Associations between Inflammatory Biomarkers, Clinical Parameters of Periodontitis, and COVID-19 History 

Introduction

Periodontitis is a multifactorial disease caused by interactions between oral microorganisms and the host, leading to chronic inflammation and destruction of tooth-supporting tissues.^1,2 The Indonesian Ministry of Health’s Basic Health Research (Riskesdas) 2018 data shows the prevalence of periodontitis is 74.1% in Indonesia, as determined by bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment loss (CAL).³ Contributing factors to periodontitis include plaque, calculus, genetics, environment, systemic health, lifestyle, and smoking habits.^1,4 Periodontopathogens trigger an immune response that releases inflammatory mediators, causing soft tissue destruction and bone resorption. The upregulation of proinflammatory cytokines from monocytes and polymorphonuclear leukocytes, such as Interleukin-8 (IL-8) and Prostaglandin E2 (PGE2) play roles in this process.^2,4–6 PGE2 suppresses lymphocyte production, reducing collagen synthesis by fibroblasts and triggering osteoclast activity which can cause bone resorption.⁶ Neutrophils, fibroblasts, macrophages, lymphocytes, and endothelial cells are among the cells in periodontal tissues that produce IL-8. Its primary role is to recruit other inflammatory cells to the infection site. IL-8 levels are elevated in individuals with chronic periodontitis and positively correlate with pocket depth and periodontal status.^7–9 Elevated IL-8 levels in inflamed periodontal tissues can induce hematologic responses, including elevated C-reactive protein (CRP) levels. Chronic inflammation may raise CRP expression by 25% or more. Compared to healthy individuals, periodontitis patients show higher CRP levels, which can be considered a marker for the connection between periodontitis and systemic disease.^5,10,11

Through systemic inflammation, the inflammatory responses in periodontitis and COVID-19 suggest a potential association. Systemic conditions, including hypertension, diabetes, cardiovascular diseases, chronic obstructive pulmonary disease, and autoimmune diseases, are risk factors for both COVID-19 and periodontitis.^12,13 Poor oral hygiene and periodontitis can further exacerbate inflammatory biomarkers and COVID-19 infection. Elevated ACE2 levels from periodontal pathogens may increase pro-inflammatory cytokines, supporting the hypothesis that periodontitis may contribute to COVID-19.^14,15

While most patients recover fully from acute SARS-CoV-2 infection, studies suggest post-infection risks to the heart, lungs, liver, and other organs. Prolonged inflammation, immune dysregulation, disease progression, and ongoing cytokine release are among the potential consequences. Individuals with a history of COVID-19 showed significant differences in inflammatory markers, and COVID-19 survivors may face long-term effects from persistently elevated inflammatory markers. Compared to controls without a history of the disease, COVID-19 survivors have elevated levels of CRP, neutrophils, and fibrinogen, and their ACE2 levels persist for up to 114 days post-infection.^16,17

Despite prior research linking COVID-19 to periodontitis, the post-infection impact on periodontal tissue is unclear. Long-term inflammation in COVID-19-infected periodontitis patients has not been widely studied. In order to further understand the association between ACE2, IL-8, PGE2, CRP, and periodontitis clinical parameters in individuals with and without COVID-19, additional research is necessary.

Methods

Results

Comparison of inflammatory biomarkers between subjects with and without a history of COVID-19

The ACE2 levels of the 23 subjects with and 24 subjects without a history of COVID-19 showed a statistically significant difference (p = 0.000) ( Figure 1a). Those with a history of COVID-19 had higher ACE2 levels (5.50 ± 1.63 ng/l) compared to those without a history of COVID-19 (3.78 ± 1.28 ng/l). Similarly, the COVID-19 history group had significantly higher CRP levels (11.32 ± 3.23 ng/mL) than the non-COVID-19 group (8.16 ± 2.27 ng/mL) (p = 0.000). IL-8 levels were also significantly higher in the groups with a history of COVID-19 (144.81 ± 48.99 ng/ml) compared to those without (107.80 ± 38.41 ng/ml) (p=0.006). For PGE2, although the group with a history of COVID-19 showed higher levels (22.77 ± 22.54 pg/ml) compared to those without a history of COVID-19 (10.82 ± 10.75 pg/ml), this difference did not reach statistical significance (p = 0.065) ( Figure 1a).

Figure 1. Comparison of ACE2, IL-8, CRP and PGE2 Levels.

a) Between COVID-19 History and No COVID-19 History Groups; b) Between Periodontitis and Non-Periodontitis Groups Based on COVID-19 History.

Comparison of clinical periodontal parameters between subjects with and without a history of COVID-19

No statistically significant differences in clinical periodontal parameters were observed between groups with and without a history of COVID-19 ( Figure 2a). The mean BOP was higher in the group with a history of COVID-19 (24.63 ± 18.94) compared to the group without a history of COVID-19 (17.69 ± 15.83), but this difference was not statistically significant (p = 0.191). Similarly, PPD was slightly higher in the COVID-19 group (4.17 ± 1.56) compared to the non-COVID-19 group (3.83 ± 1.27), but the difference was not significant (p = 0.466). In addition, CAL was higher in the COVID-19 group (2.61 ± 2.08) compared to the non-COVID-19 group, but this difference did not reach statistical significance (p = 0.249).

Figure 2. Comparison of BOP, PPD and CAL Mean Scores.

a) Between Groups Based on COVID-19 History; b) Between Periodontitis and Non-Periodontitis Groups Based on COVID-19 History.

Abbreviations: BOP = Bleeding on Probing; PPD = probing pocket depth; CAL = clinical attachment level.

