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Research Article
Revised

Evaluation of Salivary HLA-DR4 and MMP-8 Levels Along with Porphyromonas gingivalis in Periodontitis Patients with Rheumatoid Arthritis

[version 2; peer review: 2 not approved]
Shaymaa Abdulkareem Nori
https://orcid.org/0009-0003-3621-4094
1, Batool Hassan Al-Ghurabi1, Nik Nairan Abdullah2
Shaymaa Abdulkareem Nori
https://orcid.org/0009-0003-3621-4094
1, Batool Hassan Al-Ghurabi1, Nik Nairan Abdullah2
PUBLISHED 12 Sep 2025
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REVIEWER STATUS

Abstract

Background

Periodontitis (PD) is a chronic infectious inflammatory disease that affects the tissues supporting teeth and results in a progressive deterioration of the alveolar bone. Rheumatoid arthritis (RA), a systemic autoimmune disease that has been associated with increased severity of periodontal disease. This study aimed to assess the clinical and immunological characteristics of PD in individuals with and without RA and to compare these characteristics with a healthy control group.

Methods

The study included three groups; thirty patients with PD, thirty patients with PD and RA, and twenty healthy control participants. Clinical periodontal parameters [plaque Index (PLI), bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment loss (CAL)] were assessed. Salivary biomarkers, including human leukocyte antigen-DR4 (HLA-DR4), matrix metalloproteinase-8 (MMP-8), and anti-citrullinated protein antibody (ACPA), were analyzed by enzyme-linked immunosorbent assay (ELISA), and the microbial load of Porphyromonas gingivalis (P. gingivalis) was determined using quantitative real time polymerase chain reaction (qRT-PCR).

Results

Our findings showed that salivary HLA-DR4 levels were significantly lower in patient groups compared with healthy controls (P = 0.000), whereas salivary MMP-8 and ACPA levels were significantly elevated in patients (P = 0.034) and (P = 0.001) respectively, with no significant differences between the patient groups(P > 0.05). On the other hand, microbial load was significantly higher in the PD with and without RA groups than that in controls (P = 0.001), with a positive correlation between microbial load and CAL in the PD-RA group.

Keywords

Periodontitis, Rheumatoid arthritis, Porphyromonas gingivalis , human leukocyte antigen-DR4, Matrix metaloprotienase-8.

Corresponding author: Shaymaa Abdulkareem Nori
Competing interests: No competing interests were disclosed.
Grant information: The author(s) declared that no grants were involved in supporting this work.
Copyright:  © 2025 Nori SA et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Nori SA, Al-Ghurabi BH and Abdullah NN. Evaluation of Salivary HLA-DR4 and MMP-8 Levels Along with Porphyromonas gingivalis in Periodontitis Patients with Rheumatoid Arthritis [version 2; peer review: 2 not approved]. F1000Research 2025, 14:615 (https://doi.org/10.12688/f1000research.164552.2) First published: 23 Jun 2025, 14:615 (https://doi.org/10.12688/f1000research.164552.1) Latest published: 12 Sep 2025, 14:615 (https://doi.org/10.12688/f1000research.164552.2)

Revised Amendments from Version 1

The revised version of the manuscript differs from the original in several important ways. The language has been polished for clarity, conciseness, and grammar, improving overall readability and flow. In the Methods section, redundant details (such as repetitive pipetting steps) were condensed, while key methodological information was retained and better organized in bullet points and tables for easier comprehension. The Results section now includes clearer statistical reporting and expanded interpretation of ROC curve analyses, which were previously presented without sufficient discussion. In the Discussion and Conclusion, repetitive points—particularly regarding MMP-8 and ACPA—were reduced, and findings are now more cautiously framed (e.g., “in this cohort”) to avoid overstating causality. Finally, references were corrected and standardized, with DOIs added where available, ensuring consistency with journal requirements.

