Periodontal Status, Oral Inflammatory Biomarker, and Relative Expression of Red Complex Bacteria in Periodontitis with Rheumatoid Arthritis Treated with Methotrexate

Periodontal Status, Oral Inflammatory Biomarker, and Relative Expression of Red Complex Bacteria in Periodontitis with Rheumatoid Arthritis Treated with Methotrexate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Periodontal Status, Oral Inflammatory Biomarker, and Relative Expression of Red Complex Bacteria in Periodontitis with Rheumatoid Arthritis Treated with Methotrexate Minessa Mahardika, Widya Rakhmawati, Benso Sulijaya, Sandra Olivia Kuswandani, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7644672/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives To determine the differences in periodontal status—Probing Depth (PD), Clinical Attachment Loss (CAL), Bleeding on Probing (BOP); oral inflammatory biomarkers—Tumor Necrosis Factor Alpha (TNF-α), Interleukin 1 Beta (IL-1β), Prostaglandin E2 (PGE2); and the relative expression of red complex bacteria— Porphyromonas gingivalis ( Pg ), Treponema denticola ( Td ), and Tannerella forsythia ( Tf ) among groups with periodontitis, periodontitis accompanied by rheumatoid arthritis (RA) before treated with methotrexate (MTX), and periodontitis with RA treated with MTX for one month. Material and Methods A cross-sectional study by analyzing periodontal clinical parameters, gingival crevicular fluid (GCF) inflammatory biomarker levels, and subgingival plaque relative expression of red complex bacteria. Samples were taken from patients with periodontitis, periodontitis with RA without MTX, and periodontitis with RA treated with MTX. Samples were collected from the Rheumatology Clinic of Dr. Cipto Mangunkusumo General Hospital, and the Periodontology Clinic of the Dental Teaching Hospital, Faculty of Dentistry, Universitas Indonesia patients. Biomarkers were analysed using the Enzyme-Linked Immunosorbent Assay (ELISA) and bacterial expression was measured using real-time polymerase chain reaction (RT-PCR). Statistical Analysis One-way ANOVA with Tukey’s post-test and Kruskal-Wallis with Dunn’s post-test were used to analysed periodontal parameters, GCF biomarkers, and bacterial expression among the groups. Results No statistically significant differences (p > 0.05) were found for PD and CAL. BOP ​​were significantly higher in the periodontitis group than the RA without MTX group. All biomarkers level were significantly increased in the RA without MTX group compared to the periodontitis group. No significant difference in bacterial expression among groups. Pg and Td expression were significantly higher in the periodontitis group compared to RA without MTX group. Conclusions Patients with periodontitis and RA without MTX exhibited significantly higher inflammatory biomarkers levels, indicating an amplified inflammatory response and tended to be reduced by the MTX. Pg and Td expression was higher in the periodontitis-only group. These findings suggest that RA and its treatment influence both the host inflammatory response and microbial profile in periodontitis. Health sciences/Biomarkers Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Biological sciences/Microbiology Health sciences/Rheumatology inflammatory biomarker methotrexate periodontal parameter periodontitis red complex bacteria rheumatoid arthritis Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Periodontitis is a chronic inflammatory disease that causes progressive periodontal tissue damage with a fairly high prevalence globally and also in Indonesia. 1 , 2 , 3 This disease results from the interaction between pathogenic bacteria as etiological factors, host immune responses, and environmental influences as risk factors. 1 , 4 , 5 , 6 The World Health Organization (WHO), in the Global burden of severe periodontal disease in the Global oral health status report (2022), stated that the global prevalence of periodontitis is around 19% in humans aged over 15 years, starting in late adolescence, peaking at age 55 years and remaining high into old age. 2 Based on the 2023 Indonesian Health Survey (SKI), the prevalence of oral health problems for cases of easily bleeding gums in the age group ≥ 15 years reached 42.4%. 7 Several systemic diseases act as risk factors for periodontitis, and RA is thought to be one of them. Rheumatoid Arthritis is a chronic autoimmune disease that causes morbidity in the joints. If it affects the hand joints, it can lead to limitations in motor movement, specifically inhibiting the process of cleaning the oral cavity, which has the potential to worsen periodontal conditions. 8 Periodontitis and RA have overlapping molecular inflammatory pathways. In both diseases, local tissue damage occurs involving the production of inflammatory mediators such as TNF-α, IL-1ß, and PGE2. 9 – 15 Rheumatoid arthritis (RA) and periodontitis share several pathological features, including bone and soft tissue destruction, as well as elevated levels of circulating inflammatory proteins. 2 Numerous studies and systematic reviews have explored the bidirectional relationship between periodontitis and RA, from both the microbial perspective as a causative agent and the inflammatory response as a host reaction. Periodontitis in RA patients has been reported, with a prevalence of two out of three patients experiencing moderate to severe forms of the disease. Severe periodontitis symptoms in RA patients have been significantly associated with increased levels of Anti-Citrullinated Protein Antibody (ACPA), alterations in the subgingival bacterial profile, and elevated levels of both systemic and oral inflammatory mediators. Methotrexate (MTX) therapy is used as an anchor drug in RA treatment, either as monotherapy or in combination therapy, due to its favourable efficacy-to-toxicity ratio. Conventional Disease Modifying Arthritis Rheumatoid Drugs (csDMARD) therapy needs to be given immediately after the diagnosis of RA is confirmed, with MTX as the primary choice, unless there are contraindications or intolerance. 16 MTX therapy affects the levels of inflammatory mediators such as TNF-α, IL-1ß, and PGE2. 17 Several systematic reviews have reported the effect of reducing TNF-α in Gingival Crevicular Fluid (GCF) in RA therapy using anti-Tumour Necrosis Factor alpha (anti-TNF-α), while studies reporting the effects of MTX use are still limited. 9 , 15 , 17 One study on the relationship between periodontitis and RA in Indonesia showed a relationship between the prevalence and severity of periodontitis in RA patients based on clinical parameters. 18 There has been no study in Indonesia that has observed the effect of RA therapy at the molecular level, especially inflammatory biomarkers in GCF. This study will provide an overview of the differences in TNF-α, IL-1ß, and PGE2 levels in GCF, as well as CAL and BOP values ​​in patients with periodontitis with RA. MATERIAL AND METHOD Study Design The study was approved by the ethics committee for dental research of the Faculty of Dentistry Universitas Indonesia (No. 19/Ethical Approval/FKGUI/III/2024) and the ethics committee for health research, Dr. Cipto Mangunkusumo General Hospital - the Faculty of Medicine Universitas Indonesia (No. KET-758/UN2FI/ETIK/PPM.00.02/2024). The study was performed at the Periodontology Clinic, Dental Teaching Hospital Faculty of Dentistry, Universitas Indonesia and the Rheumatology Clinic, Dr. Cipto Mangunkusumo General Hospital, from September to December 2024. All participants were informed of the aims and methods of the study, and written informed consent was obtained in advance. In total, the study population included 25 participants: 12 systemically healthy periodontitis patients (P group; 2 men and 10 women each; mean age 48.17 ± 6.32; range 41–59 years), 9 RA patients with periodontitis (P + RA group; 9 women; mean age 42.44 ± 8.23; range 35–59 years), and 4 RA patients with periodontitis receiving MTX (P + RA + MTX group; 4 women; mean age 49.50 ± 9.75; range 38–59 years). The inclusion criteria for all groups were age of 35–59 years and diagnosis of periodontitis with interdental CAL detectable at ≥ 2 non-adjacent teeth or buccal CAL ≥ 3 mm with pocketing ≥ 3 mm detectable at ≥ 2 teeth, but the observed CAL cannot be ascribed to non‐periodontitis‐related causes. For the RA groups, a confirmed diagnosis of RA was required by a rheumatologist according to the 2010 ACR/EULAR classification. For the P + RA + MTX group, patients have received 1 month of MTX therapy. The exclusion criteria for all groups were diabetes mellitus, hypertension, endocarditis, other autoimmune disease, HIV/AIDS, hepatitis, tuberculosis, pregnancy, smoking, use of antibiotic drugs and/or periodontal treatment within the last 3 months, and inability to perform oral hygiene. All participants underwent a full-mouth periodontal examination by calibrated periodontists (MM and WR), assessing PD, CAL, and BOP. The PD and CAL measurements were performed with a UNC-15 periodontal probe (Hu-Friedy Co., Chicago, IL, USA) at 6 sites per tooth (mesiobuccal, mid buccal, distobuccal, mesiolingual, midlingual, and distolingual) for all teeth except the third molar, and the deepest one was recorded. The BOP was recorded as present or absent within 30 seconds of probing at 6 sites per tooth for all teeth. GCF Sampling GCF for sampling was taken at the deepest PD site. The site was isolated with cotton rolls to avoid saliva contamination. A paper point was inserted into the pocket for 30 seconds. Paper with blood contamination was discarded. Subsequently, the paper point was removed and immediately frozen at -80℃ in an Eppendorf tube. Subgingival Biofilm Sampling The sampling area was isolated with a cotton roll, supragingival plaque was eliminated using an excavator, scraped gently at the deepest PD site, and then placed on a paper point no. 15. Then it was put into an Eppendorf tube containing Phosphate Buffer Saline solution and labelled. Statistical Analysis Power analysis was performed with the statistical software (G*Power, version 3.1.9.7, Heinrich Heine Universität Düsseldorf, Germany), based on previous research by Yuce et al. (2017). 