19 © 2022 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License Evaluation of Salivary Interleukin 2 (IL-2) and Interleukin 12(IL-12) in Recurrent Aphthous Stomatitis Sura Farhan Saeed1,* and Zainab Abduljabar Aldhaher2 1,2Department of Basic Sciences, College of Dentistry, University of Baghdad, Baghdad, Iraq. *Corresponding Author. Received: 14 March 2023 Accepted: 4 May 2023 Published: 20 October 2024 doi.org/10.30526/37.4.3338 Abstract Recurrent aphthous stomatitis (RAS) is a common kind of excruciating mouth mucosal disease. Despite their high prevalence, etiopathogenesis remains unclear. This study was to investigate the concentration of Interleukin 2 (IL-2) and Interleukin 12 (IL-12) in the saliva of RAS. Eighty people were involved In this study (forty RAS patients and forty healthy controls), their ages ranged (from 16 - 60) years. The sandwich enzyme-linked immune-sorbent assay was used to assess Interleukin 2 (IL-2) and Interleukin 12 (IL-12) concentrations in saliva in each group. The salivary concentrations of Interleukin 2 (IL-2) and Interleukin 12 (IL-12) was significantly higher in the patient group (159.06±20.37), (331.32±94.42) respectively in comparison to the control group at (p≤0.05). These results indicate that saliva provides the perfect medium for the detection of pro- inflammatory markers of the oral cavity, Additionally, salivary Interleukin 2 (IL-2) and Interleukin 12 (IL-12) may play a significant role in the pathophysiology of this illness. Keywords: Recurrent aphthous stomatitis, salivary evaluation, interleukin. 1. Introduction Until now, the etiology of recurrent aphthous stomatitis (RAS) has remained unknown, although genetic susceptibility, infectious agents, and alterations in immune mechanics have been implicated [1, 2]. Recurrent aphthous stomatitis (RAS) is a prevalent oral mucosal disease characterized by a yellowish-gray base with elevated margins and an erythematous halo. RAS typically manifests in the lining or mucosa that is not keratinized [3, 4]. It has a prevalence of 50– 66% and it is one of the most prevalent conditions affecting the oral mucosa [5, 6]. Recurrent aphthous stomatitis is frequently observed and is immune-mediated [7, 8] . RAS can be managed through a wide variety of preventative measures and therapies, intending to reduce ulcer pain, stimulate ulcer healing, and/or prevent ulcer recurrence [9]. First-line treatment options include topical medications in the form of corticosteroids (triamcinolone acetonide), anti-inflammatory drugs (amlexanox), antibiotics (doxycycline), and antiseptics (lidocaine) [10]. In more severe cases of RAS where local treatment is insufficient, systemic drugs in the form of corticosteroids (prednisone), immunomodulatory drugs (thalidomide), and antibiotics/antimicrobials (clofazimine) can prove effective [11]. Because they are essential mediators of the immune https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0009-0007-2122-8046 mailto:surafarhan6@gmail.com https://orcid.org/0000-0002-8977-8761 mailto:Zainab.aldhahir@codental.uobaghdad.edu.iq IHJPAS. 2024, 37(4) 20 response, cytokines have received a lot of attention in the immunological pathophysiology of RAS [12]. Recent studies suggest that RAS may be brought on by a cellular defect in the oral mucosa membrane, which results in a cellular immune response focused on the portion of the oral mucosa membrane at its center [13-16]. Two different types of cytokines pro-inflammatory cytokines that induce cell-mediated immunity and are produced by Th1 (IL-2, IL-12, IFN-g and TNF-a) and anti- inflammatory cytokines (IL-4, IL-5, IL-10 and IL-13) produced by Th2 and promote humoral immunity and suppression of cell-mediated