See the authors' detailed response to the review by Ali A Abdulkareem
See the authors' detailed response to the review by Aram Mohammed Sha

Introduction

Periodontitis is a profound and irreversible condition affecting the periodontium, marked by a complicated relationship among infections that incite inflammation, ultimately resulting in the loss of vital supporting connective tissue and alveolar bone (Mahmood and Al-Ghurabi, 2020; Domokos et al., 2024 and Al-Daragi et al., 2024). Severe PD is considered the main cause of edentulism among adults, affecting about 11% of the global adult demographics (Kwon et al., 2021 and Daily et al., 2023a). RA is a chronic, systemic autoimmune disease that presents significant challenges due to its progressive nature, leading to functional impairment and increased risk of premature mortality. It influences around 1% of the worldwide population, with higher occurrence rates noted among women and the elderly (Kang et al., 2024 and Abdullah et al., 2024). Epidemiological studies investigating the connection between PD and RA present a spectrum of findings, showcasing a diverse prevalence of PD in RA patients, ranging from 28% to 85%, with 11-14% diagnosed with severe PD (Punceviciene et al., 2021). PD and RA exhibit parallels in their pathophysiological progression, immunological regulation, hereditary susceptibility, infiltration of inflammatory cells, and the involvement of enzymes and cytokines in immunological reactions (Nguyen et al., 2020 and Nori and Al-Ghurabi, 2025). P. gingivalis, a pathogenic bacterium associated with periodontal disease, is the sole inhabitant of the oral cavity possessing the unique enzyme that transforms arginine into citrulline. It has been demonstrated that upon infection with P. gingivalis, a periodontal tissue protein undergoes citrullination by this enzyme, leading to the formation of ACPA, which then creates an immune complex with citrullinated proteins in the joints, consequently triggering RA (Mauramo et al., 2021).

The connection between PD and RA is further underscored by a specific genetic predisposition and associated environmental risk factors, such as smoking. Both conditions share genetic susceptibility, particularly the HLA-DRB1 alleles encoding HLA-DR4. Additionally, polymorphisms in genes that encode inflammatory cytokines, along with interleukin-1 combined risk alleles, may create a synergistic effect on bone destruction in joints and the periodontium, potentially increasing susceptibility to both RA and PD (Naaom et al., 2017; Sorsa et al., 2020; and Ancuta et al., 2017). Furthermore, the alleles of HLA-DRB1 that encode class II major histocompatibility complex’s beta chain possess the ability to bind citrullinated peptides, potentially enhancing the auto-antigenic citrullinated peptide’s immunogenicity associated with RA (de Molon et al., 2019). Sandal et al. revealed a promising association of HLA-DRB1 in the production of ACPA following P. gingivalis oral infection in a mouse model, suggesting a potential causal relationship between PD and RA (Sandal et al., 2016).

A multitude of inflammatory cells release MMP enzymes. In PD and RA, genetically separate but structurally similar MMPs destroy almost all extracellular matrix constituents. Increased concentrations of MMP-8 have been linked to PD and expedited disease development (Mauramo et al., 2021). It is significant that specifically MMP-8 has been identified as an effective biomarker for PD. Besides PD, new research indicates that MMP-8 is linked to chronic inflammation and inflammatory conditions such as RA and cardiovascular illnesses (Raheem & Ahmed, 2014; Äyräväinen et al., 2018). Interestingly, there is no study that has investigated the connection among the salivary levels of HLA-DR4, MMP-8, ACPA, and P. gingivalis altogether in PD subjects with and without RA. Thus, our finding illuminates the current study, which seeks to assess the accuracy of salivary HLA-DR4, MMP-8, ACPA, and P. gingivalis levels in differentiating patients with PD with and without RA from clinically healthy individuals and to correlate these biomarkers with the periodontal parameters for better understanding the immune response and inflammation mechanisms involved in the development and progression of both diseases.

Methods

Study design

It is a case-control research which relies on observational data. This research was conducted within the College of Dentistry/University of Baghdad, spanning the period from 15 October 2024 to 15 January 2025. Ethical considerations were paramount in the present study, guided via the World Medical Association’s Helsinki Declaration, and ethical approval was obtained from the ethical committee at the Dentistry College/University of Baghdad (Reference Number: 940, Project number: 940824, Date: 14-10-2024).

Research instrument

Three ELISA kits were used in this study for the analysis of salivary biomarkers. These kits include: HLA-DR4 ELISA kit (AFG™ Scientific company / USA), MMP-8 and ACPA ELISA kits (SunLong Biotech™ company/China). For P. gingivalis analysis, a bacterial DNA isolation kit (Presto™ Mini gDNA Bacteria Kit, Geneaid Biotech Ltd/Taiwan) was used, primers for P. gingivalis (Macrogen™ /South Korea), and qRT-PCR Master Mix (Luna® Universal/New England Biolabs/USA).

