Higher interleukin-33 levels in aggressive periodontitis cases.

CONTEXT: Interleukin-33 (IL-33) is a novel alarmin that warns immune cells of tissue destruction in injury or infection. AIMS: This study is aimed at analyzing and correlating the concentration of IL-33 in the gingival crevicular fluid (GCF) and plasma of healthy subjects and chronic and aggressive periodontitis patients with intronic variant single nucleotide polymorphisms (SNPs), rs1157505 (C/G) and rs7044343 (C/T) in the IL-33 gene. SETTINGS AND
DESIGN: This is a cross-sectional, biochemical, genetic study.
MATERIALS AND METHODS: Ninety subjects were divided into Group H: subjects with healthy periodontium, Group CP: chronic periodontitis patients, and Group GAgP: generalized aggressive periodontitis patients, based on clinical parameters of periodontal inflammation. IL-33 concentration in GCF, as well as plasma, was quantified using enzyme-linked immunosorbent assay. The SNPs rs1157505 and rs7044343 were analyzed using polymerase chain reaction-restriction fragment length polymorphism.
RESULTS: A significant difference was found in IL-33 concentration in GCF and plasma between the three groups. GG genotype of IL-33 SNP rs1157505 was associated with the highest GCF and plasma IL-33 concentration and was significantly more in GAgP than healthy or CP groups. IL-33 SNP rs7044343 did not show any such association. All GAgP patients had the highest GCF and plasma concentration of IL-33.
CONCLUSIONS: IL-33 may be a potential inflammatory marker of periodontitis. GG genotype of SNP rs1157505 may be associated with GAgP.

INTRODUCTION

Periodontal disease is the sequel to interplay between the host's defense mechanisms and plaque microorganisms. Cytokines are glycoprotein mediators that help immuno-inflammatory cells inter-communicate to affect an adequate immune response. Cytokine immunobiology pivots the development of numerous destructive inflammatory diseases such as autoimmune arthritis, Crohn's disease, and periodontitis.[1]

The classical interleukin-1 (IL-1) cytokine clan especially is recognized to orchestrate disease processes and is identified as therapeutic targets. Excessive or abnormal activity of these agents, along with tissue destruction, is a hallmark of periodontitis.[23]

IL-33 is the newest entrant to the IL-1 family.[4] IL-33 (IL-1F11 [IL-1 Family member 11]) is a bi-functional alarmin that not only links innate and adaptive immune responses as a cytokine but is also a nuclear factor.[56] It may function similar to the archetypal alarmin IL-1α, as an endogenic “red flag” to notify the innate immune system of tissue damage, as it can be released extracellularly following endothelial cell damage or mechanical injury.[7] Reports from preceding studies delving into the part played by IL-33 in periodontal disease have been inconclusive.

Cytokines are the primary mediators of immunoregulation; therefore, the corresponding genetic regulatory factors concerned with cytokine function and periodontitis have been probed. Association between a composite genotype of the IL-1 gene with severe periodontitis has been found.[8] Polymorphisms in the IL-33 gene have been found to be associated with Alzheimer's disease (AD) and rheumatoid arthritis.[910]

The aim of this investigation was to estimate IL-33 in gingival crevicular fluid (GCF) and plasma and to study the association of IL-33 gene single-nucleotide polymorphisms (SNPs) rs1157505 and rs7044343 in chronic periodontitis (CP) and generalized aggressive periodontitis (GAgP) patients.

MATERIALS AND METHODS

Study population

The current study comprised 90 subjects [demographic data as in Table 1] - thirty CP patients, 30 GAgP patients, and 30 healthy subjects, enlisted from the outpatient department of periodontics from June 2017 to April 2018. The purpose and procedure of the study were thoroughly explained to each participant before recruitment for the study, and informed consent was procured in writing. Approval for the same was given by the Institutional Ethical Committee and was executed according to the guidelines of the Helsinki Declaration of 1975, modified in 2013.

