Activity of antimicrobial peptide mimetics in the oral cavity: II. Activity against periopathogenic biofilms and anti-inflammatory activity

Abstract

Whereas periodontal disease is ultimately of bacterial etiology, from multispecies biofilms of gram-negative anaerobic microorganisms, much of the deleterious effects are caused by the resultant epithelial inflammatory response. Hence, development of a treatment that combines anti-biofilm antibiotic activity with anti-inflammatory activity would be of great utility. Antimicrobial peptides (AMPs) such as defensins are naturally occurring peptides that exhibit broad-spectrum activity as well as a variety of immunomodulatory activities. Furthermore, bacteria do not readily develop resistance to these agents. However, clinical studies have suggested that they do not represent optimal candidates for exogenous therapeutic agents. Small-molecule mimetics of these AMPs exhibit similar activities to the parent peptides, in addition to having low toxicity, high stability and low cost. To determine whether AMP mimetics have the potential for treatment of periodontal disease, we examined the activity of one mimetic, mPE, against biofilm cultures of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Metabolic assays as well as culture and biomass measurement assays demonstrated that mPE exhibits potent activity against biofilm cultures of both species. Furthermore, as little as 2 μg ml(-1) mPE was sufficient to inhibit interleukin-1β-induced secretion of interleukin-8 in both gingival epithelial cells and THP-1 cells. This anti-inflammatory activity is associated with a reduction in activation of nuclear factor-κB, suggesting that mPE can act both as an anti-biofilm agent in an anaerobic environment and as an anti-inflammatory agent in infected tissues.

Figure 1

Activity of mPE against Aggregatibacter actinomycetemcomitans biofilms. Strain 1005 of A. actinomycetemcomitans was grown in AAGM in 96-well plates until complete confluence. Two-fold dilutions of mPE were added for 24 h (A) or 5 min to 2 h (C). Medium was removed and replaced with RPMI-1640 with XTT and grown for a further 3 h. Metabolic activity was quantified by measuring the optical density (OD) at 450 nm and 600 nm. Results are shown as % reduction in the OD₄₅₀–OD₆₀₀ from untreated cultures. The experiment in (A) was carried out in triplicate, and error bars are ± SD. In (B) parallel wells were stained with Crystal violet, destained and quantified by reading OD₆₀₀; bacteria were removed and plated on AAGM agar plates to quantify viable colonies. Results are shown as % reduction from untreated control.

Figure 2

Activity of mPE against biofilms of Porphyromonas gingivalis. Biofilms were grown in a 96-well plate for 21 days in an anaerobic chamber in brain–heart infusion (BHI) medium. (A) The mPE was added for 24 h, and replaced with XTT in RPMI-1640 as described in Fig. 1. (B) After XTT measurement, wells were stained with Crystal violet, and staining was quantified by reading optical density at 600 nm (OD₆₀₀). Values are presented as mean ± standard deviation. The difference in both metabolic activity and OD between the lowest concentration and 8 µg ml⁻¹ was significant at P < 0.002 as determined by Student’s t-test.

Figure 3

Anti-inflammatory activity of mPE. The oral keratinocyte cell line OKF6/TERT (A) or THP-1 cells (B) were treated with 100 ng ml⁻¹ recombinant human interleukin-1β (IL-1β) in the presence of 0, 2 or 5 µg ml⁻¹ mPE for 6 h (A) or 2 h (B). The concentration of IL-8 secreted into the medium was quantified by enzyme-linked immunosorbent assay (SA Biosciences). No IL-8 was observed in either case in the absence of IL-1β (not shown). The experiment was carried out in quadruplicate; error bars represent + SD. The inhibition by mPE was significant at both concentrations with P < 0.01, as determined by Student’s t-test.

Figure 4

Mechanism of anti-inflammatory activity of mPE. (A) Effect on nuclear factor-κB-regulated genes. OKF6/TERT cells were treated with mPE as above in the presence of 100 ng ml⁻¹ interleukin-1β (IL-1β). Total mRNA was isolated and IL-8 and hBD-2 mRNA levels were quantified by quantitative polymerase chain reaction normalized to β2-microglobulin. Levels are shown relative to the no-mPE sample for each group. The experiment was carried out in triplicate; error bars represent + SD. (B) Effect on IκB phosphorylation. OKF6/TERT cells were grown in 96-well plates, treated with 100 ng ml-1β IL-1β for 2 or 4 h in the presence of 0, 2 or 5 µg ml⁻¹ mPE. Phosphorylation of IκB was measured using the CASE assay (SA Biosciences) and quantified relative to total IκB levels. Phosphorylated IκB levels/total IκB of IL-1β-treated cultures are shown compared with untreated cultures. The experiment was carried out twice in quadruplicate. Reductions in pIκB/total IκB are significant at P < 0.002.