19 A total sample of 12 subjects would be required to show a significant difference in periodontal measurements, with 95% power and 5% confidence. Considering a possible attrition rate of 15% during the study period, the total sample size of 15 subjects was calculated. Statistical analysis was performed using the statistical software GraphPad Prism (version 10.4.1). Metric data were examined according to their normal distribution using the Shapiro-Wilk test. The comparison of normally distributed data was performed using a one-way ANOVA test, and non-normally distributed data were compared using the Kruskal-Wallis test. In cases of significant results, post-hoc testing with Tukey’s or Dunn’s multiple comparisons test was applied. A p-value of < 0.05 was considered statistically significant. RESULTS Demographic data, including patient gender and age, are summarized in Table 1 . Sixteen patients diagnosed with periodontitis (P), 2 men and 10 women, ranging in age from 41 to 59 years (48.17 ± 6.32 years). The RA patients with periodontitis group (P + RA) consisted of 9 women aged 35 to 59 years (42.44 ± 8.23 years), while RA patients with periodontitis receiving MTX group (P + RA + MTX) consisted of 4 women aged 38 to 59 years (49.50 ± 9.75 years). Table 1 Demographic characteristics Characteristic P (n = 12) Mean ± SD P + RA (n = 9) Mean ± SD P + RA + MTX (n = 4) Mean ± SD Age (years) 48.17 ± 6.32 42.44 ± 8.23 49.50 ± 9.75 Sex Female 10 9 4 Male 2 - - Abbreviations: P, periodontitis; P + RA, periodontitis + rheumatoid arthritis; P + RA + MTX, periodontitis + rheumatoid arthritis + methotrexate; n, number of subjects; SD, standard deviation Table 2 presents the periodontal clinical examination. There were no statistically significant differences (p > 0.05) for PD and CAL values ​​between the three groups, as well as for BOP values ​​between the periodontitis group without RA or accompanied by RA without MTX therapy and the periodontitis group accompanied by RA that had been treated with MTX. Statistically significant differences (p = 0.016) were only found for BOP values between the periodontitis group and the periodontitis group accompanied by RA without MTX therapy as shown in Fig. 1 . Table 2 Periodontal clinical examination Clinical Parameters P (n = 12) Median (Min-Max) P + RA (n = 9) Median (Min-Max) P + RA + MTX (n = 4) Median (Min-Max) PD (mm) 6.00 (4.00–9.00) 5.00 (4.00–8.00) 4.50 (4.00–5.00) P value vs P ns ns P value vs P + RA + MTX ns CAL (mm) 5.00 (2.00–11.00) 4.00 (3.00–6.00) 3.00 (2.00–4.00) P value vs P ns ns P value vs P + RA + MTX ns BOP (%) 15.30 (8.00–58.00) 3.80 (0.00–12.00) 5.70 (4.40–18.50) P value vs P 0.0016 ns P value vs P + RA + MTX ns Post-hoc Tukey’s test for CAL, Dunn’s test for PD and BOP. Abbreviations: P, periodontitis; P + RA, periodontitis + rheumatoid arthritis; P + RA + MTX, periodontitis + rheumatoid arthritis + methotrexate; n, number of subjects; Min, minimum; Max, maximum; PD, probing depth; CAL, clinical attachment loss; BOP, bleeding on probing; ns, non-significant Table 3 shows statistically significant differences found only in TNF-α (p = 0.0052), IL-1β (p = 0.0084), and PGE2 (p = 0.0138) levels between the periodontitis group and the periodontitis group accompanied by RA without MTX therapy. Figure 2 shows the differences in each group for levels of inflammatory biomarkers. Table 3 Inflammatory biomarker level Cytokine Level P (n = 12) Median (Min-Max) P + RA (n = 9) Median (Min-Max) P + RA + MTX (n = 4) Median (Min-Max) TNF-⍺ (pg/mL) 35.95 (10.03–57.15) 102.4 (24.47–231.5) 60.89 (50.17–112.8) P value vs P 0.0052 ns P value vs P + RA + MTX ns IL-1β (pg/mL) 147.4 (49.96–267.7) 397.2 (100.5-869.7) 259.1 (254.2-504.1) P value vs P 0.0084 ns P value vs P + RA + MTX ns PGE2 (pg/mL) 28.03 (7.959–54.49) 80.43 (18.53–191.7) 49.24 (45.6-105.1) P value vs P 0.0138 ns P value vs P + RA + MTX ns Post-hoc Dunn’s test for all markers. Abbreviations: P, periodontitis; P + RA, periodontitis + rheumatoid arthritis; P + RA + MTX, periodontitis + rheumatoid arthritis + methotrexate; n, number of subjects; Min, minimum; Max, maximum; TNF-⍺, tumour necrosis factor alpha; IL-1β, interleukin 1 beta; PGE2, prostaglandin E2; ns, non-significant The relative expression of total bacteria and red complex bacteria values is presented in Table 4 . The relative expression of total bacteria in each sample group showed no significant difference. The result show statistically significant differences found only in Pg (p = 0.012) and Td (p = 0.0315) between the periodontitis group and the periodontitis group accompanied by RA without MTX therapy. Figure 3 shows the differences in each group for levels of inflammatory biomarkers. Table 4 Relative expression of total bacteria and red complex bacteria Relative Expression P (n = 12) Median (Min-Max) P + RA (n = 9) Median (Min-Max) P + RA + MTX (n = 4) Median (Min-Max) 16sRNA 17.86 (16.19–20.08) 16.83 (16.15–19.03) 17.36 (15.99–18.16) P value vs P ns ns P value vs P + RA + MTX ns Pg 63.21 (15.57–402.0) 5.79 (0.014–40.98) 22.10 (0.004–105.9) P value vs P 0.012 ns P value vs P + RA + MTX ns Td 3.37 (0.85–24.36) 0.85 (0.006–9.91) 1.41 (0.002–3.45) P value vs P 0.0315 ns P value vs P + RA + MTX ns Tf 9.67 (0.76–22.45) 1.71 (0.10-71.68) 2.77 (0.10–3.63) P value vs P ns ns P value vs P + RA + MTX ns Post-hoc Tukey’s test for 16sRNA, Dunn’s test for Pg , Td , and Tf . Abbreviations: P, periodontitis; P + RA, periodontitis + rheumatoid arthritis; P + RA + MTX, periodontitis + rheumatoid arthritis + methotrexate; n, number of subjects; Min, minimum; Max, maximum; Pg, Porphyromonas gingivalis ; Td , Treponema denticola ; Tf , Tannerella forsythia ; ns, non-significant DISCUSSION The 2023 SKI report showed a higher prevalence of oral health problems, bleeding gums, in women (7.3%) compared to men (6.4%). 7 Pavlov-Dolijanovic et al. (2023) in their review explained that the ratio of women to men in YORA was 3:1. 20 Periodontal clinical parameters in this study included PD, CAL, and BOP. The results of the comparative analysis of CAL among patients with periodontitis, patients with periodontitis accompanied by RA without MTX therapy, and patients with periodontitis accompanied by RA who have been treated with MTX showed no significant difference in CAL values. Zhao et al. (2018) in their systematic review state that periodontitis significantly increases the severity of RA, but RA does not significantly affect the development of periodontitis. 21 Research by Susanto et al. (2013) showed no significant differences in most periodontitis severity parameters between RA patients and controls. 18 Studies from Jung et al. (2018) show that conventional DMARD therapy alone has no significant effect on periodontal conditions, while significant improvement in periodontal parameters is obtained after non-surgical periodontal therapy in RA patients with conventional DMARD therapy. 22 The BOP value of periodontitis patients was significantly higher than that of patients with periodontitis accompanied by RA without MTX therapy. The BOP value of patients with periodontitis accompanied by RA without MTX therapy, which was lower than that of patients with periodontitis accompanied by RA who had been treated with MTX was possibly caused by the administration of the corticosteroid drug methylprednisolone while the patient was being examined whether the patient could be given DMARD drugs or not. Corticosteroids have vasoconstrictor and anti-inflammatory effects by inhibiting the action of histamine and inflammatory mediators. Corticosteroids increase the effect of vasoconstrictor hormones by increasing the response of blood vessel contraction to norepinephrine through increasing the sensitivity of α adrenergic receptors, increasing the number of angiotensin type 1 (AT1) receptors in vascular smooth muscle cells, thereby strengthening contractions triggered by angiotensin II, and also by stimulating the release of endothelin. 23 , 24 , 25 This study showed comparative analysis results that were significantly different in each inflammatory biomarker level between periodontitis patients and periodontitis patients with RA without MTX therapy, and no significant differences between periodontitis patients and periodontitis patients with RA who had been treated with MTX, where the lowest median value was seen in periodontitis patients. No significant differences were seen in each inflammatory biomarker level between periodontitis patients with RA without MTX therapy and periodontitis patients with RA who had been treated with MTX, where the median values ​​of TNF-α, IL-1β, and PGE2 levels were lower in periodontitis patients with RA who had been treated with MTX. The tendency for the median values ​​of inflammatory biomarker levels to increase in periodontitis patients with RA without MTX therapy and then decrease in periodontitis patients with RA who had been treated with MTX indicates a strengthening of the inflammatory effect by RA conditions in periodontitis patients and the presence of a decrease in inflammation after MTX therapy. The ‘two-hit model’ hypothesis proposed by Golub et al. in 2006 stated that in individuals who initially experience chronic inflammation in the form of periodontitis, which produces anti-cyclic citrullinated peptide antibodies, when joint inflammation occurs later, which produces citrullination, the subsequent antibody response can be very strong. 