Catalogue numbers

MMP-8 ELISA Kit = SL1156

ACPA ELISA Kit = SL2976HU

HLA-DR4 ELISA Kit = EK716715

Presto™ Mini gDNA Bacteria Kit = GBB100/101

Luna® Universal qPCR Master Mix = NEB #E3005S/L

Study population

This study included 80 subjects aged between 30 and 55 years. All participants had a body mass index <25 kg/m2. Diagnosis of the PD patients was established according to the classification criteria of periodontal diseases by Tonetti et al. (2018). While RA diagnoses was established according to the American College of Rheumatology/European Alliance of Associations for Rheumatology 2010 criteria (Aletaha et al., 2010). The chosen participants were categorised into three groups as shown below ( Table 1).

Table 1. Sample categorization.

GroupParticipants numberConditionCriteria
Healthy control20No RA, no PDHealthy periodontium BOP <10%, PPD ≤ 3 no CAL,
PD without RA30Generalized unstable PD onlyPPD ≥4 mm, CAL ≥4 mm including at least 30% of teeth, no RA diagnosis
PD with RA30Both generalized unstable PD and RADiagnosed RA who had previously been treated with non-biological Disease-modifying anti-rheumatic drugs + generalized unstable PD (PPD ≥4 mm, CAL ≥4 mm including at least 30% of teeth)

Inclusion criteria

The inclusion criteria were:

  • 1. Generalized unstable PD patients with RA.

  • 2. Generalized unstable PD patients without RA.

  • 3. Age range between (30-55) years.

  • 4. The existence of a minimum 20 natural teeth or more.

  • 5. Subjects had PPD ≥4 mm, CAL ≥4 mm including at least 30% of teeth.

Exclusion criteria

The exclusion criteria were:

  • 1. Presence of systemic disorders other than RA, such as hypertension, thyroid diseases and diabetics.

  • 2. Previous periodontal therapy for the last 6 months.

  • 3. Pregnant and menopause women.

  • 4. The use of antibiotics and/or anti-inflammatory medication in the last 3 months.

  • 5. History of smoking or alcohol drinking.

Sample size

Utilizing G power 3.1.9.7 (Program written by Franz-Faull, University of Kiel, Germany), the sample size was calculated with a power of 90% for the study, a two-sided alpha error of probability of 0.05, an effect size of F is 0.4 (large effect size), three groups, under these circumstances the sample size is approximately 80 participants. Effect size F is: Small = 0.1, medium = 0.25, large = 0.4 (Cohen, 2013).

Disease activity score-28

Each candidate in PD patients’ group with RA scored for disease activity, according to Disease activity score-28 (DAS-28) (Van der Heijde et al., 1993; Prevoo et al., 1995; and Van Gestel et al., 1998).

Saliva samples

Three ml of whole un-stimulated saliva was collected from study groups in a sterile cup. Subjects were asked to refrain from drinking and eating one hour before donation of saliva. Within one hour after collection, saliva centrifuged at 1000 × g for 15 minutes to eliminate debris and cellular matter, the supernatants were aspirated immediately, divided into three aliquots and kept at ≤-20°C until used (Costa et al., 2021).

Plaque samples

Subgingival plaque samples were collected using fine sterile Gracey curettes from the gingival sulcus in the control group and the four deepest periodontal pockets in the patient groups, placed immediately in eppendorf tube containing 0.5 ml TE buffer (10 mM TrisHCl, 1 mM EDTA, pH 7.6) and stored at (-40°C) (Borges et al., 2009).

