of the manufacturer, and the assay was duplicated. All reagents were allowed to thaw at room temperature (18°C–25°C), 30 min before use. 50 μl of each standard and 40 μl of sample were added to wells precoated with monoclonal complement factor B antibody. 10 μl of biotin conjugate was added into each sample well. Followed by which 50 μl of streptavidin-horse radish peroxidase conjugate was added into each well and incubated for 1 h at 37°C in the incubator. The solution was aspirated, and the wells were washed 4 times with wash buffer. 50 μl of substrate A and then 50 μl of substrate B were added to each well, gently mixed and incubated for 10 min at 37°C in dark. Stop solution (50 μl) was added to each well. Absorbance of the substrate color reaction was read on an ELISA reader using 450 nm as primary wavelength. The total complement factor B was determined in picograms, and the calculation of the concentration in each sample was performed by dividing the amount of complement factor B by the volume of sample (picograms per milliliters).
Full-mouth SRP was done for CP individuals at baseline. Patients were recalled after 3 months during which subgingival plaque, serum, and GCF samples were collected, and the clinical parameters were recorded. The clinical evaluation was done by the same examiner throughout the study.
Intraexaminer calibration – Intraexaminer calibration was done to avoid examiner-related bias. It was done by examining 10 sites twice, 24 h apart before beginning the study. Examiner was considered calibrated if measurements at baseline and 24 h had a difference of not more than 1 mm.
All the recorded clinical and biochemical and microbiological parameters were subjected to statistical analysis using Statistical package for social sciences (SPSS software version 16.0; SPSS, Chicago IL, USA). Descriptive analyses including mean and standard deviation (SD) were found for each parameter in two groups. Unpaired t-test was used to compare the levels of complement factors in serum and GCF and P. gingivalis in health and disease. Paired t-test was used to compare clinical parameters before and after periodontal treatment. Correlation between the values of complement proteins and P. gingivalis before and after therapy was done using Pearson correlation test. P ≤ 0.05 was considered statistically significant with confidence interval of 95%.
| Results|| |
The descriptive statistics along with mean and SD of both serum and GCF samples of all groups are tabulated in [Table 1]. The levels of C3 and factor B were higher in Group II than in Group I. This difference between the two values was statistically significant at P < 0.001. The mean values of levels of P. gingivalis of Group I and Group II individuals were 126.67 ± 221.897 and 60,000 ± 20,996.59, respectively. This difference between the two values was statistically significant at P < 0.001.
[Table 2] shows the descriptive statistics along with mean and SD values of both serum and GCF samples of C3 and factor B in the Group II and Group III at baseline and the end of 3 months. The mean values of the levels of serum C3 in Group II at baseline and Group III at 3 months were 176.67 ± 39.082 and 152.33 ± 31.002, respectively, with P = 0.069. The corresponding mean values of complement C3 levels in GCF were 135.27 ± 33.27 and 115.07 ± 27.481 with P = 0.081. This difference between the two values was statistically insignificant. The mean values of complement factor B levels in serum of Group II individuals at baseline and Group III at 3 months were 225.73 ± 30.595 and 175.73 ± 20.240, respectively, with P < 0.001. The corresponding mean values of complement factor B levels in GCF were 329.60 ± 42.695 and 288.73 ± 28.078 with P = 0.004. This difference between the two values was statistically significant. The mean values of levels of P. gingivalis of Group II at baseline and Group III at 3 months were 60,000 ± 20,996.598 and 21,066.67 ± 9535.398, respectively, with P < 0.001. This difference between the two values was statistically significant.
|Table 2: Comparison of the descriptive values between Group 2 and Group 3|
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Pearson correlation coefficient between serum and GCF levels of C3 and factor B and P. gingivalis is tabulated in [Table 3]. Serum C3 and C3 in GCF showed positive and very strong correlation with P. gingivalis levels in Groups II and III whereas Group I did not show any statistical significant correlation (P > 0.01).
|Table 3: Groupwise correlation between Porphyromonas gingivalis with biochemical variables in serum and gingival crevicular fluid|
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Factor B serum and factor B GCF showed strong positive correlation with P. gingivalis in all the three groups (P< 0.01). Pearson correlation coefficient between clinical parameters and P. gingivalis is tabulated in [Table 4]. The correlation between P. gingivalis and PI showed to be weak and negative in all the three groups; however, correlation between GI and P. gingivalis showed positive and moderate statistically insignificant correlation in Groups I and III. However, Group II showed very weak and negative correlation (statistically significance with P > 0.01).
|Table 4: Groupwise correlation between Porphyromonas gingivalis and clinical parameters|
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The correlation between PD and P. gingivalis showed strong positive statistically significant (P< 0.01) in all the three groups.
