Keywords
periodontitis - inflammasomes - caspase - saliva - interleukin
Introduction
Periodontitis is a chronic inflammatory disorder affecting the gingiva and adjacent
periodontal tissues (the bone, connective tissue, adjacent oral mucosa, periodontal
ligament [PDL], and gingiva).[1]
[2] Bacterial biofilms form on dental surfaces and provoke inflammatory reaction.[3] From 2011 to 2020, the prevalence of periodontitis among dentate adults was around
62%, with severe periodontitis affecting 23.6% of this population. This indicates
an abnormally elevated prevalence of periodontitis.[4]
Inflammasomes are molecular signaling complexes that form in response to a threat.[5] They are intracellular pattern recognition receptors that activate upon the recognition
of several signals.[6] Inflammasomes represent a component of the innate immune response that activates
inflammatory caspases.[7] In the initial phase of host response, different pathogen-associated molecular patterns
(PAMPs) and host-derived danger-associated molecular patterns (DAMPs) initiate inflammasome
production and the release of critical inflammatory cytokines.[8] The NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome
is a multimeric complex composed of 1- (NLRP3), 2-apoptosis-associated speck-like
protein including a caspase activation and recruitment domain (CARD) (ASC), and 3-pro-caspase-1
(pro-Casp-1).[9]
[10] Recruitment of the adaptor protein ASC and pro-Casp-1 to the inflammasome results
in the autoproteolytic activation of pro-Casp-1[11] ([Fig. 1]).
Fig. 1 The pathway of the NOD-like receptor thermal protein domain associated protein 3
(NLRP3) inflammasome activation.
[Fig. 1] shows the canonical inflammasome activation that is dependent on the formation of
a multiprotein complex that detects danger signals and recruits pro-Casp-1, thereby
facilitating the maturation of pro-interleukin (IL)-1β and pro-IL-18 into their active
forms, IL-1β and IL-18, respectively, and their subsequent release.[12] The activation of the NLRP3 inflammasome is established to necessitate two consecutive
phases: the priming phase and the assembly phase.[13] The nucleotide-binding regions and a leucine-rich repeat (NLR) family is distinguished
by its tripartite structure. All NLRs possess a CARD or pyrin domain (PYD) at the
N-terminal, a central nucleotide-binding oligomerization domain (NACHT) and a C-terminal
region characterized by leucine-rich repeats, which facilitate interactions with other
proteins and enhance inflammasome formation.[14] The activation of the inflammasome and pyroptosis constitute an evolutionarily conserved
mechanism for pathogen defense, observed in diverse cellular types and tissues, including
macrophages, monocytes, periodontal tissues, osteoclasts, and osteoblasts.[15]
The IL-1β, a member of the IL-1 cytokine family, is an essential proinflammatory cytokine
released by host cells in the inflammatory process of periodontal disease.[16] IL-1β secreted by macrophages not only recruits immune cells to enhance the inflammatory
response but also induces collagen degradation by elevating matrix metalloproteinase
secretion and facilitates receptor activator of nuclear factor kappa B ligand (RANKL)-mediated
osteoclastogenesis, ultimately leading to bone resorption in periodontitis.[17] IL-1β is higher in serum, saliva, and gingival crevicular fluid of patients with
periodontitis.[18]
The IL-18, a member of the IL-1 cytokine family, stimulates the upregulation of other
proinflammatory cytokines (tumor necrosis factor α [TNF-α], IL-1β, IL-6), perpetuating
a detrimental cycle that contributes to inflammation and tissue destruction in periodontal
tissue.[19]
IL-37 is also a member of the IL-1 cytokine family. Recent studies have identified
IL-37 as a bifunctional cytokine demonstrating significant immunosuppressive and anti-inflammatory
properties.[20] It may either penetrate the nucleus directly within the cell to perform its function
or be secreted extracellularly to interact with the membrane receptors of itself or
adjacent cells. Evidence has established that IL-37 can diminish and suppress immune
responses in inflammatory and autoimmune illnesses, reducing tissue damage.[21] Research indicates that IL-37 might regulate the expression of inflammatory cytokines
and exert an anti-inflammatory effect by regulating the NLRP3 inflammasome.[22]
The relationship among these biomarkers is based on the potential sequence of events
that may arise within the context of periodontitis.[23] Understanding the equilibrium among these cytokines is essential, as their respective
and collective impacts and their correlation with the progression of periodontitis.[24] This may provide critical insights into the fundamental causes of disease severity,
treatment efficacy, and the investigation of novel therapeutic techniques that seek
to target specific components within this intricate cytokine network.[25] This study investigates the complex network of inflammatory mediators by examining
the salivary concentrations of five specific biomarkers: NLRP3, Casp-1, IL-1β, IL-18,
and IL-37.
