DOI: https://doi.org/10.21203/rs.3.rs-3806269/v1
Secretory leukocyte protease inhibitor (SLPI), mainly secreted by epithelial cells, is abundant in saliva and other mucus secretions. In healthy periodontal tissues, SLPI maintains homeostasis by modulating immune responses and inhibiting tissue destruction through its neutrophil elastase activity. Recently, decreased SLPI levels have been found in severe periodontitis when compared to healthy individuals. In this study, we hypothesized that intragingival administration of SLPI would inhibit periodontal tissue destruction caused by periodontitis. SLPI administration significantly reduced neutrophil elastase activity in periodontal tissue and alleviated alveolar bone loss in mice. Real-time PCR analysis revealed that SLPI administration downregulated the transcription of proinflammatory cytokines and osteoclast-related factors in gingival tissue. In vitro, treatment of bone marrow macrophages with SLPI resulted in the downregulation of osteoclast differentiation. SLPI also inhibited osteoclast differentiation and promoted osteoblast mineralization in vitro. These findings suggest that SLPI prevents periodontal tissue destruction by affecting inflammation and bone metabolism.
Neutrophils are the most abundant type of leukocytes in periodontal pockets and tissue 1 2. Periodontitis is a chronic inflammatory disease of the periodontium, disrupting periodontal tissue structure, often resulting in tooth loss 3 4 5 6. It is initiated by the formation of dental plaque, which is the adhesion of oral bacteria to the superficial layers of the teeth and the subsequent appearance of pathogenic species. These pathogens enhance inflammatory responses via excessive migration of neutrophils and promotion of inflammatory cytokine production in the periodontal tissue 4. Moreover, host immune cells and overexpressed inflammatory cytokines in periodontal tissues promote osteoclast activity, leading to alveolar bone resorption 7. Therefore, in addition to eliminating pathogens, it is imperative to control excessive inflammatory responses during periodontal treatment.
Neutrophils act as host defense factors by phagocytosing and degrading pathogens using the intrinsic protease neutrophil elastase (NE). However, NE leakage from activated neutrophils destroys host tissues through proteolytic activity during inflammatory reactions 8. Several studies, including ours, have reported that leaked NE in periodontal tissues exacerbates periodontitis as it disrupts the epithelial barrier by cleaving cell adhesion molecules, thereby inducing pathogen invasion and inflammatory progression9 10 11. NE activity in the gingival crevicular fluid correlates with periodontitis and may indicate the severity of inflammation12. We hypothesized that suppressing NE activity in the periodontal tissue might prevent the exacerbation of periodontitis.
Secretory leukocyte protease inhibitor (SLPI) is an 11 kDa multifunctional protein involved in the host defense response expressed by several epithelia, including the salivary glands, skin epidermis, and respiratory and digestive organs, primarily in mucosal secretions 13 14 15 16. A critical feature of SLPI is its potent inhibition of serine proteases, including NE, and its suppression of tissue injury 17 18. SLPI expression is upregulated in response to inflammatory cytokines and bacterial products, and high local levels of SLPI have been observed in chronic inflammatory diseases, including asthma and arthritis 19 20. However, clinical findings indicate that SLPI levels decrease in the saliva and gingival crevicular fluid of patients with severe chronic periodontitis 21 22. Thus, the homeostatic balance between proteases and antiprotease factors is disrupted in patients with severe periodontitis.
Our previous study suggested that the administration of NE inhibitors suppressed inflammation and alveolar bone loss in mice with periodontitis 23. In this study, we hypothesized that decreased SLPI expression exacerbates periodontitis, owing to high levels of NE activity in the periodontal tissue. We examined the effects of SLPI administration on alveolar bone loss and transcription of inflammatory cytokines and osteoclast differentiation-related factors in a gingiva of a mouse model of periodontitis. We also investigated the effects of SLPI on osteoclast differentiation and mineralization in vitro. Consequently, SLPI administration suppresses NE activity and alveolar bone resorption in a mouse model of periodontitis. SLPI also inhibits osteoclast differentiation and promotes osteoblast mineralization in vitro, suggesting its potential to inhibit periodontal tissue destruction by affecting inflammation and bone metabolism.