15 The results of this study are in accordance with this hypothesis, where the levels of TNF-α, IL-1β, and PGE2 in GCF are higher in patients with periodontitis accompanied by RA. Various studies on TNF-α, IL-1β, and PGE2 in GCF of patients with periodontitis and RA have shown varying results. The results of this study showed significantly higher levels of TNF-α, IL-1β, and PGE2 in patients with periodontitis accompanied by RA, following several previous studies that support the ‘two-hit model’ hypothesis. 15 Research from Xiao et al. (2021), Arvikar et al. (2021), Cosgarea et al. (2019), and a literature review by Bartold and Lopez-Olivia (2020) stated that there were high levels of TNF-α, IL-1β in patients with periodontitis with RA compared to patients with periodontitis without RA. 15,26,27,28 A study by Kurgan et al. (2016) showed statistically higher PGE2 levels in the periodontitis group with RA compared to the periodontitis group without RA in their research results. 29 The immunomodulatory cytokines TNF-α and IL-1β have proinflammatory effects that play an important role in periodontitis and RA by increasing chemokine production and promoting the expression of metalloproteinases and prostaglandins. Both induce inflammatory cells to attack the infected site in periodontitis, causing bone destruction through the production of inflammatory factors and activation of cytokines that contribute to alveolar bone resorption and collagen fiber damage. 15 , 26 , 30 Tumor Necrosis Factor alpha plays a key role in the immune response to periodontitis and RA by modulating the inflammatory response in the form of an increased inflammatory response by binding to the p55 receptor (T receptor type 1; CD120a). 15 Several studies on the treatment of periodontitis and RA have shown that anti-TNF-α therapy in RA patients can reduce TNF-α levels in GCF and cause milder periodontal disease compared to patients who do not receive anti-TNF-α therapy. 15 , 31 Several studies on periodontal treatment have reported a decrease in TNF-α levels in GCF in RA patients with periodontitis who receive non-surgical periodontal treatment. 15 , 28 These results indicate the dynamics of TNF-α in inflammatory conditions and the response to its treatment. Most reports on the effects of RA therapy on periodontal disease show the benefits of using TNF-α inhibitors. Other reports on interleukin-6 receptor inhibitor therapy and anti-lymphocyte B therapy also show benefits in periodontal disease. 15 , 28 This study showed the results of a decrease in TNF-α, IL-1β, and PGE2 levels in GCF of patients with periodontitis accompanied by RA who had been treated with MTX, although not significant. MTX therapy is the most commonly used DMARD, working by inhibiting the enzyme dihydrofolate reductase, providing an inhibitory effect on thymidine synthesis, RNA transmethylation, DNA, protein, phospholipids, and de novo purine synthesis, so that it can then suppress the secretion of IL-6, IL-8, IL-17A, and TNF in RA subjects. 32 Limited supporting data from previous studies and the insignificant results in this study leave questions that cannot be answered regarding the effect of MTX therapy in RA patients on periodontitis conditions. Ziebolz et al. (2018) reported increased BOP values ​​in patients receiving combination therapy of MTX with TNF-α antagonists. 33 Several reports from previous studies have shown improvements in RA parameters and disease activity after non-surgical periodontal treatment, thus indicating the need for periodontal treatment as additional treatment for RA patients with periodontitis. 13 , 15 , 25 , 34 Most treatments for RA use DMARDs, which are included as host-modulating treatments. 16 , 35 , 36 This is interesting because host-modulating treatments are also additional treatments for periodontitis. 37 Chronic inflammation can also have the effect of suppressing the host defense system, which makes periodontal infection worse because the host loses its defense capacity, while from this study, the view arises that periodontal inflammation and infection can affect the effectiveness of DMARDs in treating RA. 38 The results of this study show that the comparative analysis of total bacteria in each sample group showed no significant difference. This can be related to the high differences in bacterial composition, with the dominant types of bacteria, whether pathogenic, commensal, or opportunistic, varying between healthy individuals, those with periodontitis, and those with systemic conditions such as rheumatoid arthritis. 39 Individuals in a healthy state are dominated by commensal bacteria that help maintain oral environment balance and prevent pathogen growth. 40 The transition from a healthy state to periodontitis involves changes in biofilm structure and microbial interactions, which trigger inflammation and periodontal tissue damage. Periodontitis occurs due to dysbiosis, i.e., changes in the composition of the microbiome that led to the dominance of pathogenic bacteria and a decrease in commensal bacteria. 40 The higher composition of pathogenic bacteria in periodontitis indicates that the microbiota ecosystem in the oral cavity undergoes major changes that support the growth of pathogens. 41 The relative expression of red complex bacteria ( Pg, Td, Tf ) shown in Fig. 3 was significantly higher in Pg and Td bacteria. The relative expression of Pg in the periodontitis sample group without accompanying RA tends to be higher. This tendency is partly caused by the antibody reaction against Pg , resulting in a decrease in Pg in the periodontitis samples with RA. A study conducted by Johansson et al. aimed to determine whether antibodies against Pg were present before the onset of RA symptoms. Pg bacteria are suspected to play a role in triggering RA by inducing the production of anti-citrullinated protein antibodies (ACPA), which are the main markers of this disease. ACPA antibodies are known to appear several years before RA symptoms manifest, indicating that an immune system imbalance occurs long before RA symptoms develop, with the likely initial site being mucosal tissues such as the gingiva. The study measured antibodies against Pg , specifically against arginine gingipain B (RgpB) and cyclic citrullinated peptide (CCP3), using the ELISA method. Anti-RgpB IgG antibody levels were significantly higher in pre-symptomatic individuals, and anti-CPP3 antibodies were detected in 5% of pre-symptomatic individuals, allowing the conclusion that high levels of antibodies against Pg can be detected years before RA symptoms appear; thus, the relative expression of Pg tends to be lower. 42 Td bacteria are often found alongside other periodontal pathogens such as Pg and Tf in RA patients. The prevalence of Td and other periodontal pathogens may vary depending on the type of RA treatment. Patients using a combination of methotrexate (MTX) and TNF-α antagonists show increased periodontal inflammation, which may influence microbial composition, including Td , resulting in a higher tendency in the periodontitis group without RA. 43,44,45 Td bacteria frequently interact with other periodontal pathogens such as Pg and Tf , which are collectively known as the red complex. The composition and microbial interactions may differ between patients with and without RA, potentially affecting the relative expression of Td detected. 46 Changes in bacterial composition due to treatment or other systemic factors may inhibit the bacterial ecosystem, so Td does not develop as rapidly in patients with periodontitis without RA. 47 Comparative analysis results of relative expression of Pg , Td , and Tf bacteria showed no significant differences between the periodontitis groups with RA before and after 1 month of MTX therapy (p > 0.05). The lack of significant differences may be related to several disease-modifying anti-rheumatic drugs (DMARDs), particularly MTX, which have a dual role in RA treatment: not only modulating immune responses but also directly targeting certain bacteria associated with RA pathogenesis. However, since this antimicrobial activity is species-dependent, the effect is not uniform across all oral pathogens. Antimicrobial evaluations of several DMARDs were conducted by Kussmann et al. Their study found that MTX exhibited antimicrobial activity against Fusobacterium nucleatum and Viridans streptococci , Aurothiomalate (ATM) showed broad-spectrum activity, Sulfasalazine (SSZ) was active against several oral pathogens ( Actinomyces spp., Capnocytophaga spp., Eikenella corrodens , F. nucleatum , and Pg ) but only at very high concentrations, while other DMARDs did not show significant antimicrobial activity. 