NLRP12 Alleviated Periodontal Destruction via Suppressing Nuclear Factor Kappa-B Signalling Pathway

Abstract

Objective

Periodontal ligament stem cells (PDLSCs) have been shown to demonstrate robust tissue regeneration capabilities. NACHT, leucine-rich repeat and PYD-containing 12 (NLRP12) could exerts negative inflammatory regulatory role in various pathological contexts. The effects of NLRP12 on the inflammatory responses and osteogenesis of PDLSCs under inflammatory microenvironment and underlying mechanisms remain unknown.

Methods

PDLSCs were isolated, cultured, and characterized, and the expression level of NLRP12 was analysed following lipopolysaccharide (LPS) treatment. Then, NLRP12 was overexpressed in PDLSCs via lentivirus transfection. Inflammatory factors were detected by quantitative real-time polymerase chain reaction, osteogenic markers were quantified using quantitative real-time polymerase chain reaction, Western blot, and the capability of osteogenesis was assessed through alkaline phosphatase staining and alizarin red staining. The underlying signalling pathways were examined by Western blot analysis and agonist treatment. Moreover, rat periodontitis models established by ligature method were used to evaluate the regulatory effects of NLRP12 on PDLSC-mediated anti-inflammation and periodontal regeneration in vivo.

Results

PDLSCs exhibited mesenchymal stem cell characteristics with multilineage differentiation. NLRP12 was downregulated in LPS-treated PDLSCs. LPS upregulated inflammatory factor expression while downregulating anti-inflammatory factors and suppressing PDLSCs’ osteogenic differentiation, which were reversed by NLRP12 overexpression. Mechanistically, nuclear factor kappa-B signalling pathway participated in these processes. Furthermore, microscopic computed tomography scanning, histological analysis, and immunohistochemical staining revealed that NLRP12 ameliorated inflammation and enhanced periodontal regeneration in periodontitis animal models.

Conclusions

NLRP12 suppressed inflammatory responses while enhancing osteogenic differentiation of PDLSCs via downregulating the activation of the nuclear factor kappa-B pathway, indicating that NLRP12 is a latent target for enhancing PDLSCs-based periodontal tissue regeneration.

Introduction

As an inflammatory disease triggered by microbial plaque, periodontitis is characterized by the continuous destruction of the periodontal supporting tissues and eventually leads to tooth loss.1, 2, 3, 4, 5, 6, 7 In recent years, due to their multipotent differentiation capabilities and excellent immunomodulatory properties, periodontal ligament stem cells (PDLSCs) have shown profound potentials in the field of periodontal tissue regeneration.8, 9, 10 However, in the inflammatory microenvironment, the biological properties of PDLSCs have undergone significant changes, not only showing marked declines in proliferation, self-renewal, and osteogenic differentiation abilities, but also accompanied by impaired immunomodulatory functions, becoming inflammatory PDLSCs.^11,12 Previous studies have confirmed that lipopolysaccharide (LPS) could inhibit the osteogenic differentiation of PDLSCs and stimulate the release of proinflammatory cytokines.^13,14 on which the nuclear factor kappa-B (NF-κB) signalling pathway was illustrated to have a crucial regulatory impact, which is responsible for LPS-mediated inflammatory responses and osteogenic differentiation inhibition of PDLSCs.^14,15

Nucleotide-binding leucine-rich repeat-containing proteins, or NOD-like receptors (NLRs), are heterogeneous oligomeric proteins within cells that induces the production of proinflammatory cytokines by recognizing signals of infection or tissue damage, thereby triggering inflammation and promoting tissue and organ repair. The abnormal activation and dysfunction of NLR inflammasomes, such as NLRP1 and NLRP3, are often considered the main participants in chronic inflammatory diseases and a health threat. As one of the key members of the NLR family, NACHT, leucine-rich repeat and PYD-containing 12 (NLRP12) is capable of exerting a negative inflammatory modulatory role mainly through the NF-κB signalling pathway in a variety of inflammatory bone loss diseases, such as periapical periodontitis and rheumatoid arthritis.16, 17, 18 However, the effects of NLRP12 on PDLSCs, especially the inflammatory responses and osteogenesis of PDLSCs under the inflammatory microenvironment, are still unclear. In this study, we explored these effects and the underlying mechanisms, and the possible role of NLRP12 in refining of periodontitis. Therefore, our present study explored the changes of NLRP12 expression levels in vitro by treating PDLSCs with LPS to mimic the inflammatory microenvironment, and how the changes affected the inflammatory responses and osteogenic differentiation of PDLSCs was further explored. In addition, we linked the NF-κB signalling pathway to this process to investigate the specific mechanism by which NLRP12 affects the inflammatory response and osteogenic differentiation of PDLSCs. Finally, we used PDLSCs overexpressing NLRP12 to intervene in periodontitis rats to validate the role of NLRP12 overexpression in the therapeutic effect of PDLSCs in experimental periodontitis in rats. The present study aimed to examine the effects and possible mechanisms of NLRP12 on the inflammatory response and osteogenic differentiation of PDLSCs in the inflammatory microenvironment through in vitro and in vivo, so as to provide a potential target for the prevention and treatment of periodontitis.

