Effects of Herpesviruses on Proinflammatory Cytokines in Chronic Periapical Lesions

Introduction

Chronic apical periodontitis is an inflammatory immune disease that is a reaction of the periapical tissues to pulp necrosis and infection. They arise due to polymicrobial endodontic infection present in the root canal, with a dominance of gram-negative anaerobic bacteria [1]. The inflammatory reaction is a consequence of interactions between microbial virulence and the host’s immune response [2]. Bacterial infections and their lipopolysaccharides stimulate the host’s immune response, which initiates the production of proinflammatory mediators and leads to destruction of connective tissue and bone resorption in the apical periodontium, and further formation of a chronic periapical lesion [3,4]. These lesions can be histologically characterized by fibrous granulomatous tissue, proliferating epithelium, or cysts, and are infiltrated by various inflammatory cells [5]. Research shows that 52% of the world’s population has at least 1 tooth with chronic apical periodontitis, and the frequency is greater in patients with systemic diseases, and also in developing countries [6]. Treatment of chronic apical periodontitis aims to remove or significantly reduce the intracanal microbes and prevent re-infection by placing a root canal filling. In cases of larger lesions or post-treatment disease, periradicular surgery is a treatment alternative to save the tooth [7].

Chronic apical periodontitis is usually asymptomatic, but acute exacerbation accompanied by swelling and pain occasionally occurs [8]. The finding of a radiolucent lesion in the periapical region on X-ray is necessary for making a diagnosis [9]. The most significant role in the pathogenesis of chronic apical periodontitis is played by polymicrobial infection of the root canal system; however, the literature indicates the great importance of herpesvirus infection, especially human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) [10]. Previous studies have indicated the role of herpesviruses in the pathogenesis of periapical lesions, and these viruses are more common in patients with symptomatic lesions compared to those with asymptomatic lesions [11]. These viruses persist after the first infection during an individual’s lifetime, and the literature indicates that in infected hosts, sites of latent infection for HCMV are periodontal macrophages and T lymphocytes, while latent EBV is present in epithelial cells, periodontal B lymphocytes, and plasma cells [12,13]. Potentially virally infected cells present a cellular infiltrate that is present in periapical lesions. In addition, in periods of acute exacerbation, there is a new wave of influx of inflammatory cells that can carry the latent virus. Herpesvirus activation can also occur in an inflammatory environment associated with local bacteria and their products [10]. The relationship between bacteria and herpesviruses is bidirectional. Bacterial virulence can activate latent herpesviruses, and reactivated herpesviruses negatively affect host defenses, leading to bacterial growth [3]. Studies have shown that herpesviruses can affect the production of proinflammatory cytokines. Cytokines primarily act through activation of osteoclasts and stimulation of mediators that destroy the extracellular matrix of bone tissue and further contribute to immune-mediated tissue destruction [14].

Therefore, this study included periapical tissue of patients with chronic apical periodontitis and aimed to evaluate the presence of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) using polymerase chain reaction (PCR) and the levels and inflammatory cytokines, IL-6, IL-8, TNF-α, and TGF-β1, using enzyme-linked immunoassay (ELISA).

Material and Methods

PATIENTS AND SAMPLES:

The study was approved by the Ethics Committee of the Faculty of Medicine, University of Niš, Serbia (no. 01-2066-5). The study included 79 patients (42 males and 37 females, mean age 45 years) at the Clinic of Dental Medicine in Niš, Serbia in whom radiological and clinical examination determined the existence of a chronic periapical lesion. Patients signed a consent form to participate in the study, and they were informed about the sample collection procedure. Since the extraction of the affected tooth was indicated due to inability to treat the tooth, the procedure itself was not harmful to the patient. The research was conducted in accordance with all applicable guidelines, and proper implementation of safety measures for persons participating in this scientific research, including the Basics of Good Clinical Practice, the Declaration of Helsinki, and the Law on Health Care of the Republic of Serbia. The researchers have pledged that the identity of the respondents will always remain anonymous.

