Nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5 affects the progression of periodontitis by regulating the function of periodontal membrane cells

Abstract

Background/purpose: Nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5 (NLRC5) plays a regulatory role in innate and adaptive immunity. However, its role in periodontitis remains unclear. This study investigated the effects of NLRC5 on periodontitis and the underlying mechanism.

Materials and methods: Experimental periodontitis models of wild-type and Nlrc5 knockout mice were established to detect alveolar bone loss. The inflammatory environment was established with Porphyromonas. gingivalis lipopolysaccharide (P. gingivalis LPS). The expression of NLRC5 in periodontal ligament stem cells (PDLSCs) were detected with P. gingivalis LPS stimulated. After knocking-down or overexpressing the NLRC5 expression level, the inflammatory cytokine level and osteogenic ability of PDLSCs were detected.

Results: The Nlrc5 knockout mice exhibited greater alveolar bone loss in periodontitis. In the presence of P. gingivalis LPS, the expression of NLRC5 decreased. Downregulating NLRC5 increased the expression of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α). Upregulated NLRC5 inhibited nuclear factor kappa-B (NF-κB) signaling and inhibited the expression of those proinflammatory factors. NLRC5 had a positive regulatory effect on the osteogenic differentiation of PDLSCs. When NLRC5 was knocked down, the ALP activity and the number of mineralized nodules in PDLSCs decreased. Conversely, overexpression of NLRC5 enhanced the osteogenic differentiation ability of PDLSCs. Overexpression of NLRC5 increased the osteogenic differentiation of PDLSCs in inflammatory environments.

Conclusion: NLRC5 affects the progression of periodontitis by regulating the function of PDLSCs. NLRC5 reduced the expression of inflammatory factors by inhibiting NF-κB, and had a positive regulatory effect on the osteogenic differentiation of PDLSCs.

Keywords: Inflammatory environments; NLRC5; Osteogenic differentiation; PDLSCs; Periodontitis.

Figure 1

Absence of NLRC5 exacerbated alveolar bone loss (A) Measurement sites for bone loss in experimental periodontitis models. (B) Maxillary micro-CT results of WT and Nlrc5^−/− mice. (C) CEJ-ABC distance of WT and Nlrc5^−/− mice in the control group. (D) The CEJ-ABC distance in WT and Nlrc5^−/− mice in the periodontitis group. WT, wild type; Nlrc5, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5; CEJ, cemento-enamel junction; ABC, alveolar bone crest.

Figure 2

P. gingivalis LPS stimulation reduces NLRC5 levels, and NLRC5 knockdown promotes the expression of P. gingivalis LPS-induced inflammatory factors in PDLSCs. (A) The expression level of NLRC5 decreased when PDLSCs were stimulated with P. gingivalis LPS for 4 h. (B–D) The mRNA levels of IL-1β, IL-6, and TNF-α were further increased in the siNLRC5 group (P = 0.001, 0.001, and 0.030, respectively) of PDLSCs treated with P. gingivalis LPS. (E) There was no significant change in the expression level of IL-8 in the siNLRC5 group (P = 0.397). (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). NLRC5, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5; NC, negative control; P. gingivalis LPS, Porphyromonas gingivalis lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor-α.

Figure 3

NLRC5 inhibited the secretion of inflammatory factors by inhibiting the NF-κB pathway. (A) Overexpression of NLRC5 alone did not affect the expression of NF-κB p-p65 in PDLSCs. (B) At 15 min to 8 h after P. gingivalis LPS treatment, the p-p65 level in the NLRC5-overexpressing group was significantly lower than that in the NC group. (C–E) The mRNA levels of IL-1β, IL-6, and TNF-α were further decreased in NLRC5-overexpressing PDLSCs (P = 0.001, 0.001, 0.003, respectively) following P. gingivalis LPS treatment. (F) There was no significant change in the expression level of IL-8 in the NLRC OE group (P = 0.817). (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). NLRC5, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5; NC, negative control; OE, overexpression group; P. gingivalis LPS, Porphyromonas gingivalis lipopolysaccharide; p-p65, phospho–NF–κB p65; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Silencing NLRC5 reduces the osteogenic differentiation ability of PDLSCs. (A) siRNA transfection efficiency; green fluorescence indicates successful transfection. (B) The expression of NLRC5 (P = 0.004) and Runx2 (P = 0.020) in PDLSCs was significantly decreased by si-NLRP5. (C) and (D) ALP activity (P = 0.014) and the number of mineralized nodules (P = 0.002) decreased in response to siNLRC5. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). NLRC5, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5; Si, small interfering RNA; NC, negative control; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; ARS, alizarin red staining; FITC, fluorescein isothiocyanate.

Figure 5

Overexpression of NLRC5 enhances osteogenic differentiation of PDLSCs. (A) Adv5-NLRC5 transfection efficiency; green fluorescence indicates successful transfection. (B) The expression of NLRC5 (P = 0.001) and Runx2 (P = 0.004) in PDLSCs was significantly increased by treatment with Adv5-NLRC5. (C) and (D) ALP activity (P = 0.001) and the number of mineralized nodules (P = 0.021) were increased by Adv5-NLRC5 interference. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). NLRC5, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5; NC, negative control; OE, overexpression group; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; ARS, alizarin red staining; FITC, fluorescein isothiocyanate.

Figure 6

Overexpression of NLRC5 enhanced the osteogenic differentiation of PDLSCs in inflammatory environments. (A) ALP-positive cells and ALP activity were significantly greater in the NLRC5 OE group than in the NC group. (P = 0.001). (B) The mineralized nodules of PDLSCs and the ARS intensity (P = 0.001) in the NLRC5 OE group were also significantly greater than those in the NC group. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). NLRC5, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5; NC, negative control; OE, overexpression group; ALP, alkaline phosphatase; ARS, alizarin red staining.

Fig. S1

Supplementary material 1: Effect of NLRC5 on the viability of PDLSCs. NLRC5, nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing protein 5; NC, negative control; OE, overexpression group.