- Email: [email protected]
Elevated Tumor Necrosis Factor-alpha Expression in Periapical Lesions Infected by Epstein-Barr Virus
Clinical Research
Elevated Tumor Necrosis Factor-alpha Expression in Periapical Lesions Infected by Epstein-Barr Virus Katinka Hern adi, DMD, PhD,* Eszter Gy€ ongy€ osi, MSc,† Be ata Mesz aros, MSc,† † † † Levente Szak acs, MSc, Anita Szalm as, MSc, Eszter Csoma, PhD, Rich ard Mogyor osi, MD,* Levente Czompa, MD,* Gy€ orgy Veress, PhD,† Istv an Varga, DMD, PhD,* Ildik o Judit M arton, MD, PhD, DSci,* and J ozsef K onya, MD, PhD† Abstract Introduction: In apical periodontitis, there is an intense inflammatory response to endodontopathogenic bacteria, an essential component of the pathogenic microbiota. The inflammation can be aggravated by herpesviruses acting as nonessential pathogens in periapical lesions. This study aimed to determine the levels of tumor necrosis factor-alpha (TNF-a) and transforming growth factor-beta (TGF-b) in periapical lesions in relation to local occurrence of Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), and human herpesvirus 8 (HHV-8). Methods: Fifty-eight samples with apical periodontitis and 20 clinically healthy gingival control tissues were collected. Viral DNA was determined with nested polymerase chain reaction, and cytokine mRNA expression was detected with real-time polymerase chain reaction assays. Results: Periapical lesions harbored EBV (75.9%) and HHV-6 (22.4%) at significantly higher frequencies compared with controls (P < .000001 and P < .05, respectively), whereas HCMV (12%) and HHV-8 (0%) occurred rarely. The median TNF-a expression was 13 times higher (P < .001) and TGF-b expression was 5 times higher in periapical lesions than in controls (P < .001). TNF-a expression was significantly higher in EBV-positive lesions than in EBV-negative lesions (P = .032). Presence of symptoms, lesion size, and infection by HCMV or HHV-6 had no significant association with either TNF-a or TGF-b expression. Conclusions: The herpesviral component of the endodontic microbiota did not correlate with TGFb expression, whereas EBV infection was associated with a median 1.5 times further elevation of the high TNF-a expression characteristic for periapical lesions. (J Endod 2013;39:456–460)
Key Words Epstein-Barr virus, human cytomegalovirus, human herpesvirus 6, periapical lesion, real time PCR, TGF-beta, TNF-alpha
A
pical periodontitis is induced by prolonged exposure of periapical tissues to microbial agents derived from the infected root canal. Anaerobic bacteria of the endogenous microflora play a primary role in the pathogenesis; nevertheless, nonbacterial components such as human herpesviruses can contribute to pathogenesis including exacerbation episodes (1–4). The mononuclear leukocytes immigrating into inflamed periapical tissues secrete various inflammatory cytokines and may import herpesviruses as well. The virus-infected host cells are able to increase the expression of several proinflammatory and immunregulatory cytokines. Recent findings have established a significant association of Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) with symptomatic and large-sized periapical lesions (1–5). Although a number of studies examined herpesvirus prevalence and cytokine levels separately in periapical lesions (3–7), only a few (8, 9) have tried to find the association between these factors through the simultaneous detection of herpesvirus prevalence and cytokine expression. Herpesviruses are important etiologic factors of many oral diseases. EBV presumably plays a role in the pathogenesis of African-type Burkitt lymphoma, nasopharyngeal carcinoma, oral hairy leukoplakia, and salivary gland tumors. It can be associated with other benign (leukoplakia, lichen oris) and malignant mucosal lesions (oral squamous cell carcinoma, oral lymphomas) (10, 11). Recent findings have established an association with irreversible pulpitis and apical and marginal periodontitis also (3, 11, 12). HCMV and human herpesvirus 6 (HHV-6) have also been implicated in the pathogenesis of marginal and apical periodontitis (2, 5, 12, 13). Human herpesvirus 8 (HHV-8) is also known as Kaposi sarcoma–associated virus because this herpesvirus is an etiologic agent of all subtypes of Kaposi sarcoma (14). This vascular tumor is predominantly found in human immunodeficiency virus–infected individuals. Oral mucosal manifestation of Kaposi sarcoma is common in acquired immunodeficiency syndrome patients and often an initial indicator of human immunodeficiency virus infection (15). HHV-8 may also be implicated in the pathogenesis of primary effusion lymphoma, multicentric Castleman disease, and salivary gland tumors (14).
From the *Faculty of Dentistry, Medical and Health Science Center, University of Debrecen, and †Department of Medical Microbiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary. Supported by the TAMOP 4.2.2.A-11/1/KONV-2012-0023 project. The project is implemented through the New Hungary Development Plan and co-financed by the European Social Fund and the European Regional Development Fund. Address requests for reprints to Dr Jozsef Konya, University of Debrecen, Department of Medical Microbiology, Medical and Health Science Center, Nagyerdei k€orut 98, H-4032 Debrecen, Hungary. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.12.028
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JOE — Volume 39, Number 4, April 2013
Clinical Research The other nucleic acid aliquot was treated with DNase (Sigma) to degrade DNA. The remaining RNA was turned to complementary DNA (cDNA) by using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) with random hexamers. The quantitative mRNA analyses from 58 periapical lesions and 20 controls were performed in a 7500 Real Time PCR System (Applied Biosystems). PCR conditions were 95 C (10 minutes), 50 cycles at 95 C (15 seconds), and 60 C (1 minute). The final volume of the PCR reaction was 20 mL containing 10 mL TaqMan Gene Expression Master Mix (Applied Biosystems), 1 mL Taqman Gene Expression Assay for TNF-a (ID no. Hs00174128_m1), TGF-b (ID no. Hs00998130_m1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ID no. 0711024), 7 mL distilled water, and 2 mL cDNA template. Determination of relative levels of TNF-a and TGF-b gene expression was performed by using the cycle threshold method in reference to GAPDH. Results are depicted as a mean mRNA expression from triplicate measurements normalized by internal control GAPDH. Significance of differences was analyzed by using Yates corrected c2 statistics. The relative cytokine mRNA levels of the patient groups were compared with each other and the control group by using Mann-Whitney test.
