Inhibition of Ctsk modulates periodontitis with arthritis via downregulation of TLR9 and autophagy

Abstract Objectives The mechanisms underlying the effects of Toll‐like receptor 9 (TLR9) and autophagy on rheumatoid arthritis (RA)‐aggravated periodontitis are unclear. We aimed to explore a novel target, cathepsin K (Ctsk)‐mediated TLR9‐related autophagy, during the progress of periodontitis with RA. Materials and Methods DBA/J1 mouse model of periodontitis with RA was created by local colonization of Porphyromonas gingivalis (Pg) and injection of collagen. The expression of Ctsk was inhibited by adeno‐associated virus (AAV). Micro‐CT, immunohistochemistry (IHC), Western blot and quantitative real‐time polymerase chain reaction (qRT‐PCR) were used to detect the expression of TLR9‐related autophagy in periodontitis with RA. Small interfering RNA (siRNA) and CpG oligodeoxynucleotides (CpG ODN) were applied in macrophages. Western blot, immunofluorescence (IF) and qRT‐PCR were used to verify the in vivo results. Results RA can promote periodontitis bone destruction in the lesion area, while inhibiting Ctsk could effectively alleviate this effect. The infiltration of macrophages, TLR9, autophagy proteins (TFEB and LC3) and inflammatory cytokines increased in the periodontitis‐with‐RA group and was reduced by the inhibition of Ctsk in the periodontal region. Macrophage stimulation confirmed the in vivo results. With the activation of TLR9 by CpG ODN, inhibition of Ctsk could suppress both TLR9 downstream signalling proteins and autophagy‐related proteins. Conclusions This study advanced a novel role for Ctsk in TLR9 and autophagy to explain the interaction between periodontitis and RA.

leading to a persistent inflammatory state. 3 Current research shows that periodontitis is closely related to autoimmune diseases. 4 Rheumatoid arthritis (RA), one of the most common autoimmune diseases, is characterized by chronic joint inflammation and bone destruction. 5 In addition to the specific autoimmune response to citrullinated protein in the body, 6 the autoimmune pathogenic mechanism of RA involves infiltration of various immune cells in the synovial tissue of diseased joints, the activation of the complement system and the maladjustment of cytokine networks. 7,8 Periodontitis and RA are both chronic inflammatory diseases with similar immune and inflammatory mechanisms, causing soft tissue inflammation and bone destruction. 9 A recent study has shown an epidemiological association between periodontitis and RA. 10 This supported previous evidence showing that chronic periodontitis is more common in patients with RA than in healthy controls 11 and that the prevalence of RA in patients with chronic periodontitis is higher than in the general population. 12 For cell physiology and health, autophagy is a necessary conservative lysosomal degradation process. 13 Autophagy can regulate cellular processes such as apoptosis, inflammation, pathogen clearance and immune response, and is a potential therapeutic target for many diseases. 14 Through mediating the survival of Pg, the autophagy pathway exerts effects on periodontal diseases. 15 It has also been shown that suppression of the autophagy pathway is related to the pathogenesis of rheumatoid arthritis and other autoimmune diseases. 16 More specifically, autophagy seems to be involved in the production of citrulline peptide and the apoptosis resistance of RA cells. 17 Therefore, the substantial progress of autophagy research provides a new understanding of the pathogenesis of periodontitis and RA.
Cathepsin K (Ctsk) is a lysosome cysteine protease that is primarily expressed in osteoclasts and plays a key role in bone resorption. 18 Ctsk inhibitors have been considered as potential therapies for diseases characterized by relative bone destruction. Our previous studies have shown that inhibition of Ctsk can effectively reduce bone resorption in periodontitis associated with arthritis, and have also found a link between Ctsk and Toll-like receptor 9 (TLR9).
Inhibition of Ctsk has been shown to downregulate the TLR9 pathway. 19 During the process of bacterial-induced inflammation, TLR9 can recognize the CpG oligodeoxynucleotide (CpG ODN) presented in bacterial DNA for innate immunity, which is a key pattern recognition receptor for the immune response. 20 Meanwhile, studies have shown that the TLR9 receptor is involved in autophagy. 21,22 For example, after TLR9 stimulation, the autophagy protein microtubule-associated protein 1A/1B-light chain 3 (LC3) can directly recruit IκB kinase α (IKKα) for type I Interferon (IFN) production. 21 There is also evidence suggesting that CpG ODN can induce autophagy in rodent and human tumour cell lines via the Toll-like receptor 9-extracellular regulated protein kinase-mammalian target of rapamycin (TLR9-ERK-mTOR) signalling pathway. 22 These data led us to the hypothesis that inhibition of Ctsk could modulate autophagy via downregulation of TLR9 and, in turn, affect the progression of RAaggravated periodontitis.
Here, we investigated the relationship between Ctsk and TLR9related autophagy in the presence of periodontitis and RA. Our findings provide a new approach and therapeutic target for investigating RA-aggravated periodontitis.

