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Table of Contents   
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 33  |  Issue : 1  |  Page : 37-40
Evaluation of serum interleukin-33 and soluble suppression of tumorigenicity 2 (sST2) receptors in patients with and without periodontal disease


1 Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), Bhopal, India
2 Department of Dentistry, Nodal Officer – Regional Training Centre for Oral Health Promotion & Oral Health Data Bank (M.P), All India Institute of Medical Sciences (AIIMS), Bhopal, India
3 Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), Bibinagar, Hyderabad, India

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Date of Submission28-Jan-2021
Date of Decision22-Feb-2022
Date of Acceptance06-Apr-2022
Date of Web Publication09-Aug-2022
 

   Abstract 


Context: Interleukin-33 and its receptor soluble suppression of tumorigenicity 2 (sST2) play an important role in inflammation and its role in periodontal disease is yet unclear. The role of both IL-33 and sST2 together in periodontal disease as biomarkers has never been studied. Aim: To assess the levels of IL-33 and sST2 in serum samples of patients with periodontitis and healthy subjects. Methods: A total of 71 subjects (30 healthy subjects and 41 patients with periodontal disease) were included in the cross-sectional study. Community Periodontal Index (CPI) was used to assess periodontal health by utilizing a mouth mirror and a CPI probe. Venous blood was collected and serum was separated. Serum levels of IL-33 and sST2 were determined by the enzyme-linked immunosorbent assay (ELISA) assay. Statistical Analysis: Graph Pad Prism 5 was used for statistical analysis. Mann Whitney test was applied to compare the two groups. Results: The level of IL-33 was not found to be elevated among healthy subjects and sST2 was found elevated among patients with periodontal disease. The serum concentration of IL-33 was found at 472 ± 114 pg/ml and 282 ± 77 pg/ml among healthy subjects and patients with periodontal disease respectively. Significantly higher values of sST2 at 28 ± 2 ng/ml were found among periodontal patients as compared to healthy subjects with values of 18 ± 1 ng/ml. No significant differences were noted between mild to moderate and severe periodontitis for IL-33 and sST2 between the two groups. Conclusion: This study shows alteration in serum levels of IL-33 and sST2 in periodontitis patients. IL-33 and sST2 may be potential inflammatory markers of periodontitis. Further studies are required on a large sample size for better understanding. This pilot study is the first to assess the serum levels of both IL-33 and sST2 together among patients with and without periodontal disease.

Keywords: Community periodontal index, interleukin-33, peridontitis, suppression of tumorigenicity 2 (sST2)

How to cite this article:
Singh H, Singh A, Saluja R. Evaluation of serum interleukin-33 and soluble suppression of tumorigenicity 2 (sST2) receptors in patients with and without periodontal disease. Indian J Dent Res 2022;33:37-40

How to cite this URL:
Singh H, Singh A, Saluja R. Evaluation of serum interleukin-33 and soluble suppression of tumorigenicity 2 (sST2) receptors in patients with and without periodontal disease. Indian J Dent Res [serial online] 2022 [cited 2022 Oct 4];33:37-40. Available from: https://www.ijdr.in/text.asp?2022/33/1/37/353545



   Introduction Top


Periodontitis is a major oral health issue characterized by the chronic inflammation of the gingival and supporting structures of teeth. Periodontitis is a gradual destruction of the periodontium, which comprises the gingiva, periodontal ligament (PDL), cementum, and alveolar bone.[1] Interleukin-33 (IL-33) is a newly discovered proinflammatory cytokine of the IL-1 super family, which includes IL- 1α, IL-1β, and IL-18. Periodontal disease develops in the presence of a host inflammatory response against bacterial pathogens.

IL-33 most likely has three roles in relation to periodontal disease: as an alarmin, a chemoattractant, and a systemic cytokine. The release of IL-33, when acting as an alarmin, results in the destruction of several cells by necrosis, mainly fibroblasts and epithelial cells.[2] IL-33 is considered an alarmin signal molecule due to its release after necrosis or tissue damage.[3]

