Exploring the Role of Interleukin-6 Receptor Inhibitor Tocilizumab in Patients with Active Rheumatoid Arthritis and Periodontal Disease

Background: The aim of our study was to explore the influence of weekly subcutaneous administration of interleukin-6 (IL-6) receptor inhibitor tocilizumab (TCZ) on periodontal status in a local longitudinal study of patients with rheumatoid arthritis (RA) and periodontal disease (PD). Methods: We performed a 6-month prospective study in 51 patients with chronic periodontitis and moderate-to-severe RA starting TCZ in accordance with local recommendations. Extensive rheumatologic (clinical activity, inflammatory, serological biomarkers) and periodontal (visible plaque index, gingival index, bleeding on probing, probing pocket depth, clinical attachment loss) assessments were done. Changes in RA activity and periodontal status were reassessed after 3 and 6 months. Results: We demonstrated significant correlations between periodontal status, disease activity, and serologic biomarkers (p < 0.05). Tocilizumab significantly improved the gingival index scores and decreased the number of sites with bleeding on probing after only 3 months (p < 0.05), while the probing pocket depth significantly decreased after 6 months; overall, clinical attachment loss presented only slight changes without any statistical significance as well as teeth count and plaque levels (p > 0.05). Conclusion: IL-6 inhibition is able to improve periodontal outcomes in patients with RA and concomitant PD, which is essentially related to a dramatic decrease in serum inflammatory mediators.


Rheumatoid Arthritis and Periodontal Disease Association
Rheumatoid arthritis (RA) is an autoimmune condition characterized by joint inflammation and destruction associated with chronic systemic inflammation accounting for significantly impaired quality of life. It is defined by the excessive activation of proinflammatory cytokines mediators, specific autoantibodies and progressive irreversible articular damage [1,2]. Considered the hallmark of RA and, perhaps, the most important step in the pathobiology of the disease, the immune response against citrullinated peptides is driven, at least in part, by antigens derived from the periodontal tissue exposed to a dysbiotic oral microbiome during periodontitis [1][2][3][4].
Periodontal disease (PD) is generally associated with a broad spectrum of chronic systemic disorders including diabetes, cardiovascular, respiratory, kidney, and neurodegenerative diseases as well as immune-mediated rheumatic conditions [1][2][3][4][5]19]. Several epidemiological studies have already communicated that PD is more prevalent during RA and vice versa [4,20,21]. Indeed, patients with PD have an increased risk to develop RA, compared to general population, particularly those with a long history of more severe periodontitis, mostly explained by excessive protein citrullination [14,21]. Furthermore, it seems that P. gingivalis positive-periodontitis is more likely to occur in ACPA-positive individuals without any arthritis, suggesting that PD may precede RA [4,11,15,20,22]. On the other hand, RA patients experience a greater risk of PD, irrespective of disease duration, especially in ACPA-positive subtype [1][2][3][6][7][8]11,14,19]; moreover, they are prone to develop moderate to severe periodontitis in established compared to early disease [6][7][8][9]14,19,23]. A detailed analysis of periodontal status in first-degree relatives of RA cases discovered a higher prevalence and severity of periodontitis in ACPA-positive RA [9,14,22,23]. Altered periodontal condition during RA seems to be multifactorial, related to increased serum concentrations of proinflammatory cytokines and altered motor skills of the rheumatoid hand which can also contribute to compromised oral hygiene [1,4,5,22].
On the other hand, several papers have addressed the effect of specific periodontal therapies (e.g., non-surgical scaling and root planning) on clinical RA activity in patients with chronic periodontitis with controversial results [37,[51][52][53][54][55][56][57]. The most recent data from the ESPERA (Experimental Study of Periodontitis and Rheumatoid Arthritis) cohort failed to demonstrate clinical improvement in established RA following aggressive and intensive periodontal treatment [57].
Considering the gap in the literature regarding the role of anti-rheumatic drugs on periodontal outcomes, the aim of our study is to assess the influence on the periodontal status of weekly subcutaneous administration of tocilizumab in a local group of patients with rheumatoid arthritis and chronic periodontitis.