The aims of this study were to determine the levels of tumor necrosis factor-alpha (TNF-a) and transforming growth factor-beta (TGF-b) in periapical lesions and to evaluate the association between these cytokines and the clinical, radiologic, and virologic characteristics of the lesions. The prevalence of EBV, HCMV, HHV-6, and HHV-8 was determined with nested polymerase chain reaction (PCR), and cytokines were identified with real-time PCR assays.
Materials and Methods The study included 58 periapical lesions and 20 clinically healthy gingival tissues used as controls. Samples were collected from patients seeking dental care at the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, University of Debrecen. The Local Ethical Committee approved the study (approval number 2885-2008). Patients were anonymously coded for identification, and they signed informed consents. Patients with apical periodontitis also filled out questionnaires that contained questions about general diseases, current medications, history of the involved tooth, and related symptoms. Pain was assessed with the help of a subjective pain score from 1–10. Inclusion criteria for patients were detailed previously (1). Teeth with apical periodontitis were classified into symptomatic and asymptomatic subgroups. Symptomatic lesions were characterized by pain ($1 in the subjective pain score), discomfort on biting, and sensitivity by percussion or palpation at the apical region of mucosa. The asymptomatic lesions did not have any clinical symptoms with the exception of periapical radiolucent area on radiographs. On the basis of the radiographic size of the lesion, samples were divided into small-sized (<5 mm) and large-sized ($5 mm) lesions. The surgical removal of apical periodontitis lesions was performed as described previously (1). All control samples were clinically healthy gingival tissues taken during the surgical removal of third molars. Each removed control sample was put into sterile Eppendorf tube with RNAlater (Qiagen, Hilden, Germany) RNA stabilization reagent and immediately frozen to 70 C. Nucleic acid was isolated from homogenized tissue samples by High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Isolated nucleic acid was divided into 2 aliquots, one for DNA and the other for mRNA expression analysis. The effectiveness of DNA isolation was controlled with PCR detection of human b-globin (16). Herpesviral DNA was identified by nested PCR; 5 mL DNA was amplified in 20 mL final volume by using 1 U Red Taq Polymerase (Sigma, St Louis, MO) on the basis of previous studies reporting PCR conditions and primer sequences for EBV (10), HCMV (17), HHV-6 (18), and HHV-8 (19) DNA detection. The HHV-6 primer set amplified 195 base pairs PCR product for subtype A and 423 base pairs for subtype B, and nucleotide sequencing of 6 randomly selected PCR amplicons of the study confirmed the conventional PCR results. The lower detection limit was 10 copies of the viral genome per PCR reaction. Prevalences of these viruses were based on the presence of their DNA genome in the clinical specimens.
Results A total of 58 apical periodontitis samples were collected from 40 patients (age, 28–55 years; mean age, 41.5 years) and 20 clinically healthy gingival tissues from 20 patients (age, 19–41 years; mean age, 30 years). Table 1 illustrates the relation between the clinical characteristics of periapical lesions and the DNA prevalence of EBV, HCMV, HHV-6, and HHV-8. Apical periodontitis lesions harbored EBV and HHV-6 at significantly higher frequencies compared with healthy gingival tissues (75.9% versus 10%, P < .000001 and 22.4% versus 0%, P < .05, respectively). Eight of the nine HHV-6B infections were detected in symptomatic lesions, and subtype A was mainly present in asymptomatic ones. Considering lesion size, all HHV-6B infections were found in large-sized lesions, whereas subtype A was linked to small-sized lesions. The prevalence of EBV was found to be significantly higher in symptomatic lesions compared with asymptomatic lesions (89.3% versus 63.3%, P < .05) and in large lesions compared with small lesions (93.55% versus 55.6%, P = .002). Similar tendency was found for HCMV, although differences were not significant. We could not detect HHV-8 DNA in either pathologic or healthy samples (Table 1), which was not unexpected on the basis of our previous data on the systemic prevalence of HHV-8 in the same geographic region (20). The real-time PCR results revealed that the median TNF-a expression was 13 times higher (6.8 10 4 versus 9 10 3, P < .001) and TGF-b expression was 5 times higher (3.7 10 2 versus 0.17, P < .001) in periapical lesions than in controls. Table 2 shows the classification of samples that is based on size, presence of symptoms, EBV, HHV-6, or HCMV infection. Independently from the classifying factor,
TABLE 1. Clinical Characteristics of Periapical Lesions and DNA Prevalence of EBV, HCMV, HHV-6, and HHV-8 Apical periodontitis (n = 58) Symptomatic (n = 28) Asymptomatic (n = 30) Large-sized ($5 mm) (n = 31) Small-sized (<5 mm) (n = 27) Healthy controls (n = 20)
EBV
HCMV
HHV-6A
HHV-6B
HHV-8
44* 25* 19† 29* 15† 2
7 4 3 5 2 0
4 1 3 1 3 0
9 8‡ 1 9‡ 0 0
0 0 0 0 0 0
*P < .0001, †P < .005, and ‡P < .05 compared with the corresponding healthy controls. The nonlabeled prevalences were not significantly different from the corresponding healthy controls.