Table 1

Descriptive statistics of the study population and gingival crevicular fluid and plasma interleukin-33 concentration

Parameters	Group H (n=30)	Group CP (n=30)	Group GAgP (n=30)	F†/χ2	P
Age (years)	26.30±5.421	39.87±9.486	27.43±6.709	31.018	<0.001*
Sex
Females (n)	14	14	11	4.727	0.094
Males (n)	16	16	19
GI	NA	1.92±0.317	2.40±0.255	42.146	<0.001*
PPD (mm)	1.93±0.740	6.30±1.264	6.63±1.189	174.066	<0.001*
PAL (mm)	NA	7.33±1.626	7.57±1.165	0.408	0.525
GCF IL-33 concentration (pg/ml)	417.13±43.244	469.27±50.803	556.65±82.737	51.262	<0.001*
Plasma IL-33 concentration (pg/ml)	13.86±4.452	16.41±8.225	204.32±147.938	58.961	<0.001*

*Statistically significant at P<0.05; †F – Kruskal–Wallis test. All values except sex have been expressed as mean±SD. SD – Standard deviation; NA – Not applicable; H – Healthy; CP – Chronic periodontitis; GAgP – Generalized aggressive periodontitis; n – Number of subjects; mm – Millimeter; pg/ml – Picogram per milliliter; GI – Gingival index; PPD – Probing pocket depth; PAL – Probing attachment level; GCF – Gingival crevicular fluid; IL – Interleukin; P – Probability; χ2 – Chi-square

Comprehensive systemic as well as dental histories of every participant were recorded. Nonsmoking dentulous subjects with a minimum of 20 teeth were selected for this study. Subjects with a significant medical history such as diabetes mellitus or autoimmune diseases; who had received any form of periodontal treatment or antibiotics or other medication in the previous half year; pregnant and lactating females formed the exclusion criteria for this study. Patients were selected as per the proposed criteria of the 1999 International World Workshop for a Classification of Periodontal Diseases and Conditions.[11]

Periodontal evaluation included gingival index (GI),[12] probing pocket depth (PPD), probing attachment level (PAL), and presence or absence of sulcular bleeding upon probing. PPD and PAL were measured using William's periodontal probe (William's periodontal probe, Hu-Friedy, Illinois, USA). Orthopantomographic examination was carried out. On the basis of the evaluation done by a single investigator, the study population selected was sorted into the aforementioned study groups.

The subjects with generalized chronic and aggressive periodontitis were selected based on the primary features and clinical presentation as defined by Lang et al.[13]

Sample site selection for gingival crevicular fluid collection

Different sites (two to four sites/subject) were sampled to obtain a suitable volume of GCF in healthy subjects.[14] In the CP and GAgP groups, a single sampling site with the greatest PAL along with radiographic evidence of alveolar bone resorption was chosen per subject.

At the subsequent appointment, GCF was collected with Periopaper strips (Periopaper, Ora Flow Inc., Smith-town, USA) by the atraumatic method described by Garg et al.[14] Blood- and saliva-contaminated samples were disposed of. The sample GCF volume in the Periopaper strip was quantified by Periotron 8000 (Periotron 8000, Ora Flow Inc., Smith-town, USA) and transferred to microcentrifuge tubes (with identification markings) containing 0.4 mL of phosphate-buffered saline and stored at −80°C till the assay was run.

DNA and plasma extraction

Ten milliliters of blood was obtained from each participant by venipuncture. Two milliliters of the blood was used for plasma separation. The plasma was stored in a plastic vial at −80°C until the time of assay. The remaining 8 ml of the whole blood sample was promptly transported in ethylenediaminetetraacetic acid-coated vacutainers in the molecular genetics laboratory for DNA extraction and subsequent SNP analysis of IL33 gene.

Interleukin-33 analysis by enzyme-linked immunosorbent assay

IL-33 concentration in GCF and plasma, obtained from the study subjects, were measured using Human IL-33 DuoSet ELISA kit (R and D Systems, USA, Imported by Biotech-India, India) and estimated using the standard curve, in accordance with the instructions provided in the kit insert.