Study Design and Population
We performed a prospective longitudinal study in fifty-one patients with moderate-tosevere RA and insufficient response to either conventional synthetic or biologic diseasemodifying antirheumatic drugs (DMARDs), starting TCZ according to the local recommendation for biologic and targeted synthetic therapy aligned with European League Against Rheumatism (EULAR) consensus statement and guidelines.
We performed extensive rheumatologic and full mouth assessments at baseline (before the first administration of TCZ) as well as after 3 and 6 months of therapy.

Inclusion Criteria
The patients were aged 18 and older, able to give informed consent themselves and to participate in the study, and willing to forgo any optional examinations.
Patients fulfilled either the old 1987 American College of Rheumatology (ACR) criteria or the new 2010 classification criteria of ACR and EULAR and were followed up in one academic rheumatology department in Northeast Romania over a period of 3 years (July 2017-January 2020).

Exclusion Criteria
Several exclusion criteria were applied before enrollment in this study because of their potential interference with a correct evaluation of periodontal status, as follows: exor current smokers, pregnant and breastfeeding women, patients with diabetes mellitus, implants, poorly fitting fixed and/or removable prosthodontics and fewer than eight evaluable teeth, patients receiving systemic or local antimicrobials, antiplatelet drugs, any type of anti-inflammatory medication or periodontal therapy within the previous 3 months.
A total of sixty-eight patients were eligible for and received TCZ for their active RA; however, among them, seventeen had no oral issues at baseline evaluation and were excluded from the study.

Ethical Considerations
The baseline clinical documentation of periodontitis cases was collected in the context of routine check-up in the dental clinic of Sanocare Medical and Research Center.
The study was approved by the local ethics committee (Sanocare Medical and Research Center, Prot. No 15/12.12.2016) and was found to conform to the guidelines of the Declaration of Helsinki. Written informed consent regarding the use of the collected data in the context of training and research was signed by all the participants before enrollment. The data used in the study were anonymized. According to the U.S. Department of Health and Human Services (HHS) definition, this investigation is not considered human subjects research.

Rheumatologic Assessments
RA-related variables comprised clinical (tender and swollen joint count based on a 28-joint assessment, 0-10 cm visual analogue scale, VAS, pain), inflammatory tests (erythrocyte sedimentation rate, ESR, and C-reactive protein, CRP) as well as disease activity scores calculated on DAS28-CRP (Disease Activity Score on 28 joints using Creactive protein) and SDAI (Simplified Disease Activity Index) were performed at all three visits.
Serological biomarkers (rheumatoid factor, RF, and ACPA) were evaluated only at baseline.

Periodontal Assessments
The periodontal status was recorded on a periodontal chart displaying the following clinical parameters for the entire dentition: number of present teeth, visible plaque index (VPI), gingival index (GI), bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment loss (CAL).
Clinical periodontal assessments were performed by a single trained examiner (C.I.) at the Sanocare Medical and Research Center, Iasi, who was blinded to the rheumatologic data. Access to previous assessment data was not allowed during the study. The examiner was considered calibrated when no statistically significant differences between measurements were obtained after the evaluation of 15 non-participant subjects on two occasions, one week apart. The mean values were assessed using paired t-test for VPI, GI, BOP, PPD, and CAL.
The periodontal evaluation was made in artificial light conditions, using a dental explorer, dental mirror, Williams probe, and air-water syringe.
BOP evaluates gingival inflammation through bleeding observed 20 s after a probe is passed along inside the gingival sulcus or pocket. It was recorded dichotomously (present/absent) at 6 sites (mesiobuccal, mid-buccal, distobuccal, mesiolingual, mid-lingual, and distolingual) around each tooth.
PPD was measured between the gingival margin and the bottom of gingival sulcus or pocket, while CAL was measured between the cemento-enamel junction and the base of the gingival sulcus or pocket. Both parameters were determined with Williams periodontal probe and recorded on 6 sites per tooth. The recorded values were to the nearest millimeter, and every reading close to 0.5 mm was rounded to the nearest integer number.
PD was considered according to the case definition proposed by the 5th European Workshop on Periodontology in 2005 [60], as follows: level 1 (mild)-CAL ≥ 3 mm in 2 or more proximal sites of non-adjacent teeth and level 2 (severe)-CAL ≥ 5 mm in 30% or more proximal sites of teeth present; level 0 was considered-for healthy periodontal status or up to one proximal site with CAL ≥ 3 mm.
Patients were instructed to maintain their oral hygiene habits throughout the 6 months of follow-up; furthermore, as we intended to assess the accurate effect of TCZ on periodontal status, any periodontal treatment was avoided.

