The expression of TNF-α in recurrent aphthous stomatitis: A systematic review and meta-analysis

Meircurius Dwi Condro Surboyo a,*, Rizky Merdietio Boedi b, c, Ninuk Hariyani d,

Arvind Babu Rajendra Santosh a, e, Ida Bagus Pramana Putra Manuaba f, Pamela Handy Cecilia g,

I Gusti Agung Dyah Ambarawati h, Adiastuti Endah Parmadiati a, Diah Savitri Ernawati a

a Department of Oral Medicine, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

b Department of Dentistry, Faculty of Medicine, Universitas Diponegoro, Semarang, Indonesia

c Centre of Forensic and Legal Medicine and Dentistry, School of Dentistry, University of Dundee, Dundee, United Kingdom

d Department of Dental Public Health, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

e School of Dentistry, Faculty of Medical Sciences, University of The West Indies, Jamaica

f Oral Medicine Specialistic Degree, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

g Graduate School of Dental Sciences Program, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia

h School of Dentistry, Faculty of Medicine, Udayana University, Jimbaran, Bali, Indonesia

A R T I C L E I N F O

Keywords:

Saliva TNF-α

Recurrent aphthous stomatitis Serum

Tissue expression

A B S T R A C T

Objective: The pathogenesis of recurrent aphthous stomatitis (RAS) is related to an increase of pro-inflammatory cytokine, namely tumor necrosis factor α (TNF-α). This cytokine plays an important role in the development of ulcer lesions, both in saliva, tissues and blood. This systematic review analyzed the differences of TNF-α in le-

sions, salivary and blood and can be used as a reliable method of diagnosis for RAS.

Methods: A comprehensive search of PubMed, Scopus databases, Web of Science, Scielo, Google Scholar and Embase with keywords. The inclusion criteria were studies that assessed the saliva, serum, and RAS lesion, with

the outcome reporting the mean of saliva, serum and tissue expression of TNF-α. The risk of bias was also

assessed.

Result: Healthy individuals showed significantly lower TNF-α than RAS (SMD = -1.517, 95% CI [-2.25, —0.78]). Although there is a significant difference between sample (i.e., saliva, serum) and detection type (i.e., cytometry bead array, ELISA), both methods can detect a significant difference in TNF-α between healthy individuals and RAS patients.

Conclusions: The TNF-α is a useful diagnostic marker for RAS. We encourage saliva to detect changes in TNF-α during ulceration as it provides accuracy, reliability, and non-invasive procedure compared to a blood draw.

Introduction

The diagnosis of recurrent aphthous stomatitis (RAS) can be given definitively if it includes four criteria: recurrence, periodic, unknown

etiology, and no systemic alteration [1–4]. Research evidence shows few

aggravating factors for RAS, and these are categorized as local (i.e., trauma, smoking) and systemic predisposing factors (i.e., periodic fever, stress) were related to RAS development. However, considering the multiple factors influencing the diagnosis, the enforcement of RAS diagnosis has not been determined.

In the current oral medicine practice, RAS diagnosis is only

determined based on the degree of recurrence, without definite etiology, ulcer period or accompanying objective examination. Since the evidence of RAS pathogenesis is related to oral bacteria changes [5,6], poly- morphism of interleukin gene [7,8], and serotonin transporter [9], observing these indicators are complex and not clinically feasible. However, studies have reported that RAS is more likely to have genetic connections and changes in the immune response, such as tumor ne-

crosis factor-α (TNF-α). TNF-α It has become a common inflammatory

marker in various mucosal abnormalities of the oral cavity, including RAS [10]. If TNF-α has an essential role in lesion development, then detecting these cytokines can be an objective reference to establish the

* Corresponding author.

