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.

Keywords: Pros1; SOCS1/3; STAT1/3; Tyro3; periodontitis.

Figure 1

Pros1 is up‐regulated in gingival tissues of chronic periodontitis patients. (A‐C) Pros1 expression in gingival tissues from periodontitis patients (periodontitis, n = 12) and healthy controls (Control, n = 8) by qRT‐PCR (A, P = 0.0063), Western blot analysis (B) and immunohistochemical staining (C)

Figure 2

p.g‐LPS down‐regulates Pros1 in hGECs. (A) hGECs were treated with p.g‐LPS at indicated concentrations for 24 hours. Cell viability was determined by the MTT assay. Untreated cells (0 ng/mL p.g‐LPS) were used as control. n = 3, **P < 0.01 vs untreated cells. (B, C) hGECs were treated with 10 g/mL p.g‐LPS for 24 hours. Pros1 mRNA and protein levels were determined by qRT‐PCR (B) and Western blot analysis (C) respectively. n = 3, **P < 0.01

Figure 3

Pros1 inhibits p.g‐LPS–induced inflammation in hGECs. hGECs were treated with p.g‐LPS (1 μg/mL) and Pros1 (10 or 50 nmol/L), alone or in combination as indicated for 24 hours. Untreated cells (Control) served as control. (A‐C, G‐I) TNF‐α (A), IL‐6 (B), IL‐1β (C), MMP‐9 (G), MMP‐2 (H) and RANKL (I) mRNA levels by qRT‐PCR. (D‐F) TNF‐α (D), IL‐6 (E) and IL‐1β (F) protein concentrations in the culture supernatants by ELISA. (J, K) The enzymatic activities of MMP‐9 and MMP‐2 in the culture supernatants by gelatin zymography. (J, L) RANKL protein levels by Western blot analysis. n = 3, *P < 0.05, **P < 0.01

Figure 4

Pros1 inhibits p.g‐LPS–induced inflammation in hGECs via Tyro3. (A‐C) hGECs were treated with p.g‐LPS (1 μg/mL) and Pros1 (10 or 50 nmol/L), alone or in combination as indicated for 24 hours. Untreated cells (Control) served as control. Tyro3 mRNA (A) and protein (B, C) levels were determined by qRT‐PCR and Western blot analysis respectively. (D‐I) hGECs were treated with p.g‐LPS (1 μg/mL), Pros1 (50 nmol/L) and anti‐Tyro3 antibody (10 μg/mL), alone or in combination as indicated for 24 hours. (D‐F) TNF‐α (D), IL‐6 (E) and IL‐1β (F) protein concentrations in the culture supernatants were determined by ELISA. (G, H) The enzymatic activities of MMP‐9 and MMP‐2 in the culture supernatants were assessed by gelatin zymography. (G, I) RANKL protein levels were determined by Western blot analysis. n = 3, *P < 0.05, **P < 0.01

Figure 5

The Pros1/Tyro3 signalling ameliorates p.g‐LPS–induced inflammation in hGECs via SOCS1/3 and STAT1/3. hGECs were treated with p.g‐LPS (1 μg/mL), Pros1 (50 nmol/L) and anti‐Tyro3 antibody (10 μg/mL), alone or in combination as indicated for 24 hours. (A‐C) SOCS1, p‐STAT1, STAT1, SOCS3, p‐STAT3 and STAT3 protein levels were determined by Western blot analysis. Representative gel images (A) and relative protein expression by densitometric analysis (B, C) are shown. n = 3; *P < 0.05, **P < 0.01 vs p.g‐LPS; P < 0.05, P < 0.01 vs p.g‐LPS +Pros1; ^$$ P < 0.01 vs p.g‐LPS + anti‐Tyro3

Figure 6

Pros1 reduces alveolar bone loss and osteoclastogenesis in periodontitis rats. Five groups of rats (n = 6 per group): Control (no‐treatment), 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. (A) Representative images of micro‐CT, HE staining and TRAP staining of the maxilla sections. Arrows indicate TRAP‐positive multinucleated cells (active osteoclasts) between maxillary first and second molar and second and third molar. B, buccal surface; P, palatal surface; scale bars = 1 mm (micro‐CT); magnification = 200 × (HE and TRAP staining). (B) Bone levels evaluated by the average distance from cemento‐enamel junction (CEJ) to the alveolar bone crest (ABC) at the palatal groove site. n = 6, *P < 0.05, ns = nonspecific. (C) Periodontal osteoclastogenesis evaluated by the density of TRAP‐positive multinucleated cells on the surface of alveolar bone around the first molar. n = 6, *P < 0.05, **P < 0.01, ns = non‐specific

Figure 7

Pros1 attenuates periodontal inflammation in periodontitis rats. Five groups of rats (n = 6 per group): Control (no‐treatment), 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. (A) Representative images of immunohistochemical staining of the maxilla sections for Pros1, Tyro3 and RANKL. GE, gingival epithelium; PL, periodontal ligament; AB, alveolar bone; R, root. (B) Pros1, Tyro3 and RANKL mRNA levels in the periodontium by qRT‐PCR. (C‐F) MMP‐9 (C), MMP‐2 (D), TNF‐α (E) and IL‐6 (F) mRNA levels in the periodontium by qRT‐PCR. n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ns = non‐specific

Figure 8

Pros1 protects against periodontitis via SOCS1/3 and STAT1/3. Five groups of rats (n = 6 per group): Control (no‐treatment), 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