Periapical lesions were taken from the roots of the teeth in which, due to the impossibility of treatment, extraction was indicated. Patients with a more developed form of marginal periodontitis were excluded. The study included only patients who had not received antibiotic or anti-inflammatory therapy in the previous 2 months and who did not have immunodeficiency conditions or diseases. According to subjective patient concerns, the lesions were divided into symptomatic (sensitivity to percussion and palpation, presence of pain or swelling) and asymptomatic (no concerns at the time of the study).

The protocol for each patient included the application of a local anesthetic, rinsing the mouth, and cleaning the soft tissue around the tooth with 0.12% chlorhexidine gluconate. After extraction, samples of periapical lesions were removed from the apex of the tooth root with a sterile scalpel, rinsed in sterile saline, dried on a sterile cotton swab, and frozen in a sterile Eppendorf tube at −70ºC. Each sample was divided into 2 halves. One half was used for PCR analysis and testing for the presence of herpesviruses, and the other half was used to analyze cytokine concentrations.

DNA EXTRACTION:

Frozen samples of periapical lesions were thawed at room temperature and immersed in 200 μl of redistilled water. DNA was isolated by treating the samples with 10% proteinase K (MBI Fermentas, Vilnius, Lithuania) at 56°C for 30 minutes, followed by inactivation of the enzyme by heating the samples at 94°C for 15 minutes. The tubes were then centrifuged for 2 minutes at 12 000× g (Eppendorf MiniSpin^®; Hamburg, Germany); obtained supernatants were transferred into sterile tubes and kept at −20ºC until PCR analysis.

HCMV AND EBV DETECTION BY PCR:

Detection of viral DNA was performed by PCR in 25 μl of reaction mixture containing 20 nM of primers, 200 μM of the 4 dNTPs, 1× buffer (10 mM KCl and 50 mM Tris-HCl), 3 mM magnesium chloride, 1.5 units of Taq DNA polymerase, and redistilled water. In each reaction mixture, 3 μl of the supernatant sample obtained by digestion of periapical lesions was added. The ATCC CCL-86 cell line was used as the positive control for EBV, while for HCMV clinical specimens previously shown to contain HCMV were used as positive controls; distilled water was added to the PCR mixture instead of the sample and served as a negative control.

For HCMV detection, the following primers were used: forward 5′-CCACCCGTGGTGCCAGCTCC-3′ and reverse 5′-CCCGCTCCTCCTGAGCACCC-3′, while the amplification profile was: initial DNA denaturation at 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C (30 seconds), annealing at 56°C (30 seconds), extension at 72°C (30 seconds), and a final extension of 5 minutes at 72°C.

For EBV detection the following primers were used: forward 5′-CTCCCGCACCCTCAACAAGCTA-3′ and reverse 5′-GAACCAGAAGGACCCAAAAGCA-3′. The amplification profile was: initial denaturation at 94°C for 10 minutes, followed by 35 cycles of denaturation at 94°C (1 minute and 30 seconds), annealing at 57°C (45 seconds), extension at 72°C (2 minutes) and a final extension at 72°C (10 minutes). Amplification was performed in a DNA Thermal Cycler (Thermo Fisher Scientific, Wilmington, DE, USA).

Amplified fragments were visualized following electrophoresis of 15 μl of the PCR product on an 8% polyacrylamide gel (20 V/cm in Tris borate EDTA buffer) and gel staining in ethidium bromide, using an ultraviolet transilluminator (Thermo Fisher Scientific, Wilmington, DE, USA) at a wavelength of 300 nm. The result was interpreted as positive if an amplicon of the expected size was present (159 bp and 494 bp for HCMV and EBV, respectively). A 100-bp DNA Ladder (Thermo Fisher Scientific, Wilmington, DE, USA) was used for molecular weight markers.