immunity [17, 18]. The imbalance of Th1/ Th2 immune response is strongly influenced by their cytokine environment [19]. To fight infections, cancers and self-antigens, the primary immune response mediators are cytokines. They have a significant impact in examining the pathophysiology of immune responses [20]. Among the cell types that make cytokines are type I and type II helper T cells. T helper cells release two types of cytokines: type 2 (IL-4, IL-5, IL-6, IL-10, and IL-13), which improve humoral immunity and tolerance, and type 1(interleukin (IL-2), IL-12, interferon (IFN), and tumor necrosis factor (TNF), which enhance cellular immunity [21]. The cytokine profile controls immune activation and tolerance [22]. IL-2 is a 15-kDa glycoprotein that was originally referred to as T-cell growth factor (TCGF). Helper T cells that have been activated are the main sources of IL-2 secretion. For controlling cellular and cellular-chronic inflammatory responses, it is essential. T cells have an IL- 2 receptor that IL-2 binds to, which causes the production of lymphokines and the promotion of cell proliferation [23, 24]. Natural killer cells, as well as antigen-presenting cells like dendritic cells (DCs) and macrophages, produce the interleukin-12 [25]. The aim of this study was to investigate the concentration of Interleukin 2 (IL-2) and Interleukin 12(IL-12) in saliva of patients with RAS. 2. Materials and methods The study included 40 patients with RAS ranged between (16-60) years., compared with 40 apparently healthy individuals ranged between (16-60years) considered as a controls. Saliva was collected from patients and control group between 9-12am to evaluate the concentrations of Interleukin 2 (IL-2) and IL-12 in saliva Were measured by enzyme-linked immunosorbent assay (ELISA) kit that is commercially available. This was completed in accordance with the booklet of the kit's instructions. Interleukin 12 (IL-12) and IL-2 ELISA kit for humans (My BioSource, USA). 2.1. Statistical analysis Data description, analysis and presentation have been performed using computerized software statistical package for social science (SPSS version-22). Shapiro Wilk test was used to test the normality distribution of the quantitative variable. Both descriptive and inferential statistics were used [26]. The descriptive statistics included: frequency, percentage, minimum, maximum, mean, standard deviation (SD) and graphical presentation by bar charts. Inferential statistics were used to accept or reject the statistical hypotheses which included: Analysis of variance student t-test and Chi-square test. The statistical significance of difference of mean between 2 groups was calculated by T-test and Chi-square test. Correlation among different parameters was calculated by the Pearson correlation coefficient test. In the statistical evaluation, the following levels of significance are used: Not significant p>0.05, Significant p≤0.05, Highly significant p≤0.01. IHJPAS. 2024, 37(4) 21 3. Results 3.1. The concentration of IL-2 in saliva The results of this study showed, there is elevation in the concentration of IL-2 in saliva of patients with (RAS)(159.06) Comparing with it is concentration in saliva of control group (Table 1), (Figure 1). Table 1. Descriptive and Analytic Statistics of Mean ±SD IL-2 concentration level for patients and Control. IL-2 pg/ml Group of the Patients N=40 Group of the Control N=40 T-test (P.value) ‘Minimum’ 125.84 65.66 0.000** ‘Maximum’ 236.21 98.95 Mean 159.06 82.09 SD 20.37 8.35 SE 3.22 1.32 Figure 1. Descriptive and Analytic Statistics of Mean Value of IL-2 concentration for patients and Control. 