Complement split product C3c in saliva as biomarker for periodontitis and response to periodontal treatment

Abstract Background and Objective The complement system is engaged in inflammatory reactions both in the periodontal pockets and in the periodontium itself, where it can mediate tissue destruction. The aim of this study was, first, to compare salivary levels of the total complement system protein C3 and its split product, fluid‐phase C3c in patients with periodontitis and periodontally healthy controls. Next, to determine if C3 and C3c levels had biomarker potential in diagnosing and monitoring periodontitis and its treatment. We hypothesized that salivary levels of total C3 and the split product C3c associated with the severity of periodontitis and reflected decreased inflammatory activity after periodontal treatment. Methods At baseline, stimulated saliva samples were collected from patients with periodontitis (n = 18) and periodontally healthy controls (n = 15). Subsequently, non‐surgical periodontal treatment was performed in the patients, and saliva sampling from patients was repeated two‐, six‐, and twelve weeks post‐treatment (NCT02913248 at clinicaltrials.gov). The patients were grouped as good and poor responders to treatment according to the achieved reduction in bleeding on probing (BOP). Salivary levels of C3 and C3c were quantified using sandwich ELISA. Results Patients with periodontitis had higher baseline levels of both total C3 and the split product C3c in saliva than did periodontally healthy controls (P < .0001). Receiver operating curve (ROC) analyses discriminated patients with periodontitis from controls based on both C3 (AUC (area under curve) = 0.91, P < .001) and C3c levels (AUC = 0.84, P < .001) in saliva. Periodontal treatment improved all clinical parameters (P < .01). Good responders (n = 10) had lower baseline levels of C3c than poor responders (n = 8), (P < .05), and baseline levels of C3c discriminated between good and poor responders (AUC = 0.80, P < .05). Conclusion In conclusion, patients with periodontitis had higher salivary levels of C3c, and the C3c levels were predictive of reductions in BOP, that is, the poor responders. This suggests that salivary C3c levels possess potential to serve as a biomarker predicting the clinical response to non‐surgical periodontal treatment.


| INTRODUC TI ON
Periodontitis is a multifactorial inflammatory disease in the tooth-supporting tissues, which affects nearly 50% of the adult population in the Western world. 1 Periodontitis is initiated and maintained by a subgingival and polymicrobial biofilm, which in susceptible individuals leads to destructive inflammation. 2 Ultimately, periodontitis can cause tooth loss and simultaneously occurrence of systemic diseases such as cardiovascular disease, diabetes, and rheumatoid arthritis. [3][4][5] Therefore, early diagnostic and treatment are essential.
The complement system consists of more than 40 proteins and plays an important role in the combat of microorganisms, recruitment and regulation of inflammatory cells, and in clearance of apoptotic host cells and immune complexes. 6 In periodontitis, bacteria modulate and activate the complement system, the latter resulting in cleavage of complement component C3 into C3a and C3b. [7][8][9] C3b becomes covalently attached to the bacterial surface and is enzymatically converted into iC3b. Subsequently, iC3b is cleaved into fluid-phase C3c and surface-bound C3dg. 7,10 C3dg and C3c may thus serve as markers of ongoing formation and degradation of the biologically active C3 fragments C3b and iC3b.
While measurement of C3 levels in blood is used to screen for specific inflammatory medical diseases such as systemic lupus erythematosus, 11,12 the inflammatory oral disease periodontitis is diagnosed based on clinical and radiographic recordings. 13 These recordings detect manifest tissue changes, which are indicative of treatment needs, but their value in diagnosing periodontitis at early disease stages and in predicting treatment responses are limited.
Screenings for complement proteins and their split products in saliva may offer earlier diagnoses and personalized treatment plans, as the content of complement proteins and their split products in saliva may reflect local production and extravasation of complement proteins, as well as their activation locally. 14,15 Previous investigations have shown associations of complement protein levels with periodontal inflammation 6,[15][16][17][18][19][20] ; however, the C3c-specificity of the assays previously used is limited. Many commercially available C3c-antibodies recognize both total C3, hydrolyzed C3 (C3 (H2O) ) and degradation fragments containing the C3c-moiety (C3b, iC3b). 21 We have raised a monoclonal antibody that only recognizes a neo-epitope in C3c, which becomes accessible when iC3b is cleaved into fluid-phase C3c and bound C3dg. 22 Consequently, our assay detects C3c detached from C3dg, which reflects local complement consumption and potentially also the ongoing inflammation due to a short half-life of C3c. 23 The aim of this study was, first, to determine the value of salivary C3 and C3c as diagnostic biomarkers for periodontitis and, secondly, to determine if C3 and C3c levels had biomarker potential in monitoring periodontal treatment. We hypothesized that salivary levels of total C3 and the split product C3c associated with the severity of periodontitis and reflected decreased inflammatory activity after periodontal treatment.

| Study population
This study is part of a series of investigations analyzing salivary biomarkers in patients with periodontitis receiving non-surgical periodontal treatment. 24,25 The clinical trial itself was performed in 2017, which is why patients with periodontitis were enrolled based on definitions from American Academy of Periodontology 2015. 26 However, when incorporating the new classification of periodontitis, all patients were categorized in stage two or three (moderate to severe periodontitis). 27 Patients with generalized moderate to severe periodontitis (n = 25) and periodon-

| Study design
The present study is an interventional study with a follow-up period of three months, which has previously been described in detail. 24,25 In brief, baseline paraffin-stimulated saliva samples were collected from patients with periodontitis and periodontally healthy controls.