E-mail addresses: meircurius@fkg.unair.ac.id, meircurius-2015@fkg.unair.ac.id (M.D.C. Surboyo).

https://doi.org/10.1016/j.cyto.2022.155946

Received 31 March 2022; Received in revised form 6 June 2022; Accepted 13 June 2022

Available online 18 June 2022

diagnosis. Several studies have reported the expression of these cyto- kines in various stages of RAS, and the level is elevated in saliva [11], serums [12], and tissue lesions [13].

Several research studies reported that TNF-α is a useful marker for

diagnosing RAS. However, a variation is observed in their results and methods for estimating TNF-α. Hence, to understand the available evi- dence on utilizing TNF-α in diagnosing RAS, the present systematic re- view analyzes the differences of TNF-α in saliva and blood and can be used as a reliable method of diagnosis for RAS.

2. Material and methods

Data sources and search strategy

The Preferred Reporting Items for Systematic Reviews and Meta- Analyses (PRISMA) guidelines were adopted for this systematic review and meta-analysis. A comprehensive search of PubMed, Scopus data- bases, Google Scholar, Scielo, Web of Science and Embase was con- ducted in March 2022. The following keyword combinations were

adopted for searching articles for recurrent aphthous stomatitis: [“recurrent aphthous stomatitis” or “recurrent aphthous ulcers” or “aphthous ulcer” or “RAS” or “RAU”] AND [“tumor necrosis factor” or “TNF” or “TNF-alpha”] AND [“cytokine”] AND [“pro-inflammatory”]. In addition, the reference lists of the eligible articles were searched

manually to identify additional relevant publications.

A search strategy was performed using the PICO model (patient, intervention, comparison, outcome), taking into consideration the following aspects: population/patient (RAS patient), diagnostic/thera-

Foundation for Statistical Computing Version 4.0.5, Vienna, Austria) with

metafor package [14]. A random-effects model was applied to pool the value of TNF-α with corresponding 95% confidence intervals (CI). The primary size effect was analyzed with the standardized mean difference (SMD) using Cohens’ D transformation, with a negative SMD value

indicating a lower amount of TNF-α in the healthy individual group and a positive SMD value indicating a higher amount of TNF-α in the RAS

group. Knapp and Hartung’s adjustment test were used to reduce the

number of unjustified significant result from the previous trans- formation. Furthermore, meta-regression analysis using a mixed-effect model was done to analyze the difference between sample acquisition (i.e., saliva or serum) and the quantification process (i.e., ELISA and cytometric bead array (CBA), etc.).

3. Result

Characteristics of included studies

A literature search with the specified keywords resulted in 5113 published articles. After title screening was done, only 247 articles were chosen for the next step. Finally, 30 studies were selected in this sys- tematic review based on abstract reading and full-text availability. The PRISMA flowchart of the study search is presented in Fig. 1.

TNF-α expression on saliva

Seven studies reported the saliva expression of TNF-α with nine ob- servations. Two hundred and seventy-six RAS patients and 190 health

peutic procedure (the saliva collection and blood drawn), comparison

patients as control were analyzed for salivary TNF-α. Six studies

(healthy individual), and outcomes (TNF-α expression or value).

Study selection

The inclusion criteria for studies were as follows: (i) the diagnostic criteria of RAS were based on an accepted clinical description, both active and remission phases (ii) RAS patient and control (health in-

dividuals) (iii) reported the TNF-α expression. Fundamental experi-

mental studies such as animal or cell studies, abstracts, narrative reviews, case reports and editorials were excluded from this analysis.

Data extraction and quality assessment

Three authors screened each study independently (MDCS, IBPPM and PHC). MDCS is an oral medicine specialist with 3 years of experi- ence, IBPPM is a final year residence of oral medicine, and PHC is the last year of the dentistry program. The authors first screened the title(s), abstracts, and full texts to determine whether the inclusion criteria had been met. The following information was then extracted from the studies

to be included in the meta-analysis: first author’s name, year of publi- cation, age, sex, sample size, study design, RAS type, and the value of TNF-α. In case of disagreement, third investigators (DSE and AEP) will act as a referral and reach a consensus through discussion.