Clinical periodontal parameters

Once saliva and plaque samples were collected, clinical parameters (PLI, BOP, PPD, and CAL) were assessed utilizing William’s periodontal probe. Each tooth was meticulously examined across six unique areas (Mesiobuccal, Buccal, Distobuccal, Mesiolingual, Lingual, and Distolingual) to assess BOP, PPD and CAL. Meanwhile, PLI scores were elegantly documented by scrutinizing just four surfaces (mesial, distal, labial/buccal, lingual/palatal). Third molar was omitted from all parameters assessment, with the exception of PLI. PLI was meticulously measured utilizing disclosing agents to ascertain dental plaque existence or non-existence (O’Leary et al., 1972), while BOP percentage was recorded as 1 present 0 absent (Mitani et al., 2024). The measurement separating the free gingival margin to the pocket’s bottom is termed PPD. In contrast, CAL refers to the measurement from cemento-enamel junction (CEJ) to the pocket’s bottom in case of gingival regression. When the gingival margin is at the CEJ, both CAL and PPD are equal. At recession, the CAL is calculated by summing the extent of the recession along with PPD. If the gingival border sits above the CEJ, the CAL is determined by subtracting the measurement from the gingival border to the CEJ from the PPD (Tonetti et al., 2018).

Detection of HLA-DR4, MMP-8 and ACPA in saliva

Using ELISA technology, salivary biomarkers, HLA-DR4, MMP-8 and ACPA levels were assessed for each subject (Aydin et al., 2025). ELISA assays were performed according to the manufacturer’s instructions, with standard volumes of samples, reagents, and wash steps applied in a 96-well format. All ELISA kits used sandwich- ELISA technique and the optical density is spectrophotometrically measured at a wave length of 450 nm. Optical density exhibited a direct proportionality to salivary biomarkers concentration.

Detection of Pophyromonas gingivalis in dental plaque

Using qRT-PCR technology, the microbial load of p. gingivalis in subgingival dental plaque was determined for each participant (Bhatsange & Rajput, 2024). Genomic DNA was isolated from dental plaque samples. For specific detection and quantification of P. gingivalis bacteria, primers were synthesized. Then target bacterial DNA sequences were amplified using qPCR, with fluorescence monitored in real-time during each amplification cycle. The cycle threshold, defined as the cycle number at which fluorescence exceeds a predetermined threshold, was recorded for each sample. Quantification was achieved by comparing the cycle threshold values of the samples to a standard curve generated from serial dilutions of known DNA concentrations (Kareem et al., 2022).

Statistical analysis

All statistical analyzes of the data were performed and processed with the computerized analysis statistical package for the social sciences (SPSS) software program (version 25, IBM, USA) and GraphPad Prism software (version 9.0). Statistical variance was deemed significant when p < 0.05. The data were presented as descriptive statistics involving mean and standard deviation. The distribution of clinical and immunological data was assessed for normality using the Shapiro Wilk test. Inferential statistics were used to accept or reject the statistical hypotheses which included: Chi-square test and one-way analysis of variance (ANOVA) parametric test were implemented to record the variations across a minimum of three distinct groups, Tukey honestly significant difference (HSD)/post hoc test was utilized to ascertain the statistical significance of the relationship between two sets of data. For non-parametric data Kruskal-Wallis test and post-hoc Dunn’s test was used to test the statistical difference between groups. Spearman correlation coefficient test was used to determine the correlation among different parameters. In addition, to show the diagnostic potential of cytokines a receiver operating characteristic (ROC) curve was established.

Results

After implementing the inclusion and exclusion criteria, 70 individuals were excluded from the study, leaving 150 subjects to be evaluated for recruiting eligibility. Μltimately, only 30 male and female PD with RA patients, 30 male and female PD patients and 20 control participants were incorporated into the study ( Figure 1).

78ac2b38-e604-4ac5-887e-fa525f0fe00c_figure1.gif

Figure 1. Flow chart of the study design.

Demographic characteristics of the study participants

The results of the present study revealed no significant differences in the age and sex of the participants between all groups. Ratio male/female between patients and healthy control group was 1:1.4. While, the mean disease duration in the RA group was 7.0 ± 1.35 years and the mean DAS-28 was 6.89 ± 1.38. Moreover, all periodontal parameters, PLI, BOP, PPD and CAL, were statistically greater for the PD groups with and without RA, compared to the healthy periodontium participants (p < 0.05), ( Table 2).

Table 2. Demographic and clinical characteristics of the study groups.

Mean percentage and values of periodontal parameters in study groups.

