Comparative evaluation of the levels of nod-like receptor family pyrin domain-containing protein (NLRP) 3 in saliva of subjects with chronic periodontitis and healthy controls.

Context: Periodontitis is an inflammatory disease which is ubiquitous. When there is an onset of infection, the innate immunity gets activated followed by the adaptive immune system. Inflammasomes identify the pathogen-associated molecular patterns or danger-associated molecular patterns and initiate inflammation. Nod- like receptor family pyrin domain-containing protein 3 (NLRP 3) is a protein belonging to the intracellular innate immune sensors that act against bacteria. The inflammasome acts along with the toll-like receptor pathways to initiate an action against pathogens. NLRP3 (also known as PYPAF-1 or cryopyrin) acts via apoptosis-associated speck-like protein (ASC). Aims: The study aimed at finding out the relation between levels of NLRP3 in chronic periodontitis and healthy subjects via the enzyme-linked immunosorbent assay (ELISA). Settings and Design: This was a Cross-sectional study. Materials and
Methods: Clinical examination and saliva sampling of the study population was done. Reagents were prepared and NLRP3 levels were estimated using ELISA analysis. Statistical Analysis: Intergroup comparison was initiated using the unpaired t-test and for within the group (intragroup), the two-way analysis of variance was used. The Pearson correlation coefficient helped to determine the strength of linear association.
Results: Increased levels of NLRP3 were seen in subjects suffering from chronic periodontitis. NLRP3 was also seen to be positively correlated to probing pocket depth, clinical attachment loss, gingival index, and plaque index. Conclusions: A positive correlation exists between NLRP3 and chronic periodontitis, and hence, NLRP3 can be a potential biomarker. Copyright:

INTRODUCTION

Host response plays an important role in disease progression, although the initiating agents are bacteria.[1] When infection sets in, the innate immunity of the body gets activated followed by acquired immunity.[2] Exaggerated inflammatory response damages periodontal tissues, and hence, the importance of host response cannot be underestimated.[34]

Routinely used diagnostic tools such as bleeding on probing, probing pocket depths, clinical attachment levels (CALs,) and radiographs are noninvasive. They are easy to use and are relatively economical. However, they do not help to evaluate current disease status.[5]

Hence, there is an urgent need to develop biomarkers to determine periodontal disease and its progression. Diagnostic approaches currently available only determine cumulative effects of previous tissue destruction whereas, what we need is determination of the present disease activity and its progression.[6]

Host cells and proteins, phenotypic markers, hormones, and bacterial products are the salivary biomarkers which are researched extensively.[789]

Inflammasomes are proteins that stimulate the caspase-1-dependent maturation and hence secrete interleukin-1 β and interleukin-18 in response to “danger.”[10] Inflammasomes detect the pathogen-associated molecular patterns or danger-associated molecular patterns.[11]

The downstream processing of IL-1 β is controlled by a cytosolic protein complex of the nucleotide-binding oligomerization domain-like receptor (NLR) protein family, called NLRP3 (NLR domain, leucine-rich, and pyrin domain (PYD)-containing protein pyrin domain (PYD)-containing protein). This is also known as cryopyrin-inflammasome.[12]

First correlation of NLRP3 with inflammation was shown by Tao et al. in subjects suffering from acute arthritis.[13]

Further research done by Isaza-Guzmán et al. in 2017 showed that NLRP3 inflammasome acts as a strong and independent indicator of periodontal damage in aggressive periodontitis and chronic periodontitis.[14]

Research has shown that biomarker expression shows variability amongst races.[15] No study has reported whether NLRP3 in saliva is correlated with the amount of periodontal damage in the Indian population. As saliva is routinely used for monitoring of microbiological, biochemical, and cellular markers of periodontal disease, this study was aimed to compare the salivary levels of NLRP3 and also to determine its relationship with the periodontal clinical status. Thus, it was aimed to compare NLRP3 levels in saliva of subjects with periodontitis of chronic origin and healthy controls.