SLPI expression is downregulated in periodontitis tissue.
We analyzed SLPI localization in periodontal tissues using a mouse model of ligature-induced periodontitis. Immunofluorescence analysis revealed that SLPI was expressed in the periodontal ligament in the unligated group, as confirmed by fluorescence immunostaining. Additionally, the fluorescence intensity of SLPI was considerably lower in the ligated group than in the unligated group (Fig. 1a). Real-time PCR analysis showed that the transcription levels of SLPI in periodontal tissues were significantly lower in the ligated group than in the unligated group (Fig. 1b). These findings are consistent with those of previous studies in humans that reported decreased SLPI expression under periodontal inflammatory conditions 21 22.
Our previous study suggested that administration of NE inhibitors suppressed inflammation and alveolar bone loss in mice with periodontitis 23. We examined the effects of SLPI in a mouse model of ligature-induced periodontitis. The maxillary second molars were ligated but the contralateral teeth were not (baseline control). As shown in Fig. 2a, the groups of mice were treated gingivally with phosphate-buffered saline (PBS), SLPI, or the NE inhibitor daily until the day of sacrifice (day 7). Figures 2b and c demonstrate that significant bone resorption was induced in the ligated + PBS group, whereas the administration of SLPI or NE inhibitor significantly attenuated bone loss. Notably, bone resorption was significantly inhibited in the ligated + SLPI group compared to the ligated + NE inhibitor group. Although NE activity in gingival tissues significantly increased after tooth ligation, SLPI or NE inhibitor administration significantly reduced NE activity (Fig. 2d). Transcription of proinflammatory cytokines, Il6 and Il1b was increased in the gingiva of the ligated + PBS group compared to that in the unligated group (Fig. 2e). Additionally, the administration of SLPI or NE inhibitor significantly downregulated the transcription of these genes in the gingiva. These findings suggest that the intragingival administration of SLPI minimizes alveolar bone loss and downregulates the transcription of proinflammatory cytokine genes.
Next, we investigated the effects of SLPI on osteoclast differentiation in periodontal tissues. Figures 3a and b show a significant increase in the number of tartrate-resistant acid phosphatase (TRAP)-positive cells around the second molars in the ligated + PBS group compared with the unligated group. Consistent with the amount of alveolar bone resorption, the administration of SLPI or NE inhibitor significantly reduced the number of TRAP-positive cells in periodontal tissue compared with that in the ligated + PBS group. Additionally, SLPI administration significantly downregulated the transcription of nuclear factor of activated T cells c1 (Nfatc1) and Rank, which are osteoclast differentiation factors, compared to the ligated + PBS group, whereas the transcription of Acp5 and Ctsk, which are bone resorption activity-related factors, was significantly downregulated by SLPI or NE inhibitor administration (Fig. 3c). The ligated + SLPI group showed significant downregulation of the transcription of RANK compared to the ligated + NE inhibitor group. Therefore, the inhibition of bone resorption by SLPI (Fig. 2b) may be associated with a decrease in the number of osteoclasts in the periodontal ligament tissue.
SLPI suppresses osteoclast differentiation and bone resorption activity in vitro.