Demographic characteristicsStudy groups P- value
PD with RA n = 30PD n = 30 Healthy control n = 20
Age (years)0.126NS
Mean ± SD44.90 ± 7.2045.33 ± 8.0441.11 ± 7.70
Disease duration of RA (years)
Mean ± SD7.0 ± 1.35---
DAS-28
Mean ± SD6.89 ± 1.38---
Study groupsSex P- value
Male N(%) Female N(%)
PD n = 6025 (41.6%)35 (58.4%)0.801NS
Healthy control n = 207 (35%)13 (65%)
Ratio male/female = 1:1.4
Periodontal parameters Mean ± SDStudy groups P- value
PD with RA n = 30PD n = 30 Healthy control n = 20
PLI68.16 ± 10.6659.53 ± 16.9710.95 ± 4.470.0000*
BOP72 ± 10.1254 ± 11.84.9 ± 1.200.0000*
PPD6.45 ± 0.896.19 ± 0.99_1.452NS
CAL5.86 ± 1.315.83 ± 1.41_0.470 NS

Immunological salivary biomarkers analysis

The current study noticed a significant decrease of salivary HLA-DR4 (P ˂ 0.05) in patient groups as compared with control group. Nevertheless, the findings indicated that there was no statistically significant difference (p > 0.05) in the salivary HLA-DR4 concentration between PD with and without RA groups, as shown in Table 3. The mean rank levels of salivary MMP-8 and ACPA in patients’ groups were significantly higher than control group, whereas, intergroup comparisons of mean values of MMP8 and ACPA between PD with and without RA groups indicated that there was no statistically significant difference (p > 0.05), as shown in Table 3.

Table 3. The disparity in the average rank values of salivary biomarkers (ng\L) and microbial load in study groups.

Immunological biomarkers and microbial loadStudy groups P value
PD with RA n = 30PD n = 30Healthy control n = 20 Kruskal Wallis Test
HLA-DR4Mean rank38.12a*30.85bNS58.55c*17.560.000*
ACPA
Mean rank		50.7a*43.93bNS20.05c*21.920.001*
MMP-8
Mean rank		44.58a*44.2bNS28.82c*6.740.034*
P. gingivalis
Mean rank		51.47a*55.40bNS20.58c*31.780.001*

a Comparison between PD with RA and healthy control group;

b Comparison between PD with and without RA groups;

c Comparison between PD without RA and healthy control group; NS: not significant;

* Significant.

Genetic analysis of Pophyromonas gingivalis

The comparison of the mean rank values of P. gingivalis microbial load in study groups showed that there was statistically significant difference between the patient groups and the control group. However, Intergroup comparisons of mean values of P. gingivalis microbial load between PD with and without RA groups indicated that there was no statistically significant difference (p > 0.05), as shown in Table 3.

Correlation between salivary biomarkers and microbial load with clinical periodontal parameters

A substantial correlation (p = 0.018) existed in PD without RA group between HLA-DR4 and CAL. Moreover, A notable connection (p = 0.025) existed in PD with RA group between P. gingivalis concentration and CAL ( Table 4).

Table 4. Correlation between biomarkers and clinical periodontal parameters in periodontitis subjects with and without rheumatoid arthritis.

GroupsPeriodontal parametersACPAHLA-DR4MMP8 Microbial load
PD with RAPLIr = 0.064r = 0.009r = 0.119r = 0.241
p = 0.736p = 0.599p = 0.528p = 0.197
BOPr = 0.239r = 0.141r = 0.176r = 0.192
p = 0.201p = 0.456p = 0.350p = 0.307
PPDr = 0.186r = 0.186r = 0.353r = 0.220
p = 0.325p = 0.309p = 0.054p = 0.241
CALr = 0.282r = 0.139r = 0.075r = 0.406
p = 0.129p = 0.463p = 0.692p = 0.025
PDPLIr = 0.073r = 0.133r = 0.212r = 0.191
p = 0.701p = 0.481p = 0.260p = 0.310
BOPr = 0.347r = 0.017r = 0.225r = 0.059
p = 0.060p = 0.926p = 0.231p = 0.756
PPDr = 0.176r = 0.055r = 0.223r = 0.162
p = 0.351p = 0.769p = 0.234p = 0.389
CALr = 0.063r = 0.425r = 0.083r = 0.162
p = 0.738p = 0.018p = 0.662p = 0.387

Diagnostic accuracy of salivary biomarkers

PD with and without RA according to ROC data had good accuracy in assessing HLA-DR4, MMP-8 and ACPA sensitivity and specificity for distinguishing PD from healthy periodontium yet, unreliable in distinguishing between PD with and without RA groups (Figure 2, Figure 3, and Figure 4).