Materials and methods

Culture and characterization of PDLSCs

Current research was approved by the Medical Ethics Committee of Shandong Second Medical University (No. 2023YX065). After healthy donors aged 18 to 24 at the Department of Oral and Maxillofacial Surgery (Supplementary Table 1), the Affiliated Hospital of Shandong Second Medical University provided written informed consent. The donor information are listed in Supplementary Table 1. The extracted third molars without caries, pulpitis, or periodontitis were collected and rinsed with phosphate‐buffered saline (PBS) (Biosharp). The middle third of the periodontal tissues on the root surface of the teeth were scraped, minced, and attached to the bottom of the culture flask. After the tissues were incubated for 3 hours at 37°C in a 5% CO2 incubator, the complete culture medium containing alpha-minimal essential medium (α-MEM) (Basal Media), 10% foetal bovine serum (FBS) (Gibco), and 10% penicillin and streptomycin solution (Beyotime) was added to the culture flask. Cells were passaged after reaching 80% confluence at a ratio of 1:2, and cells at passages 4 were used for the experiments.

Then, flow cytometry was conducted to detect cell surface markers of the cells. The BD Human Mesenchymal Stem Cell Surface Marker Assay Kit (BD Biosciences) was used according to the manufacturer’s instructions. Cells were incubated with CD14, CD29, CD34, CD45, CD73, CD90, CD105, CD146, and Stro-1 antibodies for 30 minutes at 4°C in the dark, then the cells were analysed by flow cytometry.

The PDLSCs were seeded in 6-well plates (2 × 10⁵ cells/well), and osteogenic medium was added for osteogenic induction. The osteogenic medium consists of α-MEM, 10% FBS, 10 nM dexamethasone (Solarbio), 10 mM β-glycerophosphate (Solarbio), and 50 mg/L ascorbic acid (Solarbio). After 28 days of osteogenic induction, the cells were stained with Alizarin Red.

The PDLSCs were seeded in 6-well plates (2 × 10⁵ cells/well), and adipogenic medium was added for adipogenic induction. The adipogenic medium consists of α-MEM, 10% FBS, 1 μM dexamethasone (Solarbio), 0.2 mM indomethacin (Solarbio), 0.01 g/L insulin (Solarbio), and 0.5 mM isobutylmethylxanthin (Solarbio). After 21 days of adipogenic induction, the cells were stained with oil red O.

Cell proliferation assays

Cell proliferation was examined with the CCK-8 kit (Solarbio). The PDLSCs (3 × 10³ cells/well) were seeded into the 96-well plates, and were triggered with complete medium containing 0, 1, 10, 20 and 50 μg/mL LPS for 2, 4, and 6 days, respectively. Subsequently, 100 μL of CCK-8 working solution (CCK-8 solution: blank medium = 1: 9) was added to each well, and the plate were incubated at 37°C, 5% CO₂ for 3 hours. Then the plate was measured using a microplate reader (Shanpu) with the absorbance at 450 nm.

Cell treatment

The culture medium of PDLSCs was supplemented with LPS (Sigma-Aldrich) (1-50 µg/mL) for 1 day. For some assays, phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) was used to stimulate PDLSCs. Cells were pretreated with 70 ng/mL PMA for 1 hour; subsequently, the medium was replaced with complete medium containing 10 μg/mL LPS and 70 ng/mL PMA for 1 day.

Lentiviral transduction

The lentiviral overexpression vector for human NLRP12 (Gene ID: 91662; transcript NM_144687.4) was purchased from GeneChem Co., Ltd. The construct (GV492/pGC-FU-3FLAG-CBh-gcGFP-IRES-puromycin) drives the expression of C-terminal 3 × FLAG-tagged NLRP12 under the Ubc promoter, and coexpresses enhanced green fluorescent protein (GFP) and a puromycin resistance gene via an IRES element for the selection of stably transduced cells. The viral titre was 3.5 × 10⁸ TU/mL. PDLSCs (1.2 × 10⁶ cells/well) were seeded into 6-well plates, and 24 hours after cell incubation, PDLSCs were transfected with the NLRP12-overexpressing lentivirus (oeNLRP12) or control lentivirus (oeNC) at a multiplicity of infection of 50 in the presence of 5 μg/mL HiTransG P (GeneChem), followed by a 16-hour incubation period. The culture medium was replaced 72 hours after transfection. Fluorescence microscopy (Zeiss) revealed that approximately 50% of PDLSCs exhibited successful transfection. The medium was subsequently changed to one containing 2 μg/mL puromycin (#ST551; Beyotime) to obtain the stably transfected NLRP12 overexpression cell line, which was used in follow-up experiments in vitro and in vivo. The efficiency of transfection was verified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis.

qRT-PCR

Total RNA was extracted from PDLSCs using AG RNAex Pro Reagent (Accurate Biology). The cDNA was obtained using the Evo M-MLV RT Kit with gDNA Clean for qRT-PCR (Accurate Biology). The mRNA levels were qualified by real-time PCR using AG SYBR Green Premix Pro Taq HS qRT-PCR Kit (Accurate Biology) according to the manufacture’s instruction on a FQD86A machine (Bioer Technology). The cycling conditions were as following: 95°C for 30 seconds, 40 cycles of 95°C for 5 seconds, and 60°C for 30 seconds. Then the 2^^–ΔΔCt method was used to quantify the relative mRNA expression of genes normalized to GAPDH. The primer sequences are listed in Supplementary Table 2.