A specific receptor is required for the binding of IL-33 is Suppression of Tumorigenicity 2(sST2). sST2 receptor exists in two major isoforms: a transmembrane form (sST2 or sST2L) and a soluble form (sST2). sST2 belongs to the toll-like receptor (TLR)/IL1R super family.[4] Soluble forms of sST2 serve as a decoy receptor for the binding of IL-33. IL-33 induces mast cell degranulation by NF-κB and MAPK activation, which is responsible for the activation of proinflammatory cytokine. This proinflammatory cytokine is responsible for the generation of immune response and inflammation. IL-33 is also responsible for bone destruction in periodontitis by activation of osteoblast, which increases the production of receptor activator of nuclear factor κBligand (RANKL) and decreases osteoprotegerin (OPG).[5]

Few studies have been conducted to assess serum levels of Interleukin-33 as biomarkers for periodontal disease. However, none to date have studied the role of both Interleukin-33 and sST2 together in periodontal disease. This is the first study to assess variation in serum levels of IL-33 and sST2 among patients together with and without periodontal disease. The aim of the study is to assess serum levels of IL-33 and sST2 among adult patients with and without periodontal disease.


   Methods Top


The target population for the cross-sectional study was patients clinically diagnosed with periodontal disease cases and healthy adults visiting the Department of Dentistry at All India Institute of Medical Sciences (AIIMS), Bhopal as controls. For the sample size estimation, we have used G Power 3.1 software. The significance level was set at 5% and the power to 80%. A total of 71 study participants (30 healthy subjects, 41 patients with periodontal disease) were involved in the study from November, 2017 to December, 2018; 80% of the total participants responded to the study.

Inclusion criteria for cases: All individuals above age 18 with periodontal problems posed by periodontal diseases. Individuals who were already suffering from inflammatory pathologies were excluded. Inclusion criteria for control: All individuals above age 18 with no periodontal problems. Individuals suffering from other inflammatory pathologies were excluded.


   Clinical Examination Top


Oral examinations were performed at Department of Dentistry, All India Institute of Medical Sciences (AIIMS), Bhopal as per WHO guidelines. Community Periodontal Index (CPI was used to determine the periodontal health status using a mouth mirror and a CPI probe. Dentition was divided into six tooth-numbered sextants namely: 18-14, 13-23, 24-28, 38-34, 33-43, and 44-48. Examinations were conducted only if a minimum of two or more teeth were present in a sextant without being indicated for extraction. Detection of sub-gingival calculus, gingival bleeding, and assessment of periodontal pockets was conducted using the CPI probe as a sensing instrument on the index teeth. Healthy gingiva (0), bleeding on probing (1), calculus detected during probing (3) pocket 4 to 5 mm, and pocket 6 mm or more (4) were the codes used for the recording of CPI scores. The highest recorded values from the index teeth is the sextant score.[6] Based on CPI value, patients were classified with healthy periodontium (CPI score of 0) with pockets of 4 to 5 mm (Mild/Moderate periodontal disease; CPI score 3) and pockets of more than 6 mm (severe periodontal disease; CPI score 4).

The Kappa statistic was conducted and an intra-examiner agreement score of 0.88 was achieved. Intra-examiner agreement scores between 0.88 – 0.90 were obtained. The Institutional Ethics Committee of AIIMS, Bhopal granted the ethical approval (IHEC-LOP/2016/EF0032). Prior to inclusion in the research, informed written consent was taken from the study participants. Duplicate examinations were performed by the two examiners throughout the survey on approximately 10% of the study participant's and the kappa statistics ranged between 0.88 – 0.90.

Measurement of serum level of IL-33 and sST2

Venous blood was collected in a BD vacutainer (Ref 367954) and serum was separated. The serum levels of IL-33 (R& D System, DY 3625-05) and sST2(R& D System, DST-200) were checked by sandwich ELISA using the standard kit protocol. The lowest detection limit for IL-33 was 23.4 pg/ml and for sST2 is 31.3 pg/ml.

Statistical evaluation

Data were collected, entered, and analyzed using Graph pad prism 5.'D' Agostino-Pearson normality test was performed. Data was not found normally distributed and was expressed as mean ± SEM. Mann Whitney test was applied to compare the two different independent groups. Significance was adjusted at P < 0.01.