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Clinical Research TABLE 2. TNF-a and TGF-b Gene mRNA Expression Relative to Endogenous GAPDH mRNA in Periapical Lesions and Controls Statistical analysis Cytokine TNF-a
Group Controls Periapical lesions Lesion subgroup Asymptomatic Symptomatic Small size Large size EBV EBV+ HCMV HCMV+ HHV-6 HHV-6+ Controls Periapical lesions Lesion subgroup Asymptomatic Symptomatic Small size Large size EBV EBV+ HCMV HCMV+ HHV-6 HHV-6+
TGF-b
Relative expression (median) 6.8 10 9 10
4
9.3 10 8.2 10 9.4 10 8.7 10 6.8 10 1.1 10 8.7 10 1.5 10 9.2 10 7 10 3.7 10 0.17
3
0.185 0.168 0.164 0.174 0.28 0.15 0.176 0.173 0.173 0.205
3
3 3 3 3 2 3 2 3 3 2
P value related to controls
P value between lesion subgroups
Reference <.001* <.001* <.001* <.001* <.001* <.001* <.001* <.001* <.001* <.001* <.001* Reference <.001*
.59
.001* <.001* .001* <.001* <.001* <.001* <.001* .017* <.001* .003*
.95
.25 .032* .21 .75
.75 .11 .99 .85
*Significant difference (P < .05).
each lesion subgroup had significantly higher expression for both TNFa and TGF-b than in the controls. It is of note that one clinically healthy control specimen had both EBV infection and remarkably high relative TNF-a expression (Fig. 1). The latter did not affect quartile or median value or the statistical analysis by the nonparametric Mann-Whitney test. When the patient subgroups were compared, the only significant difference was that EBV-positive lesions had a further elevation of TNF-a expression in relation to EBV-negative lesions (Fig. 1). The other disease parameters were not associated with TNF-a expression (P values ranging .21–.75); small-sized, asymptomatic, and HHV-6–negative lesions had even slightly higher median TNF-a expression. HCMVpositive lesions showed higher TNF-a expression than HCMV-negative lesions, although the difference was not significant (8.7 10 3 versus 1.47 10 2, P = .21).
Regarding TGF-b, large-sized lesions showed slightly but not significantly higher cytokine expression compared with small-sized lesions (0.164 versus 0.174, P = .75). Of note, the median TGF-b expression was higher in EBV-negative subgroup than in EBV-positive subgroup (Fig. 1), although this difference also was not significant (P = .11). HHV-6–positive lesions showed higher TGF-b expression than HHV-6–negative lesions, although the difference was not significant (0.173 versus 0.205, P = .85). The subgroups divided by the remaining disease parameters had very similar median values of TGFb expression (P values ranging .75–.99) (Table 2).
Discussion Apical periodontitis is characterized by local production of proinflammatory TNF-a and anti-inflammatory TGF-b in apical periodontitis
Figure 1. TNF-a and TGF-b gene mRNA expression relative to endogenous GAPDH mRNA in EBV-negative and EBV-positive periapical lesions and controls. Boxes (gray) represent lower, median, and upper quartiles; bars represent the range of the results. *TNF-a value of an outranging (EBV-positive) control case. *P <.001; † P =.03; ‡P = .11.
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Clinical Research (6, 7, 21–23). TNF-a is a major bone resorptive mediator through osteoclast activation and the stimulation of mediators that destroy the extracellular matrix of bone tissue (24). It can induce powerful hyperalgesia by interfering with nociceptive signaling (25, 26). TGF-b is a cardinal regulator of oral wound healing through the stimulation of fibroblast proliferation, connective tissue fiber synthesis, and angiogenesis (27). It also has an important immunoregulatory effect through the repression of lymphocyte proliferation, cytotoxic T-cell functions, and toll-like receptor signaling (28). These cytokines were addressed in this study because EBV and HCMV infection is known to induce both of them (29, 30), and HHV-6 infection is able to elevate TNF-a expression (31). Our real-time PCR results also revealed that TNF-a expression and TGF-b expression were significantly higher in periapical lesions compared with healthy gingival tissues. These findings are in agreement with previous studies that used noninflamed periodontal ligaments (21) and healthy gingival tissues (32) as control and applied real-time PCR for cytokine detection. Within periapical lesions, we studied the effect of different clinical and virologic parameters on TNF-a and TGF-b expression. The latter correlated with none of the investigated parameters. EBV and HCMV also had no effect on TGF-b expression in periapical pathosis of deciduous teeth (9), indicating a pathogenesis similar to that of permanent teeth. On the other hand, TNF-a expression was increased in the presence of EBV. It should be emphasized that endodontopathogenic bacterial involvement is essential in periapical inflammation. These bacteria are potent TNF-a inducers in cultured macrophages, in which both endotoxin concentration and biodiversity of gram-negative bacteria correlate with TNF-a secretion (33). Indeed, we also observed approximately 10-fold increase in TNFa expression in the absence of EBV infection, which was plausibly caused by inflammatory response to endodontopathogenic bacteria. Nevertheless, EBV infection further increased TNF-a expression by 1.5 times (Table 2). Our findings on EBV are in accordance with a recent study by Sabeti et al (8), although they also revealed an association between symptomatic manifestation and the expression of several cytokines including TNF-a. Nevertheless, there are many differences between the populations of our study and their study. The latter (8) included a limited asymptomatic group consisting of only virus-uninfected lesions, which makes it difficult to determine whether virus infection or symptomatic