MATERIALS AND METHODS

Eighty participants (44 males and 36 females aged 18–60 years) were recruited for the study which was conducted between January 2019 and October 2019. The institutional ethics board gave approval for this research to be conducted and it was undertaken as per the Helsinki Declaration of 1975 (revision 2013). Complete medical history and demographic data (including age, gender, systemic health, medications, tobacco, and smoking habits) were obtained from all participants. Inclusion criteria were (1) systemically healthy subjects, (2) subject with age group range from 18 to 60 years, and (3) Subjects consenting to be part of the study. Exclusion criteria were (1) the consumption of tobacco in any form and alcoholics, (2) pregnant and lactating females, (3) subjects having any systemic diseases, and (4) subjects who have received anti-inflammatory drugs in previous 3 months.

Measurement of probing depth (PD), CAL, gingival index (GI),[16] and plaque index (PI)[17] was performed. PD and CAL were measured in millimeter using a sterile mouth mirror and University of North Carolina number 15 probe (calibrated at 1 mm, 15 mm long) (Williams, Hu-Friedy, Chicago, IL). Radiographic evaluation was done to assess the presence and extent of alveolar bone loss in each subject. Clinical attachment loss was recorded as the distance from the cementoenamel junction (CEJ) to the base of the clinical periodontal pocket. This was calculated by measuring the distance from the gingival margin to CEJ and deducting it from the PD measurement. The same operator carried out all the measurements.

Based on the GI, CAL, and PD, subjects were divided into two groups: (1) Group I (healthy) consisting of 40 subjects with clinically healthy periodontium (GI = 0; PD ≤4 mm; CAL = 0 mm) and (2) Group II (chronic periodontitis) consisting of 40 subjects with chronic periodontitis (GI ≥ 1; PD ≥4 mm; CAL ≥1 mm). Group I comprised 21 (52.5%) males and 19 (47.5%) females, whereas Group II comprised 23 males (57%) and 17 females (43%).

All saliva samples were obtained between 8 am and 10 am to weaken the influence of circadian rhythm on biomarker levels. No psychological or physiological gustatory stimulus was used to stimulate the salivary flow and subjects were instructed to let the saliva pool in the oral cavity and drool into the Eppendorf safe-lock tubes [Figure 1]. Two milliliter of undiluted whole saliva sample was collected. To accommodate for salivary expansion during freezing, reserve air space was left in the vial.

Figure 1

Graduated polypropylene Eppendorf safe-lock tubes®

Estimation of NLRP3 was done by the quantitative sandwich enzyme immunoassay technique using the Elabscience® human NLRP3 Immunoassay Kit [Figure 2]. This kit had a microplate lined with a human monoclonal antibody specific for NLRP3. Samples from both the groups were pipetted into the wells and an immobilized antibody coated any NLRP3 present. Any unbound substance was washed away. Further, what was put in the wells was an enzyme-linked polyclonal antibody specific for NLRP3 conjugated to biotinylate, with preservative. Unbound antibody-enzyme reagent was washed away. The wells were filled with a substrate solution and a color was achieved to the same amount of NLRP3 bound in the first step. After stoppage of color development, a microplate reader [Figure 3] set to 450 nm was used to check the color intensity.

Figure 2

Elabscience® human nod-like receptor family pyrin domain-containing protein (NLRP3) The enzyme-linked immunosorbent assay kit (Catalog number: E-EL-H2557)

Figure 3

The enzyme-linked immunosorbent assay plate reader

At the onset of reagent preparation, a room temperature of 18°C–25°C was set. Wash buffer (30 ml) was added to wash buffer (750 ml) with distilled water. Solution that was not used was kept back at 4°C (If crystallization occurred, the solution was warmed at 40°C in water bath and was mixed till the crystals dissolved and cooled). Fifteen minutes before usage, the standard was made. It was centrifuged at ×10,000g for 1 min and reconstituted with 1 ml of reference standard and sample diluent. After 10 min, it was turned upside down several times and dissolved fully using a micropipette. This reconstitution produced a stock solution of 10 ng/ml and serial dilutions of 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0 ng/ml. The tube with biotinylated antibody was centrifuged before use and was diluted to the working concentration using Biotinylated Detection Antibody Diluent (1:100). Concentrated HRP Conjugate was diluted to the working concentrated HRP Conjugate Diluent (1:100). Since the substrate reagent is photosensitive and can be easily contaminated, it was only opened when it was required. Washing steps throughout the experiment was followed by the addition of 350 μl wash buffer into each well and soaking it for 1–2 min. Wash buffer was decanted by turning the plate and drying it by tapping it on an absorbent paper.

