The Pros1/Tyro3 axis protects against periodontitis by modulating STAT/SOCS signalling

Abstract Periodontitis, an oral inflammatory disease caused by periodontal pathogen infection, is the most prevalent chronic inflammatory disease and a major burden on healthcare. The TAM receptor tyrosine kinases (Tyro3, Axl and Mertk) and their ligands (Gas6 and Pros1) play a pivotal role in the resolution of inflammation and have been associated with chronic inflammatory and autoimmune diseases. In this study, we evaluated the effects of exogenous Pros1 in in vitro and in vivo models of periodontitis. We detected higher Pros1 but lower Tyro3 levels in inflamed gingival specimens of periodontitis patients compared with healthy controls. Moreover, Pros1 was mostly localized in the gingival epithelium of all specimens. In cultured human gingival epithelial cells (hGECs), Porphyromonas gingivalis LPS (p.g‐LPS) stimulation down‐regulated Pros1 and Tyro3. Exogenous Pros1 inhibited p.g‐LPS–induced production of TNF‐α, IL‐6, IL‐1β, MMP9/2 and RANKL in a Tyro3‐dependent manner as revealed by PCR, Western blot analysis, ELISA and gelatin zymography. Pros1 also restored Tyro3 expression down‐regulated by p.g‐LPS in hGECs. In rats treated with ligature and p.g‐LPS, administration of Pros1 attenuated periodontitis‐associated gingival inflammation and alveolar bone loss. Our mechanistic studies implicated SOCS1/3 and STAT1/3 as mediators of the in vitro and in vivo anti‐inflammatory effects of Pros1. Collectively, the findings from this work supported Pros1 as a novel anti‐inflammatory therapy for periodontitis.

and matrix metalloproteinases (MMPs) by various resident cells of the periodontium through activation of Toll-like receptor 2 (TLR2) or TLR4. 2 The combined inflammatory responses stimulate osteoclastogenesis, resulting in alveolar bone loss. 3 Periodontitis is the most prevalent chronic inflammatory disease and a major burden on healthcare systems. Severe periodontitis, which may result in tooth loss, affects 5%-20% of most populations worldwide. 4 In addition, periodontitis is an independent risk factor for several chronic diseases such as diabetes, cardiovascular disease and cancer. [5][6][7] For instance, periodontitis is associated with increased risk for total cerebrovascular incidents, and in particular, non-haemorrhagic stroke. 8 The TAM receptor tyrosine kinases (RTKs) TYRO3, AXL and MERTK were identified as a distinct RTK subfamily in 1991. 9,10 Although TAM receptors have important functions in the adult nervous, reproductive and vascular systems, they are best known for their pivotal roles in the negative regulation of the immune system, functioning at the interface of innate and adaptive immunity.
Together with their ligands GAS6 and Pros1 (also called protein S), they inhibit innate inflammatory response to pathogens by dendritic cells and macrophages, stimulate the phagocytic activity of antigen-presenting cells and promote the maturation of natural killer cells. 11 Not surprisingly, the TAM signalling is implicated in a number of chronic inflammatory and autoimmune diseases such as multiple sclerosis (MS) 12 and systemic lupus erythematosus (SLE). 13,14 Mechanistically, the TAM receptors can associate with interferon (IFN)-receptor 1 (IFNAR1), and thereby, activate the suppressor of cytokine signalling proteins SOCS1 and SOCS3, subsequently inhibiting IFN-I production. 15 Accumulating evidence has suggested a role of IFN-I in the development of periodontitis. Elevated levels of IFN-were detected in gingival tissues and plasma of periodontitis patients. 16,17 Moreover, the periodontitis-associated pathogen P gingivalis or its LPS (p.g-LPS) can stimulate IFN-production by macrophages through TLR signalling. 18 Interestingly, loss of negative regulation on IFN-I by TAM was reported to be responsible for the uncontrolled IFN-1 production in a murine model of P gingivalis-induced periodontitis, 19 supporting a protective role of the TAM signalling against this oral inflammatory disease. However, the mechanisms underlying immunoregulation of TAM receptor tyrosine kinases or their ligands during periodontitis, especially for Pros1, was yet to be elucidated.
In this study, we evaluated the effects of exogenous Pros1 on P gingivalis LPS (p.g-LPS)-induced inflammation in human gingival epithelial cells (hGECs) in vitro as well as on ligature-induced, p.g-LPSaugmented periodontal inflammation and alveolar bone loss in vivo.
Also, the mechanisms involving SOCS1/SOCS3 and STAT1/STAT3 were also investigated. The aim of the study was to clarify the specific role of Pros1/Tyro3 axis in regulating oral inflammatory disease such as periodontitis.