manifestation is the primary determinant for inflammatory cytokine expression. Our study involved virus-infected asymptomatic lesions too and revealed the following pattern of associations; EBV infection was linked to both symptomatic manifestation and increased TNF-a expression, but these 2 parameters appeared independent from each other. In agreement with latter results, measurements of TNF-a on protein level also did not find association with symptomatic manifestation (25). Altogether, the TNF-a data of the above mentioned studies indicate that EBV infection can aggravate the basically bacterial pathogenesis in periapical lesions. Study size is critical to identify this type of pathogenic association. This study is one of the biggest among the ones targeting herpesviral involvement in periapical lesions. The size of the patient group allowed finding an association between EBV infection and TNF-a expression in periapical lesions, even though there were 3 times more EBVpositive lesions than EBV-negative lesions. To assess the statistical power related to size of the patient group, we classified TNF-a expression into low and high categories and performed sample size calculation by using the discrete type data, which revealed that less than 2fold increase of the present size of the patient group would reproduce the association of EBV infection and TNF-a expression with as high as 80% probability. JOE — Volume 39, Number 4, April 2013
This study also focused on further viral components of the endodontic microbiota. The ratio between the median TNF-a expression of HCMV-positive and HCMV-negative lesions was similar to that of EBV; however, the low prevalence did not allow establishing a statistically significant association. The same association was significant in the study by Sabeti et al (8), which had higher HCMV prevalence compared with our study population. It should be mentioned that in both studies, most HCMV-infected lesions were coinfected with EBV. Because EBV infection itself tends to increase TNF-a expression, the independent effect of HCMV cannot be determined. Subtypes A and B of HHV-6 possess different biological features and disease associations. HHV-6B is more commonly associated with exanthema subitum, a pediatric febrile illness with skin rashes (34). In contrast, HHV-6A is more frequently detected in immunodeficient patients and seems to play a role in chronic fatigue syndrome and other neurologic or immunologic diseases (12, 35, 36). We also observed a characteristic subtype distribution; whereas subtype A was found in small or asymptomatic lesions, subtype B was associated with large-sized symptomatic lesions, suggesting the biological diversity of the 2 subtypes appeared also in apical periodontitis. Correlation of HHV-6 infection with cytokine expression could not be assessed because of the low prevalence and the tendency to occur in coinfection with EBV. In summary, the investigated clinical parameters of periapical lesions, ie, radiologic size and symptomatic manifestation, did not correlate with either TNF-a or TGF-b expression in agreement with other studies (22, 25). The herpesviral component of the endodontic microbiota did not correlate with TGF-b expression, and EBV infection was associated with a median 1.5 times further elevation of the high TNF-a expression characteristic for periapical lesions.
Acknowledgments The authors deny any conflicts of interest related to this study.
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Clinical Research 12. Braun DK, Dominguez G, Pelleti PE. Human herpesvirus 6. Clin Microbiol Rev 1997; 10:521–67. 13. Rotola A, Cassai E, Farina R, et al. Human herpesvirus 7, Epstein-Barr virus and human cytomegalovirus in periodontal tissues of periodontally diseased and healthy subjects. J Clin Periodontol 2008;35:831–7. 14. Ablashi DV, Chatlynne LG, Whitmann JE Jr, et al. Spectrum of Kaposi’s sarcoma associated virus, or human herpesvirus 8, diseases. Clin Microbiol Rev 2002;15: 439–64. 15. Webster-Cyriaque J, Edwards RH, Quinlivan EB, et al. Epstein-Barr virus and human herpesvirus 8 prevalence in human immunodeficiency virus-associated oral mucosal lesions. J Infect Dis 1997;175:1324–32. 16. Konya J, Veress G, Juhasz A, et al. Additional human papillomavirus types detected by the hybrid capture tube test among samples from women with cytological and colposcopical atypia. J Clin Microbiol 2000;38:408–11. 17. Yazdi KA, Sabeti M, Jabalameli F, et al. Relationship between human cytomegalovirus transcription and symptomatic apical periodontitis in Iran. Oral Microbiol Immunol 2008;23:510–4. 18. Yalcin S, Karpuzoglu T, Suleymanlar G, et al. Human herpesvirus-6 and human herpesvirus-7 infections in renal-transplant recipients and healthy-adults in Turkey. Arch Virol 1994;136:183–90. 19. Ferreira DC, Paiva SSM, Carmo FL, et al. Identification of herpesviruses types 1 to 8 and human papillomavirus in acute apical abscesses. J Endod 2011;37: 10–6. 20. Juhasz A, Remenyik E, Konya J, et al. Prevalence and age distribution of human herpesvirus-8 specific antibodies in Hungarian blood donors. J Med Virol 2001; 64:526–30. 21. Menezes R, Garlet TP, Trombone APF, et al. The potential role of suppressors of cytokine signaling in the attenuation of inflammatory reaction and alveolar bone loss associated with apical periodontitis. J Endod 2008;34: 1480–4. 22. Colic M, Gazivoda D, Vucevic D, et al. Proinflammatory and immunoregulatory mechanisms in periapical lesions. Mol Immunol 2009;47:101–13. 23. Pezelj-Ribaric S, Magasic K, Prpic J, et al. Tumor necrosis factor-alpha in periapical tissue exudates of teeth with apical periodontitis. Mediators Inflamm 2007;2007:69416. http://dx.doi.org/10.1155/2007/69416.
