NLRP1 and NLRC4 inflammasomes are not responsible for the induction of inflammation in pulp tissues from carious teeth.

INTRODUCTION: Inflammation is a risk factor for dental complications. Inflammasomes are a set of intracellular sensors which participate in the induction of inflammation. As the main factors involved in the induction of pulp inflammation in the carious teeth are yet to be clarified, this study was aimed to evaluate NLRP1 and NLRC4 expression levels in the inflamed and healthy pulps.
MATERIALS AND METHODS: Fifty inflamed and fifty healthy pulps were evaluated regarding the multiRNA levels of NLRP1 and NLRC4 using real-time polymerase chain reaction technique.
RESULTS: Results demonstrated that expression of NLRP1 (P = 0.985) and NLRC4 (P = 0.581) did not significantly differ in inflamed in comparison to healthy pulps. NLRP1 (P = 0.989) and NLRC4 (P = 0.170) did not change in males when compared with females in inflamed pulps. Furthermore, NLRP1 (P = 0.133) and NLRC4 (P = 0.642) were not altered in males in comparison to females in healthy pulps.
CONCLUSION: Although NLRP1 and NLRC4 are the main inflammasomes, it appears that they are not the responsible molecules involved in the human pulp inflammation in the carious teeth.

INTRODUCTION

Inflammation is an important complication which is induced by dental disease.[1] The inflammation in the dental caries may lead to pulp necrosis.[2] Several inflammatory molecules can participate in the induction or stimulation of inflammation in the pulp.[3] The main factors which participate in the induction or stimulation of inflammation are the innate immunity sensors which are famous as pathogen recognition receptors (PRRs). PRRs are the main factors which start inflammation via recognition of “damage-associated molecular patterns” (DAMPs) and “pathogen-associated molecular patterns” (PAMPs) on or in the immune and also nonimmune cells.[4]

Inflammasomes are a family of PRRs which are located in the cell cytoplasm and can recognize microbial PAMPs and also endogenous DAMPs.[5] The sensors contain several molecules such as nucleotide-binding domain, leucine-rich family (NLR), pyrin-containing 1 gene (NLRP1), NLRP3, NLRC4, and absent in melanoma-2 (AIM 2).[5] Inflammasomes induce the activation of pro-inflammatory cytokines, interleukin (IL)-18, and IL-1β, after interaction with their ligands.[6] Based on the fact that IL-18 and IL-1 β are the main factors that participate in the pulp inflammation,[7] it has been hypothesized that inflammasomes may be the main parts of the inflammation puzzle in the dental pulps derived from dental caries. Interestingly, the roles played by inflammasomes in the induction and stimulation of inflammation, which is induced following exposure to infectious agents, in the human pulps had been reported previously.[8] However, the main inflammasomes which participate in the induction or stimulation of pulp inflammation are yet to be clarified.

Due to the fact that the roles played by NLRP3 and AIM 2 in the pathogenesis of pulp inflammation have been evaluated previously,[89] but the roles of NLRP1 and NLRC4 were not explored, the current study aimed to evaluate the NLRP1 and NLRC4 expression levels in the inflamed and noninflamed pulps from carious and healthy teeth, respectively.

MATERIALS AND METHODS

Subjects

The samples in this cross-sectional study were derived from carious tooth (fifty inflamed pulps) and healthy tooth (fifty healthy pulps) from the patients who referred to Rafsanjan Dental Faculty. The sample size was calculated using the following formula:

The p1 and p2 were obtained from a study on the expression of inflammasomes in Rafsanjan population.[10] After collecting the demographic data from the participants, the samples were examined regarding the multiRNA (mRNA) levels of NLRP1 and NLRC4. The pulps' vitality was evaluated by the expert endodontists using cooling, heating, and electric pulp testing tests. Accordingly, the asymptomatic carious teeth with depth about 1–2 mm, which are confirmed by radiography, were collected from the patients. The carious teeth were extracted from the patients with gum diseases or the cases who need orthodontic operation. The samples that were obtained from the participants with infectious diseases, autoimmunity, allergy, pregnancy, smoking, immune-modulator and immunosuppressor drug consumers, addiction, and surgery in the last 2 weeks were excluded from the project. The inflamed pulps were derived from asymptomatic and decayed teeth which suffered from decay depth of 1 to 2 mm in radiography and were extracted for reasons of prosthetic and periodontal diseases, while the orthodontic reasons were the main causes of the extraction of healthy teeth (control group). The patients with decayed and healthy teeth were not different regarding age, sex, and ethnicity. The participants filled out the consent for their voluntary contributions in the investigations.

Pulp preparation

The root tips (apical 3–5 mm) were quickly removed and gathered in artificial cerebrospinal fluid (CSF) and kept at −20°C for RNA purification. The solution contained diethylpyrocarbonate to inactivate mRNA degrading enzyme, RNase. After transferring the teeth to the laboratory, based on the longitudinal format, the teeth were split into two equal parts and the pulps were extracted in sterile condition using excavator and put in artificial CSF. (For more information regarding pulp departure and preparation, please refer to previous studies[111213]).

RNA purification, synthesis of cDNA, and real-time polymerase chain reaction

Total RNA was purified using ethanol precipitation method with minor modification. Accordingly, the pulp tissues have to be made fragile via 30-min incubation in liquid nitrogen to be homogenized. The conditions of total mRNA extraction, synthesis of appropriate cDNA, and real-time polymerase chain reaction (PCR) were described in previous investigation.[14] Accordingly, RNA was extracted using RNX solution from Sinaclon Company (Tehran, Iran), cDNA was synthesized using a commercial kit from Parstous Company (Mashhad, Iran), and real-time PCR was performed in a thermocycler machine from Bio-Rad Company (New York, USA) by using a commercial master mix from Parstous Company (Mashhad, Iran). Primer sequences for NLRP1, NLRC4, and β-actin (as housekeeping gene) were as follows:

NLRP1 forward primer: 5′-ACTCTCCCTCATTCCCCTAC -3'

NLRP1 reverse primer: 5′-GCTGTCTCAAAACCCT TCTC-3'

NLRC4 forward primer: 5'-TTCGTCTTCTTCCTCC GTCT-3'

NLRC4 reverse primer: 5'-ATGTCTGCTTCCTGAT TGTG-3'

β-actin forward primer: 5'-GGCACCCAGCACAAT GAAG-3'

β-actin reverse primer: 5'-CCGATCCACACGGAGTA CTTG-3'.

Statistical analysis

Based on the normality of the raw data, using one-sample Kolmogorov–Smirnov test, the differences between groups regarding sex and age were analyzed using t-test, and the relative mRNA levels of NLRP1 and NLRC4 were analyzed using Mann–Whitney U-test under SPSS software version 18 (IBM Company, USA, New York). Thus, the data were reported as the median (1st quartile and 3rd quartile). P < 0.05 was considered statistically significant.

RESULTS

Statistical analysis demonstrated that the patients with decayed teeth have the same age (P = 0.271) and sex (P = 1.0) distribution when compared to the participants with healthy teeth.

The results showed that NLRP1 relative expression levels in the inflamed and healthy pulps and healthy noninflamed pulp sample were 0.0095 (0.0016–0.1386) and 0.0103 (0.0009–0.1037), respectively, differences of which were not statistically significant [P = 0.985, Figure 1].

Figure 1

MultiRNA expression levels of NLRP1 and NLRC4 in inflamed and noninflamed pulps. MultiRNA levels of NLRP1 and NLRC4 did not significantly differ between inflamed and healthy pulps

Relative expression levels of NLRC4 in the inflamed (0.0880 [0.0087–14.0592]) and healthy (0.4888 [0.0433–1.3722]) pulps were also not statistically significantly different [P = 0.581, Figure 1].

The results also demonstrated that relative expression levels of NLRP1 (P = 0.989) and NLRC4 (P = 0.170) did not significantly differ in male and female patients who suffered from inflamed pulps [Figure 2]. Pulp mRNA levels of NLRP1 (P = 0.133) and NLRC4 (P = 0.642) also did not differ between healthy male and female participants [Figure 3].

Figure 2

NLRP1 and NLRC4 multiRNA levels in male versus female inflamed pulps. MultiRNA levels of NLRP1 and NLRC4 did not significantly differ in males when compared to females with inflamed pulps

Figure 3

MultiRNA levels of NLRP1 and NLRC4 in male and female noninflamed pulps. The figure shows that the differences were not significant

DISCUSSION

It has been documented that inflammasomes are the main factors that participate in the induction or stimulation of tissue inflammation.[1516] NLRP1 and NLRC4 are two important members of the inflammasomes whose roles in the inflammation-related tissue injuries have been demonstrated by several investigators.[17] Based on the fact that inflammation in the dental pulps is a risk factor for dental decay, it has been hypothesized that inflammasomes may participate in the inflammatory pathogenesis of the pulps.

Moreover, as the pulps which derived from decayed teeth suffered from inflammation, and due to the results presented in the current investigation which show that expression levels of NLRP1 and NLRC4 do not change in the inflamed pulp, it may be concluded that more investigations with higher sample size need to be performed to determine the roles played by NLRP1 and NLRC4 in the induction or stimulation of pulp inflammation. Considering that IL-1β and IL-18 are two important cytokines which are activated by inflammasomes,[17] and due to previous investigations which reported an elevated expression of the cytokines in the inflamed pulps,[718] it seems that inflammasomes are the key parts of the inflammation puzzle in the carious teeth. However, based on the results of the current study, NLRP1 and NLRC4 expressions do not alter in the inflamed pulps. According to the fact that inflammasomes contain other molecules such as NLRP3 and AIM 2, therefore, it may be hypothesized that NLRP3 and AIM 2 may be the responsible molecules for activation of IL-1β and IL-18 in the inflamed pulps, which has been confirmed by previous investigations.[89] To the best of our knowledge, our investigation is a unique study which examines NLRP1 and NLRC4 expression levels in the inflamed pulps. However, several investigations have evaluated the relation between NLRP3/AIM 2 and the inflammation in the dental pulps. Accordingly, Zhang et al. reported that bacterial infection in dental pulps leads to interaction between the bacterial PAMPs with toll-like receptor 4 on the human dental pulp fibroblasts, which results in the upregulation of NLRP3 and consequently activation of IL-1β and, subsequently, induction of inflammation in the pulps.[19] Involvement of NLRP3 in the induction of inflammation in the dental pulps has also been documented by Wang et al.,[20] Jiang et al.,[8] Liu et al.,[21] and Song et al.[22] Interestingly, Lee et al. not only proved the roles played by NLRP3 in the pulp inflammation, but also reported that local inhibition of NLRP3 may decrease destructive processes in pulpitis.[23] Additionally, the roles played by AIM 2 in the pathogenesis of pulp inflammation have also been documented by investigators.[924]

CONCLUSION

According to the results presented in the current study and also based on the previous investigations, it may be concluded that the roles of NLRP1 and NLRC4 in the human pulp inflammation are disputable and the key roles of the other inflammasomes need to be proved by further investigations. Due to the fact that NLRP3 and AIM 2 are the most common candidates of inflammasomes which are increased in the inflamed tissues, in the case of NLRP1 and NLRC4, no change was observed,[10] thus it appears that NLRP3 plays important roles in the pathogenesis of pulp inflammation.

The results also demonstrated that male and female patients and controls also have the same expression levels of NLRP1 and NLRC4. Thus, it seems that gender differences also cannot be considered as a risk factor for induction or stimulation of pulp inflammation in NLRP1- and NLRC4-dependent manner.

Financial support and sponsorship

Rafsanjan University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.