The Joanna Briggs Institute Critical Appraisal Tools, including the 10-item Checklist for Case-Control Studies, 10-item Checklist for Analytical Cross-Sectional Studies, and 13-item Checklist for Random- ized Control Study, were used to assess the methodological quality of the

included studies. Each item was scored as “yes”, “no”, “unclear”, or “not applicable”. One point was assigned to the answer “yes”, and zero points were assigned to “no”. The total point of each study was categorized into < 50%, 51–75% and > 75% for high, moderate, and low risk of bias. Furthermore, each study assessed the publication bias using Begg’s rank correlation test, and a p-value of < 0.05 indicated no publication bias.

Data synthesis and analysis

The data extracted from the included articles were entered into R (R

analyzed the salivary TNF-α using ELISA [11,15–19], and one study analyzed using CBA methods [20]. These methods resulted in a higher salivary expression of TNF-α in RAS patients compared to the healthy individual’s cohort [11,15–18,20]. In contrast, only one study showed lower salivary expression of TNF-α in RAS patients compared to healthy individuals [19] (Table 1).

TNF-α expression on serum

The serum TNF-α was reported by seven studies with twelve obser- vations. A total of 283 RAS patients and 351 health patients as control were analyzed for serum expression of TNF-α. Six studies analyzed the serum expression of TNF-α using ELISA methods [19,21–24], and three studies analyzed using CBA [25–27]. The ELISA method showed a higher serum expression of TNF-α in RAS patients compared to healthy individuals. One study showed lower salivary expression of TNF-α in RAS patients compared to the healthy individuals [24] (Table 2).

Risk bias assessments

The risk assessments provide in Table 3, Table 4 and Table 5. Pub- lication bias was not detected in the current study sample (p < 0.05).

Meta-Analysis

Fourteen studies with 21 observations were included in the meta–

= —

analysis. The SMD value reported from the random effect model favored the healthy individual group (SMD -1.376, 95% CI [-2.05, 0.7]) Fig. 2. High heterogeneity was observed with a significant Q-test (I2

91.68%, Tau2 1.19) (Table 6).

—

The mixed-effect model for meta-regression analysis found a signif- icant difference on SMD between saliva and serum sample acquisition, with saliva samples giving a higher SMD value (SMD -1.618, 95% CI [-2.64, 0.59]). In the detection type, ELISA and CBA significantly different from each other, CBA gives higher SMD value (SMD -1.881, 95% CI [-3.11, 0.84]). High heterogeneity was detected in each meta– regression model with a significant Q test (Table 6).

Fig. 1. PRISMA flow chart of the literature search and study selection.

Table 1

TNF-α expression on saliva.

Author Reference Type of RAS Subject TNF-α expression (Mean ± SD) Method of detection Samples

Health patient RAS Health patient RAS

Boras et al [15] MiRAS 26 26 7.88 ± 8.45 28.00 ± 26.19 ELISA Saliva

Chaudhuri et al Valle et al Hegde et al Seifi et al

Borra et al

Deng et al

[15] MiRAS – remission 26 13 7.88 ± 8.45 54.31 ± 49.63 ELISA Saliva [11] RAS 30 30 47.85 ± 17.48 86.30 ± 18.59 ELISA Saliva [16] RAS 10 20 26.03 ± 7.66 53.59 ± 20.05 ELISA Saliva [17] MiRAS 30 30 23.09 ± 6.95 58.82 ± 15.24 ELISA Saliva

[18]	MiRAS	18	18
[18]	MiRAS	18	18

10.76 ± 1.83 34.9 ± 11.35 ELISA Saliva

[19] RAS 17 20

10.76 ± 1.83 28.09 ± 9.07 ELISA Saliva

[20] MiRAS 15 101

21.90 ± 42.90 11.50 ± 14.00 ELISA Saliva

0.14 ± 0.18 8.87 ± 20.86 CBA Saliva

RAS: Recurrent aphthous stomatitis; MiRAS: Minor recurrent aphthous stomatitis; CBA: Cytometric bead array.