DETERMINATION OF CYTOKINE LEVELS:

The concentrations of TNF-α, IL-6, IL-8, and TGF-β1 in culture supernatants were detected by ELISA kits specific for human cytokines (R&D Systems, Inc. Minneapolis, USA). The kits used were TNF-α (DTA00D), IL-6 (D6050), IL-8 (D8000C), and TGF-β1 (DB100C). The detection of these cytokines was based on a sandwich ELISA method, where a particular anti-cytokine antibody was coated on a 96-well plate. After incubation of plates with supernatants and subsequent washing away of any unbound substances, an enzyme-linked polyclonal antibody specific for cytokines was added to the wells. After incubation and additional washing, substrate solution was added to the wells and color developed in proportion to the amount of bound cytokine in the initial step. Stopping the color development was achieved with 2N sulfuric acid and the intensity of developed color was measured at 450 nm by a microplate spectrophotometer (Multiskan Ascent 96/384 Plate Reader, Thermolab Systems, USA). For the determination of TGF-β1 concentration, the samples were previously activated by the addition of 1N HCl, and then, after incubation for 10 minutes, they were neutralized with 1.2N NaOH/0.5 M HEPES buffer to pH 7.2–7.6. Cytokine levels were determined based on standard curves, constructed according to known concentrations of cytokines (positive controls) and samples without cytokines (negative controls). The sensitivity of the ELISA test for TNF-α was 2.09–6.23 pg/ml, for IL-6 0.7 pg/ml, for IL-8 1.5–7.5 pg/ml, and for TGF- β1 0.889–5.50 pg/ml.

STATISTICAL ANALYSIS:

Comparison of cytokine values between symptomatic and asymptomatic lesions was performed using the t test with a significance level set at P<0.05.

One-way ANOVA with a significance of P<0.05 was used to determine if there was a significant difference in cytokines in groups of symptomatic and asymptomatic lesions according to the presence of viruses. A post hoc Tukey test was used to determine between which groups this difference was significant.

The chi-square test was used to determine whether HCV, EBV, and dual infections have a significant impact (P<0.05) on symptomatology of lesions.

All statistical analyzes were calculated in IBM SPSS version 26.0.

Results

PRESENCE OF HERPESVIRUSES IN CHRONIC PERIAPICAL LESIONS:

Out of a total of 79 periapical lesions examined, 43 were symptomatic and 36 were asymptomatic. PCR analysis showed that in patients with symptomatic lesions, 34.9% were positive for HCMV, 11.6% were positive for EBV, and 20.9 had double HCMV/EBV infection. The analysis of asymptomatic lesions proved the presence of HCMV in 22.2% of lesions, the presence of EBV in 5.6% of lesions, and 5.6% of lesions had dual HCMV/EBV infection. Statistical analysis showed that HCMV (P<0.05) and dual HCMV/EBV infection (P<0.05) significantly affect the symptomatology of the lesion, however, EBV infection had no significant effect (Table 1).

CONCENTRATION OF IL-6 IN CHRONIC PERIAPICAL LESIONS:

The concentration of examined cytokines in virus-positive and -negative lesions is shown in Table 2. By analyzing the concentration of IL-6 in the tissue of periapical lesion samples, it was observed that its concentration was significantly higher in symptomatic lesions compared to asymptomatic in virus-negative ones (P<0.001), HCMV-positive (P<0.001), EBV-positive (P<0.05), and in dual HCMV/EBV (P<0.001). A statistically significant difference was also observed between virus-negative lesions compared to groups of lesions that were positive for HCMV, EBV, and dual HCMV/EBV individually, both in symptomatic (P<0.05) and asymptomatic lesions (P<0.05). Symptomatic lesions with dual HCMV/EBV infection had a significantly higher concentration of IL-6 compared to lesions with single HCMV infection (P<0.05), while compared to EBV the difference was not statistically significant. Asymptomatic lesions with dual HCMV/EBV infection also had significantly more IL-6 compared to lesions with single HCMV infection (P<0.05), while compared with single EBV infection the difference was not statistically significant (Table 2).