The sample of 80 subjects was included keeping the alpha error under 5% and the power of the study was 80%. Microsoft Excel (MS office version 2016) was used for data entry and tabulation. Data analysis was done using the IBM SPSS software version 25, (Illinois, Chicago, USA). Intergroup comparison was initiated with unpaired t-test and within the group (intragroup) comparison was undertaken using the two-way analysis of variance (ANOVA). The Pearson correlation coefficient was used to measure the strength of linear association.

RESULTS

This was a clinico-biochemical in vitro study done to explore the association, if any, between the levels of NLRP3 in saliva of normal subjects and subjects with chronic periodontitis.

The present research investigation comprised 80 participants who were categorized into two groups: subjects with good periodontal health and diseased subjects with periodontitis in chronic form, with 40 participants in each category.

Table 1 represents the comparison of mean values of age in the study population. When the mean age of both the groups was compared by unpaired t-test, the result was statistically significant (P < 0.05) revealing and authenticating the fact that periodontal disease is associated with aging.

Table 1

Comparison of mean values of age using unpaired t-test in the study population

	Healthy control	Chronic periodontitis
Mean	39.75	44.85
SD	9.50	10.05
SEM	1.50	1.58

SD – Standard deviation; SEM – Standard error of mean

Table 2 represents a comparison of mean values of PI, GI, pocket PD, and clinical attachment loss in the two groups. On statistical analysis by unpaired t-test, difference in the mean PI score of both the groups was statistically significant (P < 0.0001). The mean PI clearly reflects elevated plaque scores in diseased subjects when compared to subjects with good periodontal health. When the mean GI of the two groups was compared, it was found that chronic periodontitis group had a greater mean GI and the difference between them was statistically significant with P < 0.0001. The differences in mean pocket probing depth (PPD) and CAL were statistically significant (P < 0.0001).

Table 2

Comparison of mean values of all clinical parameters using unpaired t-test

	Group A (healthy)	Group B (chronic periodontitis)	Probability value (P)
Mean PI	0.21	2.06	<0.0001
Mean GI	0.09	1.79	<0.001
Mean PPD	2.025	6.925	<0.001
Mean clinical attachment loss	0.0	4.95	<0.001

Probability value (P) <0.05 was considered statistically significant. PPD – Probing pocket depth; GI – Gingival index; PI – Plaque index; P - probability value

Table 3 compares mean values of concentration of NLRP3 (ng/ml) in both the groups. The mean value of NLRP3 in the test group was 0.285 and that in the control group was 0.230. On statistical analysis by unpaired t-test, a difference in the mean value for NLRP3 concentration was observed between both the groups which was statistically significant (P < 0.0001), thereby disproving null hypothesis.

Table 3

Mean values of Nod-like receptor family pyrin domain-containing protein concentration in nanograms/ milliliter (ng/ml) in both the groups using unpaired t-test

NLRP3
Group A (healthy)	Group B (chronic periodontitis)	Probability value (P)
0.230	0.285	<0.0001

Probability value (P) <0.05 was considered statistically significant. NLRP3 – Nod-like receptor family pyrin domain-containing protein; P - Probability value

On statistical analysis using two-way ANOVA, the PI, GI, PPD, and CAL were statistically significant (P < 0.0001) [Table 4].

Table 4

P values using two-way ANOVA and values for Pearson’s correlation with Nod-like receptor family pyrin domain-containing protein of all parameters in both the groups

	P	Pearson’s correlation (r)
Age	0.0324	0.077
PI	0.0001	0.5016
GI	0.0001	0.5016
Pocket depth	0.0001	0.689
Clinical attachment loss	0.0001	0.659
Concentration of NLRP3	0.0001

Probability value (P)<0.05 was considered statistically significant. GI – Gingival index; PI – Plaque index; NLRP3 – Nod-like receptor family pyrin domain-containing protein; P - Probability value; r - Pearson’s correlation

Two-way ANOVA test comparing the concentration of NLRP3 (ng/ml) of males and females in both the groups showed that

When NLRP3 levels of males in both the groups were compared to NLRP3 levels of females in both the groups, the difference was statistically insignificant

When the levels of NLRP3 in males and females chronic periodontitis group were compared to levels of NLRP3 in males and females of healthy control group, a statistically significant difference was found with P < 0.0001.

This suggests that irrespective of the gender of the patient, the NLRP3 levels were increased in subjects with periodontitis of chronic origin to those compared to subjects with good periodontal health and this difference was statistically significant with P < 0.0001.

The Pearson correlation coefficient is used to measure the strength of a linear association between two variables, where the value r = 1 means a perfect positive correlation and the value r = −1 means a perfect negative correlation. Pearson's correlation analysis was used to test for any correlation between the NLRP3 concentration in saliva with the disease severity measures, i.e. PI, GI, PPD, and CAL in both the groups. In our study population, the concentration of NLRP3 (ng/ml) in all subjects showed slight positive correlate (r = 0.5016), (r = 0.6112), (r = 0.689), and (r = 0.659) with the PI, GI, PPD, and CAL scores, respectively, of the subjects. Pearson's correlation coefficient suggests that the concentration of NLRP3 is influenced by the increase in the plaque scores, gingival scores, pocket depth and clinical attachment loss. [Table 4].

The null hypothesis which stated that there is no relation between the NLRP3 levels in subjects with periodontitis in chronic form and subjects with good periodontal health was rejected after analyzing the results.

DISCUSSION

The key to successful treatment lies in early detection. Molecular disease biomarkers are being researched so that systemic diseases are diagnosed early.[18]

In 2002, the National Institute of Dental and Craniofacial Research advised research on oral fluids which can aid in advanced diagnosis. Saliva contains proteins and genetic molecules, is easily accessible, and thus can be used in detecting periodontal diseases in its early stage.[19] Despite all the studies done on existing salivary biomarkers tested for chronic periodontitis, none was conclusively established as a diagnostic marker for detecting periodontitis.

Inflammasomes are multi-proteins that first stimulate the caspase-1-dependent maturation. This leads to the secretion of interleukin-1 β and interleukin-18 when there is danger.[10] Research has shown that modulation of host response is strongly associated with inflammasomes and host-derived small “danger molecule ATP.” There is also alteration of the microbiome structure and intermicrobial associations. Hence, chronic inflammatory disease sets in.[10]

Liu et al. conducted an in vitro study on cerebellar granule neurons (CGN) upon injury and transient focal ischemia and found elevated levels of NLRP1 in injured and ischemic CGN.[20]

Pontillo et al. carried out a study in which genomic DNA was extracted from peripheral whole blood of 467 subjects affected by leprosy and found that the NLRP1-combined haplotype was associated with leprosy susceptibility.[21]

Enzyme-linked immunosorbent assay (ELISA) analysis conducted by Li et al. on human liver samples including nonsteatosis and steatohepatitis suggested that NLRP2 was decreased in steatotic liver tissues. Although NLRP1 and NLRP2 showed great potential for diagnosis of a large array of medical conditions, they did not correlate with periodontal pathologies.[22]

With the discovery of NLRP3, an association between NLRP3 and inflammation was established.[14] Periodontitis being an inflammatory pathology was researched for the presence of NLRP3 and a direct correlation of increased NLRP3 levels and presence of periodontitis was seen.

NLRP3 has three main domains: PYD, NBD, and leucine-rich repeat (LRR), which respond to triggers, by assembling into a functional inflammasome implying that it acts as a sensor causing cell damage.[23]

A sensor NLRP3, an adaptor (ASC: Also known as PYCARD), and an effector (caspase-1) are the parts of an NLRP3 inflammasome. The sensor protein NLRP3 has an amino-terminal on one end with a PYD, a carboxy-terminal with LRR domain on the other end, and a NACHT domain in the center. The LRR domain folds back onto the NACHT domain inducing autoinhibition.

The adaptor ASC has a carboxy-terminal caspase recruitment domain (CARD) and amino-terminal PYD. The effector caspase-1 has a small catalytic subunit domain (p10) at the carboxy-terminal and amino-terminal CARD and P20 which is a central large catalytic domain.

Oligomerization of NLRP3 occurs, upon stimulation, with homotypic interactions between NACHT domains. This oligomerized NLRP3, through homotypic PYD-PYD interactions, recruits ASC and nucleates the helical ASC filament formation. These multiple ASC filaments unify into ASC speck – a single macromolecular focus.[24]

Once assembled, ASC recruits caspase-1 which enables proximity-induced caspase-1 self-cleavage and activation. The linker between P20 and P10 is then self-cleaved by the caspase-1 clustered on ASC. This generates a complex of P33 (CARD and P20) and P10, which is proteolytically active and bound to ASC.[25]

Thus, NLRP3, ASC, and caspase-1 initiate an inflammatory response along with activation of another inflammatory mediator interleukin-1 β.[26] The detection that NLRP3 mutations cause the autosomal dominant disease called cryopyrin-associated periodic syndrome is a great advancement in inflammatory research.[27]

The first corelation of NLRP3 with inflammation was shown by Tao et al. in subjects suffering from acute arthritis.[13] A study by Isaza-Guzmán et al. showed that NLRP 3 levels increased in saliva of subjects suffering from periodontitis in both chronic and aggressive forms.

Research has shown that biomarker expression shows variability amongst races.[15] None of the previous studies have correlated the salivary levels of NLRP3 with the degree of periodontal breakdown in the Indian population. Hence, this study was undertaken. In this study, the Indian population was selected to evaluate the probable role of NLRP3, as a mediator of inflammation in the periodontium. This was accomplished by measuring NLRP3 levels in saliva and a comparison was made between the levels of salivary NLRP3 in subjects suffering from chronic periodontitis and healthy controls.

Whole unstimulated saliva samples were collected from subjects in Eppendorf safe-lock tubes® and were transported at 0°C for further storage at −80°C at the laboratory. The collected samples were then analyzed for concentrations of NLRP3 levels using Elabscience® ELISA kit which uses sandwich technique and colored markers to detect the antigen–antibody complex. The results were recorded using BioRad® microplate 65 reader** [Figure 3] which uses optical density to determine the concentration of NLRP3 in the samples. The results were obtained as nanogram per milliliter, i.e., (ng/ml).

Higher levels of NLRP3 were observed in subjects suffering from periodontitis of chronic origin as compared to subjects with good periodontal health. The plaque scores of subjects suffering from chronic periodontitis were found to be comparatively higher and were also statistically significant and also the chronic periodontitis group showed a higher mean PPD than the control group and the difference was found to be statistically significant. Furthermore, when intragroup comparisons were made between males and females in healthy group and chronic periodontitis group, respectively, no statistically significant differences were observed, suggesting that NLRP3 has the potential to be used as a biomarker irrespective of the patient's gender.

The results of this study show that IL-1 β processing and secretion mediated by NLRP3 inflammasome activation, and induced by endotoxin and whole bacteria, may stimulate an immune response, exaggerating the inflammatory process, thus leading to tissue destruction and bone resorption.

The results of our study were similar to the research conducted by Isaza-Guzmán et al. in which significantly increased levels of NLRP3, ASC, and IL-1 β were detected in periodontitis group as compared to healthy controls.[14] The results are also similar to the research conducted by García-Hernández et al., in which, higher levels of NLRP3 were found in periodontitis group as compared to healthy controls.[28]

Our study implies that NLRP3 could be used as a potential biomarker for detecting periodontitis.

CONCLUSIONS

This study was carried out to detect the presence of NLRP3 in human saliva and to evaluate the potential of NLRP3 to be a marker of active periodontal disease. The results suggested that levels of NLRP3 were significantly higher in subjects suffering from periodontitis of chronic origin as compared to subjects with good periodontal health. The difference in the levels of NLRP3 at different time intervals can also be used to determine the rate of disease progression. NLRP3 levels showed no significant differences between the gender groups indicating that it may well be effectively used as a biomarker marker. Further prospective interventional clinical trials, detecting the levels of NLRP3 postperiodontal therapy, with a bigger sample size, will further elucidate the role of NLRP3 in the diagnosis of periodontal disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.