48 , 49 Prospective studies are needed to obtain a causal relationship between periodontitis and RA or vice versa, along with the effects of therapy for each disease. Studies with a longer follow-up period are needed to further observe the effects of MTX therapy on periodontal clinical parameters, RA parameters, and inflammatory biomarker levels. Metagenomics-based studies or comprehensive microbiome analyses can be conducted to obtain a more comprehensive understanding of oral microbiota changes resulting from periodontitis and RA. CONCLUSIONS Within the limitations of this study, the following conclusions can be drawn. Significant differences in clinical parameters among the groups were only seen in the lower BOP values of the periodontitis group compared to the periodontitis group with RA without MTX therapy. This study showed a significant increase in the level of all three inflammatory biomarkers in the periodontitis group compared to the periodontitis group with RA without MTX therapy. However, the decrease in the level of inflammatory biomarkers in the group of periodontitis with RA without MTX therapy compared to the group of periodontitis with RA with MTX therapy was not significant. The relative expression of red complex bacteria ( Pg , Td ) was significantly higher in Pg and Td bacteria in the periodontitis sample group without accompanying RA. However, comparative analysis results of relative expression of Pg , Td , and Tf bacteria showed no significant differences between the periodontitis groups with RA before and after 1 month of MTX therapy. Declarations Author Contribution Statement Conceptualization: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W, Takahashi N, Tabeta K; Formal analysis: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Investigation: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Methodology: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Writing—original draft: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Writing—reviewing and editing: Mahardika M, Rakhmawati W, Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Soeroso Y, Masulili SLC, Tadjoedin FM, Rahdewati H, Takahashi N, Tabeta K; Project administration: Sulijaya B; Supervision: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA. Conflict of Interest The authors state no conflict of interest. Funding Information This study was supported by a grant from the Directorate of Research and Community Engagement, Universitas Indonesia (Research Grant PUTI Q1 No: NKB-320/UN2.RST/HKP.05.00/2024 to B.S.). Author Contribution Conceptualization: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W, Takahashi N, Tabeta K; Formal analysis: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Investigation: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Methodology: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Writing—original draft: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Writing—reviewing and editing: Mahardika M, Rakhmawati W, Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Soeroso Y, Masulili SLC, Tadjoedin FM, Rahdewati H, Takahashi N, Tabeta K; Project administration: Sulijaya B; Supervision: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA; All authors reviewed the manuscript. Acknowledgement The authors gratefully acknowledge Universitas Indonesia for supporting the publication of this study. We also extend our sincere appreciation to the Dental Teaching Hospital, Faculty of Dentistry, Universitas Indonesia, and Dr. Cipto Mangunkusumo General Hospital (RSCM) for providing the facilities and support necessary for conducting the research Data Availability All data generated or analysed during this study are included in this published article. References Papapanou, P. N. et al. Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J. Periodontol . 89 (Suppl 1), S173–S182. 10.1002/JPER.17-0721 (2018). Global oral health. status report: towards universal health coverage for oral health by 2030 (World Health Organization, 2022). Licence: CC BY-NC-SA 3.0 IGO. Suratri, M. A. L., Jovina, T. A. & Andayasari, L. Pengaruh Hipertensi Terhadap Kejadian Penyakit Jaringan Periodontal (Periodontitis) pada Masyarakat Indonesia (Data Riskesdas 2018). 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Kussmann, M., Obermueller, M., Spettel, K., Winkler, S. & Aletaha, D. In vitro evaluation of disease-modifying antirheumatic drugs against rheumatoid arthritis associated pathogens of the oral microflora. RMD Open. 7 (3), e001737. 10.1136/rmdopen-2021-001737 (2021). Inchingolo, F. et al. The Effects of Periodontal Treatment on Rheumatoid Arthritis and of Anti-Rheumatic Drugs on Periodontitis: A Systematic Review. Int. J. Mol. Sci. 24 (24), 17228. 10.3390/ijms242417228 (2023). Published 2023 Dec 7. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Post-hoc Tukey’s test for CAL, Dunn’s test for PD and BOP. N: 4-12 subjects per group. Note: * and **, \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05; ns, non-significant\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7644672/v1/df5e80440f9a615bd9fbfd7e.png"},{"id":93637955,"identity":"861c8536-a61f-4cfd-96b3-0b4048482402","added_by":"auto","created_at":"2025-10-16 01:56:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25116,"visible":true,"origin":"","legend":"\u003cp\u003eGCF inflammatory biomarkers. Post-hoc Dunn’s test for all markers. N: 4-12 subjects per group. Note: * and **, \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05; ns, non-significant\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7644672/v1/327fbc03ee04a3d3add36b92.png"},{"id":93637958,"identity":"f2389b00-dbff-4fca-aa14-fa70d384293f","added_by":"auto","created_at":"2025-10-16 01:56:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69443,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of total bacteria (16sRNA) and red complex bacteria. Post-hoc Tukey’s test for 16sRNA, Dunn’s test for \u003cem\u003ePg\u003c/em\u003e, \u003cem\u003eTd\u003c/em\u003e, and\u003cem\u003e Tf\u003c/em\u003e. N: 4-12 subjects per group. Note: * and **, \u003cem\u003eP \u003c/em\u003e\u0026lt;0.05; ns, non-significant\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7644672/v1/561f9fdf4b703d9e7a1769ff.png"},{"id":94065657,"identity":"4f658d87-2c4e-4ed3-b1ae-3ee217546795","added_by":"auto","created_at":"2025-10-22 07:53:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1122430,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7644672/v1/738ed605-d074-4ba6-937a-cf24a719e14f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Periodontal Status, Oral Inflammatory Biomarker, and Relative Expression of Red Complex Bacteria in Periodontitis with Rheumatoid Arthritis Treated with Methotrexate","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePeriodontitis is a chronic inflammatory disease that causes progressive periodontal tissue damage with a fairly high prevalence globally and also in Indonesia.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e This disease results from the interaction between pathogenic bacteria as etiological factors, host immune responses, and environmental influences as risk factors.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e The World Health Organization (WHO), in the Global burden of severe periodontal disease in the Global oral health status report (2022), stated that the global prevalence of periodontitis is around 19% in humans aged over 15 years, starting in late adolescence, peaking at age 55 years and remaining high into old age.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Based on the 2023 Indonesian Health Survey (SKI), the prevalence of oral health problems for cases of easily bleeding gums in the age group\u0026thinsp;\u0026ge;\u0026thinsp;15 years reached 42.4%.\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eSeveral systemic diseases act as risk factors for periodontitis, and RA is thought to be one of them. Rheumatoid Arthritis is a chronic autoimmune disease that causes morbidity in the joints. If it affects the hand joints, it can lead to limitations in motor movement, specifically inhibiting the process of cleaning the oral cavity, which has the potential to worsen periodontal conditions.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Periodontitis and RA have overlapping molecular inflammatory pathways. In both diseases, local tissue damage occurs involving the production of inflammatory mediators such as TNF-α, IL-1\u0026szlig;, and PGE2.\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Rheumatoid arthritis (RA) and periodontitis share several pathological features, including bone and soft tissue destruction, as well as elevated levels of circulating inflammatory proteins.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Numerous studies and systematic reviews have explored the bidirectional relationship between periodontitis and RA, from both the microbial perspective as a causative agent and the inflammatory response as a host reaction. Periodontitis in RA patients has been reported, with a prevalence of two out of three patients experiencing moderate to severe forms of the disease. Severe periodontitis symptoms in RA patients have been significantly associated with increased levels of Anti-Citrullinated Protein Antibody (ACPA), alterations in the subgingival bacterial profile, and elevated levels of both systemic and oral inflammatory mediators. Methotrexate (MTX) therapy is used as an anchor drug in RA treatment, either as monotherapy or in combination therapy, due to its favourable efficacy-to-toxicity ratio. Conventional Disease Modifying Arthritis Rheumatoid Drugs (csDMARD) therapy needs to be given immediately after the diagnosis of RA is confirmed, with MTX as the primary choice, unless there are contraindications or intolerance.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e MTX therapy affects the levels of inflammatory mediators such as TNF-α, IL-1\u0026szlig;, and PGE2.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Several systematic reviews have reported the effect of reducing TNF-α in Gingival Crevicular Fluid (GCF) in RA therapy using anti-Tumour Necrosis Factor alpha (anti-TNF-α), while studies reporting the effects of MTX use are still limited.