Analysis of the expression of NLRP3 and AIM2 in periapical lesions with apical periodontitis and microbial analysis outside the apical segment of teeth

Highlights

• For the first time, we reported the NLRP3 and AIM2 proteins were highly expressed in periapical lesions.

• Most of the samples that showed an up-regulation of NLRP3 also demonstrated an up-regulation of caspase-1.

• Anaerobeswere the main detected pathogenic species outside of root canal infections.

• LPS of P. gingivalis induced IL-1β secretion and inflammasome activation.

Abstract

Objective

To detect the distribution and expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and the absent in Melanoma 2 (AIM2) inflammasomes in periapical lesions and to analyse the possible microbial stimuli outside of teeth.

Design

The distribution of NLRP3 and AIM2 inflammasomes in sixteen periapical lesions was investigated by immunohistochemistry. Meanwhile, the relative gene expression levels of NLRP3 and AIM2 in sixteen periapical lesions and three health periodontal tissue were quantified by real-time polymerase chain reaction (PCR). Moreover, forty-seven teeth without sinus tracts were obtained in the clinic and included in bacterial analysis using PCR. Then, the mRNA levels of apoptosis-associated speck-like protein (ASC), caspase-1, interleukin-1β (IL-1β), NLRP3 and AIM2 in THP-1-derived macrophages treated with lipopolysaccharides (LPS) of Porphyromonas were also quantified by real-time PCR, and the IL-1β secretion level was investigated using enzyme-linked immunosorbent assay (ELISA).

Results

NLRP3 and AIM2 were positively expressed in periapical lesions and were mainly distributed in inflammatory cells. Most of the samples that demonstrated up-regulation of NLRP3 mRNA also demonstrated up-regulation of caspase-1 mRNA. Microbial analysis revealed that Porphyromonas endodontalis was the most commonly detected species and was detected in 27 of 47 cases (57.4%), followed by Fusobacterium nucleatum (20/47, 42.6%), Porphyromonas gingivalis (19/47, 40.4%), Tannerella forsythia (19/47, 40.4%), Actinomyces sp. (17/47, 36.17%), Treponema denticola (10/47,21.28%), Actinomyces israelii (9/47,19.15%), Prevotella intermedia (6/47, 12.77%), Enterococcus faecalis (1/47,2.13%) and Enterococcus faecium (0/47,0). Furthermore, we found that LPS of P. gingivalis induced THP-1 cells to produce IL-1β and to activate NLRP3 and AIM2 inflammasomes.

Conclusions

Our results suggest that the NLRP3 and AIM2 proteins play a part in the pathogenesis of periapical periodontitis. Anaerobes, such as P. endodontalis, P. gingivalis, F. nucleatum and T. forsythia, were the main detected microbial stimuli that might activate inflammasomes in periapical tissues.

Introduction

Apical periodontitis is commonly seen in dental clinics, and it is frequently caused by bacterial invasion from root canals (Rechenberg, Bostanci, Zehnder, & Belibasakis, 2014). In the complex interaction between bacteria and host defence, neutrophils play an important role against advancing bacterial invasion. In the immediate aftermath of the recruitment of neutrophils, the overwhelming release of pro-inflammatory cytokines is unavoidable (Nair, 1997). Among the cytokines secreted, interleukin-1β (IL-1β) is the most frequently detected form in apical granulomas and their exudates and is of great importance in the host defence against pathogens (Barkhordar, Hussain, & Hayashi, 1992). However, excessive secretion of IL-1β enhances inflammation, eventually leading to tissue damage (Bergsbaken, Fink, & Cookson, 2009).

The mechanism of IL-1β secretion is very complex. The process includes two main steps: the induction of pro-IL-1β and the maturation of IL-1β conducted by caspase-1 (Franchi, Eigenbrod, Munoz-Planillo, & Nunez, 2009). The activation of caspase-1 requires the involvement of the inflammasome (Martinon & Tschopp, 2007). The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), an important component of the inflammasome, can recognize many various microbial stimuli, including muramyl dipeptide (MDP), lipopolysaccharides (LPS), bacterial RNA and other stimuli (Kanneganti, Lamkanfi, & Nunez, 2007). During this activation, a NOD-like receptor (NLR) family member binds to procaspase-1 through the apoptosis-associated speck-like protein (ASC) (Park et al., 2014). The inflammasome complex is assembled by pyrin domain (PYD)-containing NLR family members and ASC; then, it recruits and activates caspase-1 (Schroder & Tschopp, 2010). NLRP3 is not involved in the cellular response to cytosolic double-stranded DNA, which is mainly recognized by absent in Melanoma 2 (AIM2) and ASC (Roberts et al., 2009).

There have been many recent advances in our knowledge concerning the role of the inflammasome in autoimmune disease and tumours (Lukens, Dixit, & Kanneganti, 2011; Shaw, McDermott, & Kanneganti, 2011). Some studies have reported that inflammasomes are involved in the progression of periodontal inflammation (Bostanci et al., 2009; Xue, Shu, & Xie, 2015). However, few studies are available on whether inflammasomes contribute to the pathogenesis of apical periodontitis. Thus, the objective of this research was to analyse the expression profiles and distributions of NLRP3 and AIM2 in periapical lesions and to investigate the diversity of the extraradicular microbial community, and to determine whether the products of the most commonly detected species in these lesions can activate inflammasomes.