Milk fat globule epidermal growth factor 8 inhibits periodontitis in non-human primates and its gingival crevicular fluid levels can differentiate periodontal health from disease in humans

Abstract

Aim: We have previously shown that the secreted glycoprotein milk fat globule epidermal growth factor 8 (MFG-E8) has anti-inflammatory and anti-osteoclastogenic properties. Our objective was to investigate the potential of MFG-E8 as a diagnostic or therapeutic agent in periodontitis.

Materials and methods: Periodontitis was induced in non-human primates (NHPs) by placing ligatures around posterior teeth on both halves of the mandible for a split-mouth design: one side was treated with MFG-E8-Fc and the other with Fc control. Disease was assessed by clinical periodontal examinations, radiographic analysis of bone loss, and analysis of cytokine mRNA expression in gingival biopsy samples. Gingival crevicular fluid (GCF) was collected from human healthy volunteers or subjects with gingivitis, chronic moderate periodontitis, or chronic severe periodontitis. Additionally, GCF was collected from a subset of severe periodontitis patients following scaling and root planing (SRP) and after pocket reduction surgery. GCF was analysed to quantify MFG-E8 and periodontitis-relevant cytokines using multiplex assays.

Results: In NHPs, sites treated with MFG-E8-Fc exhibited significantly less ligature-induced periodontal inflammation and bone loss than Fc control-treated sites. In humans, the GCF levels of MFG-E8 were significantly higher in health than in periodontitis, whereas the reverse was true for the proinflammatory cytokines tested. Consistently, MFG-E8 was elevated in GCF after both non-surgical (SRP) and surgical periodontal treatment of periodontitis patients.

Conclusion: MFG-E8 is, in principle, a novel therapeutic agent and biomarker of periodontitis.

Keywords: cytokines; inflammation; milk fat globule epidermal growth factor 8; non-human primates; periodontitis.

Figure 1. MFG-E8-Fc decreases inflammatory clinical parameters of NHP periodontitis

Starting 3 days after initiation of ligature-induced periodontitis, MFG-E8-Fc or Fc control (both at 50 μg) were injected locally into the mandibular interdental papillae from the first premolar to the second molar, three times weekly, in opposite sides of the mouth (split-mouth design). The animals were clinically examined at the indicated timepoints and the effects of MFG-E8-Fc on the indicated inflammatory clinical parameters were recorded: (A) probing pocket depth (PPD), (B) gingival index (GI), (C) bleeding on probing (BOP), (D) mobility index (Mob), and (E) plaque index (PI). Data are means ± SD (n = 3 monkeys). *P < 0.05; **P < 0.01 compared with time-matched control (paired t test).

Figure 2. Inhibition of periodontal bone loss after treatment of NHP periodontitis with MFG-E8-Fc

Three monkeys were treated as described in the legend to figure 1, and their mandibular bone heights (CEJ-ABC distance) were measured using Nikon Imaging System software and standardized X-ray images (taken at baseline and at week 6). Measurements were made at six points (first premolar, distal; second premolar, mesial and distal; first molar, mesial and distal; second molar, mesial) and the data in A and B reflect the 6-site total at baseline and at week 6, respectively. For each pair of Fc control and MFG-E8-Fc treatments, bone loss was calculated as bone height at baseline minus bone height at 6 week (C); the difference between Fc control and MFG-E8-Fc treatments was significant (P < 0.05; paired t test).

Figure 3. Effect of MFG-E8-Fc treatment on cytokine expression in the NHP periodontal tissue

Dissected gingiva were processed for real-time PCR to determine mRNA expression of the indicated cytokines. Results were normalized to those of GAPDH mRNA and were presented as fold change relative to the mRNA transcript levels of the Fc control group, which were assigned an average value of 1. The bar graphs indicate the means ± SD (n = 3 animals, each of which is represented with a distinct symbol). *P < 0.05; **P < 0.01 versus Fc control (paired t test).

Figure 4. Flowchart showing distribution of human subjects in health, disease and treatment groups

GCF was collected at baseline from all subjects. GCF collection was repeated for a subset of chronic severe periodontitis patients following non-surgical treatment (SRP) and after surgical treatment for pocket reduction.

Figure 5. GCF levels of MFG-E8 and indicated cytokines in periodontal health and disease

GCF was collected from 40 subjects classified as either periodontally healthy or as having plaque-induced gingivitis, chronic moderate periodontitis, or chronic severe periodontitis. GCF was sampled from a total of 200 distinct sites (5 sites per individual; see Fig. 4 for site distribution among groups). Bead-based multiplex assays on a Bio-Plex system were used to assay the following molecules: (A) MFG-E8, (B) IL-1β, (C) IL-6, (D) IL-17A, (E) RANKL and (F) OPG. Dots represent individual values from distinct sampled sites and each box in the box-and-whisker plots extends from the 25th to the 75th percentiles. **P < 0.01; ****P < 0.0001 between indicated groups (Repeated measures analysis as implemented in SAS Proc GENMOD). ND, not detectable; NS, not significant.

Figure 6. GCF levels of MFG-E8 and indicated cytokines after treatment of severe periodontitis

GCF was collected from 5 subjects classified as having chronic severe periodontitis at baseline, at a re-assessment appointment following non-surgical periodontal treatment (SRP) and after pocket reduction surgery. GCF was sampled from a total of 30 distinct sites (6 sites per individual). Bead-based multiplex assays on a Bio-Plex system were used to assay the following molecules: (A) MFG-E8, (B) IL-6, (C) IL-17A, and (D) RANKL. (E) Measured deep probing pocket depths (PPD) before and after treatments. In A-D, dots represent individual values from distinct sampled sites and each box in the box-and-whisker plots extends from the 25th to the 75th percentiles. In E, data are means ± SD (n = 30 sites). **P < 0.01; ****P < 0.0001 between indicated groups (Repeated measures analysis as implemented in SAS Proc GENMOD).