CONCENTRATION OF IL-8 IN CHRONIC PERIAPICAL LESIONS:

Analysis of the concentration of IL-8 showed that its concentration was significantly higher in symptomatic lesions compared to asymptomatic ones in virus-negative (P<0.001), HCMV-positive (P<0.001), EBV-positive (P<0.001), and dual HCMV/EBV-infected lesions (P<0.05). Within the group of symptomatic lesions, a significantly higher concentration of IL-8 was observed in lesions infected with HCMV, EBV, and dual HCMV/EBV-infected compared to virus-negative lesions, as well as between lesions that had dual HCMV/EBV infection compared to HCMV single infection, and HCMV-infected compared to EBV-infected lesions (P<0.05). In patients with asymptomatic lesions, a significant difference in IL-8 concentration was observed between virus-negative lesions and lesions infected with HCMV (P<0.05), EBV (P<0.05), and HCMV/EBV (P<0.05), as well as between individual HCMV infections compared to single EBV (P<0.05) and dual HCMV/EBV (P<0.05) (Table 2).

CONCENTRATION OF TNF-α IN CHRONIC PERIAPICAL LESIONS:

Analysis of the examined lesions revealed a higher concentration of TNF-α in all symptomatic lesions compared to asymptomatic ones. The difference was statistically significant in virus-negative (p<0.05), HCMV-positive (p<0.05), and EBV-positive (p<0.05) lesions, while in lesions with dual HCMV/EBV infection, the difference was not statistically significant. Within the group of symptomatic lesions, a significantly higher concentration of TNF-α was observed in lesions infected with HCMV, EBV, and dual HCMV/EBV compared to virus-negative lesions, as well as in lesions with HCMV/EBV compared to single HCMV infection (P<0.05). Asymptomatic lesions showed a significantly higher concentration in all virus-positive subgroups compared to virus-negative lesions (P<0.05) (Table 2).

CONCENTRATION OF TGF-β1 IN CHRONIC PERIAPICAL LESIONS:

The study showed that the difference in TGF-β1 concentrations was significantly higher in symptomatic lesions within the groups of virus-negative (p<0.05) and HCMV-positive lesions (P<0.05), while in lesions with EBV and dual HCMV/EBV infection, the difference was not statistically significant. However, within the group of symptomatic lesions, there was a statistically significant difference between HCMV-positive, EBV-positive, and dual HCMV/EBV-positive lesions compared to virus-negative lesions (P<0.05). Within the group of asymptomatic lesions, a significant difference in TGF-β1 concentration was observed only between HCMV/EBV dual-infected lesions and virus-negative ones (P<0.05) (Table 2).

Discussion

The results of this study indicate the importance of HCMV and EBV infection in the pathogenesis of chronic periapical lesions. HCMV, EBV, and dual HCMV/EBV infections were more common in symptomatic lesions, and the concentrations of IL-6, IL-8, TNF-α, and TGF-β1 were higher in virus-positive lesions compared to virus-negative ones.

Numerous studies have investigated the relationship between human herpesvirus infection and endodontic pathologies, and it has been proven that their activity can influence the outcome of endodontic treatment [15]. Their role in pathogenesis has not yet been fully elucidated. According to the literature, herpesviruses primarily affect the production of proinflammatory cytokines in periapical lesions, which can change the chronic course of the lesion [16]. Understanding the immunosuppressive and immunomodulatory effects on periapical tissues in active herpesvirus infection may contribute to a better understanding of the pathogenesis of chronic inflammatory processes in this region.

In different stages of the periapical lesion development, different levels and ratios of inflammatory cells and mediators can be observed, which indicates the complexity of the interactions between the herpesvirus and the host [18]. HCMV and EBV affect inflammatory cells by being able to infect and alter monocytes, macrophages, lymphocytes, and polymorphonuclear leukocytes. Changes at the level of cellular defense can lead to growth of endodontic pathogenic bacteria. Herpesviruses can activate a host response that includes dysregulation of macrophages and lymphocytes that participate in the regulation of the antiviral response. HCMV can inhibit the expression of macrophage surface receptors for lipopolysaccharide, thereby negatively affecting the defense response to gram-negative bacterial infection [16].