| Animals
A total of 80 six-week-old male DBA/J1 mice (purchased from the Nanjing Biomedical Research Institute) were used for this study.

| Experimental periodontitis and collageninduced arthritis (CIA)
We established our mouse model of periodontitis with CIA following previous protocols. 19 The bacterial strain used was Pg (ATCC: 33277), which was grown in an anaerobic chamber (80% N 2 , 10% H 2 and 10% CO 2 ) at 37°C. Before the formal oral bacterial colonization, kanamycin (0.5 mg/mL) was added to drinking water for three consecutive days in order to remove other bacteria. The bacterial pellet obtained by centrifugation was mixed with an equal volume of sterile 3% carboxymethylcellulose (CMC) and topically applied in the oral cavity and anus eight consecutive times. The dose of the mixture was 100 μL (5 × 10 10 cells/mL of Pg) per mouse. 23 Following bacterial application, chicken type II collagen (Cat#20011, Chondrex) was emulsified in 100 μg complete Freund's adjuvant (Cat#7023, Chondrex). Fifty microlitres of the emulsion was slowly injected intradermally at a point about 1.5 cm distal from the base of the tail.
The primary immunization was performed one day after bacterial application, and the booster immunization was made after 14 days. 24
Ten microlitres of AAV working solution was injected into each periodontal region and knee joint using a 100-μL microsyringe.
Mice were transfected with AAV once a day and rested for four days after two consecutive days. The procedure of AAV transfections was repeated eight times during the experimental period. 26

| Micro-CT scanning
Micro-CT scanning of maxillae and knee samples was applied by vi-vaCT 80 (Scanco Medical) with the resolution of 12 μm. 28 Threedimensional reconstruction and data analysis were performed by Scanco Evaluation and Mimics Research 19.0 software. The alveolar bone and subchondral bone area of tibia and femur were defined as the region of interest (ROI), and bone volume/total volume was calculated for quantification. 29 The region from the cemento-enamel junction (CEJ) of the molar to the alveolar bone crest (ABC) was defined as the vertical alveolar bone resorption.

| Immunohistochemistry (IHC) and immunofluorescence (IF) analysis
The animal specimens for IHC or IF analysis were demineralized and embedded in paraffin wax, and then sectioned into 5 μm thicknesses.
Sections were submitted to analysis with F4/80 (Cat#70076, Cell Signaling Technology), TFEB (Cat#A303-673A, Bethyl Laboratories) and GFP (Cat#2956, Cell Signaling Technology) rabbit monoclonal primary antibody according to the manufacturer's instructions. The sections of IF were then stained with DAPI (Cat#4083, Cell Signaling Technology) to identify the nuclei. 30 All samples in vitro were fixed by 4% paraformaldehyde solution and then treated with 0.5% Triton X-100 for 15 minutes and 5% serum for 1 hour. The cells were incubated with TLR9 and TFEB primary antibodies at 4°C overnight.
On the second day, cells were incubated with the secondary antibody and stained using phalloidin and DAPI successively. Finally, the samples were sealed with the anti-fluorescence quenching agent, and the images were observed and captured under the fluorescence microscope.

| RNA extraction and qRT-PCR analysis
Total RNA was extracted from tissue homogenate and cultured cells  Table 1. According to the manufacturer's instructions, the concentration of primary antibodies was 1:1000. On the second day, HRP-conjugated antibodies (Cat#L3012-2, SAB) were applied to the membranes and the signals were detected using the ChemiDoc™ MP Imaging System (Bio-Rad). 31

| Safranin O staining (SO)
According to the manufacturer's instructions (Cat#TMS-009, Sigma Aldrich), after dewaxing and dehydrating the sample sections, the specific process of safranin O staining was performed as follows: slides were stained with 0.002% fast green solution and washed with 1% acetic acid. After stained with haematoxylin solution for 10 minutes and rinsed, the slides were dyed with 0.1% SO solution for 6 minutes. Articular cartilage was evaluated by OARSI grading. 32

| Statistical analysis
Data were shown as mean ± SD of different groups and analysed using two-tailed Student's t test and one-way ANOVA test followed by Tukey's multiple comparison test and non-parametric Mann-Whitney U test/Kruskal-Wallis test. P values < .05 or U values > 1.96 were considered significant.