| Patients and tissue samples
Gingival specimens containing both epithelial and connective tissues were obtained during tooth extraction from 12 patients with chronic periodontitis and 8 healthy controls with non-inflamed gingiva. All participants were 23-62 years of age and had no history of smoking. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Shanghai Chang Zheng Hospital (Shanghai, China). All study participants gave written informed consent. Gingival tissues were either fixed for histologic examinations or promptly frozen in liquid nitrogen and stored at −80°C until further use. The clinical features of the patients are presented in Table 1.

| Cell culture, reagents and antibodies
Primary hGECs were obtained from CELLnTEC (Stauffacherstrasse, Switzerland) and maintained in CnT-PR media (CELLnTEC) supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C, 5% CO 2 in a humidified incubator. p.g-LPS was obtained from
The rats were anaesthetized with an intraperitoneal injection of Zoletil (0.4 mL/kg, Virbac Laboratories, Carros, France) and Rompun (10 mg/kg, Bayer Korea Ltd., Seoul, South Korea). An elastic ligature was placed between the first and second right maxillary molars to TA B L E 1 Clinical data of periodontitis patients and control participants with non-inflamed gingiva

| Quantitative real-time PCR
Total RNA was isolated using TRIzol reagent (Invitrogen) according to manufacturer's instructions and reverse-transcribed into complementary DNA (cDNA). Quantitative PCR was carried out on a LightCycler system (Roche LifeScience) with SYBR Green master mix (Roche Applied Science). Primer sequences used in the PCR reactions are listed in Table 2. Data were normalized to that of β-actin in the same reaction. Relative expression was calculated using the 2 −∆∆Ct method.

| Western blot analysis
Cells were lysed using RIPA lysis buffer containing protein kinase and phosphatase inhibitors for 30 minutes on ice. Tissue samples were homogenized by sonication and proteins were extracted.
Protein concentrations were determined using a BCA kit (Thermo Fisher

Minneapolis, MN, USA) following manufacturer's instructions. Data
were normalized to cell number in each test.

| Gelatin zymography
The enzymatic activities of MMP-2 and MMP-9 in hGEC culture media were determined using a gelatin zymography system (Novex Life Technology, Carlsbad, CA, USA). In brief, proteins in the medium were separated under non-reducing denaturing conditions on a 10% SDS-polyacrylamide gel containing 1 mg/mL gelatine. After washing with 2.5% Triton X-100 and overnight incubation at 37C, the gels were stained with 0.1% Coomassie blue R-250 for 4 hours and immersed into a buffer containing 45% methanol and 10% acetic acid. Gel images were obtained on a Transilluminator (Diagnostic Instruments, Sterling Heights, MI, USA).

| Micro-CT analysis
Micro-CT imaging was performed 2 weeks after periodontitis induction on a SkyScan microCT scanner (Bruker microCT, Kontich, Belgium). The maxillary jaws were hemisected and the right half of the block samples were cut into 18-μm slices and fixed in 4.0% paraformaldehyde.
Computed tomography was conducted with a voltage of 50 kV and an electrical current of 455 μA. Three-dimensional images were acquired using the Bruker microCT version 1.1 software. The distance from the cement-enamel junction (CEJ) to the alveolar bone crest (ABC) at the palatal groove site of M2 was measured as previously described. 20

| Histology and immunohistochemistry
The maxilla specimens were fixed in 4% paraformaldehyde for at least 24 hours and decalcified in 10% EDTA solution for 6 weeks at 4°C, with the solution exchanged every other day. The decalcified specimens were dehydrated, embedded in paraffin and cut into 4-μm sections. After dewaxing and rehydration, the sections were stained with haematoxylin and eosin (HE) for histological analysis.
Expression and distribution of Pros1, Tyro3 and RANKL were detected by immunohistochemical staining.