78ac2b38-e604-4ac5-887e-fa525f0fe00c_figure2.gif

Figure 2. Receiver operating curves for salivary biomarkers in PD with RA vs. PD without RA.

78ac2b38-e604-4ac5-887e-fa525f0fe00c_figure3.gif

Figure 3. Receiver operating curves for salivary biomarkers in PD with RA vs. control

78ac2b38-e604-4ac5-887e-fa525f0fe00c_figure4.gif

Figure 4. Receiver operating curves for salivary biomarkers in PD without RA vs. control.

Correlation of Pophyromonas gingivalis microbial load counts with salivary biomarkers in Periodontitis patients with and without Rheumatoid Arthritis

Spearman correlation test coefficients (r) calculated between P.gingivalis microbial load counts with salivary biomarkers in PD subjects with and without RA. The results demonstrated significant positive correlations between MMP-8 with both P.gingivalis microbial load counts, (r = 0.540_ p = 0.002) and ACPA (r = -0.359_ p = 0.050) in PD with RA group. While in PD group the results revealed significant positive correlation between MMP-8 and ACPA only, (r = 0.433_ p = 0.016).

Discussion

The clinical periodontal assessments revealed significant differences in PLI and BOP between both PD with and without RA groups in comparison to control group, indicating increased periodontal inflammation and bacterial presence in these groups. PD with RA group had superior PLI and BOP values than the PD group, suggesting that in this cohort, RA was associated with exacerbation of periodontal inflammation. This aligns with previous studies (Daily et al., 2023b; Kim et al., 2018; Rodríguez-Lozano et al., 2019; and Hamed & Ali, 2017) showing higher periodontal indices in RA patients, indicating that RA may worsen periodontal inflammation. However, no significant differences in PPD and CAL were found between PD with and without RA groups, which was analogous to the findings of a study done by Varshney et al. (2021). The outcomes of the current study propose that RA influences the inflammatory processes in periodontal disease, leading to more pronounced inflammation but not necessarily increased tissue destruction in the short term.

Salivary biomarkers were analyzed to investigate potential immunological factors contributing to periodontal disease progression. Among the biomarkers, HLA-DR4 levels were significantly lower in PD with and without RA groups in comparison to controls, though no substantial disparity was noticed across PD with RA and PD without RA groups. However, studies investigating the role of HLA-DR4 in periodontal disease among RA patients have yielded mixed results. A previous study conducted in Sudan reported that HLA-DRB1*07, HLA-DQB1*02, and *06, which are HLA-DR4 alleles and haplotypes, were significantly higher in healthy control group and showed protection against RA (Ali et al., 2023). As for HLA-DR4 association with PD, Bonfil et al. reported that a higher frequency of at least one of the alleles compared to healthy persons: DRB1*0401, DRB1*0404, DRB1*0405, or DRB1*0408 (Bonfil et al., 1999). One can not claim that the current study does coincide with the above motioned studies, since it investigated HLA-DR4 as a protein without analyzing its allelic variants. According to Gung et al., the expression of HLA-DR is positively correlated with interleukin IL-23 (Jung et al., 2018). Interestingly, Sadeghi et al., reported that IL-23 level is usually higher in healthy control individuals than in PD (Sadeghi et al., 2018). Furthermore, Patients taking methotrexate therapy usually show decreased levels of IL-23 as reported by Elghandour et al. (2013). The above mentioned studies support the significant decrease in HLA-DR4 level in PD with and without RA groups in comparison to control group. Furthermore, the current study found positive significant correlation between HLA-DR4 and CAL in PD without RA group. This finding is consistent with Bonfil et al., study which indicated a significant relationship between HLA-DR4 and CAL in severe and rapidly progressive PD (Bonfil et al., 1999).