| Inhibition of Ctsk in the lesion area reduced bone destruction from periodontitis and comorbid rheumatoid arthritis
Since the AAVs designed in our study contained the sequence of GFP, we could detect the transfection effect of AAVs by GFP fluorescence staining. Compared with the control group, the percentage of positive GFP cells was increased significantly in the AAV-treated group (the Control + GFP group) ( Figure 1A,B), which indicated that the transfection of AAVs in vivo was successful.
To investigate the effect of Ctsk on bone destruction from periodontitis and comorbid RA, we analysed the alveolar bone and joint in vivo by micro-CT scanning for bone malformation in lesion areas.
According to the images of alveolar bones scanned by micro-CT, we quantitated and analysed periodontal bone resorption. The results showed a greater bone resorption area in the periodontitis group (Pg + GFP) than in the control group (Figure 2A,B), which indicated that the model of periodontitis had been successfully established.

| Inhibition of Ctsk in the lesion area reduced the number of macrophages and inflammatory cytokines in the periodontium with RA
In order to further explore the influence of periodontitis with RA on the inflammatory state in the lesion area and to explore whether inhibiting Ctsk alleviated the inflammatory state, we evaluated the abundance of macrophages and inflammatory cytokines in the lesion area.
We detected the infiltration of macrophages in the periodontium through IHC analysis. The specific marker of macrophages

| Inhibition of Ctsk in the lesion area reduced the expression of TFEB in the periodontium with RA
Previous studies have suggested that autophagy is involved in the development of periodontitis and arthritis. 15,16 In this study, inhibition of Ctsk effectively alleviated the process of promoting periodontitis by RA, which suggested that Ctsk might affect the autophagy response in the course of the disease. To evaluate this response, we first detected the classic autophagy-coordinating protein TFEB.
The results of IHC showed that, compared with the control group, the expression of TFEB increased significantly in the periodontitis (Pg + GFP) and arthritis (CIA + GFP) groups, and the expression of TFEB was highest in the comorbidity group (CIA + Pg + GFP). As expected, the expression of TFEB was significantly downregulated after inhibition of Ctsk ( Figure 5A). IHC revealed not only the expression of TFEB but also its distribution.
When CTSK was inhibited, the amount of TFEB entering the nucleus decreased, and quantification of the nuclear TFEB also confirms this result ( Figure 5B). The results of qRT-PCR confirmed this  Figure 5C). In summary, when periodontitis was accompanied by RA, the autophagic reaction was aggravated more severely than in periodontitis or RA alone. These data also showed an association between Ctsk and autophagy, namely that inhibition of Ctsk can reduce the autophagic reaction.

| Inhibition of Ctsk reduced the expression of the TLR9 and autophagy signalling pathway
Our previous studies also confirmed that TLR9 is highly expressed in the lesion area in periodontitis and RA. With inhibition of Ctsk, we found significantly reduced TLR9 expression in all groups. 19 This suggests that TLR9 may be related to autophagy in the process of RA-aggravated periodontitis, and Ctsk may also play an important role.
To test our hypothesis, we assessed expression of CTSK, TLR9 and Data are presented as the mean ± SD, compared with the control. *P < .05, **P < .01, ***P < .001. The asterisk (with no line connection) on the column of this group represents the statistical difference between it and the control group. Experiments were repeated three times analyses ( Figure 6B). As expected, RA can promote the expression of CTSK in periodontitis lesions. Expression of TLR9 and autophagy proteins also increased with increased CTSK protein, while expression of TLR9 and autophagy proteins significantly decreased with inhibition of Ctsk. These data indicated that the expression of Ctsk is connected to TLR9 and autophagy in periodontitis with RA. The trend of TLR9 expression change was the same in periodontitis with RA and autophagy, which suggested that TLR9 may be closely related to autophagy in periodontitis with RA.