Table 2

TNF-α expression on serum.

Author Reference Type of RAS Subject TNF-α expression (Mean±SD) Method of detection Samples

Health patient RAS Health patient RAS

Borra et al [19] RAS 20 21 0.90 ± 2.90 2.70 ± 5.70 ELISA Serum

Albinidou et al [21] MiRAS 40 32 177.6 ± 16.00 184 ± 16.00 ELISA Serum

Avci et al [22] MiRAS 25 25 3.45 ± 1.01 5.26 ± 1.21 ELISA Serum

Yamamoto et al [23] RAS-active 20 20 95.0 ± 0.00 111.7 ± 52.7 ELISA Serum

Zhu et al Elamrousy et al Shen et al Lewkowicz et al

[23] RAS-remission 20 20 95.0 ± 0.00 95.0 ± 25.00 ELISA Serum [24] RAS 70 70 278.4 ± 31.50 263.5 ± 32.70 ELISA Serum [25] MiRAS 20 20 278.20 ± 11.37 281.30 ± 11.79 CBA Serum [26] MiRAS 20 127 0.33 ± 0.63 2.26 ± 5.02 CBA Serum [27] RAS-active 12 10 391.4 ± 105.78 762.0 ± 193.74 CBA Serum [27] RAS-remission 12 8 391.4 ± 105.78 754.5 ± 258.73 CBA Serum [27] RAS-active 12 10 579.3 ± 70.54 1236.3 ± 219.89 CBA Serum [27] RAS-remission 12 8 579.3 ± 70.54 1826.3 ± 277.36 CBA Serum

RAS: Recurrent aphthous stomatitis; MiRAS: Minor recurrent aphthous stomatitis; CBA: Cytometric bead array.

Table 3

Study quality of analytical cross-sectional study.

Author Reference Type of study Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Total

Chaudhuri et al [11] Analytical cross-sectional study √ √ √ √ X X √ √ 75% Borra et al [19] Analytical cross-sectional study √ √ √ √ X X √ √ 75% Deng et al [20] Analytical cross-sectional study √ √ √ √ X X √ √ 75% Avci et al [22] Analytical cross-sectional study √ √ √ √ X X √ √ 75% Zhu et al [24] Analytical cross-sectional study √ √ √ √ √ √ √ √ 100% Shen et al [26] Analytical cross-sectional study √ √ √ √ X X √ √ 75%

Q1: Were the criteria for inclusion in the sample clearly defined? Q2: Were the study subjects and the setting described in detail? Q3: Was the exposure measured in a valid and reliable way?

Q4: Were objective, standard criteria used for measurement of the condition? Q5: Were confounding factors identified?

Q6: Were strategies to deal with confounding factors stated? Q7: Were the outcomes measured in a valid and reliable way? Q8: Was appropriate statistical analysis used?

Table 4

Study quality of case control study.

Author Reference Type of study Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Total

Boras et al [15] Case control study √ √ √ NA NA X X √ NA √ 50% Valle et al [16] Case control study √ √ √ NA NA X X √ NA √ 50% Hegde et al [17] Case control study √ √ √ NA NA X X √ NA √ 50% Seifi et al [18] Case control study √ √ √ NA NA X X √ NA √ 50% Albinidou et al [21] Case control study √ √ √ NA NA X X √ NA √ 50% Yamamoto et al [23] Case control study √ √ √ NA NA X X √ NA √ 50% Lewkowicz et al [27] Case control study √ √ √ NA NA X X √ NA √ 50%

Q1: Were the groups comparable other than the presence of disease in cases or the absence of disease in controls? Q2: Were cases and controls matched appropriately?