| Discussion|| |
The present interventional study was conducted to assess the interplay between P. gingivalis and the complement factors in CP patients and the effect of SRP on the correlation of microbiologic and host inflammatory response. The primary objective of the present study was to assess and compare the levels of complement C3 and factor B in serum and GCF in healthy and CP groups and to evaluate the effect of nonsurgical periodontal therapy on their levels in CP group. Patients with CP showed significant increase in levels of complement C3 and factor B in serum and GCF compared to healthy controls. Therapeutic intervention with SRP in CP group showed significant decrease in the levels of factor B at 3 months when compared to the baseline values, whereas decrease in the levels of C3 was not statistically significant. The results of the study could be supported by the evidence provided by the previous studies, which showed that P. gingivalis depends on the continuous flow of inflammatory serum exudates to obtain essential nutrients for survival in the periodontal niche. To achieve this, P. gingivalis inhibits the complement cascade by enzymatic breakdown of C3, mediated by a group of cysteine proteases called gingipains., Hence, following SRP although the levels of C3 were reduced, it was not statistically significant. Factor B is continuously recruited for the formation of the alternative pathway C3-convertase, C3bBb, which further cleaves numerous C3 molecules and is known as the amplification loop. This could be the reason for the increased levels of factor B in CP group compared to healthy group and their statistically significant decreased levels following SRP.
In addition to constituting a “ first line of defense”, complement is acknowledged as a factor promoting an adaptive immune response and a key to maintaining host–microbial homeostasis, by virtue of regulator proteins. It can differentiate between healthy, diseased and foreign intruders and respond accordingly., On one hand, complement eradicates invasive pathogens, On the other hand, continuous complement activation and modulation by the sub-gingival microorganisms, may enhance local tissue destruction, thereby providing them with essential nutrients and ensuring its persistence., Periodontitis is characterized by the presence of a hyperactivated phenotype of neutrophils with enhanced pro-inflammatory activity., Complement also activates neutrophils via complement receptor 3 and the C5a receptor, leading to exaggerated inflammatory response, release of reactive oxygen species, proteolytic enzymes, and cytokines which manifest clinically as edematous gingiva, loss of attachment, and ultimately bone resorption. The present study results were in accordance with previous studies on concentrations of complement fragments in GCF., A recent study has identified C3 among the top 21 most promising candidate genes involved in periodontitis. Although a number of candidate susceptibility genes have been projected, it remains uncertain whether individual genes play important roles in periodontal disease pathogenesis.,, In this regard, CP is considered as polygenic disease influencing the host immune response and the microbiota. Despite limited numbers of studies, evidence indicates that treatment of periodontitis results in a significant downregulation of RNA coding for C3 in the periodontium. In addition, treatment of periodontitis markedly decreases the concentration of the final split product of C3, C3d, in the periodontium, implying that complement is involved in the pathogenesis. To elucidate the mechanisms of complement activation occurring in the periodontal pocket, evidence for presence of the alternate pathway protein C3 proactivator (factor B) was sought. The findings of our study showed that C3-proactivator was elevated in CP group which indicates that the accumulation of C3 conversion products may be in part attributable to alternate pathway proteins. These findings were consistent with the previous study.
The secondary outcome measure was to determine the levels of P. gingivalis in plaque samples from periodontally healthy and CP patients and to evaluate the effect of nonsurgical periodontal therapy on their levels in CP patients. P. gingivalis showed significant increase in their levels in CP group when compared to the healthy group, and their levels were significantly decreased after SRP in CP patients.
Studies on P. gingivalis and complement have stated that P. gingivalis can effectively evade complement action by various proposed strategies breakdown C3 by gingipains and selectively generate biologically active C5a.
The proactive release of C5a enhances P. gingivalis-induced IL-6 production, and the inflammatory reaction that follows contributes to nutrient procurement and deepening of the pockets, providing more room for bacterial growth., Moreover, cleavage of C5 to C5a by gingipains produced by P. gingivalis induces migration of neutrophils,, but the high C5a concentrations in the periodontal tissues “paralyze” neutrophils and protect bacteria from being phagocytized by macrophages. This could help us substantiate as to why in our study we found significant correlation between P. gingivalis and PD at both baseline and 3 months.
Taken together, these findings we would like to conclude by stating that our study supports the role of P. gingivalis in modulating complement C3 and factor B activation in CP. The significant finding of this study was that it showed variations in the levels of C3, factor B, and P. gingivalis before and after periodontal therapy indicating that complement is an important player in the pathogenesis of periodontitis. However, it should be regarded as a double-edged sword, which under normal physiological conditions protects the host, but under pathological circumstances can be destructive to host tissues. Thus, the present study found a significant correlation between P. gingivalis levels and factor B levels in GCF and serum, thus suggesting possible modulation of alternate complement pathway by P. gingivalis. While the present study has focused on the interaction between complement and single species bacteria, further longitudinal studies addressing the ability of complement to attack or sustain complex communities of bacteria within biofilms are warranted. Studies investigating whether and how complement facilitates systemic spread of bacteria associated with periodontitis would enhance our understanding on periodontitis and systemic diseases. Eventually, such knowledge may provide a basis for new therapeutic strategies in periodontitis and other chronic inflammatory diseases.
| Conclusion|| |
Within the limitations of the current study, P. gingivalis levels correlated strongly with factor B values in CP and SRP resulted in reduction of these values suggesting a possible modulation of alternate complement pathway by P. gingivalis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]