| Analysis of complement factors
Established sandwich ELISAs were used to assess total C3 and split product C3c levels in saliva samples from patients and controls, as described. 22 Maxisorp plates (Nunc) were coated with either 100 μL capture antibody mAb clone F1-23 anti-C3 total (2 μg/mL) or mAb clone

| Statistical analyses
All data were visually assessed for normal distribution using box-plot illustrations and histograms. Differences between groups in total C3 and C3c concentrations were analyzed using Mann-Whitney tests.
Alterations in clinical parameters were analyzed using repeated measures ANOVA (PI and BOP) and paired-samples t tests (Log10 transformed PD and CAL). Alterations in total C3 and C3c concentrations before and after treatment were analyzed using Friedman test. Area under curve (AUC) was used to discriminate between periodontal health and periodontitis, and between good and poor responders, both with respect to C3 and C3c values. A P-value < .05 was considered statistically significant in all analyses. GraphPad Prism version 8 (GraphPad software) and SPSS-statistics version 26, (IBM) have been used as statistical software.

| Salivary levels of C3 and C3c in patients with periodontitis and periodontally healthy controls
Significantly higher baseline levels of both C3 and C3c were observed in the patients with periodontitis, as compared to the periodontally healthy controls (P < .0001), (Figure 1

| Effect of non-surgical periodontal treatment on clinical parameters and salivary levels of C3 and C3c
The effect of the non-surgical periodontal treatment on clinical parameters in this cohort has previously been described. 24,25 In short, the periodontal treatment improved all periodontal registrations of PI, BOP, PD, and CAL throughout the study period (Table 1).
The improved periodontal status was reflected by a decrease in both C3 and C3c levels at week two; however, the changes were not statistically significant (Figure 3).

| Baseline salivary levels of C3 and C3c in good and poor responders
Patients with periodontitis were grouped into good (n = 10) and poor responders (n = 8) based on a reduction in BOP above or below mean (mean level of the total BOP reductions from baseline to week 12 = 22%). Baseline values of total C3 did not differ significantly between the good and poor responders ( Figure 4A). In contrast, the good responders had significantly lower baseline levels of C3c than the poor responders (P < .05), ( Figure 4B).
In line, baseline values of total salivary C3 could not distinguish the patients who responded poorly to treatment from patients who responded well ( Figure 5A), but salivary baseline levels of C3c were able to discriminate between the two groups. Using a ROC analysis, an AUC = 0.80, CI = [0.59;1.00] was found for C3c (P < .05), ( Figure 5B).

| D ISCUSS I ON
The aim of the study was to determine if measurements of salivary levels of total C3 and C3c had diagnostic and predictive value in regard to periodontitis and treatment thereof. Previously, salivary levels of total C3 and C4 have been associated with periodontitis. 30 However, so far activation-related complement split products may better reflect ongoing inflammation but have not been reported in saliva from patients with periodontitis. To our knowledge, this longitudinal study is the first to include measurements of salivary C3c split from the C3dg moiety in relation to periodontitis. We were able to do so by using an in-house raised monoclonal antibody recognizing a neo-epitope in C3c. 22 Salivary levels of total C3 and its split product C3c were higher in patients with untreated periodontitis than in periodontally healthy controls. Moreover, both parameters were able to discriminate periodontitis from periodontal health. The increased salivary levels of C3 can be explained by both an inflammation-related increase of vascular permeability and by an increase in local production of complement proteins by macrophages and dendritic cells. 31 In line with our findings, increased levels of intact complement proteins have been related to gingival inflammatory activity and periodontitis. 16,17 For example, total C3 and C5 have been demonstrated present in gingival biopsies from patients with periodontitis but absent in biopsies from healthy individuals. 16 Also, in gingival crevicular fluid (GCF) higher levels of total C3, C4, and C5 have been associated with inflamed periodontal tissues. 17 Thus, beside a re-demonstration of the association between intact complement proteins and periodontitis, our study shows a potential of split product C3c, as a marker of local and ongoing complement activation, and periodontitis.
In the present study, improvements of all clinical parameters were accompanied by decreases in salivary levels of total C3 and C3c levels. At week 2, a decrease in the inflammatory parameter BOP was reflected by decreases in levels of both total C3 and split product C3c. As in our study, associations of clinical improvements with complement proteins have been shown previously. 32,33 These findings, however, are based on assays using polyclonal antibodies, which cannot exclude detection of some intact C3, C3b, and iC3b fragments, which also contain the C3c moiety, in addition to isolated C3c. Nevertheless, they can indicate decreased complement levels, as a result of decreased vascular permeability and/or local TA B L E 1 Clinical parameters before and after non-surgical periodontal treatment in patients with periodontitis Baseline Week 2 Week 6 Week 12  The observed associations of C3 and C3c with periodontitis suggest that inhibition of these proteins can improve the treat- It should be noted that C3c is not to a specific marker for periodontitis, but rather a marker indicating increased inflammatory activity. Thus, a recent study found an association of increased salivary levels of the split product C3c with presence of oral lichen planus, using the same monoclonal antibody as employed in the present study. 38 In conclusion, high salivary baseline levels of complement component C3c were predictive of periodontitis and poor responses to treatment. C3c therefore has potential to diagnose periodontitis and predict treatment responses. In the future, clinical measurement of baseline C3c levels may contribute to risk assessment and monitoring of periodontitis, which may result in a better and more personalized treatment.

This study was supported by the Danish Foundation for Mutual
Efforts in Dental Care.

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.