The ligated + SLPI group showed decreased alveolar bone resorption compares to the ligated + NE inhibitor group, despite no significant difference in the number of osteoclasts around the periodontal ligament. These findings promoted us to determine whether SLPI affected osteoclasts and osteoblasts. Bone marrow-derived macrophages (BMMs) differentiated into multinuclear osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and nuclear factor-κB ligand activating factor (RANKL). Figures 4a and b demonstrate that the treatment of BMMs with SLPI resulted in a reduction in the number of TRAP-positive cells. Subsequently, using fluoresceinamine-labeled sodium chondroitin poly-sulfate/calcium phosphate (FACPS/CaP)-coated plates, we examined the effect of SLPI on the bone resorption activity of BMMs. When cultured on labeled CaPs stimulated with M-CSF and RANKL, BMMs differentiate into osteoclasts, and the fluorescent intensity of the culture supernatant is consistent with the bone resorption activity of BMMs 24.The fluorescence intensity was significantly decreased in the culture supernatant from the SLPI-treated group compared to that in the medium-only group (Fig. 4c). Additionally, in the SLPI-treated group, the transcription of Nfatc1 and Rank was significantly downregulated, whereas no significant differences were observed for Acp5 and Ctsk (Fig. 4d).
SLPI promotes osteoblast differentiation and mineralization in vitro.
We have investigated the effect of SLPI on osteoblast differentiation and bone formation. MC3T3-E1 cells are most commonly used as an in vitro model of bone mineralization, and the collagenous extracellular matrix synthesized by the cell line and its organization and mineralization is very similar to what occurs in the bone 25 26. After MC3T3-E1 cells were cultured in osteoblast differentiation medium for 5 days, staining for alkaline phosphatase (ALP), a marker of osteoblast maturation, was performed to evaluate osteoblast differentiation. ALP activity was enhanced in the SLPI-treated group compared to that in the untreated and NE inhibitor-treated groups (Fig. 4e,f). Additionally, when mineralized nodule formation were detected in MC3T3-E1 cells cultured for 22 days by Alizarin Red staining, the amount of mineral nodule formation significantly increased in the SLPI-treated group (Fig. 4g,h). In the SLPI-treated group, there was a significant increase in the expression of master osteogenic transcription factor Runx2 and Sp7, which are typical early and middle osteogenic markers27, compared to the medium-only group; however, no significant difference was observed in the transcription of bone γ-carboxyglutamic acid-containing protein (Bglap), a late-stage osteoblast marker (Fig. 4i). These results suggest that SLPI is enhanced in the early to mid-stage of osteoblast differentiation of MC3T3-E1 cells to promote mineralization.
In this study, we found that SLPI significantly inhibited NE activity and alveolar bone loss in experimental periodontal tissues. These findings support the concept that SLPIs exert periodontitis-preventive effects via protease inhibition. Although SLPI is a multifunctional protein that has recently been reported to be involved in bone metabolism28, its function remains unclear. We found that SLPI suppressed osteoclast differentiation of BMMs and promoted osteoblast differentiation and mineralization of MC3T3-E1 cells. Therefore, we suggest that the inhibitory effect of SLPI on periodontal tissue destruction is due to its protease inhibitory effect and ability to regulate bone-resorbing osteoclasts and bone-forming osteoblasts.
Excessive extracellular NE released from neutrophils exacerbates by destroying epithelial tissues and alveolar bone via the spread of bacterial infections 11. Additionally, clinical evidence suggests that NE induces inflammation progression and contributes to the severity of periodontitis 10. Selective NE inhibition using Sivelestat in a ligature-induced periodontitis mouse model suppressed the transcription of inflammatory cytokine genes in periodontal tissues and reduced alveolar bone loss 9 23. Therefore, NE inhibition holds promise as a potential treatment for inflammatory diseases such as periodontitis. Consistent with these findings, our study demonstrated that SLPI suppressed NE activity, transcription of inflammatory cytokine genes, and alveolar bone loss in periodontal tissues.