WB analysis

Total proteins were extracted using RIPA lysis buffer (Solarbio) containing protease inhibitor and phosphatase inhibitor (Solarbio). The protein concentration was measured using a BCA Kit (Solarbio). Protein samples were denatured at 100°C for 5 min with sodium dodecyl sulphate polyacrylamide gel electrophoresis loading buffer (Beyotime). Then, 30 µg of each protein sample was separated through 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Merck Millipore). After blocking with rapid sealing fluid for 30 minutes, the membranes were incubated with primary antibodies, including COL1 (#12256; CST), RUNX2 (#8486; CST), NLRP12 (#DF14960; Affinity Biosciences), phospho-p65 (p-p65) (#3033; CST), p65 (#8242; CST), p-IκBα (#2859; CST), IκBα (#4814; CST), GAPDH (#60004; Proteintech) overnight at 4°C. After binding with secondary HRP-labelled Goat Anti-Rabbit IgG (H+L) (#A0208; Beyotime) or HRP-labelled Goat Anti-Mouse IgG (H+L) (#A0216; Beyotime) for 2 hours at room temperature, the protein bands were tested by the imaging system (Bio-Rad) with enhanced chemiluminescent ECL reagent (Biosharp). The grey values were quantified by ImageJ software (v1.53).

Immunofluorescence staining

The PDLSCs (1 × 10⁵ cells/well) were seeded in 12-well plates, and treated at various different conditions. These cells were fixed with 4% paraformaldehyde, incubated with 0.5% TritonX-100, and blocked with 5% bovine serum albumin, then the cells incubated with primary antibodies NLRP12 (#DF14960; Affinity) overnight at 4°C and incubated with fluorescent secondary antibodies Goat Anti-Rabbit IgG/FITC (#SF134; Solarbio) for 1 hour at room temperature, avoiding light exposure. After staining with DAPI (Solarbio) for 10 minutes at room temperature in the dark, the cells were observed and captured with a fluorescence microscope (Olympus).

Animals models

Animal experiments were approved by the Animal Ethics Committee of Shandong Second Medical University (No. 2025SDL413) and conformed to ARRIVE checklist. A total of 24 male SD rats (8-week-old, 270-300 g) were purchased from Pengyue Laboratory Animal Breeding Co., Ltd. All animal were housed in specific pathogen-free conditions with constant temperature and humidity, with a 12-hour light/dark cycle and fed with sufficient diet and water.

The rats were randomly divided into four groups (n = 6): (1) The control group: without treatment; (2) the periodontitis group: ligature-induced experimental periodontitis model; (3) oeNC PDLSCs group: the periodontitis models treated with oeNC PDLSCs; (4) oeNLRP12 PDLSCs group: periodontitis models treated with oeNLRP12 PDLSCs.

After 1 week of acclimation, the rats were anaesthetized with the intraperitoneal injection of 3% pentobarbital sodium (40 mg/kg body weight). The 4-0 nonabsorbable surgical suture (Ming’an Kang) was inserted between the first and second molar in the left maxillary to generate periodontitis models for 4 weeks, and the ligatures were checked every other day to ensure they remained in place during the experiment.¹⁹ After 4 weeks of ligation, the ligature suture was removed. According to the preliminary experiments, 1 × 10⁶ PDLSCs diluted in 20 μL PBS was injected into gingival sulcus on the buccal and palatal sides between the left maxillary first and second molars, as well as at the middle of the first and second molars in rats, respectively, according to the group. The periodontitis group was injected with the same volume of PBS. The rats were injected once a week for a total of 2 times. Two weeks later, the rats were sacrificed, and the specimens were collected.

Microscopic computed tomography (micro-CT) analysis

The maxillae of rats were dissected and fixed in 4% paraformaldehyde for 24 hours, rinsed with PBS, then scanned with a Skyscan 1172 micro-CT system (Bruker MicroCT). 3D image reconstruction was performed using software (Brucker MicroCT) to assess alveolar bone resorption between the first and second molars in the left maxillae of rats.

Histological and immunohistochemical analysis

The maxilla specimens were decalcified at room temperature with 10% disodium ethylenediamine tetraacetate in phosphate buffer for 1 month. The specimen was dehydrated with a series of graded ethanol and dimethylbenzene and then embedded with paraffin. Serial 5 μm sections were stained with the H&E Staining Kit (Solarbio) and the Masson Trichrome Stain Kit (Solarbio), respectively. To detect multinucleated osteoclasts, tartrate-resistant acid phosphatase (TRAP) staining was performed with a TRAP kit (Servicebio).