Statistical Analysis
Statistical analysis was performed with the IBM SPSS Statistics for Windows, Version 19.0. (IBM Corp., Armonk, NY, USA), with p-values less than 0.05 being considered as statistically significant; data at 3 and 6 months were summarized as means ± SD, or percentages (%) as appropriate, and correlations by Spearman rank tests; comparisons between baseline and 3 and 6 months of TCZ were assessed by Wilcoxon test.

Baseline RA and Periodontal Assessments
Demographics, rheumatologic and periodontal characteristics, as well as RA-related drugs (concomitant glucocorticoids and immunosuppressives) taken at baseline are summarized in Table 1.
Most patients included in our study had seropositive established RA, with moderateto-severe activity despite background medication. Eight patients (15.68%) received TCZ as their first biologic agent (bio-naïve), while the majority were bio-experienced patients, with failure (either insufficient response or adverse reactions) to previous biologics-15 (29.41%) to one biologic, 20 (39.21%) to two biologics, and 8 (15.68%) to three biologic agents.
We detected impaired oral health in all patients included in the final analysis, as follows: all had gingivitis (abnormal GI and increased prevalence of sites with BOP), and different degrees of chronic periodontitis (mainly level 1 and 2); advanced loss of attachment was reported in up to 23.52% of cases, while increased prevalence of sites with dental plaques in 21.56% of cases.

Changes in Rheumatologic and Periodontal Parameters with Tocilizumab
Changes in RA activity and periodontal status were reassessed after 3 and 6 months of TCZ; at follow-up visits, we reported significant improvement as compared to baseline (p < 0.05), although the results at 6 months were only slightly different from data obtained at 3 months (p > 0.05).

Changes in Rheumatologic Status
Patients displayed consistent improvements in clinical activity meaning a significant decrease in the number of tender and swollen joints, VAS pain, and morning stiffness as rapid as 3 months; as expected, clinical response was maintained 3 months later in all patients, at the final monitoring visit. Similarly, we reported a dramatic decline in inflammatory biomarkers (both ESR and CRP), as well as a considerable immunologic response, particularly for serum levels of ACPA, but also for RF (Table 2). DAS28-CRP and SDAI strongly improved during monitoring visits reaching either low disease activity or, even, remission (EULAR responders) vs. baseline, irrespective of the severity of periodontitis.

Changes in Periodontal Status
Clinical data showed improvement in periodontal inflammation after only 3 months of TCZ and maintained over 6 months, as supported by an important decrease in gingival index and sites with bleeding of probing (p < 0.05). However, the improvement of specific periodontal parameters such as probing pocket depth becomes evident after prolonged treatment (6 months); overall, clinical attachment loss presented only slight changes without any statistical significance; teeth count and bacterial plaque scores were also not significantly influenced by medication (p > 0.05) ( Table 3). No significant correlations between changes in periodontal parameters and changes in RA activity were described in our study (p > 0.05).
We assumed that all the modifications in the degree of local gingival and periodontal inflammation is related to IL-6 blockade as no local periodontal treatment was allowed during follow-up.

