Abstract
Given the spread of antibiotic resistance in bacterial pathogens, antimicrobial peptides that can also modulate the immune response may be a novel approach for effectively controlling periodontal infections. In the present study, we used a three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) to investigate the anti-inflammatory properties of human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) and to determine whether these antimicrobial peptides can act in synergy. The 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to LPS stimulation compared to fibroblasts and epithelial cells alone. The 3D co-culture model was stimulated with non-cytotoxic concentrations of hBD-3 (10 and 20 µM) and LL-37 (0.1 and 0.2 µM) individually and in combination in the presence of A. actinomycetemcomitans LPS. A multiplex ELISA assay was used to quantify the secretion of 41 different cytokines. hBD-3 and LL-37 acted in synergy to reduce the secretion of GRO-alpha, G-CSF, IP-10, IL-6, and MCP-1, but only had an additive effect on reducing the secretion of IL-8 in response to A. actinomycetemcomitans LPS stimulation. The present study showed that hBD-3 acted in synergy with LL-37 to reduce the secretion of cytokines by an LPS-stimulated 3D model of gingival mucosa. This combination of antimicrobial peptides thus shows promising potential as an adjunctive therapy for treating inflammatory periodontitis.
Highlights
Periodontitis is a multifactorial chronic inflammatory disease of polymicrobial origin that causes the destruction of the toothsupporting tissues, including the periodontal ligament and alveolar bone [1]
To determine whether the interactions between the two cell types modified the response to LPS stimulation, the secretion of IL-6 and IL-8 by each cell type and by the 3D co-culture model was determined by enzyme-linked immunosorbent assay (ELISA)
While the stimulation of the fibroblasts with LPS did not significantly increase IL-6 and IL-8 secretion, the stimulation of the epithelial cells with LPS resulted in the secretion of higher amounts of both cytokines compared to the unstimulated cells (Figure 3)
Summary
Periodontitis is a multifactorial chronic inflammatory disease of polymicrobial origin that causes the destruction of the toothsupporting tissues, including the periodontal ligament and alveolar bone [1]. The lipopolysaccharide (LPS) of A. actinomycetemcomitans is a major virulence factor that can promote adhesion to oral cells and can activate the host immune response, resulting in the secretion of large amounts of pro-inflammatory cytokines, including interleukin-6 (IL-6) and interleukin-8 (IL-8) that contribute to the destruction of periodontal tissues [4,5,6]. Epithelial cells and fibroblasts are the predominant cells of periodontal tissues and serve as a first line of defense against periodontopathogens. They act as a mechanical barrier against bacterial invasion in addition to secreting different classes of inflammatory mediators and tissue-destructive enzymes in response to pathogen stimulation. Higher levels of IL-8 and monocyte chemo-attractant protein 1 (MCP-1) have been found in gingival crevicular fluid (GCF) from periodontitis sites than in GCF from healthy control sites, while their levels decrease after periodontal treatments [8,9,10,11,12]
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