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24. Chang YC, Yang SF, Huang FM, et al. Induction of tissue plasminogen activator gene expression by proinflammatory cytokines in human pulp and gingival fibroblasts. J Endod 2003;29:114–7. 25. Prso IB, Kocjan W, Simic H, et al. Tumor necrosis factor-alpha and interleukin 6 in human periapical lesions. Mediators Inflamm 2007;2007:38210. http: //dx.doi.org/10.1155/2007/38210. 26. Rittner HL, Brack A. Leukocytes as mediators of pain and analgesia. Curr Rheumatol Rep 2007;9:503–10. 27. Derynck R, Feng XH. TGF-beta receptor signaling. Biochim Biophys Acta 1997;1333: F105–50. 28. Li MO, Wan YY, Sanjabi S, et al. Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol 2006;24:99–146. 29. Mogensen TH, Paludan SR. Molecular pathways in virus-induced cytokine production. Microbiol Mol Biol Rev 2001;65:131–50. 30. Rochford R, Cannon MJ, Sabbe RE, et al. Common and idiosyncratic patterns of cytokine gene expression by Epstein-Barr virus transformed human B cell lines. Viral Immunol 1997;10:183–95. 31. Flamand L, Gosselin J, Daddario M, et al. Human herpesvirus-6 induces interleukin1-beta and tumor-necrosis-factor-alpha, but not interleukin-6, in peripheral-blood mononuclear cell-cultures. J Virol 1991;65:5105–10. 32. Fukada SY, Silva TA, Garlet GP, et al. Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases. Oral Microbiol Immunol 2009;24:25–31. 33. Martinho FC, Chiesa WM, Leite FR, et al. Antigenic activity of bacterial endodontic contents from primary root canal infection with periapical lesions against macrophage in the release of interleukin-1beta and tumor necrosis factor alpha. J Endod 2010;36:1467–74. 34. Dewhurst S, Mcintyre K, Schnabel K, et al. Human herpesvirus 6 (HHV-6) variant B accounts for the majority of symptomatic primary HHV-6 infections in a population of U.S. infants. J Clin Microbiol 1993;31:416–8. 35. Knox KK, Carrigan DR. Disseminated active HHV-6 infection in patients with AIDS. Lancet 1994;343:577–8. 36. Razonable RR, Fanning C, Brown RA, et al. Selective reactivation of human herpesvirus 6 variant a occurs in critically ill immunocompetent hosts. J Infect Dis 2002; 185:110–3.
JOE — Volume 39, Number 4, April 2013
Elevated Tumor Necrosis Factor-alpha Expression in Periapical Lesions Infected by Epstein-Barr Virus Katinka Hern adi, DMD, PhD,* Eszter Gy€ ongy€ osi, MSc,† Be ata Mesz aros, MSc,† † † † Levente Szak acs, MSc, Anita Szalm as, MSc, Eszter Csoma, PhD, Rich ard Mogyor osi, MD,* Levente Czompa, MD,* Gy€ orgy Veress, PhD,† Istv an Varga, DMD, PhD,* Ildik o Judit M arton, MD, PhD, DSci,* and J ozsef K onya, MD, PhD† Abstract Introduction: In apical periodontitis, there is an intense inflammatory response to endodontopathogenic bacteria, an essential component of the pathogenic microbiota. The inflammation can be aggravated by herpesviruses acting as nonessential pathogens in periapical lesions. This study aimed to determine the levels of tumor necrosis factor-alpha (TNF-a) and transforming growth factor-beta (TGF-b) in periapical lesions in relation to local occurrence of Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), and human herpesvirus 8 (HHV-8). Methods: Fifty-eight samples with apical periodontitis and 20 clinically healthy gingival control tissues were collected. Viral DNA was determined with nested polymerase chain reaction, and cytokine mRNA expression was detected with real-time polymerase chain reaction assays. Results: Periapical lesions harbored EBV (75.9%) and HHV-6 (22.4%) at significantly higher frequencies compared with controls (P < .000001 and P < .05, respectively), whereas HCMV (12%) and HHV-8 (0%) occurred rarely. The median TNF-a expression was 13 times higher (P < .001) and TGF-b expression was 5 times higher in periapical lesions than in controls (P < .001). TNF-a expression was significantly higher in EBV-positive lesions than in EBV-negative lesions (P = .032). Presence of symptoms, lesion size, and infection by HCMV or HHV-6 had no significant association with either TNF-a or TGF-b expression. Conclusions: The herpesviral component of the endodontic microbiota did not correlate with TGFb expression, whereas EBV infection was associated with a median 1.5 times further elevation of the high TNF-a expression characteristic for periapical lesions. (J Endod 2013;39:456–460)
Key Words Epstein-Barr virus, human cytomegalovirus, human herpesvirus 6, periapical lesion, real time PCR, TGF-beta, TNF-alpha
A
pical periodontitis is induced by prolonged exposure of periapical tissues to microbial agents derived from the infected root canal. Anaerobic bacteria of the endogenous microflora play a primary role in the pathogenesis; nevertheless, nonbacterial components such as human herpesviruses can contribute to pathogenesis including exacerbation episodes (1–4). The mononuclear leukocytes immigrating into inflamed periapical tissues secrete various inflammatory cytokines and may import herpesviruses as well. The virus-infected host cells are able to increase the expression of several proinflammatory and immunregulatory cytokines. Recent findings have established a significant association of Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) with symptomatic and large-sized periapical lesions (1–5). Although a number of studies examined herpesvirus prevalence and cytokine levels separately in periapical lesions (3–7), only a few (8, 9) have tried to find the association between these factors through the simultaneous detection of herpesvirus prevalence and cytokine expression. Herpesviruses are important etiologic factors of many oral diseases. EBV presumably plays a role in the pathogenesis of African-type Burkitt lymphoma, nasopharyngeal carcinoma, oral hairy leukoplakia, and salivary gland tumors. It can be associated with other benign (leukoplakia, lichen oris) and malignant mucosal lesions (oral squamous cell carcinoma, oral lymphomas) (10, 11). Recent findings have established an association with irreversible pulpitis and apical and marginal periodontitis also (3, 11, 12). HCMV and human herpesvirus 6 (HHV-6) have also been implicated in the pathogenesis of marginal and apical periodontitis (2, 5, 12, 13). Human herpesvirus 8 (HHV-8) is also known as Kaposi sarcoma–associated virus because this herpesvirus is an etiologic agent of all subtypes of Kaposi sarcoma (14). This vascular tumor is predominantly found in human immunodeficiency virus–infected individuals. Oral mucosal manifestation of Kaposi sarcoma is common in acquired immunodeficiency syndrome patients and often an initial indicator of human immunodeficiency virus infection (15). HHV-8 may also be implicated in the pathogenesis of primary effusion lymphoma, multicentric Castleman disease, and salivary gland tumors (14).
