Effects of Monolaurin on Oral Microbe-Host Transcriptome and Metabolome

Abstract

The aim of this in vitro study was to evaluate the effects of monolaurin against Aggregatibacter actinomycetemcomitans (Aa) and determine their effects on the host transcriptome and metabolome, using an oral cell/bacteria co-culture dual-chamber model to mimic the human periodontium. For this, the Aa, was applied to cross the monolayer of epithelial keratinocytes (OBA-9) to reach the fibroblasts layer (HGF-1) in the basal chamber. The Monolaurin treatments (25 or 50 μM) were added immediately after the inoculation of the dual-chamber with Aa. After 24 h, the transcriptional factors and metabolites produced were quantified in the remaining cell layers (insert and basal chamber) and in supernatant released from the cells. The genes IL-1α, IL-6, IL-18, and TNF analyzed in HGF-1 concentrations showed a decreased expression when treated with both concentration of Monolaurin. In keratinocytes, the genes IL-6, IL-18, and TNF presented a higher expression and the expression of IL-1α decreased when treated with the two cited concentrations. The production of glycerol and pyruvic acid increased, and the 2-deoxytetronic acid NIST, 4-aminobutyric acid, pinitol and glyceric acid, presented lower concentrations because of the treatment with 25 and/or 50 μM of Monolaurin. Use of monolaurin modulated the immune response and metabolite production when administered for 24 h in a dual-chamber model inoculated with A. actinomycetemcomitans. In summary, this study indicates that monolaurin had antimicrobial activity and modulated the host immune response and metabolite production when administered for 24 h in a dual-chamber model inoculated with A. actinomycetemcomitans.

Keywords: Aggregatibacter actinomycetemcomitans; fibroblast; immune system; keratinocyte; monolaurin; periodontal disease.

FIGURE 1

Cytotoxicity assay of cells treated with different doses of monolaurin in an oral cell/bacteria co-culture inoculated with A. actinomycetemcomitans: (A) HGF-1 cell (fibroblasts); (B) OBA-9 cell (keratinocytes). Cell viability was presented in percentage (%) ± standard deviation. Control group = 100% viability, n = 6. Non-treated cells were considered as 100% viability; DMSO was used as positive control to demonstrate an appropriate system-cell response (100% cytotoxicity – data not shown).

FIGURE 2

Percent concentrations of GM-CSF, IL-1α, IL-1-β, IL-6, and IL-10 by ELISA, obtained from cell culture supernatant. Oral cell co-culture inoculated with A. actinomycetemcomitans and treated with 25 μM of monolaurin (Mono 25) or 50 μM of monolaurin (Mono 50) compared with the control group. Control group is oral cell co-culture inoculated with A. actinomycetemcomitans and no treatment. The control group mean is expressed as 100% and treated groups have their mean relative to the control group. Mean ± standard deviation; n = 4.

FIGURE 3

Relative expression of genes from HGF-1 cells (fibroblasts) by quantitative PCR: (A) IL-1α; (B) IL-6; (C) IL-18; (D) CASP3; (E) MMP-1; (F) TNF. Oral cell/bacteria co-culture inoculated with A. actinomycetemcomitans and treated with different doses of monolaurin (25 μM – Mono 25 or 50 μM – Mono 50). Control group is oral cell co-culture inoculated with A. actinomycetemcomitans and no treatment. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. Different letters (a, b, and c) indicate statistical difference between groups. The results were expressed by mean ± standard deviation; n = 6 and p < 0.05.

FIGURE 4

Relative expression of genes from OBA-9 cells (keratinocytes) by quantitative PCR: (A) IL-1α; (B) IL-6; (C) IL-18; (D) CASP3; (E) MMP-1; (F) TNF. Oral cell/bacteria co-culture inoculated with A. actinomycetemcomitans and treated with different doses of monolaurin (25 μM – Mono 25 or 50 μM – Mono 50). Control group is oral cell co-culture inoculated with A. actinomycetemcomitans and no treatment. The control group has their mean expressed equal to 1 and treated groups have their mean relative to the control group. Different letters (a, b, and c) indicate statistical difference between groups. The results were expressed by mean ± standard deviation; n = 6 and p < 0.05.

FIGURE 5

Metabolites obtained from cell culture supernatant (HGF-1 and OBA-9 co-culture cells): (A) 2-deoxytetronic acid NIST; (B) 4-aminobutyric acid; (C) glyceric acid; (D) glycerol; (E) pinitol; (F) pyruvic acid. Oral cell/bacteria co-culture model inoculated with A. actinomycetemcomitans and treated with different doses of monolaurin (25 μM – Mono 25 or 50 μM – Mono 50). Control group is oral cell co-culture inoculated with A. actinomycetemcomitans and no treatment. Different letters (a, b, and c) indicate statistical difference between groups. The data is expressed in relative peak heights (mAU) from HPLCMS analysis, which are unitless (mean ± standard deviation); n = 4 and p < 0.05.