Immunohistochemistry was performed using a standard immunoperoxidase staining procedure to detect IL-6, IL-8, IL-10, COL1, and RUNX2 expression in paraffin-embedded murine maxilla sections. The samples were deparaffinized and rehydrated in a series of graded alcohol and dimethylbenzene, and then boiled in 10 mM sodium citrate buffer (Zsbio), pH 6.0, by microwave for 10 minutes for antigen retrieval. After blocking with 10% normal goat serum (Beyotime), sections were covered overnight at 4°C with primary antibodies against IL-6 (#21865-1-AP; Proteintech), IL-8 (#94407; CST), IL-10 (#ab290735; Abcam), COL1 (#12256; CST) and RUNX2 (#8486; CST), then covered with secondary antibodies Goat Anti-Rabbit IgG (Zsbio), and then stained with DAB (Zsbio) for 20 to 60 seconds and counterstained with haematoxylin.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 9 software (GraphPad Software). All quantitative results are expressed as mean ± standard deviation, and t test was applied for comparison between two groups. The One-way and two-way ANOVA with Tukey post hoc was applied for comparison between multiple groups. A value of P < .05 was considered statistically significant.

Results

Characterization of PDLSCs

In order to get primary PDLSCs, we used tissue explant culture methods (Figure 1A). The flow cytometry analysis showed that the PDLSCs expressed mesenchymal stem cell markers such as CD29, CD73, CD90, CD105, CD146, and Stro-1, but did not express hematopoietic lineage markers such as CD14, CD34, and CD45 (Figure 1B). With osteogenic induction for 28 days, alizarin red staining-positive mineralized nodules were observed (Figure 1C), confirming the osteogenesis potential of PDLSCs. With adipogenic induction for 21 days, lipid droplets with oil red O staining-positive were observed (Figure 1D), confirming the adipogenesis potential of PDLSCs. All above results indicated that the PDLSCs maintained the characteristics of mesenchymal stem cells.

Fig. 1.

The characterization of PDLSCs. (A) Representative images of PDLSCs showed the isolation and culture of PDLSCs (scale bar = 500 μm). (B) Flow cytometry analyses showed that the PDLSCs positively expressed mesenchymal stem cell markers such as CD29 (100%), CD73 (99.9%), CD90 (100%), CD105 (78.6%), CD146 (95.5%) and Stro-1 (26.4%), but did not express hematopoietic lineage markers such as CD14 (2.36%), CD34 (2.99%) and CD45 (3.54%). (C) Representative images of alizarin red staining-positive mineralized nodules were observed, which is pointed using black arrow (scale bar = 500 μm). (D) Representative images of oil red O-positive lipid droplets were observed, which is pointed using white arrow (scale bar = 50 μm).

LPS triggered an inflammatory response and suppressed the osteogenic differentiation potential of PDLSCs

To assess the effects of LPS concentration on PDLSCs’ viability, cells were exposed to LPS at concentrations of 0, 1, 10, 20, and 50 μg/mL for 2, 4, and 6 days separately with the CCK-8 assay. The results showed that the proliferation activity of PDLSCs treated with each concentration of LPS did not show significant differences compared with the control group (Figure 2A). qRT-PCR showed that LPS significantly upregulated IL-6 expression in a concentration-dependent manner. However, there were no statistically significant differences in the expression level of this proinflammatory cytokine among the 10, 20, and 50 μg/mL LPS groups (Supplementary Figure 1). Combined with the qRT-PCR results and relevant literature,²⁰ we chose 10 μg/mL LPS for subsequent experiments. To induce the inflammation condition, PDLSCs were stimulated with LPS treatment for 24 hours, and qRT-PCR results showed that compared with the control groups, the gene expression levels of IL-6 and IL-8 in the LPS group were significantly increased, and levels of IL-10 in the LPS group were significantly decreased (Figure 2B), which indicated that LPS could trigger the inflammatory response in PDLSCs. Furthermore, qRT-PCR and WB analysis revealed significantly reduced expression of the osteogenic markers COL1 and RUNX2 at transcriptional and protein (Figure 2C,D) levels following LPS-treated PDLSCs. In addition, the pictures of ALP staining appeared that the group with LPS disposing had lower staining than the NC group (Figure 2E). Alizarin red staining showed that lighter staining and fewer the mineralized nodules were observed in the LPS group than those in the NC group (Figure 2F). These results confirmed that the osteogenic differentiation of PDLSCs was restrained with LPS exposing.

Fig. 2.

LPS triggered an inflammatory response and suppressed the osteogenic differentiation potential of PDLSCs. (A) The proliferative capacity of PDLSCs exposed to varying LPS concentrations over 2, 4, and 6 days was measured with CCK-8 assay. (B) The qRT-PCR analysis revealed elevated mRNA expression of inflammatory mediators IL-6, IL-8, and IL-10 in PDLSCs following treatment with 10 µg/mL LPS. The qRT-PCR (C) and WB (D) analysis revealed significantly lower expression of osteogenic markers (COL1 and RUNX2) in PDLSCs exposed to 10 µg/mL LPS. The ALP (E) and alizarin red (F) staining assays showed that 10 µg/mL LPS significantly inhibited the osteogenic differentiation capacity of PDLSCs compared to the control group. Data were presented as mean ± SD (n = 3). ***P < .001, ****P < .0001.