Discussion
We aimed to assess the influence of the IL-6 receptor inhibitor on periodontal status in active RA associated with periodontitis, assuming that TCZ might be able not only to improve clinical and biochemical RA-related parameters but also to ameliorate chronic periodontitis as a result of decreased IL-6 in the periodontal microenvironment via declining systemic inflammation.
Although our target is to demonstrate the ability of TCZ to modulate periodontal inflammation and subsequent damage, firstly, we emphasized its role in controlling RA activity. We reasonably confirmed a consistent response to TCZ in real-life settings, which was achieved in as rapid as three months and continued after six months of therapy, irrespective of background medication and clinical scenario (mono-or combined therapy, bio-naïve or bio-experienced patients); it is more than clear that even in the short-term, IL-6 blockade displays significant clinical, biological, as well as serologic disease improvement. Although we found no consistent difference in clinical response in seropositive vs. seronegative RA, we noticed a significant impact on ACPA serum concentration after six months, which is an improvement that parallels the decrease in periodontal inflammation, suggesting a role of IL-6 in both systemic and local inflammation (synovial and periodontal) and the potential implications via citrullination. Therefore, our results stand by as a proof of the effectiveness of subcutaneous TCZ in managing inflammatory and immune pathways in RA [53].
We also focused on the magnitude of compromised oral health in RA; most patients in our initial group presented a high rate of mild and severe periodontal disease, validating/reinforcing the already known risk of periodontitis in such patients, particularly in established, longstanding disease [2][3][4]9,37]. We have included in the final analysis only those cases with overt periodontal disease, meaning that up to 75% had at baseline altered periodontal status in a group of consecutive patients starting TCZ for their active disease. Indeed, recent reviews and meta-analyses have already discussed periodontal disease in various RA settings (independent of age, disease duration, serology profile, and disease activity) compared to general population [1,3,5,11,[19][20][21]23].
Finally, we demonstrated that short-term tocilizumab significantly reduced gingival as well as periodontal inflammation as supported by decreased levels of gingival index, bleeding on probing, and probing pocket depth, paralleling the articular improvement. Indeed, only minor changes in clinical attachment loss were detected in our enrolled patients, and the supragingival plaque remained stable after 3 and 6 months of biological treatment (p > 0.05).
An interesting trial compared periodontal condition in patients with RA and periodontitis before and after biological therapy in two cohorts: one under tocilizumab and the other receiving medication with TNF inhibitors [16]. After 6 months, both tocilizumab and TNF inhibitors demonstrated a consistent improvement of oral health with significantly reduced periodontal inflammation (gingival index, bleeding on probing, and probing depth) compared to baseline, with similar results in both cohorts unless there was a greater decrease in gingival index and less gingival inflammation with tocilizumab; however, plaque levels remained the same irrespective of medication, while periodontal clinical attachment loss decreased only after TCZ but not after TNF inhibitors [16,17,27,28]. These observations were partially supported by the results of another study about an excessive inflammatory response against oral pathogens essentially based on high levels of IL-6 [16,17,27,28,31].
Recent meta-analyses reviewed the most important studies on TNF and non-TNF biologics in patients with RA and PD [5,20]. The critical difference between the class of TNF inhibitors and TCZ or B-cell depletive agent rituximab is that infliximab, an anti-TNF monoclonal antibody, may negatively address gingival inflammation although it may also improve alveolar bone destruction [5][6][7]19,37] resulting in a dissociated response for patients with severe periodontitis [5], while both tocilizumab and rituximab associate with significant a down regulation of gingival inflammation and damage in RA associated with periodontitis [16,17,27,28,50].
Additional research is necessary to clearly differentiate between the direct effects of TCZ on local periodontal inflammation and IL-6 or its receptor levels in the gingival crevicular fluids and periodontium of patients and the indirect effect via dramatically decreasing systemic inflammation, which may impact also oral health [16,17,27,28]. Indeed, numerous studies indicated a rapid and significant decline in typical inflammatory parameters (ESR and CRP), but also in serological RA biomarkers (RF, ACPA) as well as inflammatory cytokines (TNF, IL-6) and mediators (serum-amyloid A, matrixmetaloproteinases 1, 3), supporting the indirect role of TCZ in periodontitis [16,17,27,28].
In our study, we assessed specific gingival and periodontal parameters before and after short-term TCZ therapy. We demonstrated successful RA as well as periodontal outcomes with TCZ and independent of potential confounding factors (such as smoking, diabetes, hematological conditions, sex steroid hormones elevations, pharmacological agents) related to periodontal disease, as such patients were excluded from the final analysis.
We concluded that tocilizumab decreased gingival inflammation since no periodontal therapy was permitted and the dental hygiene behavior remained unchanged in our enrolled patients. Unfortunately, we were not able to assess either the serum or gingival crevicular fluid levels of IL-6 or its receptor in all patients; we assumed that tocilizumab indirectly contributes to modulate local (gingival and periodontal) inflammation by limiting systemic inflammation. Indeed, the biofilm plaque accumulation was not consistently diminished with tocilizumab, and we were not able to depict a spectacular impact on clinical attachment loss, but we arrived to demonstrate a positive effect of IL-6 blockade on exuberant gingival inflammation.
Further studies are necessary to confirm the benefits of Il-6 inhibitors in larger populations and longer follow-ups also focusing on IL-6/IL-6 receptor levels in gingival crevicular fluid. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Conflicts of Interest:
The authors declare no conflict of interest.