3.2. The concentration of IL-12 in saliva The results of this study showed ,there is elevation in the concentration of IL-12 in saliva of patients with (RAS)(331.32) Comparing with it is concentration in saliva of control group (Table 2), (Figure 2). Table 2. Descriptive and Analytic Statistics of Mean ±SD IL-12 concentration for patients and Control. IL-12 pg/ml Group of the Patients N=40 Group of the Control N=40 T-test (P.value) Minimum 228.93 79.29 0.000** Maximum 811.55 199.98 Mean 331.32 130.31 SD 94.42 34.38 SE 14.93 5.43 Patients Control IL-2 159.06 82.09 0 20 40 60 80 100 120 140 160 180 M e a n v a lu e IL-2 pg\ml IHJPAS. 2024, 37(4) 22 Figure 2. Effect of Age groups in parameters of sample studied. 4. Discussion 4.1. The concentration of IL-2 and IL-12 in saliva In this study, the patient group had significantly greater concentrations of IL-2 and IL-12 in saliva than the control group. This is essential because RAS frequently affects the oral mucosa and is caused by alterations in cellular and humoral immunity. These results are in accordance with other results The level of IL-2 and IL-12 is highly increased while IL-10 is decreased in patients. The age group 20 to 40 years showed a higher prevalence [27]. These observations may be explained by the fact that The pathogenesis of RAS involves cell-mediated responses, involving T cells and tumor necrosis factor (TNF)-α production by these and other leucocytes. TNF is a pro- inflammatory cytokine .Secreted by activated monocytes, causing activation of cytotoxic T lymphocytes and neutrophils; epithelial necrosis and eventually the development of an aphthous lesion [28]. This study may also be explained by the fact that type-1 Th1 cytokines include pro- inflammatory cytokines like IL-2, IL-12, IFN, and TNF, wihich activate cell-mediated immunity [19]. Significant elevation in secretion of Th1 cytokine that produces pro-inflammatory cytokines (IL-2, IL-12 and IFN-y) increased in patients with RAS in comparison to healthy patients was demonstrated in earlier studies [29, 30]. 5. Conclusions According to these results, saliva is the optimal medium for detecting oral pro-inflammatory markers. The concentration of the inflammatory interleukin 2 (IL-2) and interleukin 12 (IL-12) in the saliva may also have a substantial effect on the pathogenesis of this disease. Acknowledgment Many thanks to editor-in-chief and members of the editorial committee in Ibn Al-Haitham Journal for Pure and Applied Sciences. Conflict of Interest There is no conflict of interest. Funding There is no funding for the article. Patients Control IL-12 331.32 130.31 0 50 100 150 200 250 300 350 M e a n v a lu e IL-12 pg\ml IHJPAS. 2024, 37(4) 23 References 1. Al-Ghurabei, B.H. Role of salivary tumor necrosis factor-alpha and immunoglobulin-a in recurrent aphthous stomatitis. Journal of the Faculty of Medicine Baghdad 2011, 53(2), 207-210. https://doi.org/10.32007/jfacmedbagdad.532876. 2. Akintoye, S.O.; Greenberg, M. S. (2014). Recurrent aphthous stomatitis. Dental clinics of North America 2014, 58(2), 281–297. https://doi.org/10.1016/j.cden.2013.12.002. 3. Al-Ani, R.H.; Shaker, A.W.; Ibrahim, L.M. Correlation between Salivary Cotinine Levels and Cigarette Smoking with Recurrent Aphthous Stomatitis. Indian Journal of Forensic Medicine & Toxicology 2021, 15(2), 3258-3263. 4. Plewa, M.C.; Chatterjee, K. Recurrent Aphthous Stomatitis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024. Available from: https://www.ncbi.nlm.nih.gov/books/NBK431059/. 5. Ali, N.S.M. Serum ferritin level and B12 in a sample of Iraqi re-current aphthous stomatitis patients. Journal of Baghdad College of Dentistry 2022, 34(3), 1-6. https://doi.org/10.26477/jbcd.v34i3.3211. 