Q3: Were the same criteria used for identification of cases and controls? Q4: Was exposure measured in a standard, valid and reliable way?

Q5: Was exposure measured in the same way for cases and controls? Q6: Were confounding factors identified?

Q7: Were strategies to deal with confounding factors stated?

Q8: Were outcomes assessed in a standard, valid and reliable way for cases and controls? Q9: Was the exposure period of interest long enough to be meaningful?

Q10: Was appropriate statistical analysis used?

Table 5

Study quality of randomized control study.

Author	Reference	Type of study	1	2	3	4	5	6	7	8	9	10	11	12	13	Total
Elamrousy et al	[25]	RCT	√	√	√	√	√	√	√	√	√	√	√	√	√	100%

Q1: Was true randomization used for assignment of participants to treatment groups? Q2: Was allocation to treatment groups concealed?

Q3: Were treatment groups similar at the baseline? Q4: Were participants blind to treatment assignment?

Q5: Were those delivering treatment blind to treatment assignment? Q6: Were outcomes assessors blind to treatment assignment?

Q7: Were treatment groups treated identically other than the intervention of interest?

Q8: Was follow up complete and if not, were differences between groups in terms of their follow up adequately described and analyzed? Q9: Were participants analyzed in the groups to which they were randomized?

Q10: Were outcomes measured in the same way for treatment groups? Q11: Were outcomes measured in a reliable way?

Q12: Was appropriate statistical analysis used?

Q13: Was the trial design appropriate, and any deviations from the standard RCT design (individual randomization, parallel groups) accounted for in the conduct and analysis of the trial?

4. Discussion

TNF-α plays a significant role in mediating acute inflammation. A similar relation between TNF-α and RAS is observed. Our current finding suggests that multiple types of research explore the quantified amount of TNF-α produced when various predisposing factors were accounted. Nevertheless, all findings agreed that TNF-α changes between healthy individuals and RAS patients. These differences in TNF-α provide

evidence that a reliable and easy to enforce RAS diagnostic is through TNF-α. Unfortunately, no published literature explains the role of mol- ecules that allow elevation of TNF-α expression in saliva and blood pa- tients with RAS.

TNF-α becomes an important marker for the occurrence and devel- opment of RAS lesions. TNF-α was found to be consistently higher in

active lesions [15,27], and recurrent [15,27], even when the lesion has healed [28]. In the formation and development of RAS lesions, trauma

Fig. 2. TNF-α expression on saliva. n: Number of patients in each group, S.D: standard deviation, SMD: Standardized Mean Difference, 95% CI: Confidence Interval, Q(df): Q test for homogeneity and degrees of freedom, I2: Total Heterogeneity.

Table 6

Meta-Regression Models.

n SMD 95% CI Q(df) Tau2 I2

Sample Type

Saliva 9 —1.618 —2.64 —0.59 194.46(19) 1.02 90.23%

Serum 12 —1.151 —2.05 —0.25

Detection Type

CBA 7 —1.881 —3.11 —0.65 239.92(19) 1.29 92.08%

ELISA 14 —1.165 —1.98 —0.35

SMD: Standardized Mean Difference, 95% CI: Confidence Interval, Q(df): Q test for homogeneity and degrees of freedom. Tau2: estimated amount of total het- erogeneity, I2: Total Heterogeneity; n: number of observations.

frequently plays a role in RAS onset. In this context, trauma is a local factor that can occur in the oral cavity due to masticatory or occluding forces or other harmful habits. In addition, immunological abnormal- ities (deficiencies/suppressed) can assist during traumatic episodes by triggering an immunological response to develop RAS. During this immunological response, an abnormal cytokine cascade is activated in the oral mucosal environment, which leads to a cell-mediated immune response in a focal area of the oral mucosa [29]. During the development

of the lesion, the CD4+/CD8 + ratio is disrupted, recruitment lympho- cytes and macrophages in the lesion — therefore, increasing the cyto- kine production of TNF-α [30]. The immune response occurs not only in the local region of ulcerated tissue but also triggers an increased blood flow and capillary permeability. Thus, the systemic influence of TNF-α is noted in the bloodstream. Increased TNF-α in the blood and saliva does

not cause clinical manifestation in RAS patients but represents a sign of damage to oral tissue triggered by an immunological alteration in the body.

