LL-37 quantification in gingival crevicular fluid (GCF) from periodontitis patients

Background: LL-37, an anti-microbial peptide belonging to the cathelicidin family, derives its name from two N-terminal leucine residues and the 37 amino acids comprising the peptide. LL-37 is the only known cathelicidin to exist in humans. It exhibits both anti-bacterial and immunomodulatory properties. Objectives: In the current study, LL-37 was quantified in GCF from periodontitis patients. Previous studies have relied on qualitative results from Western blotting to detect LL-37 in GCF. This study aims to quantitatively determine LL-37 levels in GCF. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 sites in 12 patients presenting with advanced periodontitis. Plaque samples were analysed by QPCR for the presence or absence of the periodontopathic bacterium Porphyromonas gingivalis (P. gingivalis). The concentrations of LL-37 in patient samples pre- and post-treatment were deduced by indirect enzyme linked immunosorbent assay (ELISA). Results: Concentrations of LL-37 in samples varied between a minimum and maximum of 1 and 40 ng/ml. LL-37 levels were shown, pre-treatment, to be higher in deep pockets (6-9 mm) compared with shallower pockets (3-5 mm) and highest in those sites which were positive for P. gingivalis. Non-surgical therapy resulted in a significant improvement in clinical indices while expression levels of P. gingivalis were reduced. Following treatment, LL-37 levels in GCF decreased from an average of 6.5 ± 1 - 5.8 ± 1.2 ng/ml. The most interesting observation however was the reduction in LL-37 levels, from an average of 7 ± 1.3 – 2.5 ± 1.1 ng/ml in those sites where P. gingivalis infection was eradicated post-treatment. Conclusions: LL-37 levels are increased at sites showing advanced periodontal disease, reduce following treatment and appear to be linked to the presence of P. gingivalis. This study will further our knowledge of host defence in chronic diseases such as periodontitis.

The role of LL-37 in periodontal wound healing

Introduction: As a result of chronic inflammation during periodontal disease the junctional epithelium becomes micro-ulcerated. The inflammatory process is mediated by both bacterial and host cell products. Host defence peptides such as defensins, secretory leucocyte protease inhibitor (SLPI) and the sole human cathelicidin, LL-37, are secreted by both periodontal cells and neutrophils into gingival crevicular fluid (GCF). They have the ability to modulate the immune response in periodontitis and are thought to have a potential role in periodontal wound healing. Objectives: The aims of this study were to determine the role of LL-37 in the production of Interleukin (IL)-8, IL-6, hepatocyte growth factor (HGF) and basic-fibroblast growth factor (bFGF) by gingival fibroblasts. The role of LL-37 in modulating total matrix metalloproteinase (MMP) activity and expression of tissue inhibitors of metalloproteinase (TIMP)-1 and -2 by gingival fibroblasts was also investigated. Methods: Primary gingival fibroblasts were co-cultured with concentrations of LL-37 (1, 5 and 10µg/ml) for 24 hours and their supernatants tested for levels of IL-8 and IL-6, HGF, bFGF, TIMP-1 and TIMP-2 by ELISA. Rates of MMP turnover in the supernatants were tested by fluorogenic assay using fluorescence resonance energy transfer (FRET) peptide substrates. Cytotoxicity was measured by MTT assay. Statistical significance was measured using the independent t-test and p<0.05 was considered significant. Results: LL-37 significantly upregulated levels of IL-8, IL-6, HGF, bFGF and TIMP-1 (p<0.05) in a dose-dependent fashion. LL-37 significantly decreased the total MMP activity (p<0.05). None of the LL-37 concentrations tested were cytotoxic to gingival fibroblasts. Conclusion: These results indicate that LL-37 is involved in periodontal wound healing. LL-37 increased levels of proinflammatory cytokines and increased levels of growth factors involved in re-epithelialisation. LL-37 has the ability to regulate remodelling of the periodontium by controlling MMP overactivity both directly and by stimulating production of inhibitors by gingival fibroblasts.

LL-37 Degradation by GCF: A Role for Porphyromonas gingivalis Proteinases?

Background: LL-37, composed of 37 amino acid residues, is an innate host defence peptide of the cathelicidin family. It is expressed by neutrophils, monocytes and epithelial cells and exhibits both anti-bacterial and immunomodulatory properties. LL-37 is however prone to proteolytic degradation by proteinases, thus potentially limiting its inherent host defence properties in the inflammatory milieu. Objectives: The present study was designed to determine whether LL-37 was degraded by components of gingival crevicular fluid (GCF) from healthy subjects or those with periodontitis. In addition, we aimed to deduce whether degradation of the peptide was accelerated in GCF samples which were determined to be positive for the periodontopathic bacterium Porphyromonas gingivalis. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 individual sites in patients presenting with advanced periodontitis. In healthy subjects, GCF samples only were collected. Plaque samples were analysed by QPCR for the presence or absence of P. gingivalis. Pooled GCF samples from healthy sites; periodontitis sites which were P. gingivalis negative (Pg-); or periodontitis sites which were P. gingivalis positive (Pg+), were incubated with synthetic LL-37 for 0 – 180 min. The degradation products were then analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Results: LL-37 was not degraded when incubated with GCF from healthy subjects. In contrast, LL-37 was degraded after 30 min when incubated with Pg- GCF. However degradation of LL-37 was apparent after only 2 min incubation with Pg+ GCF and the parent molecule was almost completely degraded after 30 min. Conclusions: The rapid degradation of LL-37, particularly in Pg+ sites, highlights the limited role which this host defence peptide may play in the presence of biologically active proteinases. It also underscores a potent virulence mechanism of P. gingivalis used to circumvent innate host responses.