Interleukin-33 gene single-nucleotide polymorphism genotyping by polymerase chain reaction

DNA analysis

Miller's method[15] was used to extract DNA after processing the blood sample. The DNA was quantified using a NanoDrop spectrophotometer (NanoDrop 1000 Spectrophotometer, Thermo Fisher Scientific, Wilmington, DE, USA), and the quantified DNA was analyzed on 0.8% agarose gel and 150–200 ng of DNA was used for the polymerase chain reaction (PCR) reaction.

Primer designing and polymerase chain reaction-restriction fragment length polymorphism

The SNPs analyzed were rs1157505 (C/G) and rs7044343(C/T), both of which are intronic variants. Genotyping was carried out by PCR-restriction fragment length polymorphism as described by Chapuis et al.[9] The PCR products were analyzed by electrophoresis and visualized under ultraviolet illumination (Box, Syngene, Frederick, MD, USA).

Statistical analyses

Statistical Package for the Social Science (SPSS® version 18.5, SPSS Inc., Chicago, USA) software was applied for statistical analyses. The distribution of data was assessed for normality using Shapiro–Wilk test. Chi-square test was employed to analyze the descriptive data, allelic frequencies of IL33 SNPs, and its deviation from the Hardy–Weinberg equilibrium[16] in the study population. The intergroup genotypes and clinical parameters in relation to IL-33 concentration in GCF and plasma were analyzed by Kruskal–Wallis test and Mann–Whitney U-test. Correlation between clinical and biochemical parameters was evaluated using the Spearman rank test.

RESULTS

Clinical and biochemical findings

Descriptive statistics of the three groups are tabulated as seen in Table 1. IL-33 was detected in all the samples. The GAgP group had the highest mean IL-33 concentration in GCF and plasma. The GCF concentration of IL-33 differed significantly between the three groups. The plasma concentration of IL-33 differed significantly between groups H and GAgP (P < 0.001) and the groups CP and GAgP (P < 0.001). GCF and plasma concentration of IL-33 was found to be statistically independent of GI, PPD, or PAL in any group.

Genotype frequencies

The observed genotype distribution for both the SNPs was in accordance with the Hardy–Weinberg equilibrium. The distribution of genotypes in subjects of the three groups is given in Figures 1 and 2. The frequency of IL33 SNP rs1157505 GG allele was greatest in GAgP (26.7%) compared to other groups. IL33 SNP rs7044343 TC genotype correlated with the lowest PPD in the healthy group (P = 0.030). The GG genotype subjects had the highest levels of IL-33 in GCF and plasma. IL33 rs7044343 TT genotype subjects of the healthy group had the highest plasma concentration of IL-33 [Tables 2–5]. The CC and CG variants of rs1157505 SNP, when divided into individual groups, varied significantly in the levels of GCF and plasma IL-33, whereas the GG variant did not show a statistically significant inter-group difference in IL-33 concentration in GCF and plasma. The CC, TC, and TT variants of rs7044343 SNP, when divided into individual groups, showed a statistically significant difference in the levels of GCF IL-33 and plasma IL-33 between the groups.

Figure 1

Distribution of interleukin-33 allelic variants of single-nucleotide polymorphism rs1157505 in the study population

Figure 2

Distribution of interleukin-33 allelic variants of single-nucleotide polymorphism rs7044343 in the study population

Table 2

Correlation of genotype rs1157505 (C/C, C/G, G/G) with respect to gingival crevicular fluid and plasma interleukin-33

Group	rs1157505	n	GCF IL-33 concentration (pg/ml), mean±SD	χ2*	P	Plasma IL-33 concentration (pg/ml), mean±SD	χ2*	P
Group H	CC	13	410.32±43.93	0.954	0.621	14.57±3.242	3.045	0.218
	CG	16	423.88±44.248			13.57±5.285
	GG	1	397.8			9.19
Group CP	CC	19	471.89±56.851	0.275	0.872	17.26±9.48	1.431	0.489
	CG	10	462.64±41.934			15.39±5.616
	GG	1	485.8			10.39
Group GAgP	CC	7	523.01±57.688	2.247	0.325	199.36±185.592	1.509	0.47
	CG	15	559.12±72.785			175.28±131.091
	GG	8	581.46±114.299			263.13±144.885