| The downregulation of autophagy induced by activation of the TLR9 pathway was mediated by Ctsk inhibition
To further investigate the roles of Ctsk and TLR9 in the immune response and autophagy, we conducted an in vitro experiment.
Transfection of siRNA was used to inhibit Ctsk expression in macrophage cell lines. The expression of Ctsk in each group was evaluated by qRT-PCR and Western blotting, and it was verified that To assess the relationship between TLR9 and autophagy in vitro, CpG ODN, a specific ligand of TLR9, was employed to stimulate RAW264.7 cells. With this stimulation of TLR9, the level of TLR9 protein, its downstream protein IKKβ ( Figure 7A,B) and the mRNA expression of Myd88 ( Figure 7C) were increased, despite which Ctsk did not appear to be affected by CpG ODN. According to previous studies, the TLR9-downstream proteins IKKβ and MYD88 are related to autophagy activation. 33,34 As we predicted, the autophagy proteins, TFEB and LC3A/B, and inflammatory cytokines were increased with the activation of TLR9. However, these proteins and inflammatory cytokines still showed a downward trend with inhibition of Ctsk ( Figure 8A,B).
For the key protein of autophagy, TFEB, the expression of TFEB not only varied with TLR9 and CTSK, but also its location in cells.
After the activation of TLR9 by CpG ODN, IF showed that the expression of TFEB increased, and almost all of TFEB entered the nucleus to function. When the CTSK was inhibited by siRNA, the expression of TFEB decreased with the number of nucleus entering ( Figure 8C), and the quantification of TFEB distribution also confirms this result ( Figure 8D). It is worth noting that, when inhibiting Ctsk under the condition of TLR9 activation by CpG ODN, autophagy proteins were expressed more highly than without TLR9 activation ( Figure 8).
Based on these data from in vitro experiments, we infer that TLR9 is associated with autophagy in macrophage cell lines. Activation of TLR9 can activate the downstream autophagy response, while Ctsk can modulate this response. TRAF6 and IRAK1 decreased with inhibition of Ctsk ( Figure 9A,B), while the mRNA expression levels of Traf6, Irak1 and Irak4 did not appear to be affected by TLR9 ligand ( Figure 9A).

| D ISCUSS I ON
Previous studies have demonstrated a mutually promoting relationship between periodontitis and RA, 19,35 and studies on autophagy in periodontitis 36 and arthritis are gradually accumulating. 37 However, the role of autophagy in the mechanism underlying the link between RA and periodontitis has not been explored.
Our in vivo experiment found that RA can increase the expres- Representative images and the normalized quantification are shown. Data are presented as the mean ± SD, repeated three times, compared with the control. *P < .05, **P < .01 and ***P < .001. The asterisk (with no line connection) on the column of this group represents the statistical difference between it and the control group that elevated autophagy, and its associated signalling pathways, is an important regulator of the aggravation of periodontitis by RA.
Ctsk is usually studied as a functional molecule related to osteoclasts. 38 Our previous study showed that Ctsk could affect the TLR9 signalling pathway in periodontitis and arthritis. 19  We also focused on the possible role of Ctsk in the TLR9 and autophagy response in macrophages. Macrophages are important components of the innate immune system, which play the role of antigen presentation and immune regulation, 45 and are known to play a critical role during the progress of autophagy. 46,47 Macrophages can also produce cytokines and promote the activation of T cells to secrete pro-inflammatory cytokines, which cause tissue damage during chronic inflammation. 48 We found that RA could aggravate the infiltration of macrophages in periodontitis lesions, while the number of macrophages was significantly decreased after inhibiting Ctsk.
In conclusion, our research provides a novel role of Ctsk-mediated, TLR9 and autophagy in the development of periodontitis aggravated by RA. This provides a new potential therapeutic target to treat periodontitis with RA.

ACK N OWLED G EM ENTS
We would like to thank Dr L.

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

AUTH O R CO NTR I B UTI O N S
Wei Wei contributed to conception and design; contributed to acquisition, analysis and interpretation; drafted the manuscript; critically revised the manuscript; gave final approval; and agreed to be accountable for all aspects of work ensuring integrity and accuracy.
Jie Ren contributed to conception and design; contributed to acquisition, analysis, and interpretation; drafted the manuscript; critically revised the manuscript; gave final approval; and agreed to be accountable for all aspects of work ensuring integrity and accuracy.
Wuwei Yin contributed to design, contributed to acquisition, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy.
Handong Ding contributed to conception, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Qiuyu Lu contributed to conception, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Liangyu Tan contributed to conception and design, contributed to analysis, drafted the manuscript, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Shibing Deng contributed to conception, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Jie Liu contributed to conception, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Qin Yang contributed to conception, contributed to analysis, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Jiajia Wang contributed to conception, contributed to analysis, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Min Wang contributed to conception and design, contributed to analysis, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Yuan Yue contributed to conception and design, contributed to analysis and interpretation, drafted the manuscript, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy.
Liang Hao contributed to conception and design, contributed to analysis and interpretation, drafted the manuscript, critically revised the manuscript, gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.