From the *Faculty of Dentistry, Medical and Health Science Center, University of Debrecen, and †Department of Medical Microbiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary. Supported by the TAMOP 4.2.2.A-11/1/KONV-2012-0023 project. The project is implemented through the New Hungary Development Plan and co-financed by the European Social Fund and the European Regional Development Fund. Address requests for reprints to Dr Jozsef Konya, University of Debrecen, Department of Medical Microbiology, Medical and Health Science Center, Nagyerdei k€orut 98, H-4032 Debrecen, Hungary. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.12.028
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Clinical Research The other nucleic acid aliquot was treated with DNase (Sigma) to degrade DNA. The remaining RNA was turned to complementary DNA (cDNA) by using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) with random hexamers. The quantitative mRNA analyses from 58 periapical lesions and 20 controls were performed in a 7500 Real Time PCR System (Applied Biosystems). PCR conditions were 95 C (10 minutes), 50 cycles at 95 C (15 seconds), and 60 C (1 minute). The final volume of the PCR reaction was 20 mL containing 10 mL TaqMan Gene Expression Master Mix (Applied Biosystems), 1 mL Taqman Gene Expression Assay for TNF-a (ID no. Hs00174128_m1), TGF-b (ID no. Hs00998130_m1), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ID no. 0711024), 7 mL distilled water, and 2 mL cDNA template. Determination of relative levels of TNF-a and TGF-b gene expression was performed by using the cycle threshold method in reference to GAPDH. Results are depicted as a mean mRNA expression from triplicate measurements normalized by internal control GAPDH. Significance of differences was analyzed by using Yates corrected c2 statistics. The relative cytokine mRNA levels of the patient groups were compared with each other and the control group by using Mann-Whitney test.
The aims of this study were to determine the levels of tumor necrosis factor-alpha (TNF-a) and transforming growth factor-beta (TGF-b) in periapical lesions and to evaluate the association between these cytokines and the clinical, radiologic, and virologic characteristics of the lesions. The prevalence of EBV, HCMV, HHV-6, and HHV-8 was determined with nested polymerase chain reaction (PCR), and cytokines were identified with real-time PCR assays.
Materials and Methods The study included 58 periapical lesions and 20 clinically healthy gingival tissues used as controls. Samples were collected from patients seeking dental care at the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, University of Debrecen. The Local Ethical Committee approved the study (approval number 2885-2008). Patients were anonymously coded for identification, and they signed informed consents. Patients with apical periodontitis also filled out questionnaires that contained questions about general diseases, current medications, history of the involved tooth, and related symptoms. Pain was assessed with the help of a subjective pain score from 1–10. Inclusion criteria for patients were detailed previously (1). Teeth with apical periodontitis were classified into symptomatic and asymptomatic subgroups. Symptomatic lesions were characterized by pain ($1 in the subjective pain score), discomfort on biting, and sensitivity by percussion or palpation at the apical region of mucosa. The asymptomatic lesions did not have any clinical symptoms with the exception of periapical radiolucent area on radiographs. On the basis of the radiographic size of the lesion, samples were divided into small-sized (<5 mm) and large-sized ($5 mm) lesions. The surgical removal of apical periodontitis lesions was performed as described previously (1). All control samples were clinically healthy gingival tissues taken during the surgical removal of third molars. Each removed control sample was put into sterile Eppendorf tube with RNAlater (Qiagen, Hilden, Germany) RNA stabilization reagent and immediately frozen to 70 C. Nucleic acid was isolated from homogenized tissue samples by High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Isolated nucleic acid was divided into 2 aliquots, one for DNA and the other for mRNA expression analysis. The effectiveness of DNA isolation was controlled with PCR detection of human b-globin (16). Herpesviral DNA was identified by nested PCR; 5 mL DNA was amplified in 20 mL final volume by using 1 U Red Taq Polymerase (Sigma, St Louis, MO) on the basis of previous studies reporting PCR conditions and primer sequences for EBV (10), HCMV (17), HHV-6 (18), and HHV-8 (19) DNA detection. The HHV-6 primer set amplified 195 base pairs PCR product for subtype A and 423 base pairs for subtype B, and nucleotide sequencing of 6 randomly selected PCR amplicons of the study confirmed the conventional PCR results. The lower detection limit was 10 copies of the viral genome per PCR reaction. Prevalences of these viruses were based on the presence of their DNA genome in the clinical specimens.