Meanwhile, the pathogen recognition receptor (PRR) releases

phagocytic-chemokines cells (i.e., macrophages, dendritic and mast cells) to secrete pro-inflammatory cytokines such as TNF-α. These in- flammatory mediators cause an increase in vascular permeability

expression of cell adhesion molecules (CAM) and chemokines. Hence, the epithelium becomes an inflamed form of ulceration [31].

High TNF-α levels in the blood serum in patients with active disease indicate a polarized Th1 response [32]. Therefore, Th1 will be seen in the RAS due to the release of TNF- α. This event will stimulate cytotoxic T lymphocytes and increase endothelial expression, causing inflamma-

tory cells migration to the inflammation site, which causes ulcer development [30,33].

Several cytokines that are linked with RAS pathogenesis have also been studied, however there were no confirmatory observations on those cytokines in RAS. Clinical studies that focused on estimation of

cytokine levels in RAS are interferon and Interleukins, such as IFN [34,35], IL-8 [36], IL-1β [37–40], IL-1 [35,41,42], IL-2 [34,43–46], IL-4

[44,45,47,48], IL-6 [37,42,46,49], IL-10 [39,45,50–53], IL-12 [50,51],

IL-13 [35,45], IL-17 [35,54], IL-17C [55], IL-17F [56]. Unfortunately,

these findings on cytokines were not able to achieve any clinically reliable application in RAS while comparing TNF-α.

One of the invasive methods that assist in analyzing TNF-α levels is

obtaining tissue samples of RAS lesions. However, current evidence in-

dicates that tissue sampling from RAS does not yield good results for estimating TNF-α levels. RAS lesions for immunohistochemical exami- nation [32,57], mRNA extraction [28,58], and RNA [59] showed that TNF-α is higher than in healthy individuals. However, this method is

invasive and requires surgical procedures, so that it cannot efficiently provide a favorable clinical application.

Nowadays, saliva is the most helpful component of chair side diag- nosis. Saliva can use to diagnose systemic illnesses, monitoring general health, understand the prognosis of a disease, or identify an oral sign of systemic disease. The serum component of saliva is derived originally from the vascularity of carotid arteries. Saliva has the same molecule found in systemic circulation [60]. From this review, we found that the

elevation of TNF-α expression in saliva and serum can be detected. The increased TNF-α expression in the saliva is easier to analyze in the

clinical setting of oral medicine for diagnosing RAS due to its relevance and non-invasive nature of specimen collection.

Further analysis of detection type indicates that CBA is significantly different in TNF- α detection compared to ELISA due to its better sensitivity. This assures clinicians, especially in RAS patients who have

passed the acute phase, both in the active and remission phase, that low concentrations of TNF-α can still be detected using CBA. This finding strengthened the procedures for the enforcement of diagnosis from RAS objectively and confirmed the role of TNF-α in the pathogenesis of RAS. Despite the significant difference, both methods are acceptable forms of diagnosis regarding TNF-α detection.

5. Conclusion

In the current dental or oral medicine practice, RAS cases are diag- nosed through clinical examination. However, this approach is not

completely adequate for starting RAS management. Hence, estimation of TNF-α is recommended as chair side consideration. Both systematic re- view and meta-analysis findings of this study state that TNF-α should

serve as a reliable diagnostic marker for RAS. Whilst the detection method is comparably similar, we encourage saliva to detect changes in

TNF-α during ulceration as it provides accuracy, reliability, and non-

invasive procedure compared to a blood draw.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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