   Results Top


Study participants, based on CPI values were classified as with healthy periodontium, mild/moderate periodontal diseases, and severe periodontal disease. The serum concentration of IL-33 and sST2 was evaluated and compared between 30 healthy subjects and 41 patients with periodontal disease. [Table 1] provides demographic data and serum levels of IL-33 and sST2 among healthy subjects and patients with periodontal disease.
Table 1: Serum level of IL-33 and sST2 among healthy subjects and patients with periodontal disease

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Serum concentration of IL-33 and sST2 among participants with and without periodontal disease assessed by ELISA

The protein concentrations of IL-33 and sST2 were quantified in all the samples by ELISA. Sera concentration of IL-33 was not significantly higher in patients with periodontitis than in healthy volunteers (472 ± 114 pg/ml versus 282 ± 77 pg/ml). Moreover, a significant difference in sST2 protein levels was shown between healthy volunteers and patients with periodontal disease (18 ± 1 ng/ml versus 28 ± 2 ng/ml) [Figure 1].
Figure 1: Analysis of the serum concentration of IL-33 and sST2 among healthy Control and periodontal patients. The protein levels in the serum from healthy subjects and patients were evaluated by ELISA. The data are expressed as the average level of protein estimation. The protein levels of sST2 were significantly higher (P < 0.01) in the serum of periodontal patients as compared to healthy subjects

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Serum level of IL-33 and sST2 among patients with periodontal disease classified based on CPI

Based on CPI score patients were classified into two groups: CPI score 3- Mild/Moderate periodontal disease, CPI score 4-severe periodontal disease. Level of IL-33 (CPI 3- 264 ± 88 pg/ml, CPI 4-322 ± 159 pg/ml), and sST2 (CPI-3, 26 ± 2 ng/ml, CPI-4,31 ± 5 ng/ml) were recorded respectively. No statistically significant differences were reported between mild to moderate and severe periodontitis for IL-33 and sST2 [Figure 2].
Figure 2: Serum level of IL-33 and sST2 among periodontal patients with Community Periodontal Index (CPI) score of 3 and 4. The level of protein in the serum was evaluated by ELISA. No statistically significant differences were reported between CPI 3 and 4 for IL-33 and sST2

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   Discussion Top


Periodontitis is an oral inflammatory disease with IL-33 and sST2 playing an important role in the induction of immune response. The serum concentration of sST2 was found significantly elevated among periodontal patients in our study. However, no significant differences could be found in serum levels of IL-33 among healthy subjects and periodontitis patients. Ballambettu et al.[2] 2019 observed significantly higher levels of IL-33 in gingival crevicular fluid (GCF) of chronic periodontitis patients and generalized aggressive periodontitis patients. Pai and Pradeep[7] observed significantly higher concentrations of IL-33 in plasma and GCF among patients with generalized aggressive periodontitis as compared to chronic periodontitis (CP), and healthy control.Similarly, a higher level of IL-33 in GCF and plasma was noted among patients with gingivitis and non-smoker chronic periodontitis when compared with controls.[6] Additionally, saliva levels of IL-33 were significantly higher among patients with chronic periodontitis in comparison to aggressive periodontitis, however, serum IL-33 was similar in both the groups.[8] Malcolm et al.,[9] 2019, reported higher mRNA expression of IL-33 and sST2 in gingivae tissue of chronic periodontitis patients as compared to healthy tissue. IL-33 shows a protective role by suppressing RANKL and inducing OPG in chronic apical periodontitis (CAP) lesions.[10] Beklen et al.,[11] 2014 reported that TNF-α is responsible for the production of IL-33 in gingival fibroblasts. Medara et al.,[12] 2020 reported significantly lower values of IL-33 and other Th-17 related cytokines after treatment in serum and saliva of periodontitis patients. All these findings suggest a non-conclusive association of IL-33 with periodontitis due to contradictory findings. Our study too observed, the tendency of elevated serum concentration of IL-33 among patients with periodontal disease, however, the differences were not significantly different when compared to healthy controls. The exact role of IL-33 and its receptor sST2 is still unclear. IL-33 need a specific receptor (sST2) for its binding to the different immune cell. sST2 receptor has two major isoforms: a transmembrane form (sST2) and a soluble form (sST2). The free level of IL-33 can be neutralized by binding to soluble sST2 (sST2) which may effectively decrease the concentration of IL-33 and further reduce the biological effect of IL-33.[4]

In this study, we also observed the significantly elevated serum level of sST2 among periodontitis patients as compared to healthy control. Torrungruang et al.,[13] 2018 reported similar findings and observed a higher level of sST2 and CRP among patients with severe periodontitis and poor oral hygiene. Study participants were further classified in our study into groups with mild to moderate pockets and those with severe pockets. However, no significant changes were observed between the two groups for IL-33 and sST2. Considering the available reported data, the main difference for measurement of IL-33 level exists at the site of sample collection. Most of the studies have reported the IL-33 in GCF. There are very few studies which are showing the level of IL-33 in the serum of periodontitis patients. In our study, we have investigated the IL-33 and sST2 together in serum samples of periodontitis patients. Future studies are recommended to be done with a larger sample size and at a multi-centric level.