Regarding MMP-8 and ACPA, our results showed significant differences between the patient groups and healthy controls. However, no significant differences were observed between PD with and without RA, which is consistent with Eriksson et al. (2022). This suggests that while both conditions share immunological biomarkers, the presence of RA does not appear to uniquely alter salivary MMP-8 or ACPA levels, highlighting their role as common rather than disease-specific markers.

In the analysis of P. gingivalis microbial load, the study found significant differences between the patient groups compared with healthy controls, but again without differences between PD with and without RA. Importantly, only in the PD with RA group did P. gingivalis correlate positively with CAL, suggesting that in RA patients, altered immune responses may amplify the periodontal tissue destruction driven by this pathogen. This finding aligns with prior studies (Selimov et al., 2020; Chen et al., 2023; Li et al., 2022; Moura et al., 2021; and Fiorillo et al., 2019) which highlighted the role of P. gingivalis in PD and RA considering it as a risk factor.

The significant correlation between MMP-8 and P. gingivalis in the RA+PD group further indicates that RA may enhance inflammatory responses to this pathogen, leading to elevated tissue-destructive enzyme levels. Moreover, the observed association between MMP-8 and ACPA in this group suggests that RA-related immune mechanisms, including ACPA production, may contribute to periodontal breakdown.

In contrast, in PD without RA, MMP-8 correlated only with ACPA, indicating that immune-mediated mechanisms, rather than P. gingivalis load, may play a more dominant role in periodontal destruction in this group.

These findings are supported by Kuula et al. (2009) who reported that P. gingivalis infection modulates MMP-8 expression, and by Sorsa et al. (2017) who highlighted the association of MMP-8 with periodontal destruction and ACPA in RA.

The diagnostic analysis of salivary biomarkers, including ACPA, HLA-DR4 and MMP-8, using ROC curves comparing PD with RA vs. PD without RA, demonstrated week discrimination. Although MMP-8 and ACPA levels were elevated in both groups, the overlap limited their ability to differentiate patients based on RA status, suggesting these markers primarily reflect periodontal inflammation rather than RA-specific changes.

In PD with RA vs. healthy controls, all biomarkers showed high diagnostic accuracy. HLA-DR4 and MMP-8 exhibited high discriminatory power, while ACPA demonstrated excellent sensitivity and specificity, supporting their potential as non-invasive indicators of periodontal inflammation in RA patients.

Similarly, for PD without RA vs. healthy controls, all the biomarkers maintained high accuracy, showing strong sensitivity and specificity. These findings highlight the utility of salivary HLA-DR4, MMP-8, and ACPA as non-invasive diagnostic tools for detecting periodontal disease. While their levels reflect general periodontal inflammation, the overlap between PD with and without RA suggests that additional biomarkers or combined panels may be needed to specifically identify RA-associated periodontal changes.

A limitation of the present research is the case-control design is observational and does not allow for establishing causality. It can show associations but not direct cause-and-effect relationships between PD and RA or between the biomarkers and disease outcomes. Although the study targeted 80 participants, the sample size is relatively small. A larger sample size could have provided more statistical power, improving the generalizability of the findings, especially when trying to differentiate between PD with and without RA.

Conclusions

In conclusion, this study highlights the complex relationship between PD and RA. Although RA did not significantly worsen periodontal severity in terms of PPD and CAL, both PD groups demonstrated elevated salivary MMP-8, ACPA, and P. gingivalis load, reflecting general periodontal inflammation rather than RA-specific effects. These findings underscore the need for further research into shared immune pathways between the two diseases and support the potential of salivary biomarkers as non-invasive diagnostic tools for monitoring periodontal and systemic health.

Ethical approval

The research adhered to the Declaration of Helsinki and received approval from the Ethics Committee at the University of Baghdad, College of Dentistry, Iraq (number 940824, 14-10-2024).

Consent to participate

All patients involved in this study provided their written informed consent.

Data availability

Underlying data

Zenodo: Patient raw data for paper (Evaluation of Salivary HLA-DR4 and MMP-8 Levels Along with Porphyromonas gingivalis in Periodontitis Patients with Rheumatoid Arthritis), https://doi.org/10.5281/zenodo.15376986 (Nori et al., 2025a).