LPS downregulated the expression of NLRP12 via activating NF-κB signalling pathway

To assess the expression levels of NLRP12 under inflammatory conditions, PDLSCs were treated with LPS for 24 hours. Immunofluorescence staining revealed a significant reduction in NLRP12 expression following LPS stimulation compared with the control group (Figure 3A). The qRT-PCR and WB analysis showed a significant reduction in NLRP12 expression at both transcriptional and protein levels following LPS-treated PDLSCs (Figure 3B,C). WB results demonstrated that the expression levels of p-p65 and p-IκBα increased in PDLSCs after treatment with LPS (Figure 3D). These results indicated that LPS suppressed the expression of NLRP12 and promoted the activation of the NF-κB signalling pathway in PDLSCs.

Fig. 3.

LPS inhibited the expression of NLRP12 and promoted the activation of the NF-κB signalling pathway in PDLSCs. (A) The expression level of NLRP12 in PDLSCs after LPS treatment was detected by immunofluorescence staining (scale bar = 50 μm). The qRT-PCR (B) and WB (C) analysis revealed significantly reduced expression of NLRP12 in PDLSCs after treatment with 10 µg/mL LPS. (D) WB analysis demonstrated that the expression of p-p65 and p-IκBα was significantly elevated relative to GAPDH in PDLSCs treated with 10 µg/mL LPS. Data were presented as mean ± SD (n = 3). ***P < .001, ****P < .0001.

NLRP12 overexpression reduced the inflammatory response and mitigated the LPS-induced suppression of osteogenic differentiation in PDLSCs

To examine how NLRP12 influences inflammatory response and osteogenic differentiation in PDLSCs, we overexpressed NLRP12 using lentiviral transfection coupled with GFP. Microscopy revealed that over 80% of cells exhibited green fluorescence following lentiviral transfection and subsequent puromycin selection (Supplementary Figure 1). qRT-PCR and WB analysis revealed significantly higher NLRP12 expression in the lentivirus-overexpressing NLRP12 group compared to the lentivirus control group (Figure 4A,B), confirming successful NLRP12 overexpression in PDLSCs. The qRT-PCR assays revealed the expression levels of proinflammatory factors IL-6 and IL-8 were significantly higher in the oeNC + LPS group than in the oeNC group, while the expression level of anti-inflammatory factor IL-10 was lower in the oeNC + LPS group than in the oeNC group. However, the expression levels of proinflammatory factors IL-6 and IL-8 were significantly lower, and anti-inflammatory factor IL-10 was higher in the oeNLRP12 + LPS group than in the oeNC + LPS group (Figure 4C). Moreover, WB analysis revealed reduced protein expression of COL1 and RUNX2 in the oeNC + LPS group compared to the oeNC group, which were significantly mitigated in the oeNLRP12 + LPS group (Figure 4D). In addition, ALP staining showed that the staining in the oeNC + LPS group was lighter compared to the oeNC group. After overexpression of NLRP12, the staining in the oeNLRP12 + LPS group was higher than that in the oeNC + LPS group (Figure 4E). Alizarin red staining showed fewer mineralized nodules in the oeNC + LPS group, which was reversed by the overexpression of NLRP12 (Figure 4F).

Fig. 4.

NLRP12 overexpression reduced LPS-induced inflammatory response and reversed LPS-induced suppression of osteogenic differentiation in PDLSCs. (A and B) The qRT-PCR and WB data revealed that expression level of NLRP12 in oeNLRP12 group was increased significantly compared to oeNC group. (C) The qRT-PCR data revealed that mRNA expression of IL-6 and IL-8 was suppressed, while the mRNA expression of IL-10 was increased in oeNLRP12 + LPS group compared to oeNC + LPS group. (D) The expression of COL1 and RUNX2 was increased significantly detected by WB assays in oeNLRP12 + LPS group compared to oeNC + LPS group. (E) Representative pictures of ALP staining showed lower staining in oeNC + LPS group compared to oeNC group and higher staining in oeNLRP12 + LPS group compared to oeNC + LPS group (scale bar = 500 μm). (F) Representative pictures of alizarin red staining showed fewer mineralized nodules in oeNC + LPS group compared to oeNC group and more mineralized nodules in oeNLRP12 + LPS group compared to oeNC + LPS group (scale bar = 500 μm). oeNC: PDLSCs transfected via negative control lentiviral. oeNLRP12: PDLSCs transfected via lentiviral with overexpression-NLRP12. oeNC + LPS: PDLSCs transfected via negative control lentiviral and subsequently cultured under 10 µg/mL LPS induction. oeNLRP12 + LPS: PDLSCs transfected via lentiviral with overexpression-NLRP12 and subsequently cultured under 10 µg/mL LPS induction. Data were presented as mean ± SD (n = 3). ns, no significant difference, ***P < .001, ****P < .0001.

