Expression and regulation of the NALP3 inflammasome complex in periodontal diseases

Abstract

Periodontitis is an infectious process characterized by inflammation affecting the supporting structures of the teeth. Porphyromonas gingivalis is a major oral bacterial species implicated in the pathogenesis of periodontitis. Processing of interleukin (IL)-1 family cytokines is regulated by an intracellular innate immune response system, known as the NALP3 [nacht domain-, leucine-rich repeat-, and pyrin domain (PYD)-containing protein 3] inflammasome complex. The aim of the present study was to investigate by quantitative real-time polymerase chain reaction (PCR) the mRNA expression of NALP3, its effector molecule apoptosis associated speck-like protein (ASC), its putative antagonist NLRP2 (NLR family, PYD-containing protein 2), IL-1beta and IL-18 (i) in gingival tissues from patients with gingivitis (n = 10), chronic periodontitis (n = 18), generalized aggressive periodontitis (n = 20), as well as in healthy subjects (n = 20), (ii) in vitro in a human monocytic cell line (Mono-Mac-6), in response to P. gingivalis challenge for 6 h. The clinical data indicate that NALP3 and NLRP2, but not ASC, are expressed at significantly higher levels in the three forms of inflammatory periodontal disease compared to health. Furthermore, a positive correlation was revealed between NALP3 and IL-1beta or IL-18 expression levels in these tissues. The in vitro data demonstrate that P. gingivalis deregulates the NALP3 inflammasome complex in Mono-Mac-6 cells by enhancing NALP3 and down-regulating NLRP2 and ASC expression. In conclusion, this study reveals a role for the NALP3 inflammasome complex in inflammatory periodontal disease, and provides a mechanistic insight to the host immune responses involved in the pathogenesis of the disease by demonstrating the modulation of this cytokine-signalling pathway by bacterial challenge.

Fig. 1

NALP3 [nacht domain-, leucine-rich repeat-, and pyrin domain (PYD)-containing protein 3] inflammasome complex expression levels in periodontal diseases. Distribution of NALP3 (a), NLRP2 (NLR family, PYD-containing protein 2) (b), ASC (c), interleukin (IL)-1β (d) and IL-18 (e) gene expression in gingival tissues of healthy subjects (n = 10) and patients with gingivitis (n = 10), chronic periodontitis (CP) (n = 18) and generalized aggressive periodontitis (G-AgP) (n = 20). The mRNA expression levels were measured by quantitative polymerase chain reaction (qPCR) analysis, normalized against the expression levels of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and 18S RNA.

Fig. 2

Scatter-plot showing the correlation between the NALP3 [nacht domain-, leucine-rich repeat-, and pyrin domain (PYD)-containing protein 3] and interleukin (IL)-1β (a) or IL-18 (b) mRNA expression in gingival tissues from all subjects (n = 58).

Fig. 3

Regulation of NALP3 [nacht domain-, leucine-rich repeat-, and pyrin domain (PYD)-containing protein 3] inflammasome complex expression by Porpyromonas gingivalis. Mono-Mac-6 cell cultures were challenged with ascending protein concentrations of P. gingivalis culture supernatant for 6 h. The mRNA expression levels of NALP-3 (a), NLRP2 (NLR family, PYD-containing protein 2) (b), ASC (c), interleukin (IL)-1β (d) and IL-18 (e) were measured by quantitative polymerase chain reaction (qPCR) analysis and normalized against the expression levels of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and ubiquitin C (UBC). Bars represent mean values ± standard error of the mean from three independent experiments. The asterisk indicates the groups that were significantly different to the control group (P < 0·05).