Materials and Methods
Study Settings
This observational case–control research study was conducted at the University of
Al-Muthannah College of Dentistry and the Specialized Dentistry Centre in Al-Muthannah,
Iraq.
Eighty subjects were recruited (mean age 35.39 ± 10.30 years; 1:1 female and male
ratio) from the middle of November 2023 to August 2024. The subjects were divided
into healthy periodontium (n = 40) and unstable periodontitis (n = 40) based on the 2017 classification of the periodontal disease and condition.[26]
Sample Size Calculating
The study was designed to detect a statistically significant difference between the
two groups with a 95% power and a 5% α margin of error. The sample size was calculated
using Nlrp3 (Isola et al, 2022),[27] yielding a sample size = 80.
By using this equation:
n = Number of samples that we need to find out
r = Control to cases ratio
z
1–β
= It is the desired power
Z
1–α/2 = Critical value and a standard value for the corresponding level of confidence
σ = standard deviation (SD)
d = effect size
Then, the sample was divided equally into two groups of same size. Those groups were
the unstable periodontitis group, n = 40, and the healthy control group, n = 40.
Study Population
Subjects included in this study were:
The clinical criteria for healthy periodontium on intact gingiva are: Bleeding on
probing (BOP) < 10%, periodontal pocket depth (PPD) ≤ 3 mm, and no clinical or radiographical
bone loss.[26]
Periodontitis groups were defined as[28]:
-
Interdental clinical attachment loss (CAL) is detectable at ≥ 2 nonadjacent teeth,
or
-
Buccal or oral CAL ≥ 3 mm with pocketing > 3 mm is detectable at ≥ 2 teeth.
Inclusion and Exclusion Criteria
The study participants were 18 years of age or older, of both sexes, possessed a minimum
of 20 teeth, and had no history of systemic diseases.
The study excluded several categories of participants to maintain the integrity of
the results. Individuals with systemic illnesses, active dental caries, or oral ulcers
were not eligible for inclusion. Similarly, those who had undergone periodontal treatment
or taken anti-inflammatory drugs or antibiotics within the preceding 3 months were
excluded. Patients with orthodontic appliances or fixed/removable prostheses were
also ineligible. The exclusion criteria extended to pregnant or lactating women, as
well as those using oral contraceptives. Smokers and alcohol users were likewise omitted
from the study population. It is important to note that participation in the study
was voluntary, and individuals who expressed unwillingness to participate were not
included in the research. These strict, stringent exclusion criteria were implemented
to minimize confounding factors and ensure the reliability of the study outcomes.
Ethical Approval Statement
The College of Dentistry, University of Baghdad's Ethics Committee approved the protocol
(Reference number: 884; Project number: 884623; Date 3-12-2023). The study followed
the Declaration of Helsinki in 2008.[29] Before participating in the study, all participants were asked to sign an informed
consent form that provided all information describing the study's purposes and aims.
Reliability Analysis
Before the start of the study, the investigator's performance in accurately documenting
the clinical periodontal parameters (BOP, CAL, PPD) was assessed through interexaminer
and intraexaminer sessions. Sessions were performed on randomly chosen patients, and
the interclass correlation coefficient[30] was > 0.75.
Salivary Collection Procedure
Participants were asked to rinse their mouths with water, and unstimulated whole saliva
was collected from the patients before clinical evaluation by the passive drooling
method.[31] Patients were asked to accumulate the saliva on the floor of their mouth for 5 minutes
and then expectorate into a plastic cup. Note that 3 mL of the collected saliva was
transferred into the test tubes to standardize the volume collected for each patient.