Overexpression of NLRP12 reversed LPS-induced activation of the NF-κB signalling pathway, and PMA reversed these effects of NLRP12 overexpression

Overexpression of NLRP12 reduced inflammatory response and alleviated the inhibition of LPS on the osteogenesis of PDLSCs. According to the literature reviewed, we evaluated whether NLRP12 overexpression played a role in the NF-κB signalling pathway. WB results showed that the expression levels of p-p65 and p-IκBα in the oeNC + LPS group were significantly increased compared to the oeNC group, indicating that LPS activated the NF-κB signalling pathway in PDLSCs. However, the protein expression levels of p-p65 and p-IκBα in the oeNLRP12 + LPS group were significantly decreased compared to the oeNC + LPS group, suggesting that NLRP12 restrained LPS-induced activation of the NF-κB signalling pathway (Figure 5A). Furthermore, after treating LPS-stimulated oeNLRP12 cells with PMA, an NF-κB agonist, the previously decreased protein expression level of p-p65 was significantly elevated (Figure 5B). These results confirm that PMA can effectively enhance the NF-κB activation level inhibited by NLRP12 overexpression.

Fig. 5.

Overexpression of NLRP12 alleviated the inflammatory responses and osteogenic differentiation inhibition of PDLSCs by suppressing the NF-κB pathway. (A) WB results showing the changes of protein expression levels of p-p65, p65, p-IκBα, and IκBα in PDLSCs after NLRP12 overexpression. (B) WB results showing the changes of protein expression levels of p-p65 and p65 in PDLSCs overexpressing NLRP12 after PMA treatment. (C)The qRT-PCR results demonstrating the transcriptional expression levels of IL-6, IL-8, and IL-10 in PDLSCs overexpressing NLRP12 after PMA treatment. (D)WB results demonstrating alterations in the protein expression levels of COL1 and RUNX2 in PDLSCs overexpressing NLRP12 after PMA treatment. (E) Representative pictures showing ALP staining (scale bar = 500 μm) (F) Representative pictures showing alizarin red staining (scale bar = 500 μm). oeNC: PDLSCs transfected via negative control lentiviral. oeNLRP12: PDLSCs transfected via lentiviral with overexpression-NLRP12. oeNC + LPS: PDLSCs transfected via negative control lentiviral and subsequently cultured under 10 µg/mL LPS. oeNLRP12 + LPS: PDLSCs transfected via lentiviral with overexpression-NLRP12 and subsequently cultured under 10 µg/mL LPS. Data were presented as mean ± SD (n = 3). ns, no significant difference, *P < .05, **P < .01, ***P < .001, ****P < .0001.

We further evaluated the effects of PMA on the anti-inflammatory and the osteogenic promoting of NLRP12 overexpression on LPS-treated PDLSCs. qRT-PCR results showed that lower expression levels of IL-6 and IL-8 and the higher expression level of IL-10 in the oeNLRP12 + LPS group significantly were compared with those in the oeNC + LPS group. However, after activating the NF-κB pathway with PMA, the higher expression levels of IL-6 and IL-8 and the lower expression level of IL-10 in the oeNLRP12 + LPS + PMA group significantly were compared with those in the oeNLRP12 + LPS group (Figure 5C). WB analysis showed the protein expression levels of COL1 and RUNX2 were elevated in the oeNLRP12 + LPS group compared to the oeNC + LPS group. However, after treatment with PMA, the protein expression levels of COL1 and RUNX2 were decreased in the oeNLRP12 + LPS + PMA group compared with the oeNLRP12 + LPS group (Figure 5D). In addition, ALP staining (Figure 5E) and Alizarin red staining (Figure 5F) showed that activation of the NF-κB signalling pathway with PMA appeared lower staining and fewer mineralized nodules, respectively, in the oeNLRP12 + LPS + PMA group compared to oeNLRP12 + LPS group. These results revealed that overexpression of NLRP12 alleviated the inhibitory effect of LPS on PDLSCs osteogenic differentiation, which was counteracted by PMA-mediated NF-κB pathway activation.

NLRP12 overexpression in PDLSCs reduced both the inflammatory response and promoted periodontal regeneration in rats with periodontitis