Discussion

This case-control study analyzed associations between ACE2, IL-8, PGE2 and CRP levels in GCF and clinical periodontitis characteristics in individuals with or without a history of COVID-19, with all participants having received at least two doses of a COVID-19 vaccine. GCF, a serum exudate that flows into the gingival sulcus, contains immune response components that are important in preventing tissue invasion by subgingival plaque bacteria. Its protein composition reflects the health status of periodontal tissues and has been widely utilized to explore the bidirectional relationship between oral and systemic diseases.²²

Consistent with Patel et al.’s findings of elevated plasma ACE2 levels post-COVID-19 infection, ACE2 expression was higher in individuals with a history of COVID-19 ( Figure 1a). The exact cause and pathophysiology of these findings after COVID-19 infection is still unknown, but there are several possible mechanisms by which ACE2 can affect inflammation in SARS-CoV-2 infection.¹⁷ Apart from being a receptor that facilitates viral infection, ACE2 also acts as a regulator of the renin-angiotensin system (RAAS). In conjunction with other enzymes and receptors it forms the angiotensin-converting enzyme 2-angiotensin 1-7-Mas receptor (ACE2-Ang1-7-MasR), which can downregulate inflammatory responses, block oxidative stress, and reduce hypertension, among other functions. When the ACE2 receptor interacts with SARS-CoV-2, an association occurs that potentially decreases ACE2 levels on the cell surface.²³ The higher levels of ACE2 in the group with a history of COVID-19 in this study indicate the regulation of the anti-inflammatory process through the formation of ACE2-Ang1-7-MasR. Another study by Sousa et al. related to ACE2 polymorphisms found that increased ACE2 levels could be a protective physiological mechanism affecting the outcome of COVID-19.²⁴ This increase can also be found in subjects with no history of COVID-19 who have received the COVID-19 vaccine.

A statistically significant difference in IL-8 levels between subjects with and without a history of COVID-19 was observed, with higher levels in those with a history of COVID-19 ( Figure 1b). This is consistent with the results of Phetsouphanh et al.,²⁵ who observed that IL-8 levels were elevated following COVID-19 infection, but they decreased after eight months. The substantial discrepancy in our study underscores the potential for a persistent increase in IL-8 levels following infection; however, additional research is required to comprehend the changes in IL-8 over prolonged periods.

In subjects with a history of COVID-19, CRP levels in GCF were significantly elevated, consistent with the findings of Gameil et al., who reported persistent inflammation following COVID-19 infection as indicated by elevated CRP levels in plasma.¹⁶ PGE2 levels appear to be higher in individuals with a history of COVID-19 than in those without, although the difference was not significant ( Figure 1a). This is consistent with the findings of Ricke-Hoch et al, who observed elevated PGE2 levels in COVID-19 patients.²⁶ PGE2 is believed to have two functions: it promotes the immune response during the inflammatory phase and inhibits the production of inflammatory cytokines during the resolution phase.²⁷ The nonsignificant results in our study may be due to sampling at least 6 months after the time of infection. Interestingly, although there was a large variation in levels, five individuals had very high levels, indicating that PGE2 levels may remain high in a subset of the population for extended periods after infection ( Figure 1a).

The significant difference in ACE2 levels between groups with and without a history of COVID-19 indicates the relationship between COVID-19 history and ACE2 levels. The inflammatory markers IL-8, PGE2, and CRP showed positive correlations with ACE2 levels ( Table 2), suggesting that elevated ACE2 levels are associated with elevated levels of these markers.

The non-significant differences in IL-8 levels between the periodontitis groups with and without a history of COVID-19 may be related to this positive correlation. SARS-CoV-2 infection may regulate pro- and anti-inflammatory processes by altering ACE2 levels, thereby balancing cytokine expression through the ACE2-Ang1-7-MasR pathway. Higher ACE2 levels in the COVID-19 history group may affect the production and response of IL-8, which has anti-inflammatory effects through the formation of angiotensin 1-7.²⁸ Furthermore, the significant differences observed in the non-periodontitis group with and without a history of COVID-19 could be explained by the fact that ACE2 and CRP are positively correlated, which means that there is systemic inflammation even when there isn’t any local periodontal inflammation.

Patients with and without a history of COVID-19 did not exhibit any significant differences in the clinical parameters of periodontal disease (BOP, PPD, and CAL) in this study ( Figure 2a and 2b). These findings contrast with previous research that indicated a correlation between the severity of acute COVID-19 infection and the clinical parameters of periodontal disease. For instance, Gupta et al. found a significant association between the severity of COVID-19 and clinical periodontal parameters. Specifically, higher scores of BOP, PPD, and CAL were significantly related to COVID-19 complications.²⁹ This discrepancy may be due to the period between the COVID-19 infection and the periodontal evaluation being generally greater than six months. The influence of COVID-19 infection on periodontal tissues may be transient and not significant following recovery.

There was a positive relationship between the clinical parameters PPD and CAL and IL-8 levels ( Table 3). This is in line with previous research that showed that IL-8 levels rise when periodontal tissue conditions get worse.⁴ On the other hand, the absence of correlation between PGE2, CRP levels, and clinical parameters contrasts with the findings of Gupta et al. and Sanchez et al. previous research, which indicated that patients with COVID-19 exhibited elevated CRP levels along with worsening periodontal conditions.^29,30 This finding may be attributed to the influence of COVID-19 history through the regulation of the RAS system and systemic inflammatory response, which could potentially influence CRP levels independently of local periodontal conditions.

Ethical considerations

The study was approved by the Ethics Committee of the Faculty of Dentistry at Universitas Indonesia (Approval Number: 90/Ethical Approval/FKGUI/X/2022, Protocol Number: 090640722), ensuring strict anonymity for all participants. Participants received both verbal and written explanations outlining the purpose and procedures of the study. Those who agreed to participate provided informed consent by signing a consent form.