Results A total of 58 apical periodontitis samples were collected from 40 patients (age, 28–55 years; mean age, 41.5 years) and 20 clinically healthy gingival tissues from 20 patients (age, 19–41 years; mean age, 30 years). Table 1 illustrates the relation between the clinical characteristics of periapical lesions and the DNA prevalence of EBV, HCMV, HHV-6, and HHV-8. Apical periodontitis lesions harbored EBV and HHV-6 at significantly higher frequencies compared with healthy gingival tissues (75.9% versus 10%, P < .000001 and 22.4% versus 0%, P < .05, respectively). Eight of the nine HHV-6B infections were detected in symptomatic lesions, and subtype A was mainly present in asymptomatic ones. Considering lesion size, all HHV-6B infections were found in large-sized lesions, whereas subtype A was linked to small-sized lesions. The prevalence of EBV was found to be significantly higher in symptomatic lesions compared with asymptomatic lesions (89.3% versus 63.3%, P < .05) and in large lesions compared with small lesions (93.55% versus 55.6%, P = .002). Similar tendency was found for HCMV, although differences were not significant. We could not detect HHV-8 DNA in either pathologic or healthy samples (Table 1), which was not unexpected on the basis of our previous data on the systemic prevalence of HHV-8 in the same geographic region (20). The real-time PCR results revealed that the median TNF-a expression was 13 times higher (6.8 10 4 versus 9 10 3, P < .001) and TGF-b expression was 5 times higher (3.7 10 2 versus 0.17, P < .001) in periapical lesions than in controls. Table 2 shows the classification of samples that is based on size, presence of symptoms, EBV, HHV-6, or HCMV infection. Independently from the classifying factor,
TABLE 1. Clinical Characteristics of Periapical Lesions and DNA Prevalence of EBV, HCMV, HHV-6, and HHV-8 Apical periodontitis (n = 58) Symptomatic (n = 28) Asymptomatic (n = 30) Large-sized ($5 mm) (n = 31) Small-sized (<5 mm) (n = 27) Healthy controls (n = 20)
EBV
HCMV
HHV-6A
HHV-6B
HHV-8
44* 25* 19† 29* 15† 2
7 4 3 5 2 0
4 1 3 1 3 0
9 8‡ 1 9‡ 0 0
0 0 0 0 0 0
*P < .0001, †P < .005, and ‡P < .05 compared with the corresponding healthy controls. The nonlabeled prevalences were not significantly different from the corresponding healthy controls.
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Clinical Research TABLE 2. TNF-a and TGF-b Gene mRNA Expression Relative to Endogenous GAPDH mRNA in Periapical Lesions and Controls Statistical analysis Cytokine TNF-a
Group Controls Periapical lesions Lesion subgroup Asymptomatic Symptomatic Small size Large size EBV EBV+ HCMV HCMV+ HHV-6 HHV-6+ Controls Periapical lesions Lesion subgroup Asymptomatic Symptomatic Small size Large size EBV EBV+ HCMV HCMV+ HHV-6 HHV-6+
TGF-b
Relative expression (median) 6.8 10 9 10
4
9.3 10 8.2 10 9.4 10 8.7 10 6.8 10 1.1 10 8.7 10 1.5 10 9.2 10 7 10 3.7 10 0.17
3
0.185 0.168 0.164 0.174 0.28 0.15 0.176 0.173 0.173 0.205
3
3 3 3 3 2 3 2 3 3 2
P value related to controls
P value between lesion subgroups
Reference <.001* <.001* <.001* <.001* <.001* <.001* <.001* <.001* <.001* <.001* <.001* Reference <.001*
.59
.001* <.001* .001* <.001* <.001* <.001* <.001* .017* <.001* .003*
.95
.25 .032* .21 .75
.75 .11 .99 .85
*Significant difference (P < .05).
each lesion subgroup had significantly higher expression for both TNFa and TGF-b than in the controls. It is of note that one clinically healthy control specimen had both EBV infection and remarkably high relative TNF-a expression (Fig. 1). The latter did not affect quartile or median value or the statistical analysis by the nonparametric Mann-Whitney test. When the patient subgroups were compared, the only significant difference was that EBV-positive lesions had a further elevation of TNF-a expression in relation to EBV-negative lesions (Fig. 1). The other disease parameters were not associated with TNF-a expression (P values ranging .21–.75); small-sized, asymptomatic, and HHV-6–negative lesions had even slightly higher median TNF-a expression. HCMVpositive lesions showed higher TNF-a expression than HCMV-negative lesions, although the difference was not significant (8.7 10 3 versus 1.47 10 2, P = .21).
Regarding TGF-b, large-sized lesions showed slightly but not significantly higher cytokine expression compared with small-sized lesions (0.164 versus 0.174, P = .75). Of note, the median TGF-b expression was higher in EBV-negative subgroup than in EBV-positive subgroup (Fig. 1), although this difference also was not significant (P = .11). HHV-6–positive lesions showed higher TGF-b expression than HHV-6–negative lesions, although the difference was not significant (0.173 versus 0.205, P = .85). The subgroups divided by the remaining disease parameters had very similar median values of TGFb expression (P values ranging .75–.99) (Table 2).