Alveolar bone loss in periodontitis is mediated by host immune and inflammatory responses to the pathogen 29 30. Normal bone remodeling depends on a precise balance between bone resorption and bone formation, but inflammation-induced cytokines such as IL-6 and IL-1 shift bone homeostasis toward bone resorption in periodontitis 7 31 32. Therefore, periodontitis treatment requires the arresting the inflammatory process by eliminating the infection and controlling bone metabolism by osteoclasts and osteoblasts. Although the functional role of SLPI in bone resorption has not been reported, in this study, we showed that treatment of BMMs with SLPI inhibited osteoclast differentiation in vitro. SLPI is a regulator of nuclear factor kappa B (NF-κB) in monocytic cells 17 33, and the transcription of nuclear factor of activated T cells c1 (Nfatc1), an important target of NF-κB 34, was decreased in BMMs treated with SLPI, suggesting that SLPI inhibits osteoclast differentiation via NF-κB signaling. Contrastingly, SLPI expressed in osteoblasts may promote bone formation by regulating gene transcription of osteogenic genes, including the master transcription factors runt-related transcription factor-2 (Runx2), osteolytic factor (Sp7), and bone γ-carboxyglutamic acid-containing protein (Bglap) 28 35. In the present study, the treatment of MC3T3-E1 cells with SLPI promoted osteoblast differentiation and mineralization. We investigated bone resorption by osteoclasts and osteoblastic differentiation and demonstrated that SLPI exerts distinct effects on each process.
This study had some limitations. Here, periodontitis was induced concurrently with SLPI administration. However, in clinical practice, periodontal treatment is often initiated after alveolar bone resorption due to inflammation 5, making it difficult to directly apply our study results to clinical practice. To simulate the pathophysiology of active periodontitis, acute inflammation in periodontal tissues was induced by ligating the teeth of mice. By simultaneously initiating SLPI administration, our experimental results suggest the potential of SLPI treatment to inhibit periodontal tissue destruction caused by active periodontitis. Additionally, as SLPI affected both bone resorption and formation, further analysis is needed to investigate the effects of SLPI on alveolar bone defects.
In conclusion, this study demonstrated that SLPI exhibits anti-protease activity in vivo, suppresses bone resorption in vivo and in vitro, and enhances bone formation in vitro. Further investigations into the regulatory mechanisms of SLPI and its use in bone metabolism could potentially expedite the development of therapeutic approaches for periodontitis.
Male 7–8-week-old BALB/cA mice were purchased from CLEA Japan, Inc. (Tokyo, Japan), maintained under standard conditions in individual cages, and provided with sterile food and water ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of Niigata University (approval no. SA00181). Sivelestat, a NE inhibitor, was purchased from Ono Pharmaceutical Co., Ltd. (Osaka, Japan). It was dissolved in phosphate-buffered saline (PBS) to a concentration of 10 mg/mL.
Recombinant mouse SLPI was constructed as previously described with some modifications 36. Mouse SLPI DNA (accession number: NP_035544) was synthesized by Eurofins Genomics. The ORF of SLPI was amplified from the synthetic DNA using forward primer 5’-GTCGCATGCCCCTGGACTGTGGAAGGAG-3’ and reverse primer 5’-GTTCTGCAGtcacatcgggggcag-3’. SphI–PstI site of the pQE-30 vector (QIAGEN) in Escherichia coli Rosetta-gami B (DE3) strain (Novagen) was transformed with the plasmid, and transformants were incubated at 30°C for 16 h in the presence of 1 mM isopropyl-β-D-thiogalactopyranoside (FUJIFILM Wako Pure Chemical). Recombinant SLPI expressed in the insoluble fraction was solubilized in a lysis buffer containing 6 M guanidine hydrochloride, 500 mM NaCl, 10 mM imidazole, and 20 mM sodium phosphate (pH 7.8), and recombinant SLPI was purified using Ni-NTA agarose (QIAGEN). Active recombinant SLPI was obtained by dialyzing recombinant SLPI purified under denaturing conditions in a refolding buffer containing 0.5 M urea, 0.4 M L-arginine, 0.5 mM GSSG, 150 mM NaCl, and 50 mM Tris-HCl at 4°C for 16 h and finally replacing the buffer with PBS.