To assess the impact of NLRP12 overexpression in vivo, we conducted rat periodontitis models using the silk ligature method. The experimental process flow diagram in vivo is shown in Figure 6A. Micro-CT reconstruction showed that the height of the alveolar bone was significantly reduced in periodontitis group compared to the control group. In addition, the height of the alveolar bone in the periodontitis with oeNC PDLSCs injection group was improved, and more importantly, the height of alveolar bone was significantly improved in the periodontitis with oeNLRP12 PDLSCs injection group, and the alveolar bone height was closer to that of the control group (Figure 6B). H&E staining showed that the periodontal tissues in the periodontitis group presented extensive inflammatory cell infiltration, flattened gingival papillae, obvious apical migration of the junctional epithelium, significant attachment loss, and periodontal pocket formation, accompanied by severe alveolar resorption. The periodontitis + oeNLRP12 group showed a reduction in attachment loss, a greater alveolar bone height closer to the control group, and a significant improvement in the morphology of the gingival papillae, in contrast to the oeNC PDLSCs group (Figure 6C). Masson trichrome staining showed that the collagen fibres were loosely arranged, slender, and some were broken in the periodontal tissues of periodontitis group. The degree of collagen fibre disorder was reduced in both the periodontitis + oeNC group and the periodontitis + oeNLRP12 group, and the proportion of blue-stained mature collagen fibres was increased. However, in terms of promoting new bone formation, the periodontitis + oeNLRP12 group had a better effect (Figure 6D). Additionally, these results were confirmed using analysis of CEJ–ABC distance, bone volume, loss of attachment, and Masson staining positive relative area (Figure 6E-H).

Fig. 6.

Overexpression of NLRP12 in PDLSCs alleviated the inflammatory responses and promoted periodontal regeneration in periodontitis rats. (A) The timeline demonstrated the experimental procedures. (B) The micro-CT scanning results. (C) Representative images of H&E staining (the black scale bars and white scale bars are 200 and 100 μm, respectively). (D) Representative images of Masson trichrome staining (the black scale bars and white scale bars are 200 and 100 μm, respectively). (E) Cementoenamel junction (CEJ)–alveolar bone crest (ABC) distance. (F) bone volume/total volume (BV/TV). (G) Loss of attachment in H&E staining. (H) Semiquantitative analysis of Masson trichrome staining. (I) Representative immunohistochemical images showing the expression level of inflammatory factor IL-6, IL-8, and IL-10, osteogenic factor COL1, and RUNX2, and TRAP staining showing the number of osteoclasts. Scale bar = 100 μm. (J) Quantitative analysis of IL-6-positive cells, IL-8-positive cells, IL-10-positive cells, COL1-positive cells, and RUNX2-positive cells. (K) Quantitative result of TRAP+ cells. Control group: without treatment. Periodontitis group: ligature-induced experimental periodontitis model. oeNC group: periodontitis group treated with oeNC PDLSCs. oeNLRP12 group: periodontitis group treated with oeNLRP12 PDLSCs. IHC: immunohistochemistry. IOD, integrated option density. The data are presented as mean ± SD (n = 6 rats per group). *P < .1, **P < .01, ***P < .001, ****P < .0001.

To further validate the anti-inflammation and osteogenic effects of NLRP12, the expression of inflammatory factors and osteogenic marker were evaluated. The immunohistochemical staining showed that the expression level of IL-6 and IL-8 was upregulated and IL-10 was downregulated, respectively, in the periodontitis group compared with the control group, which was reversed markedly by the PDLSCs overexpressing oeNLRP12 group (Figure 6I). In addition, the osteogenic marker, COL1 and RUNX2, was lowly expressed in periodontitis group, whereas was highly expressed in periodontitis + oeNLRP12 PDLSCs (Figure 6I,J). To further examine the osteoclastogenic effects of NLRP12, the number of osteoclasts was quantified with TRAP staining assay. The numbers of TRAP-positive osteoclasts in both the periodontitis + oeNC group and periodontitis + oeNLRP12 PDLSCs group decreased compared to periodontitis group, with lower number of osteoclasts in periodontitis + oeNLRP12 PDLSCs group, which was closer to that of the control group (Figure 6I,K).

Discussion

PDLSCs are ideal seed cells for periodontal tissue engineering and are crucial for the establishment of periodontal tissue homeostasis.²¹ Extensive studies have confirmed that the inflammatory microenvironment affects the immunomodulatory and osteogenic differentiation capabilities of PDLSCs.¹² LPS, as one of the main components of the cell wall of gram-negative bacteria, is a key virulence factor of dental plaque and could induce the infiltration of a large number of inflammatory cells in periodontal tissues.22, 23, 24 In addition, LPS is capable of upregulating the expression of multiple inflammatory factors, including IL-6 and IL-8, and the excessive production of these factors accelerates the pathological damage of periodontal tissues.²⁵ In this study, we have confirmed that LPS induced inflammatory responses in PDLSCs and inhibited osteogenesis via activating the NF-κB signalling pathway. Therefore, how to reshape the tissue regeneration ability of PDLSCs in the inflammatory microenvironment has become the focus of current research.