Discussion Apical periodontitis is characterized by local production of proinflammatory TNF-a and anti-inflammatory TGF-b in apical periodontitis
Figure 1. TNF-a and TGF-b gene mRNA expression relative to endogenous GAPDH mRNA in EBV-negative and EBV-positive periapical lesions and controls. Boxes (gray) represent lower, median, and upper quartiles; bars represent the range of the results. *TNF-a value of an outranging (EBV-positive) control case. *P <.001; † P =.03; ‡P = .11.
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Clinical Research (6, 7, 21–23). TNF-a is a major bone resorptive mediator through osteoclast activation and the stimulation of mediators that destroy the extracellular matrix of bone tissue (24). It can induce powerful hyperalgesia by interfering with nociceptive signaling (25, 26). TGF-b is a cardinal regulator of oral wound healing through the stimulation of fibroblast proliferation, connective tissue fiber synthesis, and angiogenesis (27). It also has an important immunoregulatory effect through the repression of lymphocyte proliferation, cytotoxic T-cell functions, and toll-like receptor signaling (28). These cytokines were addressed in this study because EBV and HCMV infection is known to induce both of them (29, 30), and HHV-6 infection is able to elevate TNF-a expression (31). Our real-time PCR results also revealed that TNF-a expression and TGF-b expression were significantly higher in periapical lesions compared with healthy gingival tissues. These findings are in agreement with previous studies that used noninflamed periodontal ligaments (21) and healthy gingival tissues (32) as control and applied real-time PCR for cytokine detection. Within periapical lesions, we studied the effect of different clinical and virologic parameters on TNF-a and TGF-b expression. The latter correlated with none of the investigated parameters. EBV and HCMV also had no effect on TGF-b expression in periapical pathosis of deciduous teeth (9), indicating a pathogenesis similar to that of permanent teeth. On the other hand, TNF-a expression was increased in the presence of EBV. It should be emphasized that endodontopathogenic bacterial involvement is essential in periapical inflammation. These bacteria are potent TNF-a inducers in cultured macrophages, in which both endotoxin concentration and biodiversity of gram-negative bacteria correlate with TNF-a secretion (33). Indeed, we also observed approximately 10-fold increase in TNFa expression in the absence of EBV infection, which was plausibly caused by inflammatory response to endodontopathogenic bacteria. Nevertheless, EBV infection further increased TNF-a expression by 1.5 times (Table 2). Our findings on EBV are in accordance with a recent study by Sabeti et al (8), although they also revealed an association between symptomatic manifestation and the expression of several cytokines including TNF-a. Nevertheless, there are many differences between the populations of our study and their study. The latter (8) included a limited asymptomatic group consisting of only virus-uninfected lesions, which makes it difficult to determine whether virus infection or symptomatic manifestation is the primary determinant for inflammatory cytokine expression. Our study involved virus-infected asymptomatic lesions too and revealed the following pattern of associations; EBV infection was linked to both symptomatic manifestation and increased TNF-a expression, but these 2 parameters appeared independent from each other. In agreement with latter results, measurements of TNF-a on protein level also did not find association with symptomatic manifestation (25). Altogether, the TNF-a data of the above mentioned studies indicate that EBV infection can aggravate the basically bacterial pathogenesis in periapical lesions. Study size is critical to identify this type of pathogenic association. This study is one of the biggest among the ones targeting herpesviral involvement in periapical lesions. The size of the patient group allowed finding an association between EBV infection and TNF-a expression in periapical lesions, even though there were 3 times more EBVpositive lesions than EBV-negative lesions. To assess the statistical power related to size of the patient group, we classified TNF-a expression into low and high categories and performed sample size calculation by using the discrete type data, which revealed that less than 2fold increase of the present size of the patient group would reproduce the association of EBV infection and TNF-a expression with as high as 80% probability. JOE — Volume 39, Number 4, April 2013
This study also focused on further viral components of the endodontic microbiota. The ratio between the median TNF-a expression of HCMV-positive and HCMV-negative lesions was similar to that of EBV; however, the low prevalence did not allow establishing a statistically significant association. The same association was significant in the study by Sabeti et al (8), which had higher HCMV prevalence compared with our study population. It should be mentioned that in both studies, most HCMV-infected lesions were coinfected with EBV. Because EBV infection itself tends to increase TNF-a expression, the independent effect of HCMV cannot be determined. Subtypes A and B of HHV-6 possess different biological features and disease associations. HHV-6B is more commonly associated with exanthema subitum, a pediatric febrile illness with skin rashes (34). In contrast, HHV-6A is more frequently detected in immunodeficient patients and seems to play a role in chronic fatigue syndrome and other neurologic or immunologic diseases (12, 35, 36). We also observed a characteristic subtype distribution; whereas subtype A was found in small or asymptomatic lesions, subtype B was associated with large-sized symptomatic lesions, suggesting the biological diversity of the 2 subtypes appeared also in apical periodontitis. Correlation of HHV-6 infection with cytokine expression could not be assessed because of the low prevalence and the tendency to occur in coinfection with EBV. In summary, the investigated clinical parameters of periapical lesions, ie, radiologic size and symptomatic manifestation, did not correlate with either TNF-a or TGF-b expression in agreement with other studies (22, 25). The herpesviral component of the endodontic microbiota did not correlate with TGF-b expression, and EBV infection was associated with a median 1.5 times further elevation of the high TNF-a expression characteristic for periapical lesions.
Acknowledgments The authors deny any conflicts of interest related to this study.
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