The results of this study show that HCMV infection was more common than EBV infection. These results are in agreement with the results of our previous study [13], as well as with the studies of other authors [18–20]. On the contrary, some studies found the dominant virus was EBV [21–23]. Another important criterion in the analysis of the herpesvirus presence is whether the herpesvirus infection is related to the clinical characteristics of the examined lesions. The results of this study showed that herpesviruses were more common in symptomatic lesions compared to asymptomatic ones. Our previous study found that HCMV was 7.68 times more common in symptomatic lesions [13]. Such results indicating the influence of herpesviruses in the acute exacerbation of periapical pathologies have been shown in several studies [17,19,22]. Contrary to these findings, some studies found no significant difference between symptomatic and asymptomatic lesions [18,21,24]. These differences can be influenced by several factors, which could include geographic or ethnic specificity in the distribution of herpesviruses, as well as specific laboratory procedures used and clinical features of the lesions [22,25].

The network of cytokines and their balance is a mechanism that plays an important role in the first line of defense against infection and significantly affects regulation of the acquired immune response. Herpesvirus infection has been proven to affect cytokine balance [17]. HCMV infection affects the Th1 immune response with increased production of IL-1β, IL-6, IL-12, TNF-α, INF-α,β, INF-γ, and PGE2. EBV infection can increase production of IL-1β, IL-1 receptor antagonist, IL-6, IL-8, IL-18, TNF-α, INF-α,β, and INF-γ [16].

TNF-α is the main mediator that affects bone resorption, acting through the mechanism of osteoclast activation and stimulation of mediators that destroy the extracellular matrix of bone tissue [22,26]. The results of this study show that a higher concentration of TNF-α was generally found in symptomatic lesions compared to asymptomatic ones. In both groups of lesions, a significant difference was observed between virus-negative and virus-positive lesions, which clearly indicates the role of this mediator in the pathogenesis of these pathologies. Jakovljevic et al [21] proved that there was no significant difference in the levels of TNF-α, IL-1β, and IL-6 between HCMV- and EBV-positive and negative periapical lesions, but a higher concentration of these cytokines was demonstrated between herpesvirus-positive symptomatic and asymptomatic lesion, which is consistent with our results. Another study found that TNF-α is associated with increased risk for development of apical periodontitis [27]. Hernadi et al [22] showed that high TNF-α expression was associated with herpesvirus infection in periapical lesions.

IL-6, TNF-α, and IL-1 belong to the group of major proinflammatory cytokines. IL-6 is also secreted by osteoblasts when stimulating osteoclast formation. IL-6 stimulates the formation of osteoclast precursors and increases the number of osteoclasts in vivo, leading to a systemic enhancement of bone resorption [28,29]. The concentration of IL-6 in this study was higher in symptomatic lesions compared to asymptomatic ones, which is consistent with our previous study [28]. Additionally, when analyzed in relation to the presence of viral infection, virus-positive lesions had a higher concentration of this cytokine compared to virus-negative ones and in the groups of symptomatic and asymptomatic lesions. Sabeti et al [17] studied herpesvirus infection and the levels of TNF-α, INF-γ, IL-1β, IL-6, IL-10, and IL-12, finding most of the symptomatic lesions were positive for HCMV or EBV, and high viral loads were detected in all herpesvirus-positive lesions. Similar to our results, a significant difference was also observed in the presence of virus and analyzed cytokines between symptomatic and asymptomatic lesions.

IL-8 is a chemokine that serves as a chemical signal that attracts neutrophils to the site of inflammation and is therefore known as a neutrophil chemotaxic factor. It is produced during the immune response, in which it directs immune system cells to the site of infection [30]. This chemokine is one of the main mediators of the inflammatory response. It is secreted by several types of cells, monocytes/macrophages, and fibroblasts under the influence of IL-1β and TNF-α. Little is known about the concentration of IL-8 in human periapical lesions, but its elevated concentration is associated with lesions with increased symptoms as well as with larger-diameter lesions [31], and higher levels of this cytokine are associated with EBV infection [16]. The results of the present study showed higher levels of IL-8 in all symptomatic lesions compared to asymptomatic ones. A significantly higher concentration of this chemokine was evident in all virus-positive lesions compared to virus-negative ones, and especially high values were observed in symptomatic lesions with EBV and dual HCMV/EBV.