| TRAP staining
The maxilla sections were subjected to tartrate-resistant acid phosphatase (TRAP) staining using a leukocyte acid phosphatase kit (Sigma-Aldrich) following manufacturer's instructions. The specimens were counterstained with haematoxylin. TRAP-positive multinucleated cells (active osteoclasts) on the surface of alveolar bone around the first molar were counted. The results were expressed as the total cell count from four different visual fields of each section.

| Statistical analysis
All results are presented as mean ± SD. Data were analysed using

| Pros1 is up-regulated in gingival tissues of chronic periodontitis patients
The staining was mainly detected in the gingival epithelium of both periodontitis patients and non-inflamed controls ( Figure 1C).

| Pros1 inhibits p.g-LPS-induced inflammation in hGECs via Tyro3
Stimulation of hGECs with increasing concentrations of p.g-LPS for 24 hours led to dose-dependent decreases in cell viability as

| Pros1 reduces osteoclastogenesis and alveolar bone loss in periodontitis rats
We subsequently examined the effects of Pros1 in rats subjected to combinatory treatment with ligature and p.g-LPS. Micro-CT

| Pros1 attenuates periodontal inflammation in periodontitis rats
We next assessed Pros1, Tyro3 and RNAKL levels in periodontal tissues of periodontitis and sham rats. Immunohistochemical staining and qRT-PCR analysis revealed lower Pros1 and Tyro3 and higher RANKL expression in the ligature + p.g-LPS group than the

| D ISCUSS I ON
The first evidence for the involvement of the TAM signalling in the development of periodontitis came from a recent study showing that the unrestrained IFN-I production following P gingivalis infection was due to down-regulation of TAM components. 19  In this study, we also detected increased Pros1 and decreased Tyro3, but similar levels of Gas6, Axl and Mertk in inflamed human gingiva compared with healthy controls. Based on these data, we speculated that the Pros1/Tyro3 signalling may be the main TAM signalling involved in the development of periodontitis in humans.
Indeed, results from our subsequent studies demonstrated that exogenous Pros1 inhibits p.g-LPS-stimulated inflammation in cultured hGECs and ameliorates periodontal inflammation and alveolar bone loss in rats subjected to ligature and p.g-LPS treatment.
Similar to previous findings, 19 Pros1 was mainly detected in the epithelium of human and murine gingiva. In previous cellular and animal studies, certain TAM components, and in particular, Pros1 were down-regulated by LPS stimulation or repeated P gingivalis infection. 19,34 In this study, both Pros1 and Tyro3 were down- Ligature (ligature only), Ligature + p.g-LPS, Ligature + p.g-LPS + PBS and Ligature + p.g-LPS + Pros1 were treated as described in Section 2 for 2 weeks. SOCS1, p-STAT1, STAT1, SOCS3, p-STAT3 and STAT3 protein levels in the periodontium were determined by Western blot analysis. Representative gel images (A) and relative protein expression by densitometric analysis (B and C) are shown. n = 6; *P < 0.05, **P < 0.01, ***P < 0.001 vs Control; $ P < 0.05, $$ P < 0.01 vs Ligature + p.g-LPS + PBS the expression of TNF-α, IL-6 and IL-1β by macrophages in response to LPS stimulation in a TAM receptor-dependent manner. 34 In this work, Pros1 inhibited p.g-LPs-induced production of TNF-α, IL-6, IL-1β, MMP9/2 and RANKL by hGECs in a Tyro3-dependent manner. In rats subjected to combinatorial treatment with ligature and p.g-LPS, administration of Pros1 attenuated periodontitis-mediated gingival inflammation, periodontal osteoclastogenesis and alveolar bone loss. Furthermore, our mechanistic studies implicated SOCS1/3 regulation of STAT1/3 as a mechanism of the in vitro and in vivo anti-inflammatory effects of Pros1. Collectively, the findings from this work supported Pros1 as a novel therapy to combat periodontitis.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science Foundation of China (No. 81300884) and Natural Science Funding of Shanghai (17ZR1439200).

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.