   Conclusion Top


This study demonstrates that periodontitis is associated with the serum level of IL-33 and sST2. A large sample size of studies is required for the further understanding of the cross talk between IL-33 and sST2 among periodontitis patients. This was the first study to assess the serum levels of both IL-33 and sST2 together among patients with periodontal disease.

Acknowledgements

This work was supported by Department of Biotechnology (DBT) sponsored Ramalingaswami Fellowship (BT/RLF/Re-entry/53/2013). The authors also thankful to Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), Bhopal for providing the infrastructure support.

Financial support and sponsorship

This study was supported by Department of Biotechnology (DBT) sponsored Ramalingaswami fellowship (BT/RLF/Re-entry/53/2013).

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Shewale AH, Gattani DR, Bhatia N, Mahajan R, Saravanan SP. Prevalence of periodontal disease in the general population of India-A systematic review. J Clin Diagn Res 2016;10:ZE04-9.  Back to cited text no. 1
    
2.
Ballambettu SP, Pradeep AR, Purushottam M, Sen S. Higher interleukin-33 levels in aggressive periodontitis cases. J Indian Soc Periodontol 2019;23:424-9.  Back to cited text no. 2
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Luthi AU, Cullen SP, McNeela EA, Duriez PJ, Afonina IS, Sheridan C, et al. Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases. Immunity 2009;31:84-98.  Back to cited text no. 3
    
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Saluja R, Khan M, Church MK, Maurer M. The role of IL-33 and mast cells in allergy and inflammation. Clin Transl Allergy 2015;5:33.  Back to cited text no. 4
    
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da Luz FA, Oliveira AP, Borges D, Brigido PC, Silva MJ. The physiopathological role of IL-33: New highlights in bone biology and a proposed role in periodontal disease. Mediators inflamm 2014;2014:342410.  Back to cited text no. 5
    
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Saglam M, Koseoglu S, Aral CA, Savran L, Pekbagriyanik T, Cetinkaya A. Increased levels of interleukin-33 in gingival crevicular fluids of patients with chronic periodontitis. Odontology 2017;105:184-90.  Back to cited text no. 6
    
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Pai BS, Pradeep AR. Correlations between interleukin-33 and -1alpha levels in gingival crevicular fluid and plasma in patients with chronic or aggressive periodontitis and disease-free subjects. Bull Tokyo Dent Coll 2019;60:279-89.  Back to cited text no. 7
    
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Gumus P, Nizam N, Nalbantsoy A, Ozcaka O, Buduneli N. Saliva, serum levels of interleukin-21, -33 and prostaglandin E2 in patients with generalised aggressive or chronic periodontitis. Oral Health Prev Dent 2017;15:385-90.  Back to cited text no. 8
    
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Malcolm J, Awang R, Oliver-Bell J, Butcher JP, Campbell L, Planell AA, et al. IL-33 exacerbates periodontal disease through induction of RANKL. J Dent Res 2015;94:968-75.  Back to cited text no. 9
    
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Gegen T, Zhu Y, Sun Q, Hou B. Role of interleukin-33 in the clinical pathogenesis of chronic apical periodontitis. J Int Med Res 2019;47:3332-43.  Back to cited text no. 10
    
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Beklen A, Tsaous Memet G. Interleukin-1 superfamily member, interleukin-33, in periodontal diseases. Biot Histochem 2014;89:209-14.  Back to cited text no. 11
    
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Medara N, Lenzo JC, Walsh KA, Darby IB, O'Brien-Simpson NM, Reynolds EC. T helper 17 cell-related cytokines in serum and saliva during management of periodontitis. Cytokine 2020;134:155186.  Back to cited text no. 12
    
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Torrungruang K, Katudat D, Mahanonda R, Sritara P, Udomsak A. Periodontitis is associated with elevated serum levels of cardiac biomarkers—Soluble ST2 and C-reactive protein. J Clin Periodontol 2019;46:809-18.  Back to cited text no. 13
    

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Correspondence Address:
Dr. Rohit Saluja
Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), Bibinagar, Hyderabad
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.ijdr_85_21

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