Data is available under Creative Commons Zero v1.0 Universal

Extended data

Data is available under Creative Commons Zero v1.0 Universal

Data is available under Creative Commons Zero v1.0 Universal

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Nori SA, Al-Ghurabi BH and Abdullah NN. Evaluation of Salivary HLA-DR4 and MMP-8 Levels Along with Porphyromonas gingivalis in Periodontitis Patients with Rheumatoid Arthritis [version 2; peer review: 2 not approved]. F1000Research 2025, 14:615 (https://doi.org/10.12688/f1000research.164552.2)
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Reviewer Report 08 Aug 2025
Ali A Abdulkareem, University of Baghdad, Baghdad, Baghdad Governorate, Iraq 
Not Approved
VIEWS 9
https://doi.org/10.5256/f1000research.181080.r394190
The authors aimed to assess the clinical and immunological characteristics of periodontitis in individuals with and without rheumatoid arthritis and to compare these characteristics with a healthy control group. While the efforts of authors are well appreciated; however, several concerns ... Continue reading
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Abdulkareem AA. Reviewer Report For: Evaluation of Salivary HLA-DR4 and MMP-8 Levels Along with Porphyromonas gingivalis in Periodontitis Patients with Rheumatoid Arthritis [version 2; peer review: 2 not approved]. F1000Research 2025, 14:615 (https://doi.org/10.5256/f1000research.181080.r394190)
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  • Author Response 12 Sep 2025
    Shaymaa Abdulkareem Nori, Department of Basic Science, College of Dentistry, University of Baghdad, Baghdad, Iraq
    12 Sep 2025
    Author Response
    We thank the reviewer for this valuable suggestions. All revisions have been made as requested by the reviewer and here are some answers for some of the reviewers questions 
    1. ... Continue reading
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  • Author Response 12 Sep 2025
    Shaymaa Abdulkareem Nori, Department of Basic Science, College of Dentistry, University of Baghdad, Baghdad, Iraq
    12 Sep 2025
    Author Response
    We thank the reviewer for this valuable suggestions. All revisions have been made as requested by the reviewer and here are some answers for some of the reviewers questions 
    1. ... Continue reading
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Reviewer Report 28 Jul 2025
Aram Mohammed Sha, University of Sulaimani, Sulaimani- Kurdistan Region, Iraq 
Not Approved
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https://doi.org/10.5256/f1000research.181080.r395768
General comments:
  1. The manuscript needs polishing (grammar, conciseness) by a native proofreader.
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1. Title and Abstract
Title:
  • Suggestion: Could specify “salivary levels” earlier
... Continue reading
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Sha AM. Reviewer Report For: Evaluation of Salivary HLA-DR4 and MMP-8 Levels Along with Porphyromonas gingivalis in Periodontitis Patients with Rheumatoid Arthritis [version 2; peer review: 2 not approved]. F1000Research 2025, 14:615 (https://doi.org/10.5256/f1000research.181080.r395768)
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https://f1000research.com/articles/14-615/v1#referee-response-395768
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  • Author Response 12 Sep 2025
    Shaymaa Abdulkareem Nori, Department of Basic Science, College of Dentistry, University of Baghdad, Baghdad, Iraq
    12 Sep 2025
    Author Response
    We sincerely thank the reviewer for this insightful report. all revisions have been done as requested.
    regarding the reviewer comment  on the use of mean rank, mean rank was used ... Continue reading
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COMMENTS ON THIS REPORT
  • Author Response 12 Sep 2025
    Shaymaa Abdulkareem Nori, Department of Basic Science, College of Dentistry, University of Baghdad, Baghdad, Iraq
    12 Sep 2025
    Author Response
    We sincerely thank the reviewer for this insightful report. all revisions have been done as requested.
    regarding the reviewer comment  on the use of mean rank, mean rank was used ... Continue reading
    Report a concern

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Alongside their report, reviewers assign a status to the article:

Approved
The paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations
A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved
Fundamental flaws in the paper seriously undermine the findings and conclusions

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 12 Sep 25
Version 1
23 Jun 25 		read read
  1. Aram Mohammed Sha, University of Sulaimani, Sulaimani- Kurdistan Region, Iraq
  2. Ali A Abdulkareem, University of Baghdad, Baghdad, Iraq

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    Alongside their report, reviewers assign a status to the article:
    Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
    Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
    Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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