To establish this model, a 5 − 0 silk ligature (Akiyama MEDICAL MFG. Co., Ltd., Tokyo, Japan) was tied around the maxillary second molar to induce periodontitis 37. Thereafter, 50 µg NE inhibitor in 5 µL PBS (ligature + NE inhibitor group), 500 ng recombinant mice SLPI in 5 µL PBS (ligature + SLPI group) or 5 µL PBS was injected into the palatal gingiva of the molar once daily for 3 days for the NE activity assay or 7 days for other analyses. All animal experiments were conducted by a researcher who analyzed the results and was blinded to the actual experiment. The humane endpoint was decided as 20% reduction in body weight from baseline or signs of intense pain.
NE activity in the palatal gingival tissue of mice has been described previously23. Briefly, three hours after the last injection of mouse SLPI, NE inhibitor, or PBS, the palatal gingival tissues were homogenized in Tris-HCl buffer using a BioMasher (Nippi, Tokyo, Japan). These samples were centrifuged at 300×g for 3 min, and NE activity in the supernatant was determined using N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Merck Millipore, Billerica, MA, USA).
Each maxilla was skinned, then the dry skulls were stained with methylene blue (5% in water) for 5 min. Periodontal bone loss in the maxilla was morphometrically assessed using a stereoscopic microscope (Leica Microsystems, Wetzlar, Germany). According to a previously described method 37, the distance from the cement-enamel junction to the alveolar bone crest was measured at six predetermined sites on the ligated second molar and adjacent affected regions. Bone change was calculated by subtracting the sum of the cemento-enamel and alveolar bone crest values from the six corresponding values in the unligated area. Negative values (mm) indicate bone loss relative to baseline (unligated control).
The maxillae prepared from the murine model were fixed in a 4% paraformaldehyde solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 24 h. The specimens were decalcified in decalcifying solution B (Wako Pure Chemical Industries) for 1 week at 4°C. The specimens were then embedded in the O. C. T. compound (Sakura Finetek, Torrance, CA, USA) and frozen in liquid nitrogen. The coronary sections were prepared using a cryostat (Leica Biosystems). The prepared coronal sections (10 µm) were stained with tartrate-resistant acid phosphatase (TRAP) staining (Cosmo Bio Co., Tokyo, Japan).
SLPI expression in frozen coronal sections was assessed by immunofluorescence using an SLPI-specific antibody (Novus Bio, Centennial, USA). After overnight incubation with the primary antibody at 4°C, SLPI was visualized using Alexa Fluor 488 conjugated anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA). Nuclei were identified using Hoechst33342 for 15 min at 37°C. Sections were observed under a confocal laser scanning microscope (Carl Zeiss, Jena, Germany).
As described previously38, BMMs were collected from the femurs and tibias of mice and age-matched unligated mice were used as controls under sterile conditions. BMMs were cultured in 96-well plates (1.0×105 per well) with recombinant macrophage colony-stimulating factor (M-CSF) (30 ng/mL; R&D Systems, Minneapolis, MN, USA) for 3 h in minimum essential medium alpha (MEMα; Wako Pure Chemical Co., Tokyo, Japan) with 10% fetal bovine serum (FBS) at 37°C in 5% CO2. After removing nonadherent cells, adherent cells were further cultured in MEMα media supplemented with 10% FBS, 100 ng/mL recombinant soluble receptor activator of nuclear factor-kappa B ligated (RANKL; R&D Systems), and 100 ng/mL M-CSF in the presence or absence of recombinant mice SLPI (1 or 10 µg/mL) or NE inhibitor (1 or 10 µg/mL) for 7 days. The medium was replaced with a fresh solution every 3 days during the incubation period. Multinucleation of osteoblasts was confirmed by TRAP staining (to confirm multinucleation). To determine the effect of SLPI on the absorption activity, BMMs were cultured in FACPS/CaP-coated 96-well plates (200 µL/well) (PG Research Co., Ltd., Tokyo, Japan). After 7 days, the supernatant was collected and fluorescence intensity (excitation: 485 nm, emission: 535 nm) was measured using GloMax (Promega Corporation, Madison, WI, USA)24.