Although the exact role of NLRP12 remains controversial, it is clearly a negative regulator of inflammation. NLRP12 gene knockout leads to the development of severe periapical periodontitis in mice, with significantly upregulated levels of inflammatory cytokines such as IL-1, IL-6, and TNF-α in periapical lesion tissues and increased expression of osteoclast markers, indicating that NLRP12 could reduce inflammatory responses and osteoclast formation and thereby playing a bone-protective role in periapical periodontitis.¹⁸ Additionally, compared with experimental arthritis mice, arthritis symptoms are more severe in NLRP12 gene knockout arthritis mice.²⁶ Furthermore, NLRP12 overexpression could inhibit the proliferation and inflammatory responses of fibroblast-like synoviocytes in rheumatoid arthritis.¹⁶ Our results revealed that the expression level of NLRP12 significantly decreased under LPS stimulation, and NLRP12 overexpression robustly alleviated the inflammatory response and the inhibition of osteogenesis caused by LPS in PDLSCs. More importantly, PDLSCs overexpressing NLRP12 also significantly enhanced periodontal bone regeneration, promoted the anti-inflammatory factor, and decreased the inflammatory factor in rat animal models. Therefore, overexpression of NLRP12 may be a potential method to regulate the inflammatory status and protect the osteogenesis ability of PDLSCs.

We then explored the mechanism by which NLRP12 regulates the inflammatory response and osteogenesis of PDLSCs in the inflammatory microenvironment. Previous results showed that NLRP12 effectively inhibits the generation and release of proinflammatory cytokines and chemokines by negatively regulating both the classical and nonclassical NF-κB signalling pathway,^16,27,28 which is an important inflammation-related signalling pathway. In addition, the inhibition of PDLSCs’ biological functions in the inflammatory microenvironment is corresponded to the NF-κB signalling pathway.¹⁴ LPS could increase the expression of TNF-α and IL-6 and downregulate the expression of IL-10 by activating the NF-κB pathway, thereby inhibiting the proliferation and osteogenesis of PDLSCs.^14,15 LPS triggers a series of inflammatory responses through the NF-κB inflammatory signalling pathway.29, 30, 31, 32 Moreover, inhibiting the NF-κB signalling pathway can alleviate LPS-induced inflammatory damage to PDLSCs.¹⁴ Therefore, downregulating the activation of the NF-κB signalling pathway is significant for decreasing the damage of LPS to the osteogenesis ability of PDLSCs. Our findings indicated that the phosphorylation levels of p65 and IκBα upregulated under the treatment of LPS significantly. After overexpression of NLRP12, the phosphorylation levels of p65 significantly decreased, and activation of the NF-κB signalling pathway by PMA could inhibit the anti-inflammatory and osteogenic-promoting effect of NLRP12 overexpression. These results indicated that NLRP12 could regulate the inflammatory response and osteogenesis of PDLSCs through the NF-κB signalling pathway. As for LPS-mediated regulation of the expression profiles of NLRP12, macrophage polarization and proinflammatory cytokine, such as TNF-α, may be involved in this course.³³ In response to heme in the presence of activators of TLRs, such as LPS, the transcription factor IRF1 was up-regulated, and the assembly of NLRP12 PANoptosome complex containing caspase-1, caspase-8, and RIPK3 is drived.³⁴ Furthermore, as another NLR that is part of the protein complex, NLRC5 was capable of functioning as a sensor of both NAD⁺ depletion and ROS production induced by LPS and heme.^35,36 Clinically, compared to normal controls, LPS could trigger higher production of TNF-α in the cells of patients of NLRP12-associated autoinflammatory disease, which was caused by mutations in NLRP12.³⁷ As for the underlying mechanisms, NF-κB signalling pathway was regarded to be responsible for the effects, which was consistent with our data and previous report.³⁸

However, it is worth noting that whether NLRP12 affects the osteogenesis of PDLSCs by other mechanisms remains uncover. One study has shown that NLRP12 significantly inhibits the proliferation and inflammatory responses of rheumatoid arthritis fibroblast-like synoviocytes by regulating the mitogen-activated protein kinase signalling pathways.¹⁶ Another study demonstrated that NLRP12 alleviates symptoms in mice with rheumatoid arthritis through modulation of the STAT3 signalling pathway.³⁹ In future studies, we will attempt to discover the molecular mechanism of NLRP12 extensively.

Besides, in vivo experiments proved that PDLSCs could partially alleviate the inflammatory response, tissue destruction, and alveolar bone resorption of periodontal tissues in rats with periodontitis, increase the expression of osteogenic proteins, and reduce the number of osteoclasts. Overexpression of NLRP12 in PDLSCs could further enhance the therapeutic effect of PDLSCs in periodontitis. Impaired PDLSCs under periodontitis have been confirmed to participate in promoting osteoclast differentiation and maturation.¹² More exploration is needed to explain the role of PDLSCs overexpressing NLRP12 in regulating osteoclast differentiation and maturation.

Conclusion

In conclusion, NLRP12 enhanced the anti-inflammatory and osteogenic capacity of PDLSCs via NF-κB pathway (Figure 7). Therefore, NLRP12 is a latent target for improving PDLSCs-based periodontal tissue regeneration in inflammatory microenvironments.

Fig. 7.

NLRP12-mediated anti-inflammatory and osteogenic capacity of PDLSCs via NF-κB pathway.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author contributions

Shuangshuang Xu, Weiping Wang: designed the study, performed research, analysed data, and wrote the manuscript. Yuhui Sun, Juefei Wu, Wenting Zhang: performed research. Gang Ding: designed the study, analysed data, wrote the article, and supervised the study. All authors read and approved the final manuscript.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.