*Kruskal–Wallis test, P<0.05 considered as statistically significant. - NA – Not applicable; IL – Interleukin; n – Number of subjects; pg/ml – Picogram per milliliter; GCF – Gingival crevicular fluid; SD – Standard deviation; H – Healthy; CP – Chronic periodontitis; GAgP – Generalized aggressive periodontitis; CC – Homozygous dominant genotype; CG – Heterozygous genotype; GG – Homozygous recessive genotype; P – Probability; χ2 – Chi-square

Table 5

Correlation of genotype rs7044343 (C/C, T/C, T/T) with respect to gingival crevicular fluid and plasma interleukin -33 levels irrespective of groups

Allele	n	GCF IL-33 levels (pg/ml), mean±SD	χ2	P*	Plasma IL-33 levels (pg/ml), mean±SD	χ2	P*
CC	14	520.85±112.746	2.574	0.276	141.66±149.209	3.376	0.155
TC	51	468.14±64.167			68.11±118.923
TT	25	484.99±96.524			63.23±109.818

*P values of Chi-square test, P<0.05 considered statistically significant. n – Number of subjects; GCF – Gingival crevicular fluid; IL – Interleukin; pg/ml – Picogram per milliliter; SD – Standard deviation; CC – Homozygous dominant genotype; TC – Heterozygous genotype; TT – Homozygous recessive genotype; P – Probability; χ2 – Chi-square

DISCUSSION

In the current cross-sectional research, IL-33 levels in the GCF and plasma were correlated with its SNPs rs1157505 and rs7044343. It is well documented that there is an increase and activation of several cytokines during the progression of periodontal disease.[1718] Since the IL-1 family of cytokines is affective in degenerative disorders such as periodontitis and rheumatoid arthritis,[1] our study aimed to quantify the concentration of IL-33 in GCF and plasma in periodontal disease.

The results of previous studies, by various researchers who have quantified IL-33 in periodontal disease, have been quite contradictory. While in vitro studies prove the central role of IL-33 in periodontitis,[19202122] most human studies have reported the contrary.[23242526] Initial studies reporting the absence of IL-33 in GCF attributed it to less sensitive ELISA kits and ethnic variation in their study population.[24] In the current study, IL-33 was detected in GCF and plasma, which may be due to a newer, more sensitive ELISA kit. Studies that detected IL-33 in GCF, saliva, and plasma have reported that IL-33 levels cannot discern chronic periodontitis patients from healthy individuals.[25]

IL-33 not only functions as an alarmin but also functions as a systemic chemokine.[27] The higher concentration of IL-33 in GCF compared to plasma in our study may be the result of increase in local production of IL-33 from the increased vasculature in diseased periodontal tissues or from periodontal tissue destruction.[4272829] Since we have included systemically healthy subjects, the higher IL-33 plasma concentration in periodontitis groups might be due to a spill-over from the periodontal tissues into the systemic circulation.

In our study, PPD and PAL measurements were greater in CP and GAgP groups as anticipated. The IL-33 concentration did not correlate with clinical parameters of inflammation. The quiescent or active state of the episodic periodontitis may not be portrayed accurately by PPD, PAL, and GI. Moreover, they are all soft tissue measurements and do not reflect active bone resorption. Analyses of GCF components give an accurate indication of the link between specific metabolic variation and disease status.[23] Hence, the concentration of IL-33 might be a true indicator of the periodontal disease activity in the absence of evident changes in the soft tissue parameters.

In initial stages of inflammation, IL-33 augments the Th2 response, which is anti-inflammatory.[27] However, due to high levels of proinflammatory cytokines in periodontal disease in response to the bacterial infection, IL-33 production is stimulated and osteoclastogenesis may be favored.[3031] In addition, mast cell degranulation stimulates the differentiation of circulating CD14(+) monocytes into osteoclasts.[30323334] This could explain the commensurate increase in concentration of IL-33 in periodontitis compared to healthy subjects.