The murine osteoblastic progenitor cell line, MC3T3-E1, was obtained from the RIKEN Bioresource Center (RCB1126). MC3T3-E1 cells were maintained in MEMα media supplemented with 10% FBS and penicillin-streptomycin solution (×100) (Wako Pure Chemical Co.) at 37°C in 5% CO2. To determine the osteogenic differentiation, cells were cultured with 50 µg/mL ascorbic acid (Wako Pure Chemical Co.) and 10 mM β-glycerophosphate (Wako Pure Chemical Co.) in MEMα media supplemented with 10% FBS. The cell culture medium was replaced every three days. ALP activity after 5 days was detected using an ALP staining kit (Cosmo Bio Co., Ltd. Tokyo, Japan) and photographed under a stereoscopic microscope (Leica Microsystems, Wetzlar, Germany) (25×). The ALP-positive area was quantified using ImageJ software version 1.53t (National Institute of Health, Bethesda, MD, USA). Mineralization of bone nodules was detected by staining with Alizarin Red S (Wako Pure Chemical Co.) 24 days after the differentiation of MC3T3-E1 cells. Alizarin Red S-stained calcified deposits were extracted using formic acid and quantified by measuring the optical density at 405 nm using a microplate reader.
Total RNA was extracted from mouse maxillary palatal gingiva, BMMs, or MC3T3-E1 cells using TRI reagent (Molecular Research Center, Inc., Cincinnati, OG, USA). RNA was reverse-transcribed using ReverTra Ace qPCR RT Master Mix (TOYOBO Co., Ltd., Osaka, Japan). Quantitative PCR with cDNA was performed according to the manufacturer’s protocol using a Step One Plus real-time PCR system (Thermo Fisher Scientific). The data was analyzed using the comparative CT (ΔΔCT) method. TaqMan probes and primers for the expression of housekeeping gene (Gapdh, Mm99999915_g1) along with Il6 (Mm00434228_m1), Il1b (Mm00434228_m1), Nfatc1 (Mm01265944_m1), and Tnfrsf11a (Rank, Mm00437132_m1), Acp5 (Mm00475698_m1), Ctsk (Mm00484039_m1), Runx2 (Mm00501584_m1), Sp7 (Mm00504574_m1), Bglap (Mm03413826_mH) were purchased from Thermo Fisher Scientific.
Data were analyzed by analysis of variance with Dunnett’s or Tukey’s multiple comparison test using GraphPad Prism 8.4.3 (GraphPad Software, Inc., La Jolla, CA, USA).
The authors confirm that the data supporting the findings of this study are available within the article.
Data Availability Statement
The authors confirm that the data supporting the findings of this study are available within the article.
Acknowledgments
We thank Dr Fumio Takizawa, Dr Rui Saito, and Dr Yoshihito Yasui for advice regarding our experiments.
This work was supported by grants from the Japan Society for the Promotion of Science KAKENHI (grant numbers 20H03858, 22K19614, 22K09923, 20K09903, 22H03267, 22KJ1445, and 23H00445). This work was supported by JST and Niigata University (Fellowship Program No. JPMJFS2114-156695-J23H0004). These funders had no role in the study design, data collection, and interpretation, or the decision to submit the manuscript for publication.
Author Contributions
K. S. and Y. T. conceptualization; K. S., H. D., S. H., and T. I. formal analysis; K. S. and T. I. investigation; K. S. and H. D. writing–original draft; H. D., T. M., and Y. T. writing–review and editing; M. T., K. T., and Y. T. supervision; Y. T. project administration; K. S., H. D., S. H., T. M., T. I. and Y. T. funding acquisition.
Competing interests
The authors declare no competing interest.