6. Al-Essa, H.S.; Zaidan, T. F. Assessment of serum and salivary oxidant and total antioxidant status of patients with recurrent aphthous stomatitis in a sample of Basrah city. Journal of Baghdad College of Dentistry 2013, 25(Special Is), 83-88. https://jbcd.uobaghdad.edu.iq/index.php/jbcd/article/view/204. 7. Altaei, T.; Ahmed, S.A.; Haider, A.A. Efficacy of topical flax paint for the treatment of recurrent aphthous stomatitis. Journal of Baghdad College of Dentistry 2011, 23, 100-107. 8. Saeed, S.A.; Zaidan, T. F. Clinical Evaluation of Recurrent Aphthous Stomatitis and Its Correlation with Helicobacter Pylori. Indian Journal of Forensic Medicine & Toxicology 2020, 14(4), 2154- 2159. https://doi.org/10.37506/ijfmt.v14i4.11870. 9. Shen, C.; Ye, W.; Gong, L.; Lv, K.; Gao, B.; Yao, H. Serum interleukin-6, interleukin-17A, and tumor necrosis factor-alpha in patients with recurrent aphthous stomatitis. Journal of Oral Pathology & Medicine 2021, 50(4), 418-423. https://doi.org/10.1111/jop.13158. 10. Elamrousy, W.A.; Mortada, A.; Shoukheba, M. Evaluation of Novel Topical Camel Whey Protein Gel for the Treatment of Recurrent Aphthous Stomatitis: Randomized Clinical Study. Journal of International Society of Preventive & Community Dentistry 2021, 11(5), 574–581. https://doi.org/10.4103/jispcd.JISPCD_172_21. 11. Lau, C.B.; Smith, G.P. Recurrent aphthous stomatitis: A comprehensive review and recommendations on therapeutic options. Dermatologic Therapy 2022, 35(6), e15500.https://doi.org/10.1111/dth.15500. 12. Belenguer-Guallar, I.; Jiménez-Soriano, Y.; Claramunt-Lozano, A. (2014). Treatment of recurrent aphthous stomatitis. A literature review. Journal of Clinical and Experimental Dentistry 2014, 6(2), e168–e174. https://doi.org/10.4317/jced.51401. 13. Buno, I.J.; Huff, J.C.; Weston, W.L.; Cook, D.T.; Brice, S. L. Elevated levels of interferon gamma, tumor necrosis factor α, interleukins 2, 4, and 5, but not interleukin 10, are present in recurrent aphthous stomatitis. Archives of Dermatology 1998, 134(7), 827-831. https://doi.org/10.1001/archderm.134.7.827. 14. Pärssinen, M.; Jäsberg, H.; Mikkonen, J.J.W.; Kullaa, A.M. Oral mucosal pellicle as an immune protection against micro-organisms in patients with recurrent aphthous stomatitis: A hypothesis. Medical Hypotheses 2021, 146, 110449. https://doi.org/10.1016/j.mehy.2020.110449. 15. Pelaez-Prestel, H.F.; Sanchez-Trincado, J.L.; Lafuente, E.M.; Reche, P.A. (2021). Immune Tolerance in the Oral Mucosa. International Journal of Molecular Sciences 2021, 22(22), 12149. https://doi.org/10.3390/ijms222212149. 16. Lin, D.; Yang, L.; Wen, L.; Lu, H.; Chen, Q.; Wang, Z. Crosstalk between the oral microbiota, mucosal immunity, and the epithelial barrier regulates oral mucosal disease pathogenesis. Mucosal Immunology 2021, 14(6), 1247–1258. https://doi.org/10.1038/s41385-021-00413-7. 17. Zhang, J.M.; An, J. Cytokines, inflammation, and pain. International Anesthesiology Clinics 2007, 45(2), 27–37. https://doi.org/10.1097/AIA.0b013e318034194e. https://doi.org/10.32007/jfacmedbagdad.532876 https://doi.org/10.1016/j.cden.2013.12.002 https://www.ncbi.nlm.nih.gov/books/NBK431059/ https://doi.org/10.26477/jbcd.v34i3.3211 https://jbcd.uobaghdad.edu.iq/index.php/jbcd/article/view/204 https://doi.org/10.37506/ijfmt.v14i4.11870 https://doi.org/10.1111/jop.13158 https://doi.org/10.4103/jispcd.JISPCD_172_21 https://doi.org/10.1111/dth.15500 https://doi.org/10.4317/jced.51401 https://doi.org/10.1001/archderm.134.7.827 https://doi.org/10.1016/j.mehy.2020.110449 https://doi.org/10.3390/ijms222212149 https://doi.org/10.1038/s41385-021-00413-7 https://doi.org/10.1097/AIA.0b013e318034194e IHJPAS. 