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eOne study on the relationship between periodontitis and RA in Indonesia showed a relationship between the prevalence and severity of periodontitis in RA patients based on clinical parameters.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e There has been no study in Indonesia that has observed the effect of RA therapy at the molecular level, especially inflammatory biomarkers in GCF. This study will provide an overview of the differences in TNF-α, IL-1\u0026szlig;, and PGE2 levels in GCF, as well as CAL and BOP values ​​in patients with periodontitis with RA.\u003c/p\u003e"},{"header":"MATERIAL AND METHOD","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design\u003c/h2\u003e\u003cp\u003eThe study was approved by the ethics committee for dental research of the Faculty of Dentistry Universitas Indonesia (No. 19/Ethical Approval/FKGUI/III/2024) and the ethics committee for health research, Dr. Cipto Mangunkusumo General Hospital - the Faculty of Medicine Universitas Indonesia (No. KET-758/UN2FI/ETIK/PPM.00.02/2024). The study was performed at the Periodontology Clinic, Dental Teaching Hospital Faculty of Dentistry, Universitas Indonesia and the Rheumatology Clinic, Dr. Cipto Mangunkusumo General Hospital, from September to December 2024. All participants were informed of the aims and methods of the study, and written informed consent was obtained in advance. In total, the study population included 25 participants: 12 systemically healthy periodontitis patients (P group; 2 men and 10 women each; mean age 48.17\u0026thinsp;\u0026plusmn;\u0026thinsp;6.32; range 41\u0026ndash;59 years), 9 RA patients with periodontitis (P\u0026thinsp;+\u0026thinsp;RA group; 9 women; mean age 42.44\u0026thinsp;\u0026plusmn;\u0026thinsp;8.23; range 35\u0026ndash;59 years), and 4 RA patients with periodontitis receiving MTX (P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX group; 4 women; mean age 49.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.75; range 38\u0026ndash;59 years). The inclusion criteria for all groups were age of 35\u0026ndash;59 years and diagnosis of periodontitis with interdental CAL detectable at \u0026ge;\u0026thinsp;2 non-adjacent teeth or buccal CAL\u0026thinsp;\u0026ge;\u0026thinsp;3 mm with pocketing\u0026thinsp;\u0026ge;\u0026thinsp;3 mm detectable at \u0026ge;\u0026thinsp;2 teeth, but the observed CAL cannot be ascribed to non‐periodontitis‐related causes. For the RA groups, a confirmed diagnosis of RA was required by a rheumatologist according to the 2010 ACR/EULAR classification. For the P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX group, patients have received 1 month of MTX therapy. The exclusion criteria for all groups were diabetes mellitus, hypertension, endocarditis, other autoimmune disease, HIV/AIDS, hepatitis, tuberculosis, pregnancy, smoking, use of antibiotic drugs and/or periodontal treatment within the last 3 months, and inability to perform oral hygiene.\u003c/p\u003e\u003cp\u003eAll participants underwent a full-mouth periodontal examination by calibrated periodontists (MM and WR), assessing PD, CAL, and BOP. The PD and CAL measurements were performed with a UNC-15 periodontal probe (Hu-Friedy Co., Chicago, IL, USA) at 6 sites per tooth (mesiobuccal, mid buccal, distobuccal, mesiolingual, midlingual, and distolingual) for all teeth except the third molar, and the deepest one was recorded. The BOP was recorded as present or absent within 30 seconds of probing at 6 sites per tooth for all teeth.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGCF Sampling\u003c/h3\u003e\n\u003cp\u003eGCF for sampling was taken at the deepest PD site. The site was isolated with cotton rolls to avoid saliva contamination. A paper point was inserted into the pocket for 30 seconds. Paper with blood contamination was discarded. Subsequently, the paper point was removed and immediately frozen at -80℃ in an Eppendorf tube.\u003c/p\u003e\n\u003ch3\u003eSubgingival Biofilm Sampling\u003c/h3\u003e\n\u003cp\u003eThe sampling area was isolated with a cotton roll, supragingival plaque was eliminated using an excavator, scraped gently at the deepest PD site, and then placed on a paper point no. 15. Then it was put into an Eppendorf tube containing Phosphate Buffer Saline solution and labelled.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003ePower analysis was performed with the statistical software (G*Power, version 3.1.9.7, Heinrich Heine Universit\u0026auml;t D\u0026uuml;sseldorf, Germany), based on previous research by Yuce et al. (2017).\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e A total sample of 12 subjects would be required to show a significant difference in periodontal measurements, with 95% power and 5% confidence. Considering a possible attrition rate of 15% during the study period, the total sample size of 15 subjects was calculated. Statistical analysis was performed using the statistical software GraphPad Prism (version 10.4.1). Metric data were examined according to their normal distribution using the Shapiro-Wilk test. The comparison of normally distributed data was performed using a one-way ANOVA test, and non-normally distributed data were compared using the Kruskal-Wallis test. In cases of significant results, post-hoc testing with Tukey\u0026rsquo;s or Dunn\u0026rsquo;s multiple comparisons test was applied. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eDemographic data, including patient gender and age, are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Sixteen patients diagnosed with periodontitis (P), 2 men and 10 women, ranging in age from 41 to 59 years (48.17\u0026thinsp;\u0026plusmn;\u0026thinsp;6.32 years). The RA patients with periodontitis group (P\u0026thinsp;+\u0026thinsp;RA) consisted of 9 women aged 35 to 59 years (42.44\u0026thinsp;\u0026plusmn;\u0026thinsp;8.23 years), while RA patients with periodontitis receiving MTX group (P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX) consisted of 4 women aged 38 to 59 years (49.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.75 years).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographic characteristics\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP\u0026thinsp;+\u0026thinsp;RA (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e\u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge (years)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e48.17\u0026thinsp;\u0026plusmn;\u0026thinsp;6.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42.44\u0026thinsp;\u0026plusmn;\u0026thinsp;8.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations: P, periodontitis; P\u0026thinsp;+\u0026thinsp;RA, periodontitis\u0026thinsp;+\u0026thinsp;rheumatoid arthritis; P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX, periodontitis\u0026thinsp;+\u0026thinsp;rheumatoid arthritis\u0026thinsp;+\u0026thinsp;methotrexate; n, number of subjects; SD, standard deviation\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents the periodontal clinical examination. There were no statistically significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) for PD and CAL values ​​between the three groups, as well as for BOP values ​​between the periodontitis group without RA or accompanied by RA without MTX therapy and the periodontitis group accompanied by RA that had been treated with MTX. Statistically significant differences (p\u0026thinsp;=\u0026thinsp;0.016) were only found for BOP values between the periodontitis group and the periodontitis group accompanied by RA without MTX therapy as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePeriodontal clinical examination\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical Parameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP\u0026thinsp;+\u0026thinsp;RA (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePD (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6.00 (4.00\u0026ndash;9.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.00 (4.00\u0026ndash;8.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.50 (4.00\u0026ndash;5.00)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCAL (mm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5.00 (2.00\u0026ndash;11.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.00 (3.00\u0026ndash;6.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.00 (2.00\u0026ndash;4.00)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBOP (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15.30 (8.00\u0026ndash;58.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.80 (0.00\u0026ndash;12.00)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.70 (4.40\u0026ndash;18.50)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003ePost-hoc Tukey\u0026rsquo;s test for CAL, Dunn\u0026rsquo;s test for PD and BOP.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations: P, periodontitis; P\u0026thinsp;+\u0026thinsp;RA, periodontitis\u0026thinsp;+\u0026thinsp;rheumatoid arthritis; P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX, periodontitis\u0026thinsp;+\u0026thinsp;rheumatoid arthritis\u0026thinsp;+\u0026thinsp;methotrexate; n, number of subjects; Min, minimum; Max, maximum; PD, probing depth; CAL, clinical attachment loss; BOP, bleeding on probing; ns, non-significant\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows statistically significant differences found only in TNF-α (p\u0026thinsp;=\u0026thinsp;0.0052), IL-1β (p\u0026thinsp;=\u0026thinsp;0.0084), and PGE2 (p\u0026thinsp;=\u0026thinsp;0.0138) levels between the periodontitis group and the periodontitis group accompanied by RA without MTX therapy. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the differences in each group for levels of inflammatory biomarkers.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInflammatory biomarker level\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCytokine Level\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP\u0026thinsp;+\u0026thinsp;RA (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNF-⍺ (pg/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e35.95 (10.03\u0026ndash;57.15)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e102.4 (24.47\u0026ndash;231.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60.89 (50.17\u0026ndash;112.8)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0052\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-1β (pg/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e147.4 (49.96\u0026ndash;267.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e397.2 (100.5-869.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e259.1 (254.2-504.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePGE2 (pg/mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28.03 (7.959\u0026ndash;54.49)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e80.43 (18.53\u0026ndash;191.7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e49.24 (45.6-105.1)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0138\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003ePost-hoc Dunn\u0026rsquo;s test for all markers.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations: P, periodontitis; P\u0026thinsp;+\u0026thinsp;RA, periodontitis\u0026thinsp;+\u0026thinsp;rheumatoid arthritis; P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX, periodontitis\u0026thinsp;+\u0026thinsp;rheumatoid arthritis\u0026thinsp;+\u0026thinsp;methotrexate; n, number of subjects; Min, minimum; Max, maximum; TNF-⍺, tumour necrosis factor alpha; IL-1β, interleukin 1 beta; PGE2, prostaglandin E2; ns, non-significant\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe relative expression of total bacteria and red complex bacteria values is presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The relative expression of total bacteria in each sample group showed no significant difference. The result show statistically significant differences found only in \u003cem\u003ePg\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.012) and \u003cem\u003eTd\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0315) between the periodontitis group and the periodontitis group accompanied by RA without MTX therapy. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the differences in each group for levels of inflammatory biomarkers.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRelative expression of total bacteria and red complex bacteria\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRelative Expression\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eP (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP\u0026thinsp;+\u0026thinsp;RA (n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eP\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX (n\u0026thinsp;=\u0026thinsp;4)\u003c/p\u003e\u003cp\u003eMedian (Min-Max)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16sRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e17.86 (16.19\u0026ndash;20.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.83 (16.15\u0026ndash;19.03)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.36 (15.99\u0026ndash;18.16)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePg\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e63.21 (15.57\u0026ndash;402.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.79 (0.014\u0026ndash;40.98)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e22.10 (0.004\u0026ndash;105.9)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTd\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3.37 (0.85\u0026ndash;24.36)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.85 (0.006\u0026ndash;9.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.41 (0.002\u0026ndash;3.45)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0315\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eTf\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.67 (0.76\u0026ndash;22.45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.71 (0.10-71.68)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.77 (0.10\u0026ndash;3.63)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value vs P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003ePost-hoc Tukey\u0026rsquo;s test for 16sRNA, Dunn\u0026rsquo;s test for \u003cem\u003ePg\u003c/em\u003e, \u003cem\u003eTd\u003c/em\u003e, and \u003cem\u003eTf\u003c/em\u003e.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eAbbreviations: P, periodontitis; P\u0026thinsp;+\u0026thinsp;RA, periodontitis\u0026thinsp;+\u0026thinsp;rheumatoid arthritis; P\u0026thinsp;+\u0026thinsp;RA\u0026thinsp;+\u0026thinsp;MTX, periodontitis\u0026thinsp;+\u0026thinsp;rheumatoid arthritis\u0026thinsp;+\u0026thinsp;methotrexate; n, number of subjects; Min, minimum; Max, maximum; \u003cem\u003ePg, Porphyromonas gingivalis\u003c/em\u003e; \u003cem\u003eTd\u003c/em\u003e, \u003cem\u003eTreponema denticola\u003c/em\u003e; \u003cem\u003eTf\u003c/em\u003e, \u003cem\u003eTannerella forsythia\u003c/em\u003e; ns, non-significant\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe 2023 SKI report showed a higher prevalence of oral health problems, bleeding gums, in women (7.3%) compared to men (6.4%).\u003csup\u003e7\u003c/sup\u003e Pavlov-Dolijanovic et al. (2023) in their review explained that the ratio of women to men in YORA was 3:1.\u003csup\u003e20\u003c/sup\u003e\u003c/p\u003e\u003cp\u003ePeriodontal clinical parameters in this study included PD, CAL, and BOP. The results of the comparative analysis of CAL among patients with periodontitis, patients with periodontitis accompanied by RA without MTX therapy, and patients with periodontitis accompanied by RA who have been treated with MTX showed no significant difference in CAL values. Zhao et al. (2018) in their systematic review state that periodontitis significantly increases the severity of RA, but RA does not significantly affect the development of periodontitis.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Research by Susanto et al. (2013) showed no significant differences in most periodontitis severity parameters between RA patients and controls.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Studies from Jung et al. (2018) show that conventional DMARD therapy alone has no significant effect on periodontal conditions, while significant improvement in periodontal parameters is obtained after non-surgical periodontal therapy in RA patients with conventional DMARD therapy.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe BOP value of periodontitis patients was significantly higher than that of patients with periodontitis accompanied by RA without MTX therapy. The BOP value of patients with periodontitis accompanied by RA without MTX therapy, which was lower than that of patients with periodontitis accompanied by RA who had been treated with MTX was possibly caused by the administration of the corticosteroid drug methylprednisolone while the patient was being examined whether the patient could be given DMARD drugs or not. Corticosteroids have vasoconstrictor and anti-inflammatory effects by inhibiting the action of histamine and inflammatory mediators. Corticosteroids increase the effect of vasoconstrictor hormones by increasing the response of blood vessel contraction to norepinephrine through increasing the sensitivity of α adrenergic receptors, increasing the number of angiotensin type 1 (AT1) receptors in vascular smooth muscle cells, thereby strengthening contractions triggered by angiotensin II, and also by stimulating the release of endothelin.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThis study showed comparative analysis results that were significantly different in each inflammatory biomarker level between periodontitis patients and periodontitis patients with RA without MTX therapy, and no significant differences between periodontitis patients and periodontitis patients with RA who had been treated with MTX, where the lowest median value was seen in periodontitis patients. No significant differences were seen in each inflammatory biomarker level between periodontitis patients with RA without MTX therapy and periodontitis patients with RA who had been treated with MTX, where the median values ​​of TNF-α, IL-1β, and PGE2 levels were lower in periodontitis patients with RA who had been treated with MTX. The tendency for the median values ​​of inflammatory biomarker levels to increase in periodontitis patients with RA without MTX therapy and then decrease in periodontitis patients with RA who had been treated with MTX indicates a strengthening of the inflammatory effect by RA conditions in periodontitis patients and the presence of a decrease in inflammation after MTX therapy.\u003c/p\u003e\u003cp\u003eThe \u0026lsquo;two-hit model\u0026rsquo; hypothesis proposed by Golub et al. in 2006 stated that in individuals who initially experience chronic inflammation in the form of periodontitis, which produces anti-cyclic citrullinated peptide antibodies, when joint inflammation occurs later, which produces citrullination, the subsequent antibody response can be very strong.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e The results of this study are in accordance with this hypothesis, where the levels of TNF-α, IL-1β, and PGE2 in GCF are higher in patients with periodontitis accompanied by RA. Various studies on TNF-α, IL-1β, and PGE2 in GCF of patients with periodontitis and RA have shown varying results. The results of this study showed significantly higher levels of TNF-α, IL-1β, and PGE2 in patients with periodontitis accompanied by RA, following several previous studies that support the \u0026lsquo;two-hit model\u0026rsquo; hypothesis.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Research from Xiao et al. (2021), Arvikar et al. (2021), Cosgarea et al. (2019), and a literature review by Bartold and Lopez-Olivia (2020) stated that there were high levels of TNF-α, IL-1β in patients with periodontitis with RA compared to patients with periodontitis without RA.\u003csup\u003e15,26,27,28\u003c/sup\u003e A study by Kurgan et al. (2016) showed statistically higher PGE2 levels in the periodontitis group with RA compared to the periodontitis group without RA in their research results.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe immunomodulatory cytokines TNF-α and IL-1β have proinflammatory effects that play an important role in periodontitis and RA by increasing chemokine production and promoting the expression of metalloproteinases and prostaglandins. Both induce inflammatory cells to attack the infected site in periodontitis, causing bone destruction through the production of inflammatory factors and activation of cytokines that contribute to alveolar bone resorption and collagen fiber damage.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Tumor Necrosis Factor alpha plays a key role in the immune response to periodontitis and RA by modulating the inflammatory response in the form of an increased inflammatory response by binding to the p55 receptor (T receptor type 1; CD120a).\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Several studies on the treatment of periodontitis and RA have shown that anti-TNF-α therapy in RA patients can reduce TNF-α levels in GCF and cause milder periodontal disease compared to patients who do not receive anti-TNF-α therapy.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Several studies on periodontal treatment have reported a decrease in TNF-α levels in GCF in RA patients with periodontitis who receive non-surgical periodontal treatment.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e These results indicate the dynamics of TNF-α in inflammatory conditions and the response to its treatment.\u003c/p\u003e\u003cp\u003eMost reports on the effects of RA therapy on periodontal disease show the benefits of using TNF-α inhibitors. Other reports on interleukin-6 receptor inhibitor therapy and anti-lymphocyte B therapy also show benefits in periodontal disease.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e This study showed the results of a decrease in TNF-α, IL-1β, and PGE2 levels in GCF of patients with periodontitis accompanied by RA who had been treated with MTX, although not significant. MTX therapy is the most commonly used DMARD, working by inhibiting the enzyme dihydrofolate reductase, providing an inhibitory effect on thymidine synthesis, RNA transmethylation, DNA, protein, phospholipids, and de novo purine synthesis, so that it can then suppress the secretion of IL-6, IL-8, IL-17A, and TNF in RA subjects.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Limited supporting data from previous studies and the insignificant results in this study leave questions that cannot be answered regarding the effect of MTX therapy in RA patients on periodontitis conditions. Ziebolz et al. (2018) reported increased BOP values ​​in patients receiving combination therapy of MTX with TNF-α antagonists.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Several reports from previous studies have shown improvements in RA parameters and disease activity after non-surgical periodontal treatment, thus indicating the need for periodontal treatment as additional treatment for RA patients with periodontitis.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eMost treatments for RA use DMARDs, which are included as host-modulating treatments.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e This is interesting because host-modulating treatments are also additional treatments for periodontitis.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Chronic inflammation can also have the effect of suppressing the host defense system, which makes periodontal infection worse because the host loses its defense capacity, while from this study, the view arises that periodontal inflammation and infection can affect the effectiveness of DMARDs in treating RA.\u003csup\u003e38\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe results of this study show that the comparative analysis of total bacteria in each sample group showed no significant difference. This can be related to the high differences in bacterial composition, with the dominant types of bacteria, whether pathogenic, commensal, or opportunistic, varying between healthy individuals, those with periodontitis, and those with systemic conditions such as rheumatoid arthritis.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Individuals in a healthy state are dominated by commensal bacteria that help maintain oral environment balance and prevent pathogen growth.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e The transition from a healthy state to periodontitis involves changes in biofilm structure and microbial interactions, which trigger inflammation and periodontal tissue damage. Periodontitis occurs due to dysbiosis, i.e., changes in the composition of the microbiome that led to the dominance of pathogenic bacteria and a decrease in commensal bacteria.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e The higher composition of pathogenic bacteria in periodontitis indicates that the microbiota ecosystem in the oral cavity undergoes major changes that support the growth of pathogens.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe relative expression of red complex bacteria (\u003cem\u003ePg, Td, Tf\u003c/em\u003e) shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e was significantly higher in \u003cem\u003ePg\u003c/em\u003e and \u003cem\u003eTd\u003c/em\u003e bacteria. The relative expression of \u003cem\u003ePg\u003c/em\u003e in the periodontitis sample group without accompanying RA tends to be higher. This tendency is partly caused by the antibody reaction against \u003cem\u003ePg\u003c/em\u003e, resulting in a decrease in \u003cem\u003ePg\u003c/em\u003e in the periodontitis samples with RA. A study conducted by Johansson et al. aimed to determine whether antibodies against \u003cem\u003ePg\u003c/em\u003e were present before the onset of RA symptoms. \u003cem\u003ePg\u003c/em\u003e bacteria are suspected to play a role in triggering RA by inducing the production of anti-citrullinated protein antibodies (ACPA), which are the main markers of this disease. ACPA antibodies are known to appear several years before RA symptoms manifest, indicating that an immune system imbalance occurs long before RA symptoms develop, with the likely initial site being mucosal tissues such as the gingiva. The study measured antibodies against \u003cem\u003ePg\u003c/em\u003e, specifically against arginine gingipain B (RgpB) and cyclic citrullinated peptide (CCP3), using the ELISA method. Anti-RgpB IgG antibody levels were significantly higher in pre-symptomatic individuals, and anti-CPP3 antibodies were detected in 5% of pre-symptomatic individuals, allowing the conclusion that high levels of antibodies against \u003cem\u003ePg\u003c/em\u003e can be detected years before RA symptoms appear; thus, the relative expression of \u003cem\u003ePg\u003c/em\u003e tends to be lower.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eTd\u003c/em\u003e bacteria are often found alongside other periodontal pathogens such as \u003cem\u003ePg\u003c/em\u003e and \u003cem\u003eTf\u003c/em\u003e in RA patients. The prevalence of \u003cem\u003eTd\u003c/em\u003e and other periodontal pathogens may vary depending on the type of RA treatment. Patients using a combination of methotrexate (MTX) and TNF-α antagonists show increased periodontal inflammation, which may influence microbial composition, including \u003cem\u003eTd\u003c/em\u003e, resulting in a higher tendency in the periodontitis group without RA.\u003csup\u003e43,44,45\u003c/sup\u003e \u003cem\u003eTd\u003c/em\u003e bacteria frequently interact with other periodontal pathogens such as \u003cem\u003ePg\u003c/em\u003e and \u003cem\u003eTf\u003c/em\u003e, which are collectively known as the red complex. The composition and microbial interactions may differ between patients with and without RA, potentially affecting the relative expression of \u003cem\u003eTd\u003c/em\u003e detected.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eChanges in bacterial composition due to treatment or other systemic factors may inhibit the bacterial ecosystem, so \u003cem\u003eTd\u003c/em\u003e does not develop as rapidly in patients with periodontitis without RA.\u003csup\u003e47\u003c/sup\u003e Comparative analysis results of relative expression of \u003cem\u003ePg\u003c/em\u003e, \u003cem\u003eTd\u003c/em\u003e, and \u003cem\u003eTf\u003c/em\u003e bacteria showed no significant differences between the periodontitis groups with RA before and after 1 month of MTX therapy (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The lack of significant differences may be related to several disease-modifying anti-rheumatic drugs (DMARDs), particularly MTX, which have a dual role in RA treatment: not only modulating immune responses but also directly targeting certain bacteria associated with RA pathogenesis. However, since this antimicrobial activity is species-dependent, the effect is not uniform across all oral pathogens. Antimicrobial evaluations of several DMARDs were conducted by Kussmann et al. Their study found that MTX exhibited antimicrobial activity against \u003cem\u003eFusobacterium nucleatum\u003c/em\u003e and \u003cem\u003eViridans streptococci\u003c/em\u003e, Aurothiomalate (ATM) showed broad-spectrum activity, Sulfasalazine (SSZ) was active against several oral pathogens (\u003cem\u003eActinomyces\u003c/em\u003e spp., \u003cem\u003eCapnocytophaga\u003c/em\u003e spp., \u003cem\u003eEikenella corrodens\u003c/em\u003e, \u003cem\u003eF. nucleatum\u003c/em\u003e, and \u003cem\u003ePg\u003c/em\u003e) but only at very high concentrations, while other DMARDs did not show significant antimicrobial activity.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eProspective studies are needed to obtain a causal relationship between periodontitis and RA or vice versa, along with the effects of therapy for each disease. Studies with a longer follow-up period are needed to further observe the effects of MTX therapy on periodontal clinical parameters, RA parameters, and inflammatory biomarker levels. Metagenomics-based studies or comprehensive microbiome analyses can be conducted to obtain a more comprehensive understanding of oral microbiota changes resulting from periodontitis and RA.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eWithin the limitations of this study, the following conclusions can be drawn. Significant differences in clinical parameters among the groups were only seen in the lower BOP values of the periodontitis group compared to the periodontitis group with RA without MTX therapy. This study showed a significant increase in the level of all three inflammatory biomarkers in the periodontitis group compared to the periodontitis group with RA without MTX therapy. However, the decrease in the level of inflammatory biomarkers in the group of periodontitis with RA without MTX therapy compared to the group of periodontitis with RA with MTX therapy was not significant.\u003c/p\u003e\u003cp\u003eThe relative expression of red complex bacteria (\u003cem\u003ePg\u003c/em\u003e, \u003cem\u003eTd\u003c/em\u003e) was significantly higher in \u003cem\u003ePg\u003c/em\u003e and \u003cem\u003eTd\u003c/em\u003e bacteria in the periodontitis sample group without accompanying RA. However, comparative analysis results of relative expression of \u003cem\u003ePg\u003c/em\u003e, \u003cem\u003eTd\u003c/em\u003e, and \u003cem\u003eTf\u003c/em\u003e bacteria showed no significant differences between the periodontitis groups with RA before and after 1 month of MTX therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eAuthor Contribution Statement\u003c/h2\u003e\u003cp\u003eConceptualization: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W, Takahashi N, Tabeta K; Formal analysis: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Investigation: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Methodology: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Writing\u0026mdash;original draft: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Writing\u0026mdash;reviewing and editing: Mahardika M, Rakhmawati W, Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Soeroso Y, Masulili SLC, Tadjoedin FM, Rahdewati H, Takahashi N, Tabeta K; Project administration: Sulijaya B; Supervision: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors state no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding Information\u003c/h2\u003e\u003cp\u003eThis study was supported by a grant from the Directorate of Research and Community Engagement, Universitas Indonesia (Research Grant PUTI Q1 No: NKB-320/UN2.RST/HKP.05.00/2024 to B.S.).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W, Takahashi N, Tabeta K; Formal analysis: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Investigation: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Methodology: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Writing\u0026mdash;original draft: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Mahardika M, Rakhmawati W; Writing\u0026mdash;reviewing and editing: Mahardika M, Rakhmawati W, Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA, Soeroso Y, Masulili SLC, Tadjoedin FM, Rahdewati H, Takahashi N, Tabeta K; Project administration: Sulijaya B; Supervision: Sulijaya B, Kuswandani SO, Haerani N, Kusumo SA; All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge Universitas Indonesia for supporting the publication of this study. We also extend our sincere appreciation to the Dental Teaching Hospital, Faculty of Dentistry, Universitas Indonesia, and Dr. Cipto Mangunkusumo General Hospital (RSCM) for providing the facilities and support necessary for conducting the research\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePapapanou, P. N. et al. Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. \u003cem\u003eJ. 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Published 2023 Dec 7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"inflammatory biomarker, methotrexate, periodontal parameter, periodontitis, red complex bacteria, rheumatoid arthritis","lastPublishedDoi":"10.21203/rs.3.rs-7644672/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7644672/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e\u003cp\u003eTo determine the differences in periodontal status\u0026mdash;Probing Depth (PD), Clinical Attachment Loss (CAL), Bleeding on Probing (BOP); oral inflammatory biomarkers\u0026mdash;Tumor Necrosis Factor Alpha (TNF-α), Interleukin 1 Beta (IL-1β), Prostaglandin E2 (PGE2); and the relative expression of red complex bacteria\u0026mdash;\u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e (\u003cem\u003ePg\u003c/em\u003e), \u003cem\u003eTreponema denticola\u003c/em\u003e (\u003cem\u003eTd\u003c/em\u003e), and \u003cem\u003eTannerella forsythia\u003c/em\u003e (\u003cem\u003eTf\u003c/em\u003e) among groups with periodontitis, periodontitis accompanied by rheumatoid arthritis (RA) before treated with methotrexate (MTX), and periodontitis with RA treated with MTX for one month.\u003c/p\u003e\u003ch2\u003eMaterial and Methods\u003c/h2\u003e\u003cp\u003eA cross-sectional study by analyzing periodontal clinical parameters, gingival crevicular fluid (GCF) inflammatory biomarker levels, and subgingival plaque relative expression of red complex bacteria. Samples were taken from patients with periodontitis, periodontitis with RA without MTX, and periodontitis with RA treated with MTX. Samples were collected from the Rheumatology Clinic of Dr. Cipto Mangunkusumo General Hospital, and the Periodontology Clinic of the Dental Teaching Hospital, Faculty of Dentistry, Universitas Indonesia patients. Biomarkers were analysed using the Enzyme-Linked Immunosorbent Assay (ELISA) and bacterial expression was measured using real-time polymerase chain reaction (RT-PCR).\u003c/p\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eOne-way ANOVA with Tukey\u0026rsquo;s post-test and Kruskal-Wallis with Dunn\u0026rsquo;s post-test were used to analysed periodontal parameters, GCF biomarkers, and bacterial expression among the groups.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eNo statistically significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were found for PD and CAL. BOP ​​were significantly higher in the periodontitis group than the RA without MTX group. All biomarkers level were significantly increased in the RA without MTX group compared to the periodontitis group. No significant difference in bacterial expression among groups. \u003cem\u003ePg\u003c/em\u003e and \u003cem\u003eTd\u003c/em\u003e expression were significantly higher in the periodontitis group compared to RA without MTX group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003ePatients with periodontitis and RA without MTX exhibited significantly higher inflammatory biomarkers levels, indicating an amplified inflammatory response and tended to be reduced by the MTX. \u003cem\u003ePg\u003c/em\u003e and \u003cem\u003eTd\u003c/em\u003e expression was higher in the periodontitis-only group. These findings suggest that RA and its treatment influence both the host inflammatory response and microbial profile in periodontitis.\u003c/p\u003e","manuscriptTitle":"Periodontal Status, Oral Inflammatory Biomarker, and Relative Expression of Red Complex Bacteria in Periodontitis with Rheumatoid Arthritis Treated with Methotrexate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-16 01:56:20","doi":"10.21203/rs.3.rs-7644672/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"119c5d70-0c22-46d2-819d-56b4c373015d","owner":[],"postedDate":"October 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56145586,"name":"Health sciences/Biomarkers"},{"id":56145587,"name":"Health sciences/Diseases"},{"id":56145588,"name":"Biological sciences/Immunology"},{"id":56145589,"name":"Health sciences/Medical research"},{"id":56145590,"name":"Biological sciences/Microbiology"},{"id":56145591,"name":"Health sciences/Rheumatology"}],"tags":[],"updatedAt":"2025-10-22T07:53:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-16 01:56:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7644672","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7644672","identity":"rs-7644672","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}