Mendonça et al. have reported increased IL-33 levels in the saliva of systemic lupus erythematosus patients with chronic periodontitis.[35] Gümüş et al. found that salivary levels of IL-33 are higher in chronic periodontitis groups.[36] Nizam et al. have quantified and noted increased salivary cytokines, including IL-33 in sleep apnea patients as compared to controls.[37] In recent implant studies, IL-33 levels in peri-implantar crevicular fluid were seen to be significantly higher in peri-implantitis and peri-implant mucositis group when compared to healthy group.[38] Peri-implant fluid levels of IL-33 from mucositis sites were seen to be independent of the degree of bone resorption around implants or teeth.[39]

IL33 SNPs rs7044343 and rs11792633 have been studied in various diseases such as Behçet's disease,[4041] systemic sclerosis, and rheumatoid arthritis,[1042] whereas rs1157505 is associated with the risk of developing AD.[943] There have been no studies which probe into the association of IL-33 gene polymorphism with periodontal disease, in hitherto published literature.

We found a significant difference between the distribution of the rare allelic variant (G/G) of rs1157505 SNP between the three study groups. It was seen that the rare allele variant GG genotype was associated with the highest concentrations of IL-33 in GCF and plasma and GAgP patients exhibited the highest concentrations of IL-33, irrespective of the genotype. The results of the genetic correlation are to be interpreted cautiously, until such a time that follow up studies, with larger sample sizes, of currently healthy subjects with the GG genotype can be done to ascertain if the G allele is indeed a risk allele for periodontal disease.

IL-33 appears to be a promising biomarker for periodontal health and disease and future interventional studies may help comprehend its exact role in periodontitis opening new avenues for periodontal therapy. IL33 SNPs have to be studied more closely to investigate the genetic milieu of disease progression and to assess the reciprocal influence of other closely related genes. Future multicenter genetic studies with larger sample sizes studying the potential effect of IL-33 and its gene polymorphism in periodontitis associated with AD and diabetes mellitus are required for a more complete understanding.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Table 3

Correlation of genotype rs7044343 (C/C, T/C, T/T) with respect to gingival crevicular fluid and plasma interleukin-33

Group	rs7044343	n	GCF IL-33 concentration (pg/ml), mean±SD	χ2*	P	Plasma IL-33 concentration (pg/ml), mean±SD	χ2*	P
Group H	CC	3	431.57±42.725	1.018	0.601	11.52±2.183	8.953	0.011†
	TC	18	416.24±31.347			12.37±3.695
	TT	9	414.11±64.377			17.61±4.336
Group CP	CC	2	463.43±31.643	0.949	0.622	9.72±0.936	4.064	0.131
	TC	19	462.42±49.749			15.91±7.881
	TT	9	485.03±57.118			18.95±9.293
Group GAgP	CC	9	563.38±119.275	0.07	0.966	214.36±139.742	0.302	0.86
	TC	14	542.63±37.042			210.62±155.36
	TT	7	576.06±100.887			178.83±162.622

*Kruskal–Wallis test, †Statistically significant at P<0.05. IL – Interleukin; n – Number of subjects; pg/ml – Picogram per milliliter; GCF – Gingival crevicular fluid; SD – Standard deviation; H – Healthy; CP – Chronic periodontitis; GAgP – Generalized aggressive periodontitis; CC – Homozygous dominant genotype; TC – Heterozygous genotype; TT – Homozygous recessive genotype; P – Probability; χ2 – Chi-square

Table 4

Correlation of genotype rs1157505 (C/C, C/G, G/G) with respect to gingival crevicular fluid and plasma interleukin-33 levels irrespective of groups

Allele	n	GCF IL-33 levels (pg/ml), mean±SD	χ2	P**	Plasma IL-33 levels (pg/ml), mean±SD	χ2	P*
CC	39	460.54±65.666	7.631	0.022†	49.05±102.754	7.572	0.023†
CG	41	482.81±81.603			73.18±110.442
GG	10	553.53±118.569			212.46±166.544

*P values of Chi-square test, †Statistically significant at P<0.05. n – Number of subjects; GCF – Gingival crevicular fluid; IL – Interleukin, pg/ml – Picogram per milliliter; SD – Standard deviation; CC – Homozygous dominant genotype; CG – Heterozygous genotype; GG – Homozygous recessive genotype; P – Probability; χ2 – Chi-square