2024, 37(4) 24 18. Lewkowicz, N.; Lewkowicz, P.; Banasik, M.; Kurnatowska, A.; Tchórzewski, H. Predominance of type 1 cytokines and decreased number of CD4+ CD25+ high T regulatory cells in peripheral blood of patients with recurrent aphthous ulcerations. Immunology Letters 2005, 99(1), 57-62. https://doi.org/10.1016/j.imlet.2005.01.002. 19. Albanidou-Farmaki, E.; Markopoulos, A.K.; Kalogerakou, F.; Antoniades, D.Z. Detection, enumeration and characterization of T helper cells secreting type 1 and type 2 cytokines in patients with recurrent aphthous stomatitis. The Tohoku Journal of Experimental Medicine 2007, 212(2), 101- 105. https://doi.org/10.1620/tjem.212.101. 20. Maiorino, L.; Daßler-Plenker, J.; Sun, L.; Egeblad, M. Innate Immunity and Cancer Pathophysiology. Annual Review of Pathology 2022, 17, 425–457. https://doi.org/10.1146/annurev-pathmechdis-032221- 115501. 21. Zhu J. T helper 2 (Th2) cell differentiation, type 2 innate lymphoid cell (ILC2) development and regulation of interleukin-4 (IL-4) and IL-13 production. Cytokine 2015, 75(1), 14–24. https://doi.org/10.1016/j.cyto.2015.05.010. 22. Ross, S.H.; Cantrell, D.A. Signaling and Function of Interleukin-2 in T Lymphocytes. Annual Review of Immunology 2018, 36, 411–433. https://doi.org/10.1146/annurev-immunol-042617-053352. 23. AL-Khateeb, S.M.; Tahir, N. T.; Al-bayati, A. A. H. Immune and inflammatory cytokines profile in Iraqi patients with acute and chronic myeloid leukemia. Biomedicine 2022, 42(2), 262- 267. https://doi.org/10.51248/.v42i2.1321. 24. Saud, A.M. Serum levels of tumor necrosis factor alpha and interleukin-12 in some Iraqi diabetic patients type I. International Journal of Current Microbiology and Applied Science 2014, 3(4), 260-268. 25. Liu, J.; Cao, S.; Kim, S.; Chung, E.Y.; Homma, Y.; Guan, X.; Jimenez, V.; Ma, X. Interleukin-12: an update on its immunological activities, signaling and regulation of gene expression. Current Immunology Reviews 2005, 1(2), 119–137. https://doi.org/10.2174/1573395054065115. 26. Mishra, P.; Pandey, C.M.; Singh, U.; Gupta, A.; Sahu, C.; Keshri, A. Descriptive statistics and normality tests for statistical data. Annals of Cardiac Anaesthesia 2019, 22(1), 67–72. https://doi.org/10.4103/aca.ACA_157_18. 27. Bhosale, S.S.; Rajput, B.S.; Takkar, H.; Bhagat, S.V.; Vagger, R.M.; Shaikh, M.I.K. Establishment of role of IL-2, IL-10 and IL-12 in patients with recurrent aphthous stomatitis-a clinical study. Journal of Contemporary Dental Practice 2018, 19(10), 1242-1245. 28. Scully, C.; Gorsky, M.; Lozada-Nur, F. The diagnosis and management of recurrent aphthous stomatitis: a consensus approach. Journal of the American Dental Association 2003, 134(2), 200–207. https://doi.org/10.14219/jada.archive.2003.0134. 29. Lewkowicz, N.; Banasik, M.; Tchórzewski, H.; Kurnatowska, A J.; Lewkowicz, P. Predominance of production of Th1 type cytokines in recurrent aphthous ulceration. Dental and Medical Problems 2004, 42, 655-60. 30. Borra, R.C.; Andrade, P.M.; Silva, I.D.C.G.; Morgun, A.; Weckx, L.L.M.; Smirnova, A.S.; Franco, M. The Th1/Th2 immune‐type response of the recurrent aphthous ulceration analyzed by cDNA microarray. Journal of Oral Pathology & Medicine 2004, 33(3), 140-146. https://doi.org/10.1111/j.0904-2512.2004.00089.x. https://doi.org/10.1016/j.imlet.2005.01.002 https://doi.org/10.1620/tjem.212.101 https://doi.org/10.1146/annurev-pathmechdis-032221-115501 https://doi.org/10.1146/annurev-pathmechdis-032221-115501 https://doi.org/10.1016/j.cyto.2015.05.010 https://doi.org/10.1146/annurev-immunol-042617-053352 https://doi.org/10.51248/.v42i2.1321 https://doi.org/10.2174/1573395054065115 https://doi.org/10.4103/aca.ACA_157_18 https://doi.org/10.14219/jada.archive.2003.0134 https://doi.org/10.1111/j.0904-2512.2004.00089.x Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions References