Contribution of Porphyromonas gingivalis lipopolysaccharide to experimental periodontitis in relation to aging

Abstract

Aging is associated with increased prevalence and severity of pathogenic outcomes of periodontal disease, including soft tissue degeneration and bone loss around the teeth. Although lipopolysaccharide (LPS) derived from the key periodontal pathogen Porphyromonas gingivalis (Pg) plays an important role in the promotion of inflammation and osteoclastogenesis via toll-like receptor (TLR)4 signaling, its pathophysiological role in age-associated periodontitis remains unclear. This study investigated the possible effects of Pg-LPS on RANKL-primed osteoclastogenesis and ligature-induced periodontitis in relation to aging using young (2 months old) and aged (24 months old) mice. To the best of our knowledge, our results indicated that expression of TLR4 was significantly diminished on the surface of osteoclast precursors isolated from aged mice compared with that of young mice. Furthermore, our data demonstrated that the TLR4 antagonist (TAK242) dramatically decreased the numbers of tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts differentiated from RANKL-primed young osteoclast precursors (OCPs) compared with those isolated from aged mice in response to Pg-LPS. In addition, using a ligature-induced periodontitis mouse model, we demonstrated that Pg-LPS elevated (1) secretion of senescence-associated secretory phenotype (SASP) markers, including the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, as well as osteoclastogenic RANKL, and (2) the number of OCPs and TRAP+ osteoclasts in the periodontal lesion induced in young mice. In contrast, Pg-LPS had little, or no, effect on the promotion of periodontitis inflammation induced in aged mice. Altogether, these results indicated that periodontal disease in older mice occurs in a manner independent of canonical signaling elicited by the Pg-LPS/TLR4 axis.

Keywords: Aging; Bone loss; Osteoclastogenesis; Periodontitis; Porphyromonas gingivalis lipopolysaccharide.

Fig. 1

Age-dependent expressions of Pg-LPS receptors, TLR2, and TLR4, on the surface of CD11b+CSF1r-eGFP+ OCPs. Osteoclast precursors (OCPs) were FACS sorted from the spleen of young (2 months old) and aged (24 months old) CSF1r-eGFP-KI mice and then were stimulated with macrophage colony-stimulating factor (M-CSF) (30 ng/ml) and RANKL (5 ng/ml). After 24 h of stimulation, the expression of TLR2 (a and b) and TLR4 (c and d) was evaluated by multicolor flow cytometry. ***p < 0.001

Fig. 2

The effects of Pg-LPS and TLR’s antagonists on RANKL-induced osteoclastogenesis in relation to aging. Microscopic evaluation (a) of the TRAP staining and quantification of the number of TRAP+ multinucleated cells (b) in the RANKL-stimulated young and aged OCPs exposed to Pg-LPS in vitro. Effects of TLR4 (CAS 243984-11-4) and TLR2 antagonists (CU-CPT22) on young (c) and aged (d) RANKL-stimulated OCPs exposed to Pg-LPS. OCPs were FACS sorted from the spleen of young and aged mice and then were stimulated with RANKL in the presence or absence of various concentrations of Pg-LPS as well as TLR2 and TLR4 antagonists. The cells were evaluated at day 6 after stimulation. **p < 0.01, ***p < 0.001

Fig. 3

Age-dependent impact of Pg-LPS on the incidence of CD11b+CSF1r-eGFP+ osteoclast precursors (OCPs) and the number of TRAP+ multinucleated cells in periodontal lesions induced in young versus aged CSF1r-eGFRP-KI mice. Representative contour plots and percentage of CD11b+CSF1r-eGFP+ OCPs (a and b) and histological evaluation of TRAP+ osteoclasts (c and d) in periodontal lesions of young and aged CSF1r-eGFP-KI mice that received: control (no ligature), ligature alone, and ligature/local gingival injection of Pg-LPS. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Age-dependent role of Pg-LPS on secretion of some pro-inflammatory SASP cytokines, including TNF-α (a), IL-1β (b), IL-6 (c), and osteoclastogenic factor, RANKL (d) in periodontal lesions of young and aged wild-type mice. Gingival tissues were sampled from the inflamed periodontal sites of young and aged mice and then were evaluated by ELISA. *p < 0.05, **p < 0.01; ***p < 0.001