To counteract the increased inflammation, the proinflammatory response causes release of anti-inflammatory mediators such as TGF-β and IL-10 [20]. TGF-β is a regulator of wound healing that acts through the mechanisms of fibroblast proliferation, connective tissue synthesis, and angiogenesis [22]. We found a significant difference between symptomatic and asymptomatic lesions only in the samples without the virus and with HCMV mono infection, while in EBV infection the difference did exist but was not statistically significant. Within the groups of symptomatic lesions, a significant difference was observed between lesions with and without the virus, while in the case of asymptomatic lesions, the differences were significant only between lesions without the virus and lesions with dual HCMV/EBV infection. It seems that the unbalanced increase in the concentration of TGF-β1 in this study can be explained by the fact that the lesions were analyzed exclusively in relation to viral infection, while their size was not considered, which in our previous study was connected to the increase of this cytokine [32]. In the study by Hernadi et al [22], herpesvirus infection did not correlate with TGF-β expression.

In this study, the occurrence of HCMV/EBV dual infection in periapical samples was a very important result. The higher frequency of dual infection was in symptomatic lesions, and unlike to our previous study, where the HCMV/EBV infection was not found in any of asymptomatic lesions, HCMV/EBV infection occurred in 5.6% of asymptomatic lesions [13]. A significant result of this study was that the concentration of all cytokines was higher in lesions with dual infection compared to lesions with single HCMV and EBV infection.

Considering the frequency of occurrence of HCMV and EBV, as well as their influence on cytokines, future therapeutic recommendations may be reflected in the application of antiviral therapy in resistant periapical abscesses. Although there is a need for clinical studies on the application of antiviral therapy in treatment of apical periodontitis, such studies have not yet been performed. Administration of dosed antiviral therapy should be considered in cases of herpesvirus-infected apical periodontitis [33].

The limitations of this study are due to the methodology used. Results regarding the frequency of virus occurrence can be affected by the choice of laboratory method. Jakovljevic et al [21] and Botero et al [34] performed a comparison of different herpesvirus detection methods and concluded that nested PCR shows a higher degree of herpesvirus detection than real-time PCR. The authors explain the lower level of detection of HCMV and EBV in subgingival samples in the studies by Dawson et al [35] and Nibali et al [36] when using real-time PCR in the absence of active herpesvirus infection in the tested samples. PCR detection of herpesvirus itself has limitations that must be considered when analyzing and comparing the obtained results. The high sensitivity of PCR can be misleading because there can be a problem in detecting latently infected periapical areas, as well as the fact that it is not certain whether PCR-negative regions contain viruses in the latent phase. The absence of a control group of samples is another limitation. Most relevant studies have not used control samples because periapical lesions are reactive tissue that occurs at the site of resorbed periapical bone, so there is no real tissue equivalent to serve as a negative control. According to Botero et al [37], healthy control samples are difficult to obtain due to the complexity of getting healthy pulp or periodontal tissue from nonaffected teeth.

Conclusions

We found that herpesvirus infection was more common in symptomatic lesions. The concentrations of IL-6, IL-8, and TNF-α were significantly higher in virus-positive lesions compared to virus-negative ones, and there were especially high cytokine levels in lesions with dual HCMV/EBV infection. The concentration of TGF-β1 was higher in virus-positive compared to virus-negative lesions, but the difference was significant only in lesions with dual HCMV/EBV infection. Due to the numerous discrepancies in the results of published studies, it is necessary to continue to conduct extensive research to fully clarify the role of herpesviruses in the pathogenesis of periapical lesions.