The samples were centrifuged to remove the cell debris at 4,000 × g at 40°C for 10 minutes. Moreover, the supernatants were stored at −20°C until they
were analyzed using enzyme-linked immunosorbent assay (ELISA).[32]
Periodontal Examination
A full periodontal examination was conducted, by the assessment of plaque index (PI)[33] on four surfaces, BOP, PPD, and CAL on six surfaces of all participants' dentition.
The researchers performed the examination using a University of North Carolina probe
(a calibrated periodontal probe). The six surfaces examined per tooth were mesiobuccal,
distobuccal, mid-buccal, mesiolingual, distolingual, and mid-lingual, while the four
surfaces assessed for PI were buccal, distal, mesial, and lingual. The diagnosis was
confirmed to differentiate unstable periodontitis from other conditions.
Laboratory Analysis of Salivary Biomarkers
Samples were thawed and centrifuged at 1,000 revolutions per minute for 1 minute at
4°C, and 100 µm of the supernatant was collected to analyze protein biomarkers. Salivary
levels of NLRP3, Casp-1, IL-1β, IL-18, and IL-37 were measured using the ELISA kit.
-
NLRP3: USCN ELISA kit product no. SEC034Hu (headquartered in Houston, United States)
-
Casp-1: Elabscience ELISA KIT product no. E-EL-H0016 (headquartered in Houston, United
States)
-
IL-1β: Human IL-1β ELISA Kit from Elabscience, product no. E-EL-H0149 (headquartered
in Houston, United States)
-
IL-18: Human IL-18 ELISA Kit, product no. E-EL-H0253 H0149 (headquartered in Houston,
United States)
-
IL-37: Human IL-37 ELISA Kit, product no. E-EL-H2571 (headquartered in Houston, United
States)
Statistical Analysis
For continuous variables, central tendency and dispersion were quantified using mean
values and SD. The Shapiro–Wilk test was applied to evaluate the normality of data
distribution. Intergroup comparisons were performed using an independent sample t-test, Mann–Whitney U test, and chi-square test. Within each group, Spearman's correlation analysis was
conducted to examine the relationships between variables. Positive and negative predictive
values were determined through contingency table analysis. Statistical significance
was defined as p < 0.05.
Result
The total number examined for eligibility criteria to participate in this study was
n = 185. Only 80 participants met the inclusion criteria; 40 of them had healthy periodontium
and included in the healthy group. The rest were excluded due to a variety of exclusion
criteria, as shown in [Fig. 2]
Fig. 2 Flowchart of the study.
[Table 1] illustrates age characteristics, sex distribution, and the number of missing teeth.
The average age was 35.39 ± 10.30 years, ranging between 18 and 63 years, and there
was no significant difference between the healthy and periodontitis groups (p = 0.077). In the healthy group, the mean age was 33.35 ± 6.84 years, ranging from
23 to 45 years, while in the periodontitis group, the mean was 37.43 ± 12.64 years,
ranging between 18 and 63 years. Distribution according to sex showed that males (n = 40) represented 50%, and females (n = 40) represented 50% of the total sample. There was no significant difference in
the proportions of males and females between both groups (p = 0.074) ([Table 1]).
Table 1
Study population demographic characters
|
Variables
|
Group
|
N
|
Min.
(y)
|
Max.
(y)
|
Mean
(y)
|
SD
|
p-Value
|
|
Age (y)
|
Healthy
|
40
|
23
|
45
|
33.35
|
± 6.84
|
0.077[a]
|
|
Periodontitis
|
40
|
18
|
63
|
37.43
|
± 12.64
|
|
Total
|
80
|
18
|
63
|
35.39
|
± 10.30
|
|
Sex
|
Group
|
N
|
Male
N
(%)
|
Female
N
(%)
|
Total
N
(%)
|
p
-Value
|
|
Healthy
|
40
|
16 40)
|
24 (60)
|
40 (50)
|
0.074
|
|
Periodontitis
|
40
|
24 (60)
|
16 (40)
|
40 (50)
|
|
Total
|
80
|
40 (50)
|
40 (50)
|
80 (100)
|
|
No. of missing teeth
|
Group
|
Min.
|
Max.
|
Total
|
Mean ± SD
|
p
-Value
|
|
Healthy
|
0
|
7
|
51
|
1.28 ± 1.77
|
< 0.001[a]
|
|
Periodontitis
|
0
|
8
|
162
|
4.05 ± 3.15
|
|
Total
|
0
|
8
|
213
|
4.63 ± 17.93
|
Abbreviations: %, frequency; Max, maximum; Min, minimum; SD, standard deviation.
a Mann–Whitney U test.
There was a significantly higher number of missing teeth in the periodontitis group
compared with the healthy group (p < 0.001). The total number of all missing teeth in both groups was 213, with a mean
of 4.63 ± 17.93 teeth. The lowest was in the healthy group, 51 teeth, and the mean
was 1.28 ± 1.77. In periodontitis, the total number of missing teeth was 162, and
the mean was 4.05 ± 3.15 ([Table 1]).
Analysis of Clinical Periodontal Parameters and Salivary Biomarkers
The Periodontal Parameters
The mean percentage of the PI was significantly higher (p < 0.001) in periodontitis (93.2% ± 0.17) compared with the healthy group (10.3% ± 0.06),
and the mean percentage of BOP was also significantly higher (p < 0.001) in the periodontitis group (62.5% ± 0.23) compared with the healthy group
(5.8% ± 0.03). The mean value of PPD was 4.46 ± 0.51 mm, and the CAL was 3.7 ± 1.41 mm
in the periodontitis group ([Table 2]).
Table 2
Descriptive and comparison statistics of clinical parameters and salivary biomarkers
|
Variable
|
Group
|
N
|
Mean
|
SD
|
Min.
|
Max.
|
p-Value
|
|
PI%
|
Healthy
|
40
|
10.3%
|
± 0.061
|
0.9%
|
20%
|
< 0.001[a]
|
|
Periodontitis
|
40
|
93.2%
|
± 0.168
|
44%
|
100%
|
|
BOP%
|
Healthy
|
40
|
5.8%
|
± 0.027
|
0.7%
|
9.8%
|
< 0.001[a]
|
|
Periodontitis
|
40
|
62.5%
|
± 0.237
|
27%
|
100%
|
|
PPD (mm)
|
Periodontitis
|
40
|
4.46
|
± 0.513
|
4.000
|
5.880
|
|
|
CAL (mm)
|
Periodontitis
|
40
|
3.697
|
± 1.405
|
1.390
|
10.390
|
|
NLRP3 (ng/mL)
|
Healthy
|
40
|
7.52
|
± 0.955
|
5.330
|
9.400
|
< 0.001[a]
|
|
Periodontitis
|
40
|
22.350
|
± 2.360
|
15.830
|
26.330
|
|
Casp-1 (pg/mL)
|
Healthy
|
40
|
1239.24
|
± 265.414
|
769.150
|
1796.520
|
< 0.001[a]
|
|
Periodontitis
|
40
|
2903.91
|
± 798.047
|
1672.020
|
4410.100
|
|
IL-1β (pg/mL)
|
Healthy
|
40
|
64.555
|
± 27.75
|
10.410
|
113.990
|
< 0.001[b]
|
|
Periodontitis
|
40
|
333.253
|
± 56.16
|
202.110
|
463.450
|
|
IL-18 (pg/mL)
|
Healthy
|
40
|
238.184
|
± 73.354
|
80.410
|
369.980
|
< 0.001[b]
|
|
Periodontitis
|
40
|
630.52
|
± 110.5
|
380.830
|
808.810
|
|
IL-37 (pg/mL)
|
Healthy
|
40
|
554.726
|
± 152.275
|
312.925
|
1000.000
|
< 0.001[a]
|
|
Periodontitis
|
40
|
281.926
|
± 97.653
|
161.280
|
505.880
|
Abbreviations: BOP%, bleeding on probing percentage; CAL, clinical attachment loss;
Casp-1, caspase-1; IL, interleukin; Max, maximum; Min, minimum; NLRP3, NLR family
pyrin domain containing 3; PI%, plaque index percentage; PPD, periodontal pocket depth.
a Mann–Whitney U test.
b Independent sample t-test.
The Salivary Biomarkers
The NLRP3 level was significantly higher (p < 0.001) in the periodontitis group, 22.35 ± 2.36 ng/mL, compared with the healthy
group, 7.51 ± 0.95 ng/mL. Similarly, the salivary Casp-1 level was significantly higher
(p < 0.001) in the periodontitis group, 2903.91 ± 798.04 pg/mL, compared with the healthy
group, 1239.24 ± 265.41 pg/mL.
The salivary level of IL-1β in the healthy group, 64.55 ± 27.75 pg/mL, was significantly
lower (p < 0.001) than in the periodontitis group, 333.25 ± 56.16 pg/mL, and the salivary
level of IL-18, it was significantly higher (p < 0.001) in the periodontitis group, 630.52 ± 110.5 pg/mL, compared with the healthy
group, 238.18 ± 73.35 pg/mL. Conversely, IL-37 was the only salivary biomarker that
exhibited significantly higher levels (p < 0.001) in the healthy group, 554.726 ± 152.275 pg/mL, compared with the periodontitis
group, 281.926 ± 97.653 pg/mL ([Table 2]).
Correlation Correlations between Salivary Biomarkers and Periodontal Parameters in
the Healthy Group
[Fig. 3] demonstrates the correlation of the healthy group between periodontal parameters
and the salivary biomarkers.
Fig. 3 The correlation between the healthy group's periodontal parameters and salivary biomarkers.
-
Significant (p < 0.026) positive weak correlation (0.353) was observed between Casp-1 and BOP% ([Figs. 3] and [4]).
-
Significant (p < 0.012) negative weak correlation (−0.395) was observed between IL-37 and IL-1β
([Figs. 3] and [5]).
-
Significant (p < 0.002) negative moderate correlation (−0.483) was observed between IL-18 and NLRP3
([Figs. 3] and [6]).
-
Significant (p < 0.003) negative moderate correlation (−0.455) was observed between IL-37 and IL-18
([Figs. 3] and [7]).
Fig. 4 Simple linear regression between caspase-1 (Casp-1) and bleeding on probing percentage
(BOP%).
Fig. 5 Simple linear regression between interleukin (IL)-37 and IL-1β.
Fig. 6 Simple linear regression between interleukin (IL)-18 (pg/mL) and NOD-like receptor
thermal protein domain associated protein 3 (NLRP3).
Fig. 7 Simple linear regression between interleukin (IL)-37 (pg/mL) and IL-18 (pg/mL).
Correlations between Salivary Biomarkers and Periodontal Parameters in the Periodontitis
Group
[Fig. 8] shows the correlations between the periodontal parameters and salivary biomarkers
in the periodontitis group.
Fig. 8 The correlation between the periodontitis group's periodontal parameters and salivary
biomarkers.
The result was:
-
A significant (p < 0.045) positive weak correlation (0.319) was found between salivary IL-18 and NLRP3
([Figs. 8] and [9]).
-
A significant (0.031) positive weak correlation (0.342) was found between salivary
IL-37 and NLRP3 ([Figs. 8] and [10]).
-
A significant (p < 0.001) positive moderate correlation (0.654) was found between salivary IL-18 and
Casp-1 ([Figs. 8] and [11]).
-
A significant (p < 0.001) negative moderate correlation (−0.508) was found between salivary IL-37
and IL-1β ([Figs. 8] and [12]).
Fig. 9 Simple linear regression between interleukin (IL)-18 (pg/mL) and NOD-like receptor
thermal protein domain associated protein 3 (NLRP3) (ng/mL).
Fig. 10 Simple linear regression between interleukin (IL)-37 (pg/mL) and NOD-like receptor
thermal protein domain associated protein 3 (NLRP3) (ng/mL).
Fig. 11 Simple linear regression between interleukin (IL)-18 (pg/mL) and caspase-1 (Casp-1)
(pg/mL).
Fig. 12 Simple linear regression between interleukin (IL)-37 (pg/mL) and IL-1β (pg/mL).
Discussion
In periodontium, any severe inflammatory reaction is expected to promote the resorption
of supporting bones and inhibit the process of osseointegration.[34]
[35]
The present research provides valuable insights into the role of inflammasomes and
associated cytokines in the pathogenesis of periodontitis. The findings of this study
demonstrated that NLRP3 inflammasome and its downstream products, including Casp-1,
IL-1β, and IL-18, are significantly higher in the periodontitis patients than in the
healthy group. Conversely, the IL-37, an anti-inflammatory cytokine, was significantly
lower in periodontitis patients than the healthy controls. These observations suggest
a complex interplay between proinflammatory (NLRP3, Casp-1, IL-1β, IL-18) and anti-inflammatory
pathways (IL-37) in the progression of periodontal disease.[22]
[36]
[37]
[38] This makes them particularly pertinent for examining the pathogenesis and advancement
of periodontitis. Examining the inflammasome pathway in periodontitis may enhance
periodontal research's overall domain and better understand the interconnection between
systemic inflammation and periodontal health.
Saliva was used among accessible oral fluids in this study. Saliva exhibits remarkable
efficacy in identifying periodontitis and a commendable capability in detecting nonperiodontitis.[39] Saliva collection is a noninvasive and uncomplicated procedure that facilitates
the acquisition of samples from several individuals without inducing discomfort. Passive
drooling was chosen for saliva collection in this study since it potentially reduces
bacterial contamination of the sample and other systemic mistakes linked to alternative
collection methods. Furthermore, a significant amount of saliva can be obtained briefly
using the drooling technique.[40]
[41]
[42]
The significant elevation of salivary levels of NLRP3 inflammasome in the periodontitis
group, as shown in [Table 2], indicates its role in the inflammatory process.[43]
[44]
[45] Upon activation, the NLRP3 inflammasome complex releases inflammatory cytokines.[46] These cytokines facilitate the eradication of harmful microorganisms as a defensive
response.[47] Excessive amounts of inflammatory cytokines are detrimental because they play a
role in collagen degradation, alveolar bone resorption, and loss of periodontal attachment.
The inflammation impacts the alveolar bone between the teeth and the adjacent connective
tissue.[48] NLRP3 controls bone resorption in periodontitis by facilitating osteoclast differentiation.[48] This discovery substantiates the role of NLRP3 in periodontitis.
Recent studies have also associated the overexpression and activation of the NLRP3
inflammasome with the onset of periodontitis.[49] The NLRP3 inflammasome performs distinct regulatory activities within PDL, promotes
osteoclastogenesis by elevating RANKL production or reducing osteoprotegerin levels,
simultaneously induces osteoblast apoptosis, increases proinflammatory cytokines in
PDL fibroblasts, and regulates immune cell activities.[49] Suggesting there is a correlation between the NLRP3 and periodontitis; those findings
matched the findings of this research. While other research indicated that the NLRP3
inflammasome is not significantly involved in inflammatory bone resorption.[50]
The observed increase in Casp-1 levels in periodontitis patients, as depicted in [Table 2], is consistent with its role in the activation of the inflammasome, the Casp-1 cleaves
pro-IL-1β and pro-IL-18 into their mature active cytokine forms.[51] Which makes the Casp-1 the primary downstream effector of all inflammasomes.[50] Activation of Casp-1 also causes rapid cell death, marked by plasma membrane rupture
and the release of proinflammatory intracellular components.[52] Elevated levels of Casp-1 in saliva suggest that an active inflammation occurs in
the periodontal tissues. This study agreed with a study done by Mahmood and Abbas
that found the salivary levels of Casp-1 were higher in periodontitis patients than
in healthy controls, indicating a correlation with the disease presence.[53] This indicates that salivary Casp-1 is involved in periodontitis. However, another
study showed that the concentration of Casp-1 in saliva samples makes its measurement
ineffective for detecting the presence and/or severity of periodontal disease.[36]
Both IL-1β and IL-18 showed significantly higher levels in the periodontitis group
than in the healthy group, consistent with other studies.[54] Upon cellular stimulation, PAMPs and DAMPs induce the assembly of inflammasomes.
Pyroptosis is facilitated by the activation of Casp-1 through the NLRP3.[55] Casp-1 cleaves gasdermin family members, including gasdermin D (GSDMD), leading
to cell membrane perforation by releasing its N-terminal domain. Activating Casp-1
via NLRP3 represents the typical canonical inflammasome pathway of pyroptosis. This
will culminate in the cleavage of GSDMD.[56] The mechanisms of pyroptosis fundamentally require the activation of GSDMD, resulting
in the formation of pores in the cell membrane, through which cytoplasmic chemicals,
including IL-1β and IL-18, are released, triggering a vigorous inflammatory response.[57] L-1β is an essential proinflammatory cytokine predominantly released by monocytes,
macrophages, and dendritic cells (DCs). IL-1β enhances vasodilation, and the chemotaxis
of inflammatory cells promotes collagen degradation through the upregulation of matrix
metalloproteinases and stimulates bone resorption by accelerating osteoclastogenesis.[58] The persistent activation of cytokines has been demonstrated to gradually impair
the adjacent structures, including gingival tissue, PDL, and bone. Research indicates
that IL-1β can promote the migration of proinflammatory cells from the bloodstream
to inflamed tissues; additionally, it signals the extracellular matrix and triggers
the production of other cytokines.[59] It has been proposed that additional noncanonical inflammasome pathways, including
Casp-4, Casp-5, and Casp-8, may facilitate or offer alternative mechanisms for releasing
IL-1β. This may explain the increasing levels of IL-1β in the periodontitis group.[60]
[61] A 2009 study conducted in Finland examined salivary cytokine levels to clarify periodontitis.
IL-1β was present in all samples.[62] All patients with periodontitis in this study showed significantly elevated levels
of IL-1β. Research indicates that targeting IL-1β or the NLRP3 inflammasome can significantly
reduce bone loss associated with periodontitis. This suggests that IL-1β plays a crucial
role in the development of the disease.[9]
IL-18 is predominantly secreted by DCs, which stimulate the production of interferon-γ
from Th1 cells and IL-17 from Th17 cells while also enhancing the release of IL-17,
TNF-α, and IL-1β, so facilitating increased osteoclastogenesis and bone resorption.[63]
Salivary concentrations of IL-18 were observed to be five times higher in people with
periodontitis compared with healthy individuals.[64] While another study found no difference in salivary IL-18 levels between people
with periodontitis and those without.[65]
There was a significant positive correlation between NLRP3 and IL-18 ([Fig. 9]) in the periodontitis group, supporting the belief that the NLRP3 inflammasome plays
a pivotal role in the inflammatory cascade associated with periodontitis. IL-18, in
particular, has been shown to increase the production of other proinflammatory cytokines,
such as TNF-α and IL-6, and prolong the inflammatory response.[19] This could be one of the reasons for the progressive tissue destruction observed
in periodontitis.
This investigation demonstrated an elevation in the concentration of IL37 in the saliva
of individuals with healthy periodontium compared with those with periodontitis, which
was inconsistent with a previous study that reported an increase in the salivary level
of IL-37 in the periodontitis group.[66] IL-37 has been shown to suppress the production of proinflammatory cytokines and
inhibit the activation of the NLRP3 inflammasome.[20]
[21]
The role of IL-37 in suppressing the innate immune response has been demonstrated.
It can be activated by Casp-1 cleavage, thereafter functioning as a cytokine via intracellular
or extracellular routes.[67] Both the precursor and mature forms of IL-37 can bind to IL-18 receptor alpha (IL-18Rα).
Mature IL-37 can also be associated with IL-18BP, the natural antagonist of IL-18,
in an extracellular manner.[58] The anti-inflammatory efficacy of IL-37 demands IL-1R8. IL-37 needs the receptors
IL-18Rα and IL-1R8 to inhibit innate immunity.[68] Upon binding to the IL-18Rα chain, IL-37 recruits TIR-8/IL-1R8/SIGIRR (Toll/IL-1
receptor/single Ig IL-1-related receptor), leading to the assembly of a triple complex
on the cell surface. This results in innate and acquired immunosuppression alongside
an enhancement of the anti-inflammatory pathway rather than the activation of the
IL-18 pathway.[69]
IL-37 serves a dual effect in suppressing IL-1β-mediated inflammation: it suppresses
IL-1β production in activated macrophages. And significantly reduces ASC oligomerization,
which precedes Casp-1 activation in inflammasome activity.[70] IL-37 markedly suppressed the expression of NLRP3, Casp-1, and IL-1β in periodontitis,
considerably reduced the levels of proinflammatory cytokines TNF-α and IL-6, and enhanced
the expression of the anti-inflammatory cytokine IL-10. These findings indicate that
IL-37 effectively regulates the nuclear factor kappa-B/NLRP3 signaling pathway in
periodontitis.[22] IL-37 also inhibits the production of several proinflammatory cytokines.[71] Consequently, it is reasonable to propose that other inflammatory cytokines have
a significant role in the pathogenesis of periodontitis. A study revealed that upon
stimulation of the innate immune system, macrophages generate both the proinflammatory
cytokine IL-1β and the anti-inflammatory cytokine IL-37 via NLRP3 activation, thereby
initiating a negative feedback process that reduces excessive inflammation.[72]
Recent research has unveiled an intriguing disparity regarding IL-37 levels with periodontal
health. One study indicates an increase in IL-37 levels in individuals diagnosed with
periodontitis[73]; conversely, another study demonstrates that healthy individuals possess higher
IL-37 levels,[74] which matches the current study. In another contrast with the current study showed
no significant difference in salivary IL-37 levels between the periodontitis group
and the healthy group.[75] This notable contrast presents new opportunities for advancing the understanding
of oral health and its complexities. This study revealed that IL-37 has no association
between the clinical periodontal measures and IL-37 levels in the saliva. This may
suggest that IL-37 is not a reliable marker for assessing periodontal disease (40).
The negative correlation between IL-37 and IL-1β in the periodontitis group ([Fig. 12]) suggests that IL-37 may play a protective role against the excessive inflammation
observed in periodontitis. This finding is consistent with previous studies that have
highlighted the potential of IL-37 as a therapeutic target in inflammatory diseases.[22]
[24]
The significant correlations between salivary biomarkers and clinical periodontal
parameters, such as BOP% and PPD, suggest that these biomarkers could serve as potential
indicators of disease activity. For instance, the positive correlation between Casp-1
and BOP% ([Fig. 4]) in the healthy group indicates that even mild inflammation may be associated with
activating the NLRP3 inflammasome. This finding underscores the importance of early
intervention in periodontal disease management.[76]
Many studies support the role of NLRP3, IL-1β, and IL-18 as diagnostic biomarkers.
One study found that NLRP3 is a potential biomarker for periodontitis.[77] Another study combined NLRP3 and IL-18. This study suggests that IL-18 may not serve
as an appropriate indicator for assessing the inflammatory status of periodontal tissues,
whereas NLRP3 may be regarded as a marker of inflammation in periodontitis.[78] Although a systematic review found IL-18 has the potential to be a diagnostic biomarker
for periodontitis.[19]
The role of IL-1β as a strong diagnostic biomarker has been highlighted in several
researches.[79]
[80]
[81]
A recent study done by Mahmood and Abbas found that Casp-1 showed high sensitivity
and specificity in the diagnosis of periodontitis.[53]
A limited number of studies included IL-37 as a potential biomarker. A study suggests
that IL-37 may serve as possible biomarkers that require additional longitudinal clinical
research to ascertain their efficacy as prognostic or diagnostic indicators.[74] Another study found IL-37 as an unuseful diagnostic biomarker for periodontitis.[75]
Limitations and Future Directions
Limitations and Future Directions
Although this study provides valuable insights into the role of inflammasomes and
cytokines in periodontitis, some limitations should be acknowledged. The case–control
design of this study prevents any conclusions regarding causality. Longitudinal studies
are needed to assess the relationships between inflammasome activation, cytokine production,
and the progression of periodontitis.
Future research should search for the potential of targeting the NLRP3 inflammasome
and IL-37 as therapeutic strategies in periodontitis.
This study examined periodontitis comprehensively without delving into specific stages
or grades. However, we recommend conducting further investigations to provide a more
detailed assessment and tailored recommendations
Given the developing evidence of the role of the NLRP3 inflammasome in chronic inflammation,
inhibitors of NLRP3 could be promising candidates for periodontal therapy. Similarly,
strategies to enhance IL-37 production or activity may help restore the balance between
proinflammatory and anti-inflammatory pathways in periodontitis.
Conclusion
The NLRP3 inflammasomes, along with related cytokines (Casp-1, IL-1β, and IL-18),
play an important role in promoting periodontal inflammation and tissue damage. IL-37,
on the other hand, is a cytokine that has anti-inflammatory properties. It does this
by suppressing the activity of the NLRP3 inflammasome, which has the effect of reducing
excessive inflammation. This interaction highlights the importance of targeting NLRP3
